Comparative Genomic Analysis of P-type ATPase Inhibitors: From Structural Insights to Targeted Drug Development

Abstract

P-type ATPases represent a crucial family of ion pumps targeted for therapeutic intervention in conditions ranging from cancer to fungal infections. This article provides a comprehensive analysis of P-type ATPase inhibitors through a comparative genomic lens. We explore the foundational genomic and structural characteristics of key ATPase targets, including recent cryo-EM revelations of novel binding partners. The review systematically examines methodological approaches for inhibitor identification and optimization, highlighting computational design, high-throughput screening, and structure-activity relationships. Critical challenges in achieving selectivity and overcoming resistance are addressed, alongside validation strategies through comparative inhibition profiling across ATPase families. This synthesis provides researchers and drug development professionals with an integrated framework for advancing next-generation ATPase-targeted therapeutics with improved specificity and clinical potential.

Genomic Diversity and Structural Biology of P-type ATPase Families

P-type ATPases constitute a large superfamily of primary active transporters that are found in all domains of life. These biological pumps are defined by the formation of a phosphorylated intermediate (hence "P-type") during their catalytic cycle and function in the transport of various substrates across cellular membranes. Based on phylogenetic analyses and substrate specificities, P-type ATPases are classified into five major families (P1-P5), with a sixth family (P6) more recently identified. This review provides a comprehensive comparison of these families, focusing on their evolutionary relationships, structural characteristics, substrate specificities, and physiological roles. We further synthesize current knowledge on experimental approaches for characterizing these pumps and their relevance as targets in therapeutic development, particularly in the context of comparative genomic analysis of P-type ATPase inhibitors research.

P-type ATPases, also known as E1-E2 ATPases, are a large group of evolutionarily related ion and lipid pumps found in bacteria, archaea, and eukaryotes [1]. They are primary transporters named for their ability to catalyze auto-phosphorylation of a conserved aspartate residue using adenosine triphosphate (ATP) as an energy source [1] [2]. These proteins share a common catalytic mechanism described by the Post-Albers cycle, alternating between at least two major conformational states (E1 and E2) to transport substrates across membranes [3] [2].

The first P-type ATPase discovered was the Na+/K+-ATPase, isolated by Jens Christian Skou in 1957, for which he received the Nobel Prize in 1997 [1] [2]. Since then, the family has expanded significantly, with current classifications identifying multiple distinct families based on phylogenetic analyses. The evolutionary relationship between these families remains partially unresolved, though they all appear to have originated from a common ancestor protein [2]. P-type ATPases serve crucial functions in human physiology, including nerve impulse conduction, muscle relaxation, kidney secretion and absorption, and intestinal nutrient absorption [1]. Their malfunction is linked to various diseases, making them important therapeutic targets.

Phylogenetic Classification and Comparative Analysis

Phylogenetic analysis of P-type ATPase sequences reveals their evolutionary relationships and provides the basis for their classification. Initial analysis in 1998 by Axelsen and Palmgren of 254 available sequences identified five major families (P1-P5) [2]. Subsequent research has confirmed these families and led to the identification of an additional family, P6 ATPases [2].

Table 1: Major P-type ATPase Families and Their Characteristics

Family	Subfamilies	Main Substrates	Representative Members	Organism Distribution	Transmembrane Helices
P1	P1A, P1B	K+, Cu+, Ag+, Cu2+, Zn2+, Cd2+, Pb2+, Co2+	KdpB, CopA, ZntA	Prokaryotes, Eukaryotes	7-8
P2	P2A, P2B, P2C	Ca2+, Na+/K+, H+/K+	SERCA1a, Na+/K+-ATPase	Eukaryotes	10
P3	-	H+	Plasma membrane H+-ATPase	Plants, Fungi	10
P4	-	Phospholipids	ATP8A1, ATP11C	Eukaryotes	10
P5	P5A, P5B	Misinserted helices, Polyamines	Spf1, ATP13A2	Eukaryotes	10-12
P6	-	Not fully characterized	-	-	-

Table 2: Key Structural Features of P-type ATPase Families

Family	Characteristic Motifs	Accessory Subunits	Cargo-binding Location	Unique Structural Features
P1A	DKTGT	KdpA, KdpC, KdpF	KdpA subunit	Heterotetrameric complex
P1B	DKTGT, CPx/SPC	Heavy-metal-binding domains	Transmembrane domain	N-terminal metal-binding domains
P2	DKTGT, TGES	β-subunit (P2C)	Transmembrane domain	Well-characterized E1-E2 conformations
P3	DKTGT	-	Transmembrane domain	C-terminal regulatory domain
P4	DKTGT	CDC50	Transmembrane domain	Lipid flippase activity
P5A	DKTGT	-	Between TM2,4,6	NTD, helix dislocase activity
P5B	DKTGT, VPPALP	-	Between TM2,4,6	Electronegative polyamine-binding cleft

The phylogenetic tree of P-type ATPases demonstrates that diversification occurred prior to the separation of eubacteria, archaea, and eukaryota, underlining the significance of this protein family for cell survival [1]. The classification is based primarily on conserved sequence motifs and substrate specificity rather than organismal origin, with each family having distinct structural and functional characteristics [2].

P1 ATPases: Heavy Metal and Potassium Pumps

P1 ATPases (Type I ATPases) consist of transition/heavy metal ATPases and are particularly predominant in prokaryotes, where they are approximately tenfold more abundant than other types [1]. This family is divided into two main subfamilies:

P1A ATPases function as potassium import pumps but are atypical because they operate as part of a heterotetrameric complex (KdpFABC), where the actual K+ transport is mediated by the KdpA subunit rather than the catalytic KdpB ATPase itself [1] [2]. These pumps have the simplest catalytic subunit among P-type ATPases, with only seven transmembrane helices, yet they form the most complicated quaternary structure [2].

P1B ATPases are involved in transport of "soft" Lewis acids including Cu+, Ag+, Cu2+, Zn2+, Cd2+, Pb2+ and Co2+ [1]. They are further classified based on their metal specificity, with PIB-4-ATPases having the broadest cargo scope among heavy metal transporters [3] [4]. These proteins are key elements for metal resistance and homeostasis across diverse organisms. The PIB-4-ATPases, such as sCoaT from Sulfitobacter sp. NAS14-1, lack classical heavy-metal-binding domains (HMBDs) present in other PIB-ATPases, representing some of the simplest and shortest proteins within the entire P-type ATPase superfamily [3] [4].

P2, P3, and P4 ATPases: Cation and Lipid Pumps

P2 ATPases are primarily found in eukaryotes and include some of the most well-characterized P-type ATPases. The P2A subfamily includes sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), while P2C includes Na+/K+- and H+/K+-ATPases [1] [2]. These pumps typically have 10 transmembrane helices and require accessory subunits for proper function. The structure of SERCA1a is generally considered representative of the overall fold of P-type ATPases [1].

P3 ATPases are plasma membrane proton pumps found in plants and fungi [1]. They share structural similarities with P2 ATPases but have distinct regulatory mechanisms, including a C-terminal regulatory domain that inhibits pumping when unphosphorylated [1].

P4 ATPases function as phospholipid flippases, transporting phospholipids across membranes to maintain asymmetric lipid distribution [1]. These are found exclusively in eukaryotes and typically form heteromeric complexes with CDC50 subunits [5]. They share structural similarities with P5 ATPases in their cargo-binding regions despite transporting dramatically different substrates [5].

P5 and P6 ATPases: Eukaryotic Specialists

P5 ATPases are found exclusively in eukaryotes and represent the least characterized class until recently [2] [5]. They are subdivided into P5A and P5B subfamilies. P5A-ATPases, such as yeast Spf1, function as helix dislocases that remove mis-inserted hydrophobic helices from the ER membrane [5]. P5B-ATPases, including human ATP13A2-5, export physiologically important polyamines such as spermine from lysosomes to the cytosol [5]. Mutations in ATP13A2 cause Kufor-Rakeb syndrome, a form of Parkinson's disease associated with dementia [5].

P6 ATPases were identified more recently than the other families, and limited information is available about their specific substrates and functions [2]. Their placement in the phylogenetic tree and distinct characteristics warranted their classification as a separate family.

Structural Architecture and Transport Mechanism

Conserved Structural Domains

All P-type ATPases share a common structural organization consisting of three cytoplasmic domains and a transmembrane domain [1] [2]:

• Phosphorylation (P) domain: Contains the conserved aspartate residue (in DKTGT motif) that gets phosphorylated during the catalytic cycle. This domain has a Rossmann fold characteristic of the haloacid dehalogenase (HAD) superfamily [1].

• Nucleotide-binding (N) domain: Serves as a built-in protein kinase that phosphorylates the P domain. It contains the ATP-binding pocket and consists of a seven-strand antiparallel β-sheet between two helix bundles [1].

• Actuator (A) domain: Functions as a built-in protein phosphatase that dephosphorylates the phosphorylated P domain. It uses a highly conserved TGES motif and consists of a distorted jellyroll structure with two short helices [1].

• Transmembrane (M) domain: Typically composed of 6-12 transmembrane helices (varying by family) that contain the substrate-binding sites. The core transport domain consists of six transmembrane segments (M1-M6), with additional segments providing structural support [1].

Some P-type ATPases also contain regulatory (R) domains, such as the N-terminal heavy metal-binding domains in P1B ATPases or the C-terminal regulatory domains in P3A ATPases [1].

The Post-Albers Reaction Cycle

P-type ATPases operate through the Post-Albers cycle, which involves four principal states [3] [2]:

• E1 state: Inward-facing conformation with high affinity for cytoplasmic substrates.

• E1P state: Phosphorylated state with occluded substrates following ATP hydrolysis.

• E2P state: Outward-facing conformation with low substrate affinity, allowing release.

• E2 state: Dephosphorylated state that can bind counter-substances.

The cycle completes with the return to E1 state, transporting substrates against their concentration gradient using ATP hydrolysis energy. Phosphorylation occurs at the conserved aspartate residue in the P-domain, inducing conformational changes that alternate access to substrate-binding sites [2].

Family-Specific Structural Adaptations

Different P-type ATPase families exhibit specific structural adaptations related to their substrates and functions:

P1B-ATPases feature 8 transmembrane helices and often contain N-terminal heavy-metal-binding domains (HMBDs) that regulate their activity [3] [4]. However, PIB-4-type ATPases lack these classical HMBDs, representing a structural simplification [3] [4].

P5B-ATPases contain a unique N-terminal domain and an electronegative polyamine-binding cleft formed between transmembrane segments 2, 4, and 6, lined with conserved residues critical for spermine recognition [5]. Despite transporting dramatically different cargo, P5B-ATPases share transport pathway principles with P4- and P5A-ATPases [5].

Experimental Approaches for P-type ATPase Characterization

Structural Biology Methods

X-ray crystallography has been instrumental in determining high-resolution structures of P-type ATPases. The first structure solved was SERCA1a from rabbit sarcoplasmic reticulum, which established the overall architecture of P-type ATPases [1]. More recently, structures of PIB-4-type ATPases like sCoaT have been determined using X-ray diffraction, revealing novel features such as the absence of HMBDs and unique ion-release mechanisms [3] [4].

Cryo-electron microscopy has emerged as a powerful technique for determining structures of challenging membrane proteins. Recent cryo-EM structures of the P5B-ATPase Ypk9 provided insights into polyamine recognition and transport mechanisms, depicting various transport cycle intermediates at resolutions reaching 3.4 Å [5].

Functional Assays

ATPase activity assays measure phosphate release during ATP hydrolysis. The Baginski assay and Malachite Green Phosphate Assay are commonly used to determine metal-dependent ATPase activity, as employed in characterizing sCoaT's specificity for Zn2+, Cd2+, and Co2+ [3] [4].

Transport assays directly measure substrate movement across membranes. These include radioactive tracer studies, fluorescence-based methods, and electrophysiological approaches that monitor transport kinetics and specificity.

Pharmacological inhibition studies utilize specific inhibitors to characterize ATPase function. For example, cardiac glycosides like ouabain specifically inhibit Na+/K+-ATPase, while novel inhibitory compounds identified through high-throughput screening can block PIB-4-ATPase activity with potential antibacterial applications [3] [2].

Table 3: Essential Reagents and Methods for P-type ATPase Research

Category	Specific Examples	Application/Function	Key Features
Activity Assays	Baginski assay, Malachite Green	Measure ATPase activity	Colorimetric phosphate detection
Structural Tools	Beryllium fluoride (BeF3-), Aluminum fluoride (AlF4-)	Stabilize specific conformations	Phosphate analogs for E2P states
Expression Systems	Saccharomyces cerevisiae, E. coli	Heterologous protein production	Enable recombinant ATPase study
Membrane Mimetics	Nanodiscs, Detergents (DDM)	Solubilize and stabilize proteins	Maintain native-like environment
Inhibitors	Ouabain, Orthovanadate, Novel compounds	Mechanistic and functional studies	Target specific states or families

Genomic and Bioinformatics Approaches

Comparative genomics analyzes sequence conservation and diversity across species. Pan-genome analysis of bacterial species like Enterobacter xiangfangensis and Pantoea agglomerans identifies heavy metal resistance genes, including P-type ATPases, and their distribution across strains [6] [7].

Phylogenetic analysis reconstructs evolutionary relationships using sequence alignments and tree-building algorithms. These analyses have established the major P-type ATPase families and their evolutionary history [2].

Homology modeling predicts structures based on related solved structures, allowing functional inferences for uncharacterized family members.

Research Applications and Therapeutic Implications

Agricultural and Environmental Applications

P-type ATPases have significant potential in agricultural and environmental applications. Engineering plants with enhanced P1B-4-ATPase expression could create species capable of removing heavy metals from contaminated soils through phytoremediation [3] [4]. Bacterial P-type ATPases contribute to heavy metal resistance in soil bacteria, enabling survival in polluted environments and potential use in bioremediation [6].

In plant growth promotion, rhizobacterial P-type ATPases help maintain metal homeostasis under stress conditions, contributing to the beneficial effects of Plant Growth-Promoting Rhizobacteria (PGPR) like Pantoea agglomerans [7].

Medical Applications and Drug Discovery

P-type ATPases represent valuable targets for drug discovery across multiple therapeutic areas:

Antibacterial development: PIB-4-ATPases function as virulence factors in many bacterial pathogens, including Mycobacterium tuberculosis (MtbCtpD), making them attractive targets for novel antibiotics [3] [4]. High-throughput screening has identified inhibitory compounds that block PIB-4-ATPase activity and kill bacteria [3].

Cancer therapeutics: Copper-transporting P1B-ATPases (ATP7A and ATP7B) influence sensitivity to platinum-based chemotherapeutic drugs and are linked to drug resistance in cancers like ovarian cancer [8]. Modulating their activity could overcome treatment resistance.

Neurological disorders: P5B-ATPases (ATP13A2) play crucial roles in lysosomal polyamine transport, with mutations causing Parkinson's disease-related syndromes [5]. Understanding their function provides insights into neurodegenerative disease mechanisms.

Rare genetic diseases: Malfunction of human P1B-ATPases ATP7A and ATP7B causes Menkes disease and Wilson disease, respectively - fatal neurological disorders resulting from copper homeostasis disruption [3] [4].

Future Perspectives

The study of P-type ATPases continues to evolve with several promising research directions:

Structural characterization of underrepresented families: While P2 ATPases are well-characterized structurally, other families like P5 and P6 remain less understood. Future structural studies will provide crucial insights into their unique mechanisms.

Mechanistic studies of transport diversity: Understanding how different families recognize and transport chemically diverse substrates (ions, phospholipids, polyamines) through conserved structural frameworks represents a fundamental challenge.

Therapeutic targeting: Developing specific modulators of disease-relevant P-type ATPases offers promising therapeutic opportunities, particularly for antibiotic-resistant infections, cancer, and neurodegenerative disorders.

Evolutionary analysis: More comprehensive phylogenetic analyses incorporating sequences from diverse organisms may reveal additional P-type ATPase families and clarify evolutionary relationships between existing families.

The continued investigation of P-type ATPase families from type I to VI will undoubtedly yield new biological insights and therapeutic opportunities, solidifying their importance as fundamental cellular components and valuable drug targets.

P-type ATPases constitute a large superfamily of primary active transporters that are found in bacteria, archaea, and eukaryotes [1]. These biological pumps are characterized by their ability to catalyze auto-phosphorylation of a conserved aspartate residue during their catalytic cycle, utilizing energy derived from adenosine triphosphate (ATP) hydrolysis to transport substrates across biological membranes [2] [1]. The significance of these enzymes in biology cannot be overstated—they are responsible for establishing electrochemical gradients that drive essential cellular processes, including nerve impulses, muscle relaxation, kidney secretion and absorption, and nutrient absorption in the intestine [1]. What makes this protein family particularly fascinating from a comparative genomic and drug discovery perspective is their conserved catalytic subunit architecture, which features a characteristic arrangement of four core domains: the transmembrane domain (TMD), actuator domain (A-domain), phosphorylation domain (P-domain), and nucleotide-binding domain (N-domain) [9] [2] [1]. This architectural conservation across evolutionarily distant species, despite divergent substrate specificities, positions P-type ATPases as promising targets for the development of novel therapeutic agents, including antifungal drugs and anticancer treatments [9] [10].

The P-type ATPase superfamily is phylogenetically divided into five main subfamilies (P1-P5) based on substrate specificity, with additional subgroups identified within these categories [2] [11] [1]. While these subfamilies transport remarkably diverse substrates—ranging from protons and metal cations to phospholipids—they share a structurally conserved core machinery that enables the conversion of chemical energy from ATP hydrolysis into vectorial transport [2]. This review provides a systematic comparison of the conserved domain architecture across P-type ATPase subfamilies, examines experimental approaches for investigating these proteins, and discusses the implications for inhibitor development within the context of comparative genomic analysis.

Core Domain Architecture and Mechanistic Principles

The Post-Albers Catalytic Cycle

All P-type ATPases operate through a fundamental mechanism known as the Post-Albers cycle, which describes the transition between two major conformational states termed E1 and E2 [2]. According to this model, P-type ATPases alternate between inward-facing (E1) and outward-facing (E2) conformations through a series of intermediate states [2] [1]. The cycle begins with binding of the cytoplasmic substrate to the E1 state, which has high affinity for the substrate to be exported. ATP binding and subsequent phosphorylation of the conserved aspartate residue in the P-domain induces a conformational change to E1P, which spontaneously converts to E2P. The E2P state has low affinity for the exported substrate and releases it to the other side of the membrane. Dephosphorylation returns the pump to the E2 state, which has high affinity for the substrate to be imported from the extracellular side. Finally, the pump returns to the E1 state, completing the cycle [2] [1]. This elegant mechanism allows the pump to alternate the accessibility of its substrate-binding site from one side of the membrane to the other, enabling direct coupling between ATP hydrolysis and substrate translocation against a concentration gradient.

Four Core Domains and Their Functions

The catalytic subunit of approximately 70-140 kDa contains four core domains that work in concert to execute the Post-Albers cycle [1]. These domains include:

• Transmembrane Domain (TMD): Typically composed of ten transmembrane helices (M1-M10) that form the pathway for substrate translocation across the lipid bilayer [9] [1]. The TMD contains the binding sites for transported substrates located near the midpoint of the bilayer [1]. While most subfamilies have 10 transmembrane helices, notable exceptions include P1A ATPases (predicted to have 7) and P1B heavy metal pumps (predicted to have 8 transmembrane helices) [1]. Common to all P-type ATPases is a core of six transmembrane-spanning segments (M1-M6) that harbors the binding sites for the translocated ligands [1].

• Phosphorylation Domain (P-domain): This cytoplasmic domain contains the conserved aspartate residue that becomes phosphorylated during the reaction cycle within a signature DKTGT motif [1]. The P-domain folds into a Rossmann fold characterized by a seven-strand parallel β-sheet with eight short associated α-helices [1]. The folding pattern and catalytic residues place P-type ATPases within the haloacid dehalogenase (HAD) superfamily, which operates through an SN2 reaction mechanism for aspartate ester formation [1].

• Nucleotide-Binding Domain (N-domain): Serving as a built-in protein kinase, this domain phosphorylates the P-domain [1]. It is composed of a seven-strand antiparallel β-sheet between two helix bundles and contains the ATP-binding pocket [1]. The N-domain docks into a cleft formed by the P-domain, facilitating ATP-driven conformational changes [11].

• Actuator Domain (A-domain): Functioning as a built-in protein phosphatase, this domain dephosphorylates the phosphorylated P-domain using a highly conserved TGES motif [1]. The A-domain adopts a distorted jellyroll structure with two short helices and plays a pivotal role in transposing energy from ATP hydrolysis in the cytoplasmic domains to substrate transport in the transmembrane domain [1]. It modulates the occlusion of transported ligands in the transmembrane binding sites [1].

The structural coordination between these domains ensures tight coupling between ATP hydrolysis in the cytoplasmic headpiece (40 Å away) and substrate translocation through the membrane [1]. This intricate mechanism has been conserved through evolution despite the diversification of substrate specificity across P-type ATPase subfamilies.

Table 1: Core Domains of P-type ATPases and Their Functions

Domain	Structural Features	Functional Role	Conserved Motifs
Transmembrane Domain (TMD)	6-10 transmembrane helices; forms central cavity for transport [9] [11]	Substrate binding and translocation across membrane; determines ion specificity [1]	Varies by subfamily; contains substrate-binding residues
Phosphorylation Domain (P-domain)	Rossmann fold; 7-strand parallel β-sheet with 8 α-helices [1]	Accepts phosphoryl group during catalytic cycle; energy transduction [1]	DKTGT (Aspartate phosphorylation site) [1]
Nucleotide-Binding Domain (N-domain)	7-strand antiparallel β-sheet between helix bundles [1]	ATP binding and hydrolysis; phosphorylates P-domain [1]	ATP-binding pocket [11]
Actuator Domain (A-domain)	Distorted jellyroll structure with two short helices [1]	Dephosphorylation of P-domain; energy transduction to TMD [1]	TGES (dephosphorylation motif) [1]

Diagram 1: The Post-Albers catalytic cycle of P-type ATPases, showing transitions between major conformational states during substrate transport.

Comparative Analysis of P-type ATPase Subfamilies

P1 ATPases: Potassium and Heavy Metal Pumps

P1 ATPases comprise transition and heavy metal ATPases that predominantly occur in prokaryotes [1]. The P1A subfamily (Type IA) represents atypical P-type ATPases that function as part of a heterotetrameric complex called KdpFABC, where the actual K+ transport is mediated by another subcomponent rather than the catalytic subunit itself [1]. These ATPases have the simplest catalytic subunit among P-type ATPases, with only seven transmembrane helices, yet they form the most complicated tertiary structure with four subunits [2]. Strikingly, these pumps exhibit the highest ligand affinity of any P-type ATPases and possibly the highest degree of ligand specificity [2].

P1B ATPases (Type IB) transport soft Lewis acids including Cu+, Ag+, Cu2+, Zn2+, Cd2+, Pb2+, and Co2+ [1]. They are crucial for metal resistance and homeostasis across diverse organisms [1]. Unlike most other P-type ATPases, P1B ATPases possess additional N- and C-terminal heavy metal-binding domains that regulate their function [1]. Metal binding to transmembrane metal-binding sites in Cu+-ATPases is essential for enzyme phosphorylation and subsequent transport [1]. Notably, these ATPases don't receive their metal substrates in free hydrated form but rather bound to chaperone proteins like CopZ, which delivers copper to the corresponding Cu+-ATPase CopA [1].

P2 ATPases: Cation Pumps with Diverse Specificities

P2 ATPases represent one of the most extensively studied subfamilies, including several medically important transporters. The P2A and P2C groups include the Na+/K+-ATPase and H+/K+-ATPase, respectively [12] [1]. The Na+/K+-ATPase, discovered by Jens Christian Skou in 1957, was the first P-type ATPase identified and serves as the prototype for the family [2] [1]. This pump creates the sodium and potassium gradients essential for nerve impulse transmission and other physiological processes [2]. The P2B ATPases are Ca2+ pumps with autoinhibitory domains in their N-terminal (plants) or C-terminal (animals) regions that contain binding sites for calmodulin [1]. In the presence of Ca2+, calmodulin activates these ATPases by neutralizing the terminal constraint [1].

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) represents a P2A ATPase that was the first to be crystallized, with its structure (SERCA1a) considered representative of the entire P-type ATPase superfamily [1]. SERCA is an ATP-dependent pump essential for maintaining intracellular Ca2+ homeostasis by actively transporting Ca2+ from the cytosol to the sarcoplasmic reticulum against steep concentration gradients [13]. The energy-intensive nature of this process renders SERCA highly sensitive to ATP depletion, which impairs Ca2+ sequestration and leads to endoplasmic reticulum Ca2+ depletion and cytosolic Ca2+ overload [13].

P3 ATPases: Fungal and Plant Proton Pumps

P3A ATPases are plasma membrane H+-ATPases uniquely found in fungi and plants [9] [11]. These proton pumps export protons from the cell to maintain the transmembrane electrochemical gradient and membrane potential [9]. The proton gradient generated by these enzymes provides energy for active nutrient transport and regulates intracellular pH around neutrality [9]. Fungal Pma1 is particularly notable as an attractive antifungal drug target due to its essential role in fungal physiology and absence from mammals [9].

Unlike other P-type ATPases that typically function as monomers or heterodimers, the plasma membrane H+-ATPase forms a hexameric structure [9]. Recent cryo-EM studies have revealed that hexameric assembly is mediated by three interfaces: interactions between transmembrane domains of adjacent subunits, ordered lipids within the hexamer ring, and cytoplasmic C-terminal tails [9]. The C-terminal autoinhibitory domain (termed C-tail) forms two helices in autoinhibited Pma1 and mediates interactions with neighboring P domains [9]. The activity of Pma1 is regulated by pH, with inhibition at neutral pH and activation at acidic pH [9].

Table 2: Comparative Analysis of Major P-type ATPase Subfamilies

Subfamily	Representative Members	Substrate Specificity	Unique Structural Features	Physiological Roles
P1A ATPases	KdpB (bacterial)	K+ import [2]	7 TM helices; functions as heterotetrameric complex (KdpFABC) [2] [1]	High-affinity potassium uptake in bacteria [2]
P1B ATPases	CopA (copper pump)	Cu+, Ag+, Zn2+, Cd2+, Pb2+, Co2+ [1]	8 TM helices; additional heavy metal-binding domains [1]	Metal resistance and homeostasis [1]
P2 ATPases	Na+/K+-ATPase, SERCA, PMCA	Na+/K+, Ca2+, H+/K+ [12] [13] [1]	10 TM helices; often require additional subunits for function [1]	Nerve impulses, muscle relaxation, calcium signaling [13] [1]
P3A ATPases	Pma1 (fungal), AHA2 (plant)	H+ [9] [11]	10 TM helices; forms hexameric structure; C-terminal regulatory domain [9]	Membrane potential generation, nutrient transport [9]
P4 ATPases	Drs2, Neo1, Dnf1-3	Phospholipids [14]	10 TM helices; often heterodimeric with β-subunit (except P4B) [14]	Lipid flippase activity, membrane asymmetry [14]

P4 ATPases: Lipid Flippases with Unique Architecture

P4 ATPases represent a remarkable adaptation of the P-type ATPase framework, as they transport bulky phospholipid molecules rather than ions [14]. These lipid flippases catalyze the translocation of phospholipids from the extracellular or luminal leaflet to the cytosolic leaflet of biological membranes, establishing and maintaining membrane asymmetry [14]. Phylogenetically, P4 ATPases are grouped into P4A, P4B, and P4C clades [14]. The P4A ATPases function as heterodimers composed of a catalytic α-subunit and an accessory β-subunit, while P4B ATPases (represented by S. cerevisiae Neo1 and its orthologs) function as monomeric flippases without a β-subunit [14].

Despite their divergent substrates, structural studies reveal that monomeric P4B flippases retain the conserved architecture and transport mechanism of heterodimeric P4A flippases [14]. Neo1, an essential P4B ATPase found in Golgi and endosomes, regulates membrane trafficking and establishes phosphatidylethanolamine (PE) and phosphatidylserine (PS) asymmetry [14]. Unlike other P4 ATPases, Neo1 functions without a β-subunit but is regulated by interacting proteins including Dop1, Mon2, Arl1, and Any1 [14]. Biochemical studies demonstrate that Neo1 ATPase activity is stimulated by PE, PS, and lyso-PS, with comparable stimulation extent but different binding affinities [14].

Specialized and Atypical P-type ATPases

The P5 ATPases represent another distinct group with unclear substrate specificity, though they are proposed to transport polyamines or transmembrane helices [9] [14]. These ATPases appear to have 12 transmembrane helices, unlike the typical 10 found in most P-type ATPases [1]. Recent research has also identified potential new families, such as the P6 ATPases (Ct pE) found in many prokaryotes that may be important for Ca2+ uptake [11].

The transmembrane protein 94 (TMEM94), recently proposed to be an endoplasmic reticulum Mg2+ ATPase (ERMA), represents a controversial member of the P-type ATPase family [15]. Structural analysis reveals that TMEM94 possesses cytoplasmic domains analogous to the A, P, and N domains of canonical P-type ATPases, along with ten transmembrane helices [15]. However, several features challenge its classification as a typical P-type ATPase, including the absence of the crucial dephosphorylation motif TGES in its A-domain equivalent, steric hindrance that may impede phosphorylation of the conserved aspartate, and a positively charged cytoplasmic vestibule that appears preferential for anion rather than cation entry [15].

Diagram 2: Conserved domain architecture of P-type ATPases, showing the four core domains present across all subfamilies and variable additional features.

Experimental Approaches for Structural and Functional Analysis

Structural Determination Methods

Recent advances in cryo-electron microscopy (cryo-EM) have revolutionized our understanding of P-type ATPase structure and function. The determination of high-resolution structures has provided unprecedented insights into the molecular mechanisms of these transporters. For instance, two recent cryo-EM studies in 2021 reported the first high-resolution structures of Pma1 hexamers in autoinhibited and activated states, revealing the hexameric architecture, autoinhibitory and activation mechanisms, and proton transport mechanism [9]. These structures provided new perspectives for developing antifungal drugs targeting Pma1 [9].

The structural determination of P-type ATPases typically involves overexpression of the protein in suitable host systems (such as S. cerevisiae for fungal Pma1 or heterologous systems for other ATPases), purification using affinity chromatography followed by size-exclusion chromatography, and vitrification for cryo-EM analysis [9] [14]. For example, Neo1 was overexpressed in S. cerevisiae using a multicopy plasmid with a strong GAP promoter and N-terminal triple FLAG tag, solubilized with dodecyl maltoside (DDM), and purified with anti-FLAG affinity chromatography followed by size-exclusion chromatography with lauryl maltose neopentyl glycol (LMNG) and cholesteryl hydrogen succinate (CHS) to stabilize the membrane protein [14].

To capture different conformational states, researchers often determine structures under multiple conditions that mimic intermediate states in the Post-Albers cycle. These may include conditions with different nucleotides (ATP, ADP, AMPPCP), magnesium concentrations, phosphorylation mimics (such as NaF and BeSO4 for E2P state), or specific inhibitors [15]. For instance, six different structures of human TMEM94 were determined under conditions mimicking MgE1, MgE1-ATP, MgE1-ATP(H), MgE1P-ADP, and E2P states to investigate its conformational changes [15].

Functional Assays and Activity Measurements

Functional characterization of P-type ATPases employs various biochemical and biophysical approaches to assess pump activity, substrate specificity, and inhibitor interactions. Substrate-stimulated ATP hydrolysis activity serves as a key indicator of flippase function, measured using colorimetric or coupled enzyme assays that detect inorganic phosphate release [14]. For lipid flippases like Neo1, researchers test stimulation by various phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), and lyso-PS to determine substrate specificity and kinetic parameters (Km and Vmax) [14].

Inhibitor screening represents another crucial experimental approach, particularly for drug discovery. Compounds are tested for their ability to inhibit ATPase activity, with IC50 values determined through dose-response curves [10]. For instance, the mixed-valence polyoxovanadate [Cl@VV7VIV8O36]6− (V15) showed an IC50 value of 14.2 μM against Ca2+-ATPase with a mixed type of inhibition [10]. Similarly, microscale thermophoresis (MST) assays can measure binding affinities between ATPases and potential inhibitors or nucleotides, providing quantitative data on molecular interactions [15].

Cellular assays complement in vitro biochemical studies by examining physiological effects of ATPase inhibition. These include cell viability assays (e.g., IC50 determinations against cancer cell lines), cell migration assays, morphological assessments, and gene expression analyses to identify cell death mechanisms [10]. For example, V15 exhibited cytotoxicity against MDA-MB-231 (IC50 = 17.2 μM) and MCF-7 (IC50 = 15.1 μM) breast cancer cell lines, reduced cell migration by 70-90%, and induced morphological changes and necroptosis-related gene expression [10].

Table 3: Essential Research Reagents and Methodologies for P-type ATPase Studies

Category	Specific Examples	Application/Function	Experimental Context
Expression Systems	S. cerevisiae overexpression [14]	High-yield protein production for structural studies	Neo1 purification for cryo-EM [14]
Detergents & Lipids	DDM, LMNG, CHS [14]	Membrane protein solubilization and stabilization	Protein purification and functional assays [14]
Nucleotides & Analogs	ATP, ADP, AMPPCP [15]	Trapping specific conformational states	Structural studies of different pump states [15]
Phosphorylation Mimics	NaF, BeSO4 [15]	Stabilize phosphorylated intermediate states	Cryo-EM sample preparation for E2P state [15]
Activity Assays	ATPase activity measurement [14]	Quantifying pump function and inhibition	Substrate stimulation and inhibitor studies [10] [14]
Inhibitors	Polyoxovanadates (V15) [10]	Mechanistic studies and therapeutic development	Ca2+-ATPase inhibition studies [10]

Implications for Inhibitor Development and Therapeutic Applications

The conserved domain architecture of P-type ATPases presents both challenges and opportunities for drug development. The structural conservation across subfamilies means that insights from one member can inform drug design for others, while sequence variations in substrate-binding pockets enable the development of selective inhibitors. Several P-type ATPases have been validated as drug targets for various therapeutic areas.

The fungal plasma membrane H+-ATPase (Pma1) represents a promising target for novel antifungal drugs [9]. As an essential enzyme found uniquely in fungi and plants but not mammals, Pma1 offers potential for selective antifungal activity with minimal host toxicity [9]. The recent elucidation of Pma1 hexameric structures in autoinhibited and activated states provides a structural basis for rational drug design targeting this pump [9]. The autoinhibitory C-terminal domain, which mediates interactions with neighboring P domains in the hexamer, presents a particularly attractive target for allosteric inhibitors [9].

Ca2+-ATPases have been investigated as targets for cardioprotection, immunosuppression, and antitumor agents [10]. Inhibitors of these pumps increase cytoplasmic Ca2+ levels, activating apoptotic factors that lead to cell death [10]. Polyoxometalates, particularly polyoxovanadates, have shown promise as Ca2+-ATPase inhibitors with antitumor activity [10]. The mixed-valence polyoxovanadate V15 not only inhibited Ca2+-ATPase with an IC50 of 14.2 μM but also exhibited cytotoxicity against breast cancer cell lines and reduced cell migration, suggesting its potential as an anticancer agent [10].

Heavy metal ATPases (P1B subfamily) represent targets for combating microbial resistance to heavy metals, while lipid flippases (P4 ATPases) may offer opportunities for developing antifungal and anticancer agents that disrupt membrane asymmetry [14]. The structural insights into substrate recognition and translocation mechanisms provide frameworks for designing inhibitors that target specific steps in the catalytic cycle or interfere with substrate binding.

Diagram 3: The workflow for P-type ATPase inhibitor development, from structural analysis to therapeutic application.

The conserved domain architecture of P-type ATPases represents a remarkable example of evolutionary optimization, where a structurally conserved catalytic core has been adapted to transport diverse substrates across biological membranes. The transmembrane, actuator, phosphorylation, and nucleotide-binding domains work in concert through the Post-Albers cycle to couple ATP hydrolysis with substrate translocation. While these domains maintain their fundamental structures and functions across subfamilies, variations in their detailed organization and additional regulatory elements enable the specialization of different P-type ATPases for specific physiological roles.

Recent advances in structural biology, particularly cryo-EM, have provided unprecedented insights into the molecular mechanisms of these transporters, revealing both conserved principles and unique adaptations across subfamilies. These structural insights, combined with functional studies and inhibitor screening, are paving the way for novel therapeutic agents targeting P-type ATPases for antifungal, anticancer, and other applications. The continued comparative analysis of P-type ATPase domains across species and subfamilies will undoubtedly yield further insights into their mechanisms and opportunities for therapeutic intervention.

Comparative Genomic Analysis of Fungal H+-ATPase (Pma1) Versus Mammalian ATPases

P-type ATPases constitute a large family of primary active transporters that are crucial for maintaining cellular ion homeostasis. Among these, the fungal plasma membrane H+-ATPase (Pma1) and various mammalian P-type ATPases, such as the Na+/K+-ATPase and Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), perform distinct yet essential physiological functions. This guide provides a comparative analysis of Pma1 and mammalian ATPases, focusing on structural, functional, and inhibitor-related properties. The unique position of Pma1 as an antifungal drug target, due to its absence in mammals, makes this comparison particularly relevant for drug development professionals seeking to exploit these differences for therapeutic gain [9] [16].

Structural Comparison: Fungal Pma1 vs. Mammalian P-type ATPases

Quaternary Structure and Assembly

The most striking structural difference lies in the quaternary organization. Fungal Pma1 assembles into a hexameric ring structure, a characteristic not observed in other P-type ATPase subfamilies. This hexameric assembly, confirmed in S. cerevisiae, N. crassa, and K. lactis, is stabilized by three key interfaces: transmembrane domain interactions, a central lipid patch, and cytoplasmic C-terminal tail interactions between subunits [9] [17]. In contrast, mammalian P-type ATPases such as SERCA and Na+/K+-ATPase typically function as monomers or heterodimeric complexes with accessory subunits [9].

Recent cryo-EM studies have revealed that the Pma1 hexamer encircles an ordered lipid domain composed of approximately 57 lipid molecules, which likely serves as the building block for the membrane compartment of Pma1 (MCP) in yeast plasma membranes [17]. This intimate association with specific lipids is a distinctive feature of the fungal proton pump.

Table 1: Comparative Structural Properties of Fungal Pma1 and Mammalian P-type ATPases

Structural Feature	Fungal Pma1 (P3A-subfamily)	Mammalian ATPases (e.g., SERCA, Na+/K+-ATPase)
Oligomeric State	Functional hexamer [9] [17]	Monomer or heterodimer with regulatory subunits [9]
Characteristic Domains	TMD, A-domain, N-domain, P-domain [9]	TMD, A-domain, N-domain, P-domain
Unique Structural Elements	Domain-swapped N-terminal extension (NTE), C-terminal autoinhibitory tail [9] [18]	Accessory subunits (e.g., β-subunit in Na+/K+-ATPase)
Key Stabilizing Factors	Inter-subunit TM interactions, central lipid patch, C-tail interactions [9] [17]	Protein-protein interactions with regulatory subunits
Endogenous Modulators	Ordered lipid molecules within hexameric ring [17]	Phospholamban (for SERCA), FXYD proteins (for Na+/K+-ATPase)

Domain Architecture and Unique Motifs

Despite differing quaternary structures, both fungal Pma1 and mammalian P-type ATPases share a conserved core domain architecture comprising a transmembrane domain (TMD) with ten helices, and three cytoplasmic domains: the actuator (A) domain, nucleotide-binding (N) domain, and phosphorylation (P) domain [9] [17].

However, Pma1 possesses unique regulatory elements not found in mammalian counterparts. The C-terminal autoinhibitory domain (residues 880-918 in yeast) forms two helices that bind to the phosphorylation domains of neighboring subunits in the autoinhibited state at neutral pH [9]. Furthermore, a recent study identified a domain-swapped N-terminal extension (NTE) that binds to the nucleotide-binding domain of an adjacent subunit, mediating cooperative ATPase activity within the hexamer—a mechanism essential for Pma1's physiological function [18].

Functional Mechanisms and Regulatory Properties

Ion Specificity and Transport Mechanisms

Fungal Pma1 is specialized for proton (H+) extrusion, utilizing ATP hydrolysis to pump protons out of the cell, thereby establishing both the membrane potential and the transmembrane electrochemical proton gradient that drives nutrient uptake [9] [16]. In contrast, mammalian P2-type ATPases transport diverse cations: SERCA pumps calcium ions, while Na+/K+-ATPase exchanges sodium and potassium ions [19] [17].

The proton transport mechanism in Pma1 involves conformational changes triggered by ATP binding and hydrolysis, following the Post-Albers cycle (E1–E1P–E2P–E2 states) common to all P-type ATPases. Activation at acidic pH involves a 6.7 Å downward shift and 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path [17].

Regulatory Mechanisms

Both fungal and mammalian P-type ATPases undergo sophisticated regulation, albeit through different mechanisms. Pma1 is primarily regulated by pH-dependent autoinhibition. At neutral pH (7.4), the C-terminal regulatory helix binds to the phosphorylation domains of neighboring subunits, locking the hexamer in an autoinhibited state. At acidic pH (6.0), this helix becomes disordered, leading to activation [17]. This pH-sensing mechanism allows fungi to respond to environmental changes.

Mammalian ATPases are typically regulated through different strategies, including association with regulatory subunits (e.g., the β-subunit in Na+/K+-ATPase) and reversible phosphorylation by protein kinases that modulate their affinity for specific ions [19].

Table 2: Functional Comparison of Fungal Pma1 and Representative Mammalian ATPases

Functional Property	Fungal Pma1	Mammalian SERCA	Mammalian Na+/K+-ATPase
Primary Ion Substrate	H+ [9]	Ca2+ [19]	Na+/K+ [17]
Transport Stoichiometry	H+ out (stoichiometry not fully defined)	2 Ca2+ per ATP hydrolyzed	3 Na+ out, 2 K+ in per ATP
Primary Physiological Role	Membrane potential generation, cytosolic pH homeostasis [16]	Muscle relaxation, calcium storage	Membrane potential, cellular sodium balance
Key Regulatory Mechanism	pH-dependent C-terminal autoinhibition [9] [17]	Phospholamban binding, oxidation	FXYD protein binding, phosphorylation
Energy Consumption	High (~50% of cellular ATP in yeast) [17]	Tissue-dependent	~20-30% of basal metabolic rate in some tissues
Cooperative Behavior	Positive cooperativity mediated by NTE [18]	Not observed	Not observed

Experimental Approaches for Comparative Analysis

Structural Determination Protocols

Cryo-Electron Microscopy (Cryo-EM) of Pma1:

• Protein Purification: Tag Pma1 with a C-terminal triple FLAG epitope in yeast cells. Extract the protein from yeast membranes using the mild detergent n-dodecyl-β-D-maltoside (DDM). Purify via anti-FLAG affinity chromatography followed by size-exclusion chromatography (SEC) to isolate hexameric complexes [17].
• Grid Preparation: Apply purified Pma1 to cryo-EM grids and vitrify using liquid ethane.
• Data Collection: Acquire micrographs using a high-end cryo-electron microscope (e.g., Titan Krios) equipped with a direct electron detector.
• Image Processing: Perform motion correction and contrast transfer function (CTF) estimation. Use single-particle analysis with 2D classification, ab initio reconstruction, and 3D refinement to achieve high-resolution structures (3.2 Å for autoinhibited state) [17].
• Model Building: Build atomic models into cryo-EM density maps using iterative rounds of manual building in Coot and refinement in Phenix [9] [17].

Heterologous Expression Systems for Mammalian ATPases: Mammalian P-type ATPases such as SERCA can be expressed in heterologous systems like insect or yeast cells for crystallization trials. However, some targets like PfATP4 (a P2-type ATPase from Plasmodium falciparum) resist heterologous expression, requiring alternative approaches such as endogenous purification from native sources [19].

Functional Characterization Assays

ATPase Activity Measurements:

• Colorimetric Assay: Measure inorganic phosphate (Pi) release using malachite green reagent after incubating the ATPase with ATP at defined pH conditions (e.g., pH 7.4 vs. pH 6.0 for Pma1) [17].
• Coupled Enzymatic Assay: Monitor NADH oxidation spectrophotometrically in a coupled system with pyruvate kinase and lactate dehydrogenase.

Inhibitor Screening Protocols:

• Compound Library Preparation: Dissolve candidate inhibitors in DMSO at appropriate stock concentrations.
• Activity Screening: Incubate purified ATPase with compounds across a concentration range (e.g., 0.1-100 μM) and measure residual ATPase activity.
• IC50 Determination: Plot dose-response curves and calculate half-maximal inhibitory concentrations using non-linear regression analysis [20].
• Specificity Testing: Counter-screen against mammalian ATPases (e.g., SERCA, Na+/K+-ATPase) to identify fungus-specific inhibitors.

Inhibitor Profiles and Therapeutic Targeting

Fungal Pma1 Inhibitors

The unique structural and functional properties of Pma1 make it an attractive antifungal target. Several compound classes have shown inhibitory activity:

• Si01 and its derivatives directly inhibit Pma1 ATPase activity and extend chronological lifespan in fission yeast, with the inhibitory effect correlating with lifespan extension [20].
• Compound series identified through screening show potential for development as specific Pma1 inhibitors, though many are in early research stages [9].

The discovery of the N-terminal extension (NTE) as a mediator of cooperative activity within the Pma1 hexamer presents a new, fungus-specific target for allosteric inhibitors that could disrupt hexamer function without affecting mammalian ATPases [18].

Mammalian ATPase Inhibitors

Mammalian P-type ATPases are established drug targets with well-characterized inhibitors:

• Cardiac glycosides (e.g., digoxin, ouabain) specifically inhibit Na+/K+-ATPase and are used clinically for heart failure.
• Thapsigargin is a potent and specific SERCA inhibitor used extensively in research.

The structural differences between fungal and mammalian ATPases, particularly the hexameric assembly and unique regulatory domains of Pma1, provide opportunities for developing highly specific antifungal agents that avoid cross-reactivity with human targets.

Table 3: Comparative Inhibitor Profiles of Fungal Pma1 and Mammalian ATPases

Inhibitor Property	Fungal Pma1 Inhibitors	Mammalian ATPase Inhibitors
Representative Compounds	Si01 and derivatives [20]	Cardiac glycosides (Ouabain), Thapsigargin
Known Binding Sites	Under investigation; NTE and C-tail interfaces potential targets [18]	Well-characterized (e.g., Thapsigargin binds TMD of SERCA)
Therapeutic Applications	Potential broad-spectrum antifungals [9]	Heart failure (digoxin), research tools
Specificity Challenges	Achieving selectivity over plant H+-ATPases	Avoiding off-target effects between mammalian isoforms
Resistance Mechanisms	Not yet clinically documented	Well-documented in various contexts

Research Reagent Solutions

Table 4: Essential Research Reagents for Pma1 and Mammalian ATPase Studies

Reagent/Category	Specific Examples	Research Application	Key Function
Expression Systems	S. cerevisiae with epitope-tagged Pma1 [17]	Protein production for structural studies	Provides natively assembled hexameric Pma1
Detergents	n-Dodecyl-β-D-maltoside (DDM) [17]	Membrane protein extraction and solubilization	Maintains native protein-lipid interactions and oligomeric state
Affinity Tags	Triple FLAG epitope [17]	Protein purification	Enables mild affinity purification of functional complexes
Activity Assays	Malachite green phosphate detection [20]	Functional characterization	Measures ATP hydrolysis activity under various conditions
Cryo-EM Reagents	UltrAuFoil gold grids [9]	High-resolution structural studies	Provides optimal support for vitreous ice formation
Specific Inhibitors	Si01, BeF3− [20] [17]	Mechanistic studies	Stabilizes specific conformational states (e.g., E2P state)

Visual Synthesis of Key Concepts

Structural Organization of Fungal Pma1 Versus Mammalian ATPases

Experimental Workflow for Comparative ATPase Analysis

This comparative analysis highlights fundamental differences between fungal Pma1 and mammalian ATPases that underlie their potential as therapeutic targets. The hexameric architecture of Pma1, its pH-dependent regulatory mechanism, and the recently discovered cooperative function mediated by the NTE represent fungus-specific characteristics absent from mammalian systems. These distinctions provide a robust structural foundation for developing selective antifungal agents that target Pma1 without cross-reacting with essential mammalian ATPases. Future drug discovery efforts should focus on exploiting these unique structural features, particularly the inter-subunit interfaces and regulatory domains, to create novel therapeutics that address the growing challenge of fungal resistance.

The sodium efflux pump PfATP4 in the malaria-causing parasite Plasmodium falciparum has emerged as one of the most promising antimalarial targets due to its essential role in parasite survival. As a P-type ATPase, it functions to maintain low intracellular sodium levels within the parasite by actively extruding sodium ions across its plasma membrane, a process critical for the parasite's survival in human red blood cells [19] [21]. The continual rise of drug resistance in malaria parasites has undermined progress against the disease, which kills over 600,000 people globally each year [22]. While PfATP4 is the target of several experimental antimalarial compounds, including Cipargamin and pyrazoleamides, resistance mutations in PfATP4 have readily emerged both in laboratory settings and in clinical isolates [19] [22]. Until recently, the lack of high-resolution structural information for PfATP4 has significantly hampered efforts to understand drug resistance mechanisms and guide the rational design of next-generation inhibitors [19] [21].

Methodological Breakthrough: Endogenous Structural Biology

The Challenge of Heterologous Expression

Previous attempts to determine the structure of PfATP4 followed standard structural biology approaches by expressing the protein in heterologous systems such as yeast or bacterial cells. These attempts were largely unsuccessful because P. falciparum genes often fail to express properly in non-native cellular environments [23]. This expression challenge thwarted structural studies and limited researchers to homology models that differed significantly from the actual protein structure [19].

Native Purification from Parasite-Infected Red Blood Cells

The research teams from Columbia and Drexel universities pioneered a different approach, using innovative techniques to study the parasite's proteins in their natural state [23]. The key methodological advances included:

• CRISPR-Cas9 genome engineering to insert a 3×FLAG epitope tag at the C-terminus of PfATP4 in Dd2 P. falciparum parasites [19] [21]
• Culture of engineered parasites in human red blood cells at a massive scale, requiring hundreds of liters of growth medium [22]
• Endogenous purification of PfATP4 directly from parasite-infected human red blood cells [19] [23]
• Validation of functional activity through Na+-dependent ATPase assays inhibited by established PfATP4 inhibitors (PA21A092 and Cipargamin) [19]

This endogenous approach enabled the discovery of completely new biology that would have been missed in heterologous expression systems [23].

Cryo-EM Structure Determination

The team determined a 3.7 Å resolution structure of endogenously purified PfATP4 using single-particle cryo-electron microscopy (cryo-EM) [19] [21]. The structure quality enabled de novo modeling of 982 of the 1264 residues of PfATP4, with all five canonical P-type ATPase domains visible in the cryo-EM map [19]. The structure has been deposited in the Protein Data Bank under accession code 9N10 [24].

The following diagram illustrates the key stages of the experimental workflow:

Structural Analysis of PfATP4 and Comparative Assessment

The cryo-EM structure reveals that PfATP4 maintains the canonical P-type ATPase domain organization but with several distinctive features [19]. The atomic model contains 982 residues with all five conserved domains visible:

• Transmembrane Domain (TMD): Composed of 10 helices (TM1-TM10) arranged in three clusters (TM1-2, TM3-4, TM5-10) that facilitate ion binding and transport [19]
• Nucleotide-binding (N) domain: Consists of several β-sheets connected by short helices and long loops, positioned below the P-domain [19]
• Phosphorylation (P) domain: Composed of two segments situated between TM4 and N domains, featuring β-sheets and connecting loops/helices [19]
• Actuator (A) domain: Shows flexibility in the structure but coordinates the translation of ATP hydrolysis into TMD movements [19]
• Extracellular loop (ECL) domain: Juts into the parasitophorous vacuole lumen, composed of four long β-sheets connected by long loops [19]

The structure differs significantly from previous homology models, with root-mean-square deviation (RMSD) values of 10.3-22.9 Å, highlighting the importance of empirical structure determination [19].

Key Functional Sites and Comparative Analysis

Table 1: Key Functional Sites in PfATP4 Structure

Functional Site	Structural Features	Residues Involved	Comparative Notes
Ion-binding Site	Located between TM4, TM5, TM6, TM8; conserved coordinating sidechains	Na+-coordinating residues conserved	Similar to SERCA E1-2Ca2+ state (RMSD 0.86-1.83Å); no cavity detected
ATP-binding Site	Between N- and P-domains; conserved architecture	E557, F614, K652, R703, K846, D865, N868, D451	Similar to SERCA E1.2Ca2+ ATP-free state; key differences at M620, R618, R840
Gate Closure Mechanism	Kink in TM1	P176 at kink position	Replaces Phe residue found in Na+/K+ ATPase that mediates gate closure

The ion-binding site analysis suggests the structure represents a Na+ bound state, with all Na+ coordinating sidechains conserved and positioned similarly to corresponding residues in SERCA in its calcium-bound state (E1-2Ca2+) [19]. Analysis with pyKVFinder detected no cavity at the ion-binding site in PfATP4, consistent with ion-bound states [19]. The ATP-binding site shows a conserved overall architecture with other P2-type ATPases, though key differences were observed in the sidechain arrangements of M620, R618, and R840 [19].

The PfABP Discovery: An Apicomplexan-Specific Modulator

Identification and Characterization of PfABP

The most unexpected finding from the endogenous structure was the discovery of a previously unknown binding partner, termed PfATP4-Binding Protein (PfABP) [19] [23]. This discovery was made possible only through the endogenous purification approach [23]. Key findings about PfABP include:

• PfABP corresponds to gene product PF3D7_1315500, a conserved P. falciparum protein of previously unknown function [19]
• It interacts with PfATP4 via a helical domain that binds to TM9 of PfATP4 in the transmembrane domain [19]
• Mass spectrometry analysis identified PfABP as the third most abundant protein in tryptic digests of the purified PfATP4 sample [19]
• The protein is apicomplexan-specific, meaning it is found in related parasites but absent in humans [19] [22]

Functional Significance of PfABP

Functional validation experiments demonstrated that PfABP is not merely an accessory protein but essential for parasite survival [23] [22]. The researchers found that:

• Loss of PfABP led to rapid degradation of the PfATP4 sodium pump and subsequent death of the parasite [23] [22]
• PfABP appears to stabilize and regulate PfATP4's function [22]
• The protein likely functions similarly to regulatory subunits in human ion pumps, pointing to a new class of drugs against pathogens [22]

The following diagram illustrates the structural relationship between PfATP4 and its newly discovered binding partner:

Resistance Mutation Mapping and Drug Binding Implications

Spatial Organization of Resistance Mutations

The high-resolution structure enabled precise mapping of known resistance-conferring mutations in PfATP4, providing insights into their potential mechanisms of action [19] [23]. Key resistance mutations include:

• G358S/A: Found in recrudescent parasites from Cipargamin Phase 2b clinical trials, located on TM3 adjacent to the proposed Na+ coordination site [19]
• A211V: Arose under pyrazoleamide (PA21A092) pressure, located within TM2 adjacent to the ion-binding site [19]

Table 2: Clinically Relevant Resistance Mutations in PfATP4

Mutation	Drug Selection	Structural Location	Proposed Resistance Mechanism
G358S/A	Cipargamin (+)-SJ733	TM3, adjacent to Na+ coordination site	Introduces sidechain that may block Cipargamin binding pocket
A211V	PA21A092 (pyrazoleamide)	TM2, adjacent to ion-binding site	Alters binding site conformation; increases Cipargamin susceptibility

Notably, the G358S mutation could potentially block Cipargamin binding by introducing a serine or alanine sidechain into the proposed drug binding pocket [19]. Interestingly, parasite lines with the A211V mutation showed increased susceptibility to Cipargamin, suggesting complex allosteric interactions within the protein [19].

Research Reagent Solutions for PfATP4 Studies

Table 3: Essential Research Reagents for PfATP4 Structural and Functional Studies

Reagent/Resource	Specifications	Function/Application
CRISPR-Cas9 System	Dd2 P. falciparum parasite line	Endogenous tagging of PfATP4 with 3×FLAG epitope
Culture Medium	Human red blood cells, large-scale (100+ liters)	Support parasite growth for endogenous protein purification
Affinity Purification	3×FLAG epitope tag system	Isolation of native PfATP4 complex from parasites
Cryo-EM Equipment	Single-particle electron microscopy	High-resolution structure determination at 3.7 Å
ATPase Activity Assay	Na+-dependent measurement	Functional validation of purified PfATP4
PfATP4 Inhibitors	PA21A092, Cipargamin	Control compounds for functional assays

The determination of the endogenous PfATP4 structure represents a significant advancement in antimalarial research with broad implications for drug development. The discovery of PfABP opens an entirely new avenue for therapeutic intervention that may overcome existing resistance challenges [19] [23]. Several strategic advantages emerge from these findings:

• Dual Targeting Approaches: Future inhibitors could target either PfATP4 itself or the PfATP4-PfABP interaction interface [19] [22]
• Reduced Resistance Potential: Since PfABP appears to be less prone to mutations than PfATP4, drugs targeting this interaction may be harder for parasites to evade [23]
• Species Selectivity: The apicomplexan-specific nature of PfABP offers potential for highly selective therapeutics with reduced risk of human side effects [22]

This structural breakthrough provides a blueprint for next-generation drug discovery, offering multiple strategies to design molecules that target not only PfATP4 but also its essential binding partner PfABP [23] [22]. The findings underscore the value of endogenous structural biology approaches in revealing novel biology that would remain hidden in heterologous expression systems [23].

The molecular machines that govern cellular homeostasis, ranging from ubiquitin-activating enzymes (E1) to ion-transporting P-type ATPases, operate through sophisticated catalytic cycles defined by precise conformational transitions. Although these systems belong to distinct protein families and perform different primary functions—post-translational modification versus ion translocation—they share fundamental mechanistic principles. Both systems utilize ATP hydrolysis to drive major structural rearrangements, cycle between high-energy phosphorylated intermediates, and employ domain alternation to coordinate multi-step reactions. This review examines the conformational landscapes of E1 and E2 enzymes within the ubiquitin-proteasome system, drawing insightful parallels to the well-characterized transport cycles of P-type ATPases. By comparing these systems through a structural and mechanistic lens, we aim to illuminate universal principles of energy transduction and allosteric control that can inform targeted therapeutic intervention, particularly in the context of comparative genomic analysis of P-type ATPase inhibitors.

The catalytic dwells and conformational oscillations observed in rotary ATPases like F1-ATPase [25] find surprising parallels in the adenylation-thioesterification cycle of E1 enzymes, where distinct conformational states (open, closed, proximal, distal) govern catalytic progression [26]. Similarly, the Post-Albers cycle of P-type ATPases [27], with its characteristic E1/E2 transitions and phosphoenzyme intermediates, provides a framework for understanding how E1 enzymes might coordinate their multiple catalytic steps. This comparative analysis reveals how evolution has converged on similar solutions for coupling chemical energy to mechanical work across diverse biological contexts.

Fundamental Operating Principles

Table 1: Core Mechanistic Features of E1-E2 Systems and P-type ATPases

Feature	E1-E2 System (Ubiquitin Activation)	P-type ATPases (Ion Transport)
Energy Source	ATP hydrolysis	ATP hydrolysis
Key Intermediate	Acyl-adenylate (~P) and thioester	Aspartyl-phosphate (E1P/E2P)
Cycle Representation	Adenylation → Thioester Formation → Transthioesterification	Post-Albers Cycle (E1/E2 transitions)
Conformational Transitions	SCCH open/closed, UFD proximal/distal	A-, N-, P-domain rearrangements
Chemical Steps	Two-step activation (adenylation, thioester)	Phosphorylation, dephosphorylation
Specificity Control	Domain architecture, cognate E2 recognition	Ion-binding residues, lipid interactions
Regulatory Elements	Cys-cap, 4HB domain, UFD position	A-domain, β-subunit, FXYD proteins

E1 enzymes, exemplified by ubiquitin-activating enzyme UBA1, initiate the ubiquitin cascade through a multi-step process requiring strict specificity for their cognate ubiquitin-like proteins (Ubls) and E2 conjugating enzymes [26]. This system operates through a conserved architectural framework where structural dynamics directly enable catalytic progression. Similarly, P-type ATPases such as Na+/K+-ATPase [28] and PMCA2 [27] employ precisely timed domain movements to couple ATP hydrolysis to ion transport against concentration gradients.

The UBA1 enzyme transitions through distinct conformational states characterized by rearrangement of its second catalytic cysteine half-domain (SCCH) between "open" and "closed" configurations and repositioning of its ubiquitin-fold domain (UFD) between "distal" and "proximal" orientations [26]. These transitions facilitate the transfer of ubiquitin from the adenylation site to the catalytic cysteine and ultimately to the cognate E2 enzyme. This sophisticated molecular choreography ensures the fidelity of ubiquitin activation and transfer while preventing premature or off-target reactions.

Experimental Evidence for Conformational States

Structural biology has been instrumental in elucidating these mechanisms. Single-particle cryo-EM studies of UBA1 have captured discrete states along the reaction pathway, revealing how domain rearrangements create and occlude binding surfaces [26]. Similarly, high-resolution structures of P-type ATPases like PMCA2 have been determined in eight distinct stages of its transport cycle, providing unprecedented insight into the conformational transitions that enable ion translocation [27].

Notably, single-molecule studies of F1-ATPase have revealed that its catalytic dwell involves rotational oscillations of the γ-subunit that progress through three distinct stages before proceeding to the power stroke [25]. These oscillations (-13° to 13°, then to 14° or 33°, and back to 0°) represent a "molecular checklist" mechanism ensuring all catalytic sites contain the correct substrate/product complement before cycle progression. This concept of conformational sampling until proper reaction conditions are met may extend to E1 enzymes, where domain motions could similarly verify proper substrate loading before progression to transthioesterification.

Structural Transitions in E1 Enzymes: A Domain-Based Analysis

Architectural Organization of UBA1

UBA1 possesses a multi-domain architecture organized around a core of two pseudosymmetric adenylation domains—the active adenylation domain (AAD) and inactive adenylation domain (IAD) [26]. Key inserted elements include:

• First catalytic cysteine half-domain (FCCH) inserted into the IAD
• Second catalytic cysteine half-domain (SCCH) inserted into the AAD
• Four-helix bundle (4HB) domain that blocks Ub binding to the IAD
• Ubiquitin-fold domain (UFD) at the C-terminus

This arrangement creates a structural framework that has been metaphorically described as a "left hand," with the UFD forming the thumb, SCCH as the fingers, FCCH as the pinky, and the AAD/IAD core as the palm [26]. This architectural motif facilitates the precise positioning of substrates and catalytic residues throughout the reaction cycle.

Conformational Transitions During Ubiquitin Activation

The E1 catalytic cycle proceeds through carefully orchestrated conformational states:

• Adenylation-Competent State: The SCCH domain adopts an "open" conformation, positioning the catalytic cysteine >35 Å from the adenylation active site. The UFD remains in a "distal" position relative to the SCCH [26].

• Adenylation Transition: Following Mg-ATP and ubiquitin binding, the catalytic AAD facilitates adenylation of ubiquitin's C-terminus, forming a ubiquitin-AMP intermediate.

• Thioester-Competent State: The SCCH domain undergoes a major rearrangement to a "closed" conformation, repositioning the catalytic cysteine within the restructured adenylation active site to receive the ubiquitin moiety [26].

• E2 Engagement: The UFD transitions to a "proximal" conformation, creating a binding surface for the cognate E2 enzyme.

• Transthioesterification: The thioester-linked ubiquitin is transferred from E1 to the catalytic cysteine of E2, completing the activation cycle.

These transitions are governed by specific structural elements, including the cys-cap (a flexible loop protecting the catalytic cysteine before thioester formation) and the 4HB domain (which prevents aberrant ubiquitin binding to the IAD) [26].

Diagram: E1 Enzyme Conformational Cycle during Ubiquitin Activation. The diagram illustrates the major domain rearrangements (SCCH and UFD transitions) that enable the sequential steps of ubiquitin activation and transfer to E2 enzymes.

Parallels with P-type ATPase Transport Cycles

The Post-Albers Cycle in P-type ATPases

P-type ATPases operate through the well-established Post-Albers cycle, which involves transitions between two principal conformations designated E1 and E2 [27]. The plasma membrane Ca²⁺-ATPase (PMCA) exemplifies this mechanism with distinctive adaptations for its physiological role:

• E1 State: Cytoplasm-facing conformation with high affinity for Ca²⁺
• E1-ATP State: Nucleotide binding primes the ion-binding site
• E1-Ca-ATP State: Ca²⁺ binding triggers closure of the intracellular gate
• E1~P State: ATP hydrolysis leads to aspartyl phosphorylation and major cytoplasmic domain rearrangements
• E2P State: Outward-open conformation enabling Ca²⁺ release to extracellular space
• E2-Pi State: Dephosphorylation intermediate with closed ion-release pathway
• E2 State: Counter-proton binding precedes return to E1 conformation

Recent structural studies of PMCA2 have revealed that unlike P2A-ATPases (exemplified by SERCA), the cytoplasmic domains of PMCAs undergo significantly smaller rearrangements during transitions between states [27]. For instance, the shift from E1 to E1-Ca state in PMCA2 involves N-domain and P-domain rotations of just 8.3° and 4.2° respectively, compared to 66.5° and 12.0° in SERCA1a. This minimized domain motion likely contributes to the ultrafast transport rates (exceeding 5,000 cycles per second) that characterize PMCAs [27].

Regulatory Mechanisms and Lipid Interactions

P-type ATPases are notably regulated by their lipid environment. PMCAs are specifically modulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), which acts as a "latch" promoting fast Ca²⁺ release and opening passageways for counter-ions [27]. The structural basis for this regulation has been identified through cryo-EM structures showing ordered PtdIns(4,5)P₂ densities bound to PMCA2. This lipid-protein interaction represents a potential target for pharmacological manipulation of intracellular Ca²⁺ levels.

Similarly, plant plasma membrane H⁺-ATPases are regulated by their lipid microenvironment through both specific lipid binding and general bilayer properties [29]. These regulatory mechanisms demonstrate how membrane-embedded molecular machines integrate chemical and mechanical cues from their environment to modulate activity.

Diagram: P-type ATPase Transport Cycle (Post-Albers Cycle). The diagram shows the principal conformational states and transitions during ion transport by P-type ATPases like PMCA2, highlighting phosphorylation-driven domain rearrangements.

Experimental Approaches for Studying Conformational Transitions

Key Methodologies and Protocols

Table 2: Experimental Methods for Analyzing Conformational Transitions

Method	Application	Key Insights	Technical Requirements
Single-particle cryo-EM	Trapping intermediate states	Atomic models of distinct conformations; lipid interactions [21] [27]	CRISPR-engineered tags; nanodisc reconstitution
Single-molecule FRET	Real-time domain movements	Dynamics and kinetics of transitions	Fluorophore labeling; TIRF microscopy
Hydrogen-deuterium exchange MS	Protein dynamics mapping	Solvent accessibility changes; flexible regions	High-resolution mass spectrometer
X-ray crystallography	High-resolution snapshots	Atomic details of active sites	High-quality crystals
Single-molecule rotation assays	Rotary ATPase mechanisms	Catalytic dwell oscillations; angular velocity [25]	Gold nanorod probes; polarized darkfield microscopy
Molecular dynamics simulations	Atomic-level dynamics	Transition pathways; allosteric networks	High-performance computing

Detailed Experimental Protocol: Cryo-EM Analysis of Conformational States

For structural characterization of E1-E2 complexes or P-type ATPases, the following protocol has proven effective:

• Protein Engineering and Purification

  • CRISPR-Cas9-mediated endogenous tagging (e.g., 3×FLAG epitope) [21]
  • Affinity purification from native sources (e.g., parasite-infected red blood cells for PfATP4) [21]
  • Reconstitution into lipid nanodiscs to preserve native membrane environment [30] [27]

• Sample Preparation for Cryo-EM

  • Grid preparation with ultra-thin carbon supports
  • Vitrification using liquid ethane plunge-freezing devices
  • Data collection on 300 keV cryo-electron microscopes with direct electron detectors

• Image Processing and 3D Reconstruction

  • Motion correction and dose-weighting of movie frames
  • Particle picking using deep learning-based approaches (e.g., Topaz, crYOLO)
  • 2D classification to remove junk particles
  • 3D classification to separate distinct conformational states
  • High-resolution refinement with imposed symmetry (if applicable)

• Model Building and Validation

  • De novo model building using Coot
  • Real-space refinement in Phenix
  • Validation using MolProbity and EMRinger scores

This approach has enabled determination of structures at resolutions sufficient to visualize side chains, bound lipids [27], and water molecules [30], providing unprecedented insight into mechanistic details.

Structural Determinants of Specificity and Regulation

Molecular Basis of E1 Specificity

E1 enzymes exhibit remarkable specificity for their cognate Ubls and E2 partners. In UBA1, this specificity is governed by:

• AAD-Ub interactions: Complementary surfaces ensure selective ubiquitin binding
• SCCH domain conformation: Proper positioning of the catalytic cysteine for thioester formation
• UFD-E2 interactions: The UFD creates a specific binding surface for cognate E2 enzymes [26]

The consequences of dysregulated specificity are severe, as demonstrated by disease-associated mutations. For instance, hemizygous missense mutations that impair UBA1 function cause X-linked infantile spinal muscular atrophy [26], highlighting the critical importance of precise molecular recognition in E1 function.

Allosteric Regulation in P-type ATPases

P-type ATPases employ sophisticated regulatory mechanisms, including:

• Lipid-mediated regulation: PtdIns(4,5)P₂ binding to PMCA2 facilitates ultrafast transport by promoting Ca²⁺ release and counter-ion passage [27]
• Subunit interactions: Accessory subunits like neuroplastin (NPTN) and basigin (BASI) modulate PMCA activity [27]
• Disease mutations: Autosomal dominant deafness mutations in PMCA2 cluster in cytoplasmic domains, disrupting allosteric communication [27]

These regulatory principles parallel the allosteric control mechanisms observed in E1 enzymes, where domain rearrangements (SCCH closure, UFD repositioning) gate catalytic progression [26].

Table 3: Key Research Reagents for Studying Conformational Transitions

Reagent/Resource	Application	Function/Rationale	Example Sources
CRISPR-Cas9 gene editing	Endogenous tagging	Preserves native regulation; avoids overexpression artifacts	Commercially available kits
Lipid nanodiscs	Membrane protein stabilization	Provides native-like lipid environment; enhances stability	Custom preparation
Gold nanorods	Single-molecule rotation assays	Probes angular position with high temporal resolution [25]	Synthesized nanoparticles
Transition state analogs	Trapping intermediates	Stabilizes specific catalytic states for structural studies	AlF₄⁻, BeF₃⁻, MgF₄²⁻
PtdIns(4,5)P₂ lipids	PMCA regulation studies	Investigate lipid-dependent modulation of transport [27]	Commercial lipids
FXYD subunit proteins	Na+/K+-ATPase regulation	Study subunit-mediated pump modulation [28]	Recombinant expression
Cys-cap peptides	E1 enzyme inhibition	Block thioester formation; mechanistic studies	Custom synthesis

The comparative analysis of E1-E2 conformational transitions and P-type ATPase transport cycles reveals striking convergent evolution in how biological systems harness ATP hydrolysis for mechanical work. Both systems employ:

• Multi-domain architectures that undergo precisely timed rearrangements
• Covalent catalytic intermediates (acyl-adenylate/thioester in E1; aspartyl-phosphate in P-type ATPases)
• Gated transitions between distinct conformational states
• Allosteric regulation through accessory subunits and lipid interactions
• Quality control checkpoints that verify proper reaction progression

These universal principles extend to other ATPase families, including the catalytic dwell oscillations observed in F1-ATPase [25] and the domain alternations in ABC transporters. Understanding these shared mechanistic themes provides a conceptual framework for predicting how mutations disrupt function, designing targeted inhibitors, and developing therapeutic strategies that exploit allosteric control points.

For researchers investigating P-type ATPase inhibitors, the structural insights from E1-E2 systems suggest opportunities for developing compounds that trap specific conformational states rather than simply competing with substrate binding. Similarly, the lipid-dependent regulation of PMCA2 [27] highlights the potential for targeting membrane-protein interfaces rather than traditional active sites. As structural biology continues to reveal increasingly detailed mechanistic information, these parallels between seemingly distinct enzyme systems will undoubtedly yield new insights for therapeutic intervention across a broad spectrum of diseases.

Advanced Screening and Design Strategies for ATPase Inhibitor Discovery

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, constitute a critical class of macromolecular biological catalysts that power essential cellular processes across all kingdoms of life [31]. These dynamic proteins function as molecular motors that utilize the energy from ATP hydrolysis to drive diverse reactions including protein trafficking, solute transport, cellular motility, and signal transduction [31]. The P-type ATPase family represents a particularly important subgroup characterized by the formation of a phosphorylated intermediate during their catalytic cycle [32]. These membrane transporters play vital roles in maintaining cellular homeostasis through the transport of various cations and phospholipids against electrochemical gradients [32].

Within pharmaceutical research, ATPases represent attractive molecular targets for drug discovery. The well-known pharmaceutical omeprazole exemplifies successful ATPase targeting through its selective and irreversible inhibition of the H+/K+-ATPase proton pump in gastric parietal cells [33]. The unique Bergerat ATP-binding fold found in bacterial histidine kinases presents a promising target for novel antibiotics, as it is structurally distinct from eukaryotic ATP-binding domains and absent in animals [34]. Similarly, the plasma membrane H+-ATPase of fungi represents a significant target for developing broad-spectrum antifungal drugs [32].

High-throughput screening (HTS) platforms for intrinsic ATPase activity provide indispensable tools for drug discovery campaigns targeting these enzymes. This guide objectively compares the major assay technologies currently employed in ATPase screening, detailing their principles, applications, and performance characteristics to inform researchers' experimental design decisions.

ATPase Assay Technologies: Mechanisms and Methodologies

Fundamental ATPase Reaction and Detection Strategies

ATPases catalyze the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (Pi), releasing energy that drives essential cellular work. HTS platforms detect this activity through various strategies, each with distinct advantages and limitations. The core reaction is:

ATP + H₂O → ADP + Pi + Energy

Table 1: Classification of Major ATPase Activity Assay Technologies

Assay Category	Detection Method	Measured Product	Throughput Capacity	Key Applications
Universal ADP Detection	Fluorescence Polarization/TR-FRET/Fluorescence Intensity	ADP	High (96-1536-well)	Compound screening, inhibitor potency, selectivity profiling [33]
Phosphate Release	Colorimetric (malachite green)	Inorganic phosphate	Medium (96-384-well)	Enzyme kinetic studies, functional characterization [31]
Fluorescent Analog Displacement	Fluorescence intensity	Competitive binding	Medium (96-384-well)	ATP-competitive inhibitor identification [34]
Cellular ATP Detection	Bioluminescence (luciferase)	ATP	High (96-1536-well)	Cell viability, toxicity screening [35]
FRET-Based Cellular Assays	Time-resolved FRET	Protein-protein interaction	High (96-384-well)	Protein complex modulation in live cells [36]

Universal ADP Detection Assays

The Transcreener ADP Assay platform exemplifies a universal approach for ATPase screening that directly measures ADP production rather than relying on enzyme-specific coupling steps [33]. This technology utilizes an antibody highly selective for ADP over ATP and a far-red fluorescent tracer. When ADP accumulates from ATP hydrolysis, it competes with the tracer for antibody binding, producing a detectable fluorescence signal change measurable by fluorescence polarization (FP), fluorescence intensity (FI), or time-resolved FRET (TR-FRET) [33].

Key Performance Characteristics:

• Robustness: Z' values >0.7 under initial velocity conditions
• Sensitivity: Detects ADP down to 10 nM with ATP concentrations from 0.1 μM to 1,000 μM
• Throughput: Compatible with 96-, 384-, and 1536-well formats
• Versatility: Applicable to diverse ATPase classes including motor proteins, helicases, and AAA+ ATPases

This platform's ability to operate in both endpoint and kinetic modes enables determination of initial velocities and drug-target residence times, providing crucial information for lead optimization [33]. The homogeneous, mix-and-read format requires no washing steps, facilitating automation and minimizing hands-on time. Importantly, direct ADP detection avoids interference from coupling enzymes commonly used in traditional assays, significantly reducing false positives in compound screening [33].

Phosphate Release Assays

The malachite green-based phosphate detection assay represents a traditional yet reliable method for measuring ATPase activity through quantitation of inorganic phosphate release [31]. This colorimetric approach exploits the formation of a complex between malachite green molybdate and free phosphate under acidic conditions, producing a measurable absorbance change at 650 nm.

Experimental Protocol for Phosphate Release Assay [31]:

• Reaction Setup: Combine purified ATPase (typically 5-10 μM) with ATP (100 mM stock in Tris base) and MgCl₂ (100 mM) in appropriate buffer (e.g., HEPES/NaCl/glycerol)
• Incubation: Allow ATP hydrolysis to proceed at optimal temperature (e.g., 37°C) for determined time intervals (0-60 minutes)
• Sampling: Remove aliquots at timed intervals and immediately freeze to stop reaction
• Detection: Mix samples with malachite green/molybdate detection reagent and incubate 25 minutes at room temperature
• Quantitation: Measure absorbance at 650 nm and calculate phosphate concentration using a standard curve

This method provides excellent linearity for kinetic measurements when optimized properly, with phosphate release proceeding linearly over time courses of at least 60 minutes [31]. The assay can be adapted for endpoint or kinetic measurements and requires only basic laboratory equipment, making it accessible for laboratories without specialized instrumentation. However, potential interference from phosphate contaminants requires careful reagent preparation and handling.

Fluorescent ATP Analog Displacement Assays

The TNP-ATP displacement assay provides a specialized approach for identifying compounds that compete with ATP for binding to the enzyme's active site [34]. This method utilizes 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), a fluorescent ATP analog that exhibits enhanced fluorescence upon binding to the ATPase pocket.

Implementation for Bacterial Histidine Kinases [34]:

• Protein Preparation: Express and purify catalytic domain of target ATPase (e.g., Salmonella PhoQ residues 332-487 as GST fusion protein)
• Binding Measurement: Titrate purified protein against fixed TNP-ATP concentration (150 μM) in buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl)
• Fluorescence Detection: Monitor emission at 540 nm with excitation at 403 nm using plate reader
• Competition Studies: Add test compounds to pre-formed TNP-ATP:protein complex and measure fluorescence decrease
• Data Analysis: Calculate dissociation constants using Langmuir single-site binding equation

This approach proved valuable for screening GHL inhibitors against the bacterial histidine kinase PhoQ, identifying compounds that bind to the unique Bergerat ATP-binding fold [34]. The method is particularly useful for ATPases that bind but do not hydrolyze ATP, as it directly measures ligand binding rather than catalytic turnover.

Cellular ATPase Activity and Viability Assays

Bioluminescent ATP assays measure cellular ATP levels as an indicator of cell viability and metabolic activity, providing indirect assessment of ATPase function in cellular contexts [35]. These assays utilize firefly luciferase enzymatic reactions where ATP from viable cells generates photons of light.

Mechanism and Advancements:

• Reaction Principle: Luciferase catalyzes ATP-dependent conversion of luciferin to oxyluciferin, emitting light at 550-570 nm
• Signal Stability: Modern formulations using Ultra-Glo rLuciferase produce stable "glow-type" signals lasting hours rather than historical "flash" assays lasting seconds
• Application Range: CellTiter-Glo assays for monolayer/suspension cultures, 3D microtissues, and microbial cells
• Throughput: Amenable to HTS with 96-, 384-, and 1536-well formats

These assays serve as workhorses for cell-based screening but provide indirect information about specific ATPase targets, as cellular ATP levels integrate contributions from all cellular ATPases and metabolic pathways [35].

FRET-Based Cellular Assays for Protein-Protein Interactions

Time-resolved FRET (TR-FRET) between fluorescent protein-tagged ATPases and regulatory partners enables monitoring protein-protein interactions in live cells [36]. This approach was successfully implemented for the SERCA2a-phospholamban (PLB) complex, a validated therapeutic target for heart failure.

Biosensor Implementation [36]:

• Construct Design: Fuse GFP to SERCA2a N-terminus and RFP to PLB N-terminus
• Cellular Expression: Co-express in HEK293 cells with proper endoplasmic reticulum localization
• FRET Measurement: Detect decreased donor (GFP) fluorescence lifetime when acceptor (RFP-PLB) binds
• Calcium Dependence: Demonstrate FRET decrease from E=0.14 to E=0.11 with high Ca²⁺ (10 μM)
• Inhibitor Validation: Test compounds for FRET modulation indicating complex disruption

This platform achieved exceptional quality with a 30-fold decrease in coefficient of variation compared to intensity-based detection, enabling identification of compounds that specifically alter SERCA2a-PLB structure and function [36].

Comparative Performance Analysis

Table 2: Quantitative Performance Metrics of ATPase Assay Platforms

Assay Platform	Sensitivity	Dynamic Range	Z' Factor	Signal Stability	Compound Interference	Cost Considerations
Transcreener ADP²	~10 nM ADP	0.1-1000 μM ATP	>0.7 [33]	>24 hours [33]	Low (far-red tracers) [33]	Moderate (commercial kits)
Malachite Green Pi	~0.06 μM Pi [33]	0-40 μM Pi	Not specified	25 min incubation [31]	Moderate (phosphate contamination)	Low (in-house reagents)
TNP-ATP Displacement	Protein-dependent	Limited by TNP-ATP Kd [34]	Not specified	10 min equilibrium [34]	High (fluorescent compounds)	Low to moderate
Cellular Bioluminescence	<1,000 cells [35]	4-5 log range	Not specified	>3-5 hours (glow-type) [35]	Moderate (quinones)	Moderate (commercial reagents)
TR-FRET Cellular	High (nanosecond resolution) [36]	Concentration-dependent FRET [36]	Excellent (30-fold CV improvement) [36]	Measurement-dependent	Low (time-resolved detection)	High (specialized instrumentation)

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for ATPase Screening

Reagent/Category	Specific Examples	Function/Application	Key Features
Universal ADP Detection	Transcreener ADP² Assay Kits (FP, FI, TR-FRET formats) [33]	Direct ADP measurement for any ATPase	Antibody selective for ADP over ATP, far-red tracers, HTS-ready
Phosphate Detection	Malachite green/molybdate reagent [31]	Colorimetric Pi detection	Simple protocol, accessible instrumentation, adaptable format
Fluorescent ATP Analog	TNP-ATP (2′,3′-O-(2,4,6-trinitrophenyl) ATP) [34]	Competitive binding studies	Fluorescence enhancement upon binding, specific for ATP pockets
Bioluminescent Cell Viability	CellTiter-Glo 2.0/3D, BacTiter-Glo [35]	Cellular ATP measurement, viability	"Glow-type" kinetics (3-5 hour stability), optimized for cell types
FRET Biosensors	GFP-SERCA2a, RFP-PLB constructs [36]	Protein-protein interaction monitoring	Live-cell compatible, conformation-sensitive, high specificity
Specialized Plate Readers	FLT plate readers (Fluorescence Innovations) [36]	Fluorescence lifetime measurement	Nanosecond resolution, TR-FRET capability, low CV
ATPase Enzymes	Purified PhoQcat, EpsE, SERCA2a [34] [31] [36]	Enzyme source for biochemical assays	Catalytically active, properly folded, contamination-free

Experimental Design Considerations

Platform Selection Guidelines

Choosing an appropriate ATPase assay platform requires careful consideration of research objectives, target characteristics, and available resources. For primary compound screening against purified ATPases, the Transcreener ADP² platform offers exceptional robustness and versatility with minimal interference [33]. When investigating specific ATP-competitive inhibitors, the TNP-ATP displacement assay provides direct binding information but requires a suitable fluorescent analog [34]. For kinetic characterization and mechanistic studies, the malachite green phosphate assay remains a reliable, cost-effective option despite requiring more optimization [31].

Cellular contexts introduce additional complexity. The SERCA2a-PLB TR-FRET biosensor exemplifies how specific protein-protein interactions can be monitored in live cells with high precision [36]. When cellular viability or metabolic effects are the primary interest, bioluminescent ATP assays provide robust, HTS-compatible solutions [35]. Researchers should consider target localization, endogenous expression, and relevance of cellular environment when selecting between biochemical and cellular formats.

Technical Optimization Strategies

Successful ATPase assay implementation requires meticulous optimization of several parameters:

Enzyme Concentration: Titrate to establish linear initial velocity conditions, typically using 5-10 μM purified protein for phosphate release assays [31] or nanogram quantities for Transcreener assays [33].

Time Course: Determine linear reaction period through kinetic measurements; phosphate release should demonstrate linearity for at least 60 minutes under optimal conditions [31].

ATP Concentration: Utilize physiologically relevant ATP levels while considering enzyme kinetics; universal ADP detection supports broad ATP ranges from 0.1 μM to 1,000 μM [33].

Cofactor Requirements: Identify essential metal cofactors (typically Mg²⁺ at 1:1 ratio with ATP) and potential stimulants [31].

Interference Controls: Include appropriate controls for compound autofluorescence (FRET/fluorescence assays), phosphate contamination (malachite green), and coupling enzyme inhibition (coupled assays) [33].

The expanding repertoire of high-throughput screening platforms for intrinsic ATPase activity provides researchers with powerful tools for drug discovery and mechanistic studies. Universal ADP detection assays offer robust, interference-resistant solutions for primary screening campaigns, while specialized approaches like TNP-ATP displacement and cellular FRET biosensors yield targeted information about binding and protein interactions. As comparative genomic analyses continue to reveal the diversity and medical significance of ATPase families, these assay technologies will play increasingly important roles in translating genetic insights into therapeutic advances.

In the context of comparative genomic analyses of P-type ATPase inhibitors, computational approaches have become indispensable for accelerating drug discovery. Structure-based virtual screening (SBVS) and homology modeling represent two pivotal methodologies that leverage computational power to identify and optimize novel therapeutic compounds. SBVS uses three-dimensional structural information of biological targets to computationally screen large libraries of small molecules, rapidly identifying potential hits without the initial need for resource-intensive wet-lab experiments [37]. When experimental structures are unavailable, homology modeling enables researchers to construct reliable protein models based on evolutionarily related templates, providing the structural foundation necessary for subsequent virtual screening campaigns [38]. These approaches are particularly valuable for studying P-type ATPases—a large family of membrane transporters implicated in numerous physiological processes and diseases—where obtaining high-resolution experimental structures remains challenging [39].

The integration of these methods into comparative genomic frameworks allows researchers to bridge genomic information with functional and structural insights, facilitating the identification of critical residues, binding sites, and structural features that govern inhibitor specificity and efficacy across different P-type ATPase subtypes and orthologs [40] [39]. This review comprehensively compares these computational methodologies, their performance characteristics, experimental protocols, and applications in P-type ATPase research, providing researchers with a practical guide for implementing these techniques in drug discovery pipelines.

Methodological Principles and Workflows

Structure-Based Virtual Screening: Docking at Scale

SBVS relies on computational docking to predict how small molecules interact with a target protein's binding site. The fundamental premise involves positioning each compound from a virtual library into the target binding site and evaluating the complementarity using scoring functions [37]. This approach transforms the drug discovery pipeline by enabling the efficient screening of millions of compounds while consuming minimal physical resources compared to traditional high-throughput screening.

The typical SBVS workflow encompasses several critical stages: target preparation, compound library selection and preparation, molecular docking, and post-processing analysis [37]. Target preparation involves processing the protein structure (experimentally determined or computationally modeled) by assigning proper protonation states, optimizing hydrogen bonding networks, and sometimes accounting for structural flexibility through molecular dynamics simulations or ensemble docking [37]. Library preparation focuses on curating chemically relevant compounds from databases like ZINC (containing over 13 million purchasable compounds) and preparing them for docking by generating 3D conformations and assigning proper tautomeric and protonation states [37]. During the docking phase, each compound is systematically positioned within the target binding site, and its binding affinity is estimated using scoring functions that evaluate steric, electrostatic, and hydrophobic complementarity [37]. Post-processing involves analyzing top-ranked compounds, considering factors beyond raw docking scores, such as interaction patterns, chemical novelty, and drug-likeness, before selecting candidates for experimental validation.

Table 1: Key Stages in Structure-Based Virtual Screening Workflow

Stage	Key Activities	Common Tools/Resources
Target Preparation	Protonation state assignment, hydrogen bonding optimization, structural refinement	Molecular dynamics simulations, protein preparation modules in Schrödinger, MOE
Library Preparation	Compound filtering, 3D conformation generation, tautomer and ionization state enumeration	ZINC database, ChemBridge, Filtering based on "rule of five"
Molecular Docking	Ligand conformational sampling, pose prediction, binding affinity estimation	AutoDock, Dock, Glide, GOLD, Surflex
Post-Processing	Pose clustering, interaction analysis, chemical novelty assessment, candidate selection	Visualization software (PyMOL, Chimera), interaction analysis tools

Recent advances in SBVS include the development of target-biased scoring functions optimized for specific protein classes and the application of machine learning techniques to improve binding affinity predictions [37]. These innovations address one of the fundamental challenges in docking: accurately ranking compounds by their predicted binding energies. The success of SBVS is evidenced by numerous case studies, including the discovery of potent and selective A1 receptor antagonists through the screening of 4.6 million compounds [41], and the identification of novel VPS4 inhibitors using a multi-tiered screening approach [42].

Homology Modeling: Predicting Structures from Sequences

Homology modeling, also known as comparative modeling, enables the construction of three-dimensional protein structures based on their amino acid sequences and known related structures. This approach relies on the fundamental observation that evolutionarily related proteins (homologs) share similar structural features, especially when their sequences share significant identity [38]. The method is particularly valuable for membrane proteins, such as P-type ATPases, where experimental structure determination remains challenging due to difficulties in crystallization and NMR analysis [38].

The homology modeling pipeline consists of four principal stages: template identification, target-template alignment, model building, and model validation [38]. Template identification involves searching protein structure databases (e.g., PDB) for evolutionarily related proteins with experimentally determined structures using sequence similarity search tools like BLAST. Target-template alignment is arguably the most critical step, where the target sequence is aligned with the template structure, ensuring proper placement of insertions, deletions, and conserved residues. For membrane proteins, specialized algorithms like MP-T and AlignMe incorporate membrane-specific information such as hydrophobicity profiles and transmembrane topology [38]. Model building employs various algorithms including rigid-body assembly, segment matching, or satisfaction of spatial restraints to generate the 3D coordinates of the target protein. Popular tools for this stage include Modeller (and its membrane-specific variant Medeller), I-TASSER, and Rosetta [38]. Finally, model validation assesses the structural quality using geometric checks, statistical potentials, and comparison with known structural features.

Table 2: Performance of Homology Modeling Tools Across Sequence Identity Ranges

Modeling Tool	High Sequence Identity (>40%) RMSD	Medium Sequence Identity (20-40%) RMSD	Key Methodology
Medeller	<1.5 Å	<4.0 Å	Membrane-specific constraints, segment matching
I-TASSER	<1.5 Å	<4.0 Å	Replica-exchange Monte Carlo, knowledge-based energy function
Rosetta	<1.5 Å	<4.0 Å	Fragment assembly, membrane-specific energy terms

The accuracy of homology models is strongly correlated with sequence identity between target and template. As demonstrated in comparative studies on rhodopsin family proteins, when sequence identity exceeds 40%, models with root mean square deviation (RMSD) below 1.5Å can be achieved—quality sufficient for many structure-based drug design applications [38]. Even at lower sequence identities (20-40%), models with RMSD under 4.0Å can provide valuable structural insights and serve as starting points for virtual screening [38]. For P-type ATPase research, homology modeling has proven particularly valuable, enabling studies of human P-glycoprotein (ABCB1) before experimental structures became available [43].

Comparative Performance Analysis

Applications in P-type ATPase Research

Both SBVS and homology modeling have demonstrated significant utility in research on P-type ATPase inhibitors. These membrane transporters, which include targets like PfATP6 in Plasmodium falciparum and human P-glycoprotein (ABCB1), represent important targets for antimalarial and anticancer therapies, respectively [44] [45].

For PfATP6, a calcium-transporting P-type ATPase identified as a promising antimalarial target, researchers characterized a library of phenolic compounds through combined experimental and computational approaches. The study identified di-alkylated hydroquinones, a naphthoquinone disulfide, and the disinfectant hexachlorophene as inhibitors of both parasite growth and PfATP6 activity [44]. Molecular modeling techniques including homology modeling, ligand docking, and molecular dynamics simulations revealed that hydrogen bonding and hydrophobic interactions primarily mediate binding to PfATP6 [44]. This integrated approach provided a foundation for designing small-molecule PfATP6 inhibitors with improved properties.

In cancer research, P-glycoprotein (P-gp), an ABC transporter that contributes to multidrug resistance, has been extensively studied using homology modeling and SBVS. Before the determination of human P-gp structures, researchers relied on homology models based on bacterial homologs (Sav1866, MsbA) and mouse P-gp (87% sequence identity to human) [43]. These models enabled docking studies that identified novel inhibitors targeting the nucleotide-binding domains rather than the drug-binding domains, potentially overcoming limitations of earlier generation inhibitors [45]. The successful application of these computational approaches demonstrates their power in targeting clinically relevant P-type ATPases.

Throughput, Accuracy, and Resource Requirements

When comparing SBVS and homology modeling, distinct performance characteristics emerge regarding throughput, accuracy, and resource requirements. SBVS excels at rapidly evaluating extremely large chemical spaces—modern implementations can screen millions to tens of millions of compounds using high-performance computing resources [37] [41]. However, its accuracy depends heavily on the quality of the target structure and the sophistication of the scoring function. False positive rates can be significant, often requiring post-processing filters and experimental validation [37].

Homology modeling, while computationally intensive during model building, generates reusable protein structures that can serve multiple drug discovery campaigns. Its accuracy primarily depends on target-template sequence identity, with models based on >40% sequence identity approaching near-experimental quality (RMSD < 1.5Å) [38]. For P-type ATPases with lower sequence identity to available templates, model quality decreases (RMSD > 4.0Å at 20% identity), though these models may still provide useful insights into general binding regions and conserved structural features [38].

Table 3: Performance Comparison of Computational Approaches

Parameter	Structure-Based Virtual Screening	Homology Modeling
Typical Throughput	1,000-5,000,000 compounds [37] [41]	Single model generation (reusable)
Primary Accuracy Determinants	Scoring function performance, target flexibility	Target-template sequence identity, alignment quality
Key Limitations	Scoring function inaccuracies, limited conformational sampling	Decreasing accuracy with lower sequence identity
Optimal Applications	Hit identification, lead optimization	Structure-based studies when experimental structures unavailable
Success Rate	Hit rates typically 1-30% after experimental validation [37]	RMSD < 1.5Å with >40% sequence identity to template [38]

A significant trend in both fields involves incorporating machine learning approaches to address methodological limitations. For SBVS, machine learning-enhanced scoring functions show promise in improving binding affinity predictions and reducing false positives [37]. In homology modeling, although not explicitly mentioned in the search results, deep learning-based structure prediction tools like AlphaFold have revolutionized the field by providing highly accurate protein models, potentially reducing dependence on close templates.

Experimental Protocols and Methodologies

Standard SBVS Protocol for P-type ATPase Inhibitor Identification

The following protocol outlines a standard SBVS workflow for identifying P-type ATPase inhibitors, integrating elements from successful implementations described in the literature [37] [45] [41]:

• Target Structure Preparation: Obtain the target P-type ATPase structure from experimental data or homology modeling. For homology models, validate using geometric checks and statistical potentials. Process the structure by adding hydrogen atoms, optimizing hydrogen bonding networks, and assigning partial charges using programs like AutoDockTools or Schrödinger's Protein Preparation Wizard.

• Binding Site Definition: Delineate the binding site coordinates based on known ligand interactions or predicted binding pockets. For P-type ATPases, this may include nucleotide-binding domains for inhibitors targeting ATP hydrolysis or transmembrane domains for substrates and modulators [45].

• Compound Library Preparation: Curate a screening library from databases like ZINC (http://zinc.docking.org) [37]. Apply drug-like filters (e.g., Lipinski's Rule of Five) and remove compounds with undesirable chemical properties. Generate 3D conformations, tautomers, and protonation states at physiological pH.

• Molecular Docking: Perform docking simulations using programs such as AutoDock, Glide, or GOLD. Utilize grid-based approaches for sampling efficiency. For each compound, generate multiple binding poses and score them using empirical or knowledge-based scoring functions.

• Post-processing and Hit Selection: Analyze top-ranked compounds based on docking scores, interaction patterns with key residues, chemical diversity, and commercial availability. Apply additional filters to remove pan-assay interference compounds (PAINS). Select 20-100 top candidates for experimental validation.

Homology Modeling Protocol for P-type ATPases

This protocol describes homology modeling specifically tailored for P-type ATPases, based on established methodologies with demonstrated success for membrane proteins [38] [43]:

• Template Identification and Selection: Search the Protein Data Bank (PDB) for suitable templates using sequence similarity tools (BLAST, HHblits). Prioritize templates with high sequence identity, similar function, and high-resolution structures. For P-type ATPases, consider templates like mammalian P-gp (for human orthologs) or sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) for calcium-transporting ATPases.

• Target-Template Alignment: Employ multiple sequence alignment algorithms incorporating membrane-specific information. Tools like AlignMe, MP-T, or PralineTM are particularly suited for membrane proteins as they consider hydrophobicity profiles and transmembrane topology [38]. Manually adjust alignments to ensure proper placement of conserved motifs and transmembrane helices.

• Model Building: Generate 3D models using satisfaction of spatial restraints (Modeller), replica-exchange Monte Carlo (I-TASSER), or fragment assembly (Rosetta). For membrane proteins, use specialized implementations like Medeller that apply membrane-specific constraints [38]. Generate multiple models (typically 20-100) to sample different conformational spaces.

• Model Validation: Assess model quality using multiple metrics: Ramachandran plot statistics, Verify3D, QMEAN, and MolProbity. Compare with known structural features of P-type ATPases, such as conserved residues in nucleotide-binding sites or transmembrane helix arrangements. Select the best model for further studies.

• Model Refinement (Optional): Subject the selected model to molecular dynamics simulations in a membrane environment to relax the structure and correct any structural artifacts, particularly in loop regions.

Essential Research Reagents and Computational Tools

Successful implementation of SBVS and homology modeling requires access to specific computational tools, databases, and resources. The following table catalogs key solutions mentioned in the literature with demonstrated applications in P-type ATPase research and related fields.

Table 4: Essential Research Reagents and Computational Tools

Tool/Resource	Type	Primary Function	Application in P-type ATPase Research
ZINC Database [37]	Chemical Database	Library of commercially available compounds	Source of screening compounds for virtual screening
AutoDock [45]	Docking Software	Molecular docking and virtual screening	Identifying P-gp inhibitors through NBD targeting
Modeller/Medeller [38]	Homology Modeling	Protein structure modeling	Membrane protein-specific modeling
I-TASSER [38]	Homology Modeling	Protein structure and function prediction	General and membrane protein modeling
Rosetta [38]	Homology Modeling	Protein structure prediction and design	Membrane protein modeling with specialized energy terms
MP-T/AlignMe [38]	Alignment Tools	Membrane-specific sequence alignment	Improved alignment for transmembrane proteins
PDB [38]	Structure Database	Repository of experimental protein structures	Source of templates for homology modeling

Integrated Workflows and Signaling Pathways

The most powerful applications of computational approaches in P-type ATPase research involve integrated workflows that combine homology modeling and SBVS. These pipelines begin with genomic data and progress through structure prediction to inhibitor identification, effectively bridging the gap between sequence information and functional modulation.

The following diagram illustrates a typical integrated workflow for identifying P-type ATPase inhibitors, incorporating both homology modeling and structure-based virtual screening:

Diagram 1: Integrated workflow for P-type ATPase inhibitor identification combining homology modeling and structure-based virtual screening.

This integrated approach leverages the complementary strengths of both methodologies: homology modeling provides structural insights when experimental structures are unavailable, while SBVS efficiently explores chemical space to identify potential inhibitors. Successful implementations of similar workflows have led to the discovery of novel inhibitors for various P-type ATPases, including PfATP6 and P-gp [44] [45] [43].

The workflow emphasizes the iterative nature of computational drug discovery, where initial models and screening results can be refined based on experimental feedback, leading to progressively improved inhibitors through structure-activity relationship analysis and computational optimization.

Structure-based virtual screening and homology modeling represent powerful complementary approaches in the computational toolkit for P-type ATPase research and inhibitor development. SBVS excels in its ability to rapidly explore vast chemical spaces, identifying potential inhibitors with specific binding characteristics, while homology modeling provides essential structural insights when experimental structures are unavailable. The performance of both methods has improved significantly with advances in algorithms, computing power, and specialized tools for membrane protein handling.

For researchers studying P-type ATPases, the integration of these computational approaches with comparative genomic analyses offers a powerful strategy for bridging sequence-structure-function relationships. The documented success in identifying inhibitors for targets like PfATP6 and P-gp underscores the practical utility of these methods [44] [45]. As machine learning continues to transform both fields, and as structural databases expand with contributions from cryo-EM and other advanced techniques, the accuracy and scope of these computational approaches will further increase, solidifying their role as indispensable tools in modern drug discovery pipelines.

P-type ATPases constitute a major family of ATP-hydrolyzing ion pumps that are critical for maintaining cellular ion homeostasis. From a comparative genomic perspective, these enzymes represent phylogenetically conserved targets across diverse organisms, from human to protozoan pathogens like Plasmodium falciparum [46]. Their inhibition offers therapeutic potential for conditions ranging from cardiac diseases to malaria. Polyoxometalates (POMs)—anionic metal-oxo clusters of early transition metals—have emerged as promising inorganic scaffolds for inhibiting these biologically crucial enzymes [47] [48]. This guide provides a systematic comparison of the inhibition profiles of two major POM classes, polyoxotungstates (POTs) and polyoxovanadates (POVs), against P-type ATPases, presenting key experimental data and methodologies to inform future drug development efforts.

Comparative Inhibition Profiles of POTs and POVs

Quantitative Inhibition Data for P-type ATPases

The inhibitory potency of POTs and POVs against P-type ATPases varies significantly based on their structural archetypes and physicochemical properties. The data below summarize half-maximal inhibitory concentration (IC₅₀) values and inhibition percentages from key studies.

Table 1: Inhibition Profiles of Polyoxotungstates (POTs) Against P-type ATPases

POT (Abbreviation)	POM Archetype	Net Charge	Ca²⁺-ATPase IC₅₀ (μM)	Na⁺/K⁺-ATPase Inhibition (%) at 1 μM
K₉(C₂H₈N)₅[H₁₀Se₂W₂₉O₁₀₃] (Se₂W₂₉)	Lacunary Keggin-based	14-	0.3	14%
K₆[α-P₂W₁₈O₆₂] (P₂W₁₈)	Dawson	6-	0.6	99%
K₆H₂[CoW₁₁TiO₄₀] (CoW₁₁Ti)	Mono-substituted Keggin	8-	4	Similar to Ca²⁺-ATPase inhibition
Na₁₀[α-SiW₉O₃₄] (SiW₉)	Tri-lacunary Keggin	10-	16	Similar to Ca²⁺-ATPase inhibition
K₁₂[α-H₂P₂W₁₂O₄₈] (P₂W₁₂)	Lacunary Dawson	12-	11	Similar to Ca²⁺-ATPase inhibition
K₁₄[As₂W₁₉O₆₇(H₂O)] (As₂W₁₉)	Doubled Keggin-based	14-	28	Similar to Ca²⁺-ATPase inhibition
Na₁₂[H₄W₂₂O₇₄] (W₂₂)	Isopolytungstate	12-	68	Similar to Ca²⁺-ATPase inhibition
Na₆[TeW₆O₂₄] (TeW₆)	Anderson-Evans	6-	200	Similar to Ca²⁺-ATPase inhibition
(C₂H₈N)₆[V₂Mo₁₈O₆₂] (Dawson Hybrid)	Dawson	6-	3.4	Not Tested
(C₄H₁₆N₃)₄[V₂W₄O₁₉]₃ (Lindqvist Hybrid)	Lindqvist	4-	45.1	Not Tested

Source: [47] [49] [50]

Table 2: Inhibition Profiles of Polyoxovanadates (POVs) Against P-type ATPases

POV (Abbreviation)	POM Archetype	Ca²⁺-ATPase IC₅₀ (μM)	Na⁺/K⁺-ATPase Inhibition (%) at 10 μM
Cs₅.₆H₃.₄PV₁₄O₄₂ (PV₁₄)	Bi-capped Keggin	5	78%
Decavanadate (V₁₀)	Isopolyvanadate	15	66%
Monovanadate (V₁)	Monovanadate	80	33%

Source: [48]

Structure-Activity Relationships and Selectivity Profiles

Analysis of the data reveals critical structure-activity relationships. For POTs, high negative charge density correlates with increased inhibitory potency for the most effective compounds (IC₅₀ < 16 μM), including Se₂W₂₉, P₂W₁₈, CoW₁₁Ti, SiW₉, and P₂W₁₂ [47]. However, notable selectivity differences exist; Se₂W₂₉ demonstrates high selectivity for Ca²⁺-ATPase over Na⁺/K⁺-ATPase (14% inhibition at 1 μM), whereas P₂W₁₈ potently inhibits both pumps (99% inhibition at 1 μM) [47]. For POVs, the larger phosphotetradecavanadate (PV₁₄) exhibits significantly stronger inhibition than decavanadate (V₁₀) or monovanadate (V₁) against both ATPases [48].

Figure 1: Structure-Activity Relationship of POM Inhibitors

Detailed Experimental Protocols and Methodologies

In Vitro Ca²⁺-ATPase Activity Assay

Biological Material Preparation: Sarcoplasmic reticulum (SR) vesicles are isolated from rabbit skeletal muscle via differential centrifugation. These vesicles are rich in Ca²⁺-ATPase and serve as an established in vitro model system [47] [49].

Activity Measurement: Ca²⁺-ATPase hydrolytic activity is determined using a coupled enzyme assay with pyruvate kinase (PK) and lactate dehydrogenase (LDH). The assay monitors NADH oxidation spectrophotometrically at 340 nm [47] [49]. The reaction mixture typically contains 20 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl₂, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 5-10 μg/mL SR vesicles, 5 U/mL each of PK and LDH, and 0.5 mM CaCl₂ to activate the Ca²⁺-ATPase. The reaction is initiated by adding 5 mM ATP, and the decrease in absorbance at 340 nm is monitored for 10-20 minutes [47].

Inhibition Testing: POM solutions are freshly prepared in water and kept on ice to prevent decomposition. IC₅₀ values are determined by measuring activity in the presence of increasing concentrations of POMs [47].

Ex Vivo Na⁺/K⁺-ATPase Activity Assay

Biological System: The opercular epithelium of killifish (Fundulus heteroclitus) is used as it contains a rich population of chloride cells with abundant Na⁺/K⁺-ATPase activity. This tissue is mounted in an USsing chamber system [47] [48].

Activity Measurement: The short-circuit current (Iₛ꜀), which reflects active chloride secretion energized by basolateral Na⁺/K⁺-ATPase activity, is measured. Tissues are bathed in physiological saline solution (e.g., 135 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.8) and continuously oxygenated [47] [48].

Inhibition Testing: After obtaining a stable baseline Iₛ꜀, POMs are added to the serosal side (basolateral membrane contact). The percentage inhibition is calculated from the reduction in Iₛ꜀ compared to the baseline [47] [48].

Figure 2: Experimental Workflow for P-type ATPase Inhibition Studies

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for P-type ATPase Inhibition Studies

Reagent/Material	Function/Application	Specifications/Considerations
SR Vesicles (Sarcoplasmic Reticulum)	Source of Ca²⁺-ATPase for in vitro assays	Isolated from rabbit skeletal muscle; stored at -80°C [47]
Killifish Opercular Epithelium	Ex vivo model for Na⁺/K⁺-ATPase activity	Requires specialized aquarium facilities; mounted in USsing chamber [47]
Pyruvate Kinase/Lactate Dehydrogenase (PK/LDH)	Coupled enzyme system for ATPase activity measurement	Commercial preparations; maintained on ice during use [47]
NADH (Nicotinamide Adenine Dinucleotide)	Spectrophotometric detection of ATP hydrolysis	Light-sensitive; prepared fresh daily [47]
POM Stock Solutions	Source of inhibitors for activity assays	Typically 10 mM or 1 mM in water; kept on ice to prevent decomposition [47]
USsing Chamber System	Measurement of short-circuit current in epithelia	Requires specialized equipment and technical expertise [47] [48]

Implications for Drug Development and Target Selectivity

The comparative data reveal that POM scaffolds offer diverse inhibition profiles against P-type ATPases, with significant implications for drug development. The observed selectivity of Se₂W₂₉ for Ca²⁺-ATPase over Na⁺/K⁺-ATPase demonstrates that target-specific POM-based therapeutics are achievable through structural design [47]. Furthermore, the potent inhibition of PfATP4, a malarial P-type ATPase, by compounds such as (+)-SJ733 and MMV665878 highlights the potential application of ATPase inhibitors in infectious disease treatment [46]. These findings align with the broader context of targeting ATP-binding sites in drug discovery, as exemplified by the clinical success of protein kinase inhibitors in cancer therapy [51] [52].

The charge-density-activity correlation identified for both POTs and POVs provides a valuable design principle for optimizing POM-based inhibitors. Dawson-type POMs consistently demonstrate high potency across different metal compositions (P₂W₁₈, V₂Mo₁₈), suggesting this structural archetype possesses inherent affinity for P-type ATPase binding sites [47] [49]. As research in this field advances, these fundamental principles will guide the development of increasingly selective POM-based therapeutics for disorders involving P-type ATPase dysfunction.

The development of inhibitors for P-type ATPases represents a promising frontier in antifungal and anticancer therapy. These membrane-embedded enzymes, which utilize ATP hydrolysis to pump ions across cellular membranes, are essential for numerous physiological processes. The fungal H+-ATPase (Pma1) has emerged as a particularly attractive target for novel antifungal agents because it is essential for fungal viability, present in all pathogenic fungi, and absent from mammalian cells [53] [54]. However, a significant challenge in targeting P-type ATPases lies in the structural conservation of their ATP-binding sites, which often leads to inhibitors that cross-react with mammalian ATPases, potentially causing toxicity [52]. This review uses tetrahydrocarbazoles as a case study to explore the development of multi-ATPase inhibitors, examining their broad-spectrum activity alongside their selectivity challenges, and frames these findings within the context of comparative genomic insights into P-type ATPase structure and function.

Tetrahydrocarbazoles as Novel P-type ATPase Inhibitors

Discovery and Initial Screening

The identification of tetrahydrocarbazoles began with a screening campaign of 20,240 compounds against Saccharomyces cerevisiae Pma1 ATP hydrolysis activity. From this screen, 100 compounds demonstrated significant inhibition (>30% inhibition at 20 μM), with a series of tetrahydrocarbazoles emerging as promising hits due to their simultaneous inhibition of both Pma1 activity and fungal growth in the low micromolar range [54]. Initial repurchased hits (compounds 1-3) exhibited Pma1 IC50 values between 12.9-18.2 μM and minimum inhibitory concentration (MIC) values against S. cerevisiae and C. albicans ranging from 5-20 μM, establishing a foundation for further medicinal chemistry optimization [54].

Structural Insights and Binding Mode

Crystallographic structure determination of a SERCA-tetrahydrocarbazole complex at 3.0 Å resolution revealed that these compounds bind to a region above the ion inlet channel of the ATPase [53] [54]. This binding site location suggests a mechanism where tetrahydrocarbazoles potentially block ion access or conformational changes necessary for ATPase function. A homology model of the Candida albicans H+-ATPase based on this crystal structure indicated that tetrahydrocarbazoles likely bind to a similar pocket in the fungal enzyme, with identified pocket extensions that could be exploited for future selectivity enhancement [53].

Table 1: Inhibition Profiles of Initial Tetrahydrocarbazole Hits

Compound ID	S. cerevisiae Pma1 IC50 (μM)	S. cerevisiae MIC (μM)	C. albicans MIC (μM)
1	18.2 ± 4.7	10	20
2	12.9 ± 3.2	12	10
3	14.8 ± 3.5	12	5

Comparative Inhibition Profiles Across P-type ATPases

Expanded Tetrahydrocarbazole Analogue Testing

To explore structure-activity relationships, researchers synthesized nine additional tetrahydrocarbazole analogues (compounds 4-12) and evaluated their activity against both fungal and mammalian P-type ATPases [54]. This systematic analysis revealed a critical challenge in tetrahydrocarbazole development: while several compounds exhibited potent inhibition of fungal Pma1, many showed even greater potency against mammalian ATPases, particularly SERCA and Na+,K+-ATPase.

Compounds 6, 7, and 8 demonstrated particularly potent inhibition of mammalian ATPases, with IC50 values against SERCA of 0.09 μM, 0.11 μM, and 0.26 μM respectively – significantly lower than their Pma1 IC50 values of 2.79 μM, 2.43 μM, and 3.57 μM [54]. This pattern highlighted the conserved nature of ATP-binding sites across P-type ATPases and the difficulty in achieving fungal selectivity. However, compounds 9 and 10 emerged as more balanced inhibitors, with Pma1 IC50 values of 4.73 μM and 6.69 μM respectively, while maintaining single-digit micromolar IC50 values against SERCA (3.27 μM and 5.54 μM) and Na+,K+-ATPase (9.76 μM and 5.50 μM) [54].

Table 2: ATPase Inhibition Profile of Optimized Tetrahydrocarbazole Analogues

Compound ID	S. cerevisiae Pma1 IC50 (μM)	C. albicans Pma1 IC50 (μM)	Mammalian SERCA IC50 (μM)	Mammalian Na+,K+-ATPase IC50 (μM)
4	75.0 ± 3.2	46.0 ± 14.5	1.55 ± 0.06	37.2 ± 13.6
5	85.9 ± 10.3	129.7 ± 32.2	61.7 ± 18.8	131.9 ± 35.2
6	2.79 ± 0.36	3.89 ± 1.71	0.09 ± 0.03	0.72 ± 0.30
7	2.43 ± 0.39	2.47 ± 0.26	0.11 ± 0.06	0.38 ± 0.12
8	3.57 ± 1.81	3.77 ± 1.04	0.26 ± 0.06	1.07 ± 0.38
9	4.73 ± 2.23	5.56 ± 1.55	3.27 ± 1.89	9.76 ± 4.47
10	6.69 ± 2.06	9.70 ± 2.15	5.54 ± 3.16	5.50 ± 2.74

Antifungal Activity Spectrum

The most promising tetrahydrocarbazole compounds demonstrated broad-spectrum antifungal activity against multiple Candida species, including C. albicans, C. krusei, and C. glabrata [54]. Compound 9 exhibited consistent activity across all tested species with MIC values of 7.5 μM, while compound 7 showed particularly potent activity against S. cerevisiae (MIC 1.5 μM) and C. krusei (MIC 4.7 μM) [54]. Importantly, this antifungal activity occurred through a non-cytotoxic mechanism at therapeutic concentrations, as demonstrated by higher EC50 values against human HepG2 cells (12.45-18.64 μM for compounds 6-10) compared to their antifungal MIC values [54].

Experimental and Methodological Framework

Key Experimental Protocols

ATPase Activity Assay: The primary method for evaluating tetrahydrocarbazole inhibition involved measuring ATP hydrolysis activity of purified P-type ATPases. The standard protocol included incubating enzyme preparations with test compounds across a concentration range (typically 0-20 μM) in ATP-containing buffer, then quantifying inorganic phosphate release using colorimetric methods such as malachite green assays. IC50 values were determined from dose-response curves generated from triplicate measurements [54].

Antifungal Susceptibility Testing: Minimum inhibitory concentrations (MICs) were determined using broth microdilution methods according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Fungi were incubated with serial compound dilutions for 24-48 hours, with growth inhibition assessed visually or spectrophotometrically [54].

Membrane Depolarization Assay: The functional consequence of Pma1 inhibition was measured using fluorescent membrane potential-sensitive dyes like DiSC3(5). Fungal cells were treated with compounds, and membrane depolarization was quantified by fluorescence increase due to dye release from cells [53].

Diagram Title: Tetrahydrocarbazole Discovery Workflow

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for ATPase Inhibitor Development

Reagent/Resource	Function/Application	Example Sources
P-type ATPase Preparations	Enzymatic activity assays; target-based screening	Purified from S. cerevisiae, rabbit SERCA, pig Na+,K+-ATPase
ATPase Activity Assay Kits	Quantification of ATP hydrolysis inhibition	Commercial kits measuring inorganic phosphate release
Fungal Strain Panels	Antifungal susceptibility testing	ATCC strains: C. albicans (SC5314, 90028), C. krusei (6258), C. glabrata (90030)
Membrane Potential Dyes	Functional assessment of H+-ATPase inhibition	DiSC3(5) and other fluorescent potentiometric dyes
Homology Modeling Software	Structural analysis and binding site prediction	MODELLER, SWISS-MODEL, and other comparative tools
X-ray Crystallography	Determination of inhibitor binding modes	Synchrotron facilities for high-resolution structure solving

Strategic Considerations for Selective ATPase Inhibitor Development

Selectivity Challenges and Opportunities

The structural conservation among P-type ATPases presents a fundamental challenge for selective inhibitor development. The tetrahydrocarbazole case study clearly demonstrates this issue, with most compounds showing higher potency against mammalian SERCA and Na+,K+-ATPase than against the fungal Pma1 target [54]. This cross-reactivity stems from the conserved nature of ATP-binding sites across this enzyme family, a challenge also observed in the development of ATP-competitive inhibitors for cancer therapy, where compounds must compete with high intracellular ATP concentrations (typically 1-10 mM) and navigate similar binding pockets across different protein families [52].

However, the structural biology work with tetrahydrocarbazoles has identified potential strategies for enhancing selectivity. The crystallographic data revealed subpockets and extensions near the binding site that vary between fungal and mammalian ATPases [53]. These structural differences represent opportunities for rational drug design to engineer compounds with improved selectivity profiles. Additionally, the identification of compound 12, which showed weak selectivity for Na+,K+-ATPase over other ATPases, demonstrates that scaffold-specific selectivity is achievable [54].

Comparative Genomic Insights

Comparative genomic analyses provide valuable context for understanding ATPase inhibitor selectivity challenges. Studies of the DUF71/COG2102 family, a subgroup of PP-loop ATPases, reveal how ATPase families have evolved diverse functions while maintaining conserved structural features [55]. Similarly, analyses of Woesearchaeota genomes demonstrate how evolutionary pressures shape ATPase distribution and function across species [56]. These genomic perspectives underscore why ATPase inhibitor development must navigate the balance between conserved catalytic mechanisms and species-specific structural variations.

Diagram Title: Multi-ATPase Inhibition Network

Tetrahydrocarbazoles represent a promising class of multi-ATPase inhibitors with demonstrated broad-spectrum antifungal activity. The case study of their development highlights both the therapeutic potential of targeting fungal Pma1 and the significant challenge of achieving selectivity over mammalian ATPases. The most promising compounds (9 and 10) balance potent Pma1 inhibition (IC50 ~5-7 μM) with antifungal activity (MIC ~7.5 μM across multiple Candida species) while showing reduced cross-reactivity with mammalian ATPases compared to earlier analogues [54].

Future directions in this field should leverage the structural insights gained from SERCA-tetrahydrocarbazole co-crystals to design next-generation inhibitors with enhanced selectivity. The identified binding pocket variations between fungal and mammalian ATPases provide a blueprint for rational drug design. Additionally, the application of advanced screening technologies, including structure-based virtual screening as demonstrated with Salvianolic acid B identification for Salmonella InvC ATPase [57], may accelerate the discovery of more selective ATPase inhibitors. As genomic analyses continue to reveal the diversity and evolution of ATPase families [56] [55], these insights should inform the development of increasingly targeted therapeutic agents that capitalize on species-specific structural differences while respecting the conserved nature of essential ATPase functions.

P-type ATPases constitute a large superfamily of primary active pumps that transport diverse substrates, from ions like H+, Na+, K+, and Ca2+ to phospholipids, across biological membranes. [12] [58] These enzymes are characterized by a catalytic cycle that alternates between high- and low-affinity conformations induced by phosphorylation and dephosphorylation of a conserved aspartate residue. [58] Within the comparative genomics landscape, P-type ATPases represent a fascinating example of evolutionary conservation and diversification, with their significance in pathophysiology making them prominent drug targets. [12] Notably, the sodium efflux pump PfATP4 in Plasmodium falciparum has emerged as a leading antimalarial target, while other family members are investigated for fungal infections, cardiovascular conditions, and gastroesophageal reflux disease (GERD). [12] [19]

The rise of ChemGen—the integration of chemical biology and genomics—coupled with artificial intelligence (AI) has revolutionized the approach to targeting these proteins. AI-driven methodologies are now significantly enhancing key aspects of drug discovery, including ligand binding site prediction, protein-ligand binding pose estimation, scoring function development, and virtual screening. [59] This guide provides a comparative analysis of experimental approaches and computational tools for optimizing binding affinity and specificity against P-type ATPase targets, with a particular emphasis on PfATP4 and related pumps.

Experimental Platforms for P-type ATPase Inhibitor Profiling

Functional Assays for Inhibitor Characterization

The assessment of P-type ATPase inhibitor efficacy relies on a suite of functional assays that measure compound effects on both parasite viability and enzymatic activity.

Table 1: Key Functional Assays for P-type ATPase Inhibitor Profiling

Assay Type	Experimental Readout	Application in P-type ATPase Research	Notable Findings
Parasite Viability/Growth Inhibition	Half-maximal inhibitory concentration (IC₅₀) against P. falciparum blood stages [44]	Primary screening for antiplasmodial activity	Di-alkylated hydroquinones, naphthoquinone disulfides, and hexachlorophene demonstrate growth inhibition [44]
Enzyme Activity Assay	Na+-dependent ATPase activity measurement of purified PfATP4 [19]	Direct quantification of target engagement and inhibition	PfATP4 activity is inhibited by known compounds PA21A092 and Cipargamin [19]
Ion Transport Measurement	Quantification of calcium transport or other ion concentration changes [44]	Functional characterization of transporters like PfATP6	Identifies compounds that disrupt ion homeostasis

Structural Biology and Target Characterization

High-resolution structural information is critical for understanding inhibitor binding modes and resistance mechanisms. Recent advances in cryo-electron microscopy (cryoEM) have enabled the determination of endogenous P-type ATPase structures, providing unprecedented insights.

PfATP4 Structure Determination: A landmark 3.7 Å cryoEM structure of PfATP4 purified from CRISPR-engineered P. falciparum parasites revealed several key features. [19] The structure confirmed the five canonical P-type ATPase domains: extracellular loop (ECL), transmembrane domain (TMD), nucleotide-binding (N), phosphorylation (P), and actuator (A) domains. Notably, the structure was resolved in a Na+-bound state, with the ion-binding site located between TM4, TM5, TM6, and TM8 helices. [19]

A surprising discovery was the identification of PfABP (PfATP4-Binding Protein), a previously unknown, apicomplexan-specific binding partner whose C-terminal helix interacts with TM9 of PfATP4. [19] This finding, made possible through endogenous purification, presents a novel, unexplored avenue for designing PfATP4 inhibitors that target this protein-protein interaction. [19]

Diagram 1: Endogenous PfATP4 Structure Determination Workflow. The cryoEM pipeline from parasite engineering to structural insights, highlighting key discoveries.

Resistance Mutation Mapping

Structural biology enables the spatial mapping of resistance-conferring mutations to understand their mechanistic basis. In PfATP4, mutations associated with resistance to compounds like Cipargamin (e.g., G358S/A) localize around the proposed Na+ binding site within the TMD. [19] The G358S mutation, found in recrudescent parasites from clinical trials, is located on TM3 adjacent to the Na+ coordination site and is proposed to block Cipargamin binding by introducing a bulkier sidechain into the binding pocket. [19] Interestingly, the A211V mutation, which confers resistance to pyrazoleamide PA21A092, increases susceptibility to Cipargamin, suggesting complex, compound-specific interactions within the ion transport pathway. [19]

AI-Driven Methodologies for Binding Optimization

Computational Framework for Protein-Ligand Interaction Prediction

AI-driven methodologies have transformed structure-based drug discovery, overcoming limitations of traditional empirical approaches. [59]

Table 2: AI-Driven Methods for Protein-Ligand Interaction Prediction

Computational Task	Traditional Approach	AI-Driven Advancements	Relevant Algorithms/Models
Ligand Binding Site Prediction	Geometry-based pocket detection	Enhanced by geometric deep learning and sequence-based embeddings [59]	PyKVFinder [19]
Binding Pose Prediction	Sampling-based molecular docking	Regression-based models and protein-ligand co-generation frameworks [59]	RFdiffusion, ProteinMPNN [60]
Scoring Function Development	Empirical force fields	Integration of physical constraints with deep learning [59]	Graph Neural Networks, Transformers [59]
Virtual Screening	Sequential docking and scoring	Integrated AI-powered workflows for efficient hit identification [59]	Mixture Density Networks, Diffusion Models [59]

Generative AI for Binder Design: A breakthrough approach from the Baker lab demonstrates the de novo design of high-affinity binders to challenging biomarkers, including helical peptides. [60] By extending RFdiffusion (a generative model for creating new protein shapes) to design binders for flexible targets and refining input models through successive noising and denoising (partial diffusion), the team generated picomolar affinity binders directly from computational design without experimental optimization. [60] This "build to fit" strategy is particularly relevant for targeting dynamic P-type ATPase regions.

Diagram 2: AI-Driven De Novo Binder Design Pipeline. Workflow for generating high-affinity protein binders using RFdiffusion and ProteinMPNN.

Molecular Modeling and Simulation for P-type ATPase Inhibitors

For P-type ATPase targets where high-resolution structures are unavailable, homology modeling provides valuable structural insights. Studies on PfATP6, a P. falciparum calcium ATPase, have utilized homology modeling, ligand docking, and molecular dynamics (MD) simulations to characterize inhibitor interactions. [44] Analysis of small phenolic inhibitors revealed that a combination of hydrogen bonding and hydrophobic interactions primarily mediates binding to PfATP6. [44]

Machine learning approaches are also being combined with MD simulations to predict transporter interactions. For P-glycoprotein (P-gp), an ABC transporter often studied alongside P-type ATPases, molecular dynamics fingerprints (MDFPs) have been used as descriptors for training substrate classification models, achieving high accuracy on chemically diverse datasets. [61]

Comparative Analysis of P-type ATPase Targeting Strategies

Targeting the Ion Transport Pathway

Structural insights signify that all P-type ATPases share a similar basic structure and transport mechanism, with an ion transport pathway constructed from two access channels leading to ion binding sites at a central cavity. [12] Targeting this ion transport pathway represents the most proficient strategy for developing efficient and selective drugs. [12] This approach is validated by the location of resistance mutations for PfATP4 inhibitors within the transmembrane domain adjacent to the ion-binding site. [19]

Emerging Strategies: Allosteric and Protein-Protein Interaction Modulation

The discovery of PfABP opens new avenues for allosteric modulation of PfATP4 function. [19] This apicomplexan-specific binding partner forms a conserved interaction with PfATP4's TM9 helix, suggesting a modulatory role that could be exploited therapeutically. [19] Similarly, AI-driven design of binders that target functional protein complexes rather than just active sites represents a promising frontier.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Research Reagents for P-type ATPase Studies

Reagent / Tool	Function/Application	Example Use Case
CRISPR-Cas9 Engineering	Endogenous tagging of target genes for purification	C-terminal 3×FLAG epitope tagging of PfATP4 in Dd2 P. falciparum parasites [19]
Cipargamin	PfATP4 inhibitor; reference compound	Positive control for ATPase inhibition assays; studying resistance mechanisms [19]
PA21A092	Pyrazoleamide PfATP4 inhibitor	Tool compound for validating Na+-dependent ATPase activity [19]
Phenolic Compound Libraries	Small molecule inhibitors of PfATP6	Screening for novel antiplasmodial chemotypes [44]
RFdiffusion & ProteinMPNN	AI-driven protein design software suite	De novo design of high-affinity binders to peptide targets [60]
Homology Modeling Software	Structure prediction for targets lacking experimental structures	Molecular modeling of PfATP6 for docking studies [44]

The integration of ChemGen and AI-driven design is accelerating the development of high-affinity, specific inhibitors for P-type ATPases. Comparative analysis reveals that successful targeting strategies combine structural biology (e.g., endogenous cryoEM), functional genomics (e.g., resistance mutation mapping), and computational approaches (e.g., generative AI and molecular dynamics). The ion transport pathway remains the most promising target site, while emerging opportunities include allosteric modulation through newly discovered binding partners like PfABP. As AI technologies continue to evolve, they are expected to further revolutionize molecular docking and affinity prediction, increasing both the accuracy and efficiency of P-type ATPase-targeted drug discovery. [59]

Overcoming Selectivity Challenges and Resistance Mechanisms in ATPase Inhibition

The continual evolution of drug-resistant Plasmodium falciparum parasites poses a significant threat to global malaria control efforts. Within this landscape, the P-type cation-transporting ATPase, PfATP4, has emerged as one of the most promising novel antimalarial targets. This sodium efflux pump, located on the parasite's plasma membrane, maintains sodium homeostasis and is critical for parasite survival [19]. Its validation as a drug target stems from the convergence of multiple, structurally distinct chemotypes—including spiroindolones, aminopyrazoles, and pyrazoleamides—that all share a common mechanism of action centered on PfATP4 inhibition [62] [63].

The clinical relevance of PfATP4 has been demonstrated by cipargamin, a spiroindolone that exhibited faster parasite clearance times than artemisinin in Phase II clinical trials [62]. However, the emergence of resistance-conferring mutations in PfATP4, both in laboratory settings and in clinical trials, underscores the urgent need to understand the molecular basis of resistance [63]. This guide provides a comparative analysis of resistance mechanisms across different PfATP4 inhibitor classes, offering experimental approaches and structural insights to inform the development of next-generation antimalarials.

Experimental Approaches for Mapping Resistance Mutations

In Vitro Resistance Selection and Whole-Genome Sequencing

The primary method for identifying resistance-conferring mutations involves long-term in vitro culture of parasites under sublethal drug pressure, followed by whole-genome sequencing of resistant clones.

Detailed Protocol:

• Parasite Culture & Drug Pressure: Initiate independent cultures of the multidrug-resistant P. falciparum Dd2 strain. Expose cultures to sublethal concentrations of the PfATP4 inhibitor (e.g., GNF-Pf4492) for approximately 70 days, with incremental dose increases to select for resistant populations [62].
• Clone Isolation: Limit-dilution subclone resistant parasites from each independent culture to ensure isolation of distinct clonal lines.
• Genomic DNA Preparation: Extract high-quality genomic DNA from resistant clones and the parental Dd2 strain.
• Whole-Genome Sequencing: Sequence genomes to high coverage (≥40x) using next-generation sequencing platforms. Align sequences to the P. falciparum 3D7 reference genome.
• Variant Identification: Apply quality filters to identify single-nucleotide variants (SNVs). Compare resistant clone sequences with the parental line to identify unique, resistance-associated nonsynonymous mutations [62].

Table 1: Key Reagents for In Vitro Resistance Selection

Reagent/Resource	Function	Example/Specification
P. falciparum Dd2 strain	Multidrug-resistant parasitic line for selection experiments	Clonal parasite line [62]
PfATP4 Inhibitor	Selective pressure agent	GNF-Pf4492, Cipargamin, (+)-SJ733, PA21A092 [62] [63]
Culture Media	Parasite growth and maintenance	Complete RPMI 1640 with human serum
Human Red Blood Cells	Host cells for parasite culture	Type O+ washed RBCs
LIMITATION: Specific catalog numbers for commercial reagents are not provided in the search results.

The following workflow diagram summarizes the key experimental and computational steps for identifying and characterizing PfATP4 resistance mutations.

Functional Validation of Resistance Mutations

Once candidate mutations are identified, their functional contribution to resistance must be validated through physiological and biochemical assays.

Key Functional Assays:

• Cytosolic Sodium Measurement: Use sodium-sensitive fluorescent dyes (e.g., SBFI-AM) to measure resting cytosolic [Na+] in resistant and parental parasites. PfATP4 inhibition typically causes rapid Na+ accumulation; resistant lines show attenuated responses [63].
• ATPase Activity Assays: Purify PfATP4 from engineered parasites using affinity tags (e.g., 3×FLAG). Measure Na+-dependent ATPase activity in the presence of inhibitors to confirm reduced drug sensitivity [19].
• Cross-Resistance Profiling: Determine IC~50~ values for resistant clones against multiple PfATP4 inhibitor chemotypes to assess cross-resistance patterns, which reveal insights into binding site overlaps [62] [63].

Comprehensive Atlas of PfATP4 Resistance Mutations

The convergence of resistance selection experiments with structurally diverse inhibitors has enabled the creation of a comprehensive resistance map for PfATP4. The table below summarizes key resistance-conferring mutations, their associated inhibitor classes, and functional impacts.

Table 2: Catalog of Clinically Relevant PfATP4 Resistance Mutations

Mutation	Primary Inhibitor Class	Resistance Level (Fold-Change in IC₅₀)	Cross-Resistance Profile	Functional Consequences
G358S [63]	Spiroindolones (Cipargamin)	High (>1000-fold)	Dihydroisoquinolones ((+)-SJ733)	Reduces PfATP4's affinity for Na+; increases resting cytosolic [Na+]
A211T/V [62]	Aminopyrazoles (GNF-Pf4492)	Moderate (4-6 fold)	Limited data	Located in TM2 near ion-binding site; may alter inhibitor access
I203L [62]	Aminopyrazoles	Moderate (~4 fold)	Limited data	TM2 mutation; often occurs with P990R
A187V [62]	Aminopyrazoles	Moderate (~3 fold)	Limited data	TM2 mutation; confers stable resistance without fitness cost
L263V	Not specified	Not specified	Not specified	Found in clinical isolates; location suggests potential role
P990R [62]	Aminopyrazoles	Moderate	Limited data	Often occurs as secondary mutation with I203L

Structural Mapping of Resistance Mutations

Recent breakthroughs in structural biology have provided unprecedented insights into PfATP4 resistance mechanisms. The determination of PfATP4's endogenous structure at 3.7 Å resolution using cryo-electron microscopy has enabled precise mapping of resistance mutations within functional domains [19] [21].

PfATP4 Architecture and Mutation Localization

The PfATP4 structure reveals five canonical P-type ATPase domains: Transmembrane Domain (TMD), Extracellular Loop (ECL) domain, Nucleotide-binding (N) domain, Phosphorylation (P) domain, and Actuator (A) domain [19]. The majority of resistance-conferring mutations cluster within the TMD, particularly around the ion-binding site, suggesting direct interference with inhibitor binding.

Key Structural Insights:

• G358S Mechanism: The G358S mutation maps to transmembrane helix 3 (TM3), adjacent to the proposed Na+ coordination site. Structural modeling indicates this mutation introduces a serine sidechain that likely sterically hinders cipargamin binding within the inhibitor pocket [19] [63].
• A211V Mechanism: Located on TM2 near both the ion-binding site and proposed cipargamin binding pocket, A211V may cause allosteric perturbations that reduce inhibitor affinity while paradoxically increasing susceptibility to certain other chemotypes [19].
• Ion-Binding Site Proximity: Multiple resistance mutations (G358S, A211V, I203L, A187V) localize to the ion-binding site formed by TM4, TM5, TM6, and TM8, highlighting this region as a hotspot for inhibitor binding and resistance development [19].

Discovery of PfABP: A Novel Regulatory Component

A groundbreaking discovery from the endogenous PfATP4 structure is the identification of PfATP4-Binding Protein (PfABP), a previously unknown apicomplexan-specific binding partner [19] [21]. PfABP forms a conserved interaction with TM9 of PfATP4 and appears essential for protein stability and function. Loss of PfABP leads to rapid degradation of PfATP4 and parasite death [23]. This discovery opens new avenues for antimalarial development targeting the PfATP4-PfABP interface, which may be less prone to resistance mutations than the inhibitor-binding site itself [19].

The following diagram illustrates the structural organization of PfATP4 and the spatial relationships between key resistance mutations and functional domains.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for PfATP4 Resistance Studies

Reagent/Category	Specific Examples	Research Application
PfATP4 Inhibitors	Cipargamin (Spiroindolone), GNF-Pf4492 (Aminopyrazole), PA21A092 (Pyrazoleamide), (+)-SJ733 (Dihydroisoquinolone)	Selective pressure in resistance studies; mode of action characterization [62] [63]
Parasite Lines	Dd2 (parental), Isogenic mutant lines (e.g., PfATP4-G358S, PfATP4-A211T), CRISPR-engineered parasites	Comparative studies of resistance mechanisms and fitness costs [62] [19]
Antibodies & Tags	3×FLAG epitope tag, Anti-FLAG antibodies	Affinity purification of PfATP4 for biochemical and structural studies [19]
Biochemical Assays	Na+-dependent ATPase activity assay, SBFI-AM sodium flux assay, Cytosolic pH measurement	Functional validation of PfATP4 activity and inhibitor effects [19] [63]
Structural Biology	Cryo-electron microscopy, Homology modeling, Molecular dynamics simulations	High-resolution structure determination and inhibitor binding analysis [19] [21]

The systematic mapping of resistance-conferring mutations in PfATP4 has provided crucial insights for antimalarial development. The convergence of multiple inhibitor classes on this target underscores its central role in parasite biology, while the distinct patterns of cross-resistance suggest both overlapping and distinct binding modes. The recent structural elucidation of PfATP4 complexed with its essential binding partner PfABP represents a transformative advancement, revealing novel vulnerabilities beyond the inhibitor-binding site itself [19] [23].

Future antimalarial development should focus on designing next-generation inhibitors that either target less mutable regions of PfATP4 or exploit the newly discovered PfABP interface. Combination therapies pairing PfATP4 inhibitors with compounds having unrelated mechanisms will be essential to protect against resistance emergence. The experimental frameworks and structural insights presented here provide a roadmap for these efforts, emphasizing the importance of integrating genomic, biochemical, and structural approaches in the fight against antimalarial resistance.

P-type ATPases constitute a large superfamily of primary active pumps that transport diverse substrates, from ions to phospholipids, across biological membranes. These enzymes are characterized by the formation of a phosphorylated intermediate during their catalytic cycle and follow a conserved mechanism described by the Post-Albers cycle, alternating between E1 and E2 conformations [2]. The significance of P-type ATPases in physiology and disease has made them attractive drug targets, with inhibitors already in clinical use for various conditions [12] [64]. However, a fundamental challenge persists: achieving sufficient selectivity for homologous ATPases across different species while sparing off-target human pumps.

This guide explores strategic approaches for enhancing inhibitor selectivity by exploiting structural variations in P-type ATPases across species. We focus on comparative structural analyses that reveal promising targets for selective intervention, supported by experimental data from recent studies. The objective is to provide researchers with a framework for rational design of next-generation inhibitors that leverage evolutionary divergence in these essential membrane transporters.

Evolutionary and Structural Diversity of P-type ATPases

Phylogenetic Classification and Functional Specialization

P-type ATPases are phylogenetically classified into five major families (P1-P5), which are further divided into subfamilies based on substrate specificity and sequence motifs [2] [65]. This evolutionary diversification has resulted in distinct structural features across families and species:

• P1A ATPases: Found exclusively in prokaryotes, these form heterotetrameric complexes (KdpFABC) for high-affinity potassium uptake and represent the simplest catalytic subunits with only seven transmembrane helices [2].
• P1B ATPases: Heavy metal transporters present across all kingdoms of life, further subdivided based on specificity for monovalent (Cu+, Ag+) or divalent (Zn2+, Cd2+, Pb2+) metals [65] [3].
• P2 ATPases: Cation pumps including SERCA Ca2+-ATPases (P2A), plasma membrane Ca2+-ATPases (P2B), and Na+/K+-ATPases (P2C) [65].
• P3 ATPases: H+-pumps found in plants and fungi [65].
• P4 ATPases: Phospholipid flippases unique to eukaryotes [2] [65].
• P5 ATPases: Poorly characterized ATPases restricted to inner membranes of eukaryotes [65].

This phylogenetic diversity underlies the structural variations that can be exploited for selective inhibitor design.

Conserved Core Structure and Mechanism

Despite their functional diversity, all P-type ATPases share a conserved core structure and catalytic mechanism. The minimal functional unit consists of three cytoplasmic domains (actuator (A), nucleotide-binding (N), and phosphorylation (P) domains) and a transmembrane domain (TMD) comprising 6-12 helices that form the transport pathway [2] [64]. The catalytic cycle involves autophosphorylation at a conserved aspartate residue within the DKTGT motif, with energy from ATP hydrolysis driving conformational changes that enable active transport against electrochemical gradients [2] [65].

The following diagram illustrates the evolutionary relationships between major P-type ATPase families and their characteristic structural features:

Strategic Approaches for Selective Targeting

Targeting Apicomplexan-Specific Accessory Proteins

Recent structural biology breakthroughs have revealed previously unknown accessory proteins that represent promising targets for species-selective inhibition. The 2023 cryo-EM structure of Plasmodium falciparum PfATP4 at 3.7 Å resolution revealed an apicomplexan-specific binding partner, PfABP, which forms a conserved, modulatory interaction with the transporter [19].

Key Findings:

• PfABP interacts specifically with transmembrane helix 9 of PfATP4
• This protein-protein interaction is conserved across Apicomplexan parasites but absent in mammalian systems
• Disruption of this interface could selectively impair parasite Na+ homeostasis without affecting host ATPases

The discovery of PfABP presents an entirely new avenue for designing selective PfATP4 inhibitors that target the protein-protein interaction interface rather than the conserved catalytic core [19].

Exploiting Ion Pathway Architecture Variations

Structural comparisons reveal significant differences in ion transport pathways, even among ATPases transporting similar substrates. The recent structure of a PIB-4-ATPase (sCoaT) revealed several novel features absent in other heavy metal transporters [3]:

Distinct PIB-4 Characteristics:

• Absence of classical heavy-metal-binding domains (HMBDs) present in PIB-1 and PIB-2 ATPases
• Unique coordination geometry for zinc and cobalt transport
• An invariant histidine serving dual roles in heavy-metal release and as an internal counter ion
• Potassium-independent turnover, contrasting with many other P-type ATPases

These structural differences enable the development of compounds that specifically target PIB-4-ATPases, which function as virulence factors in pathogens like Mycobacterium tuberculosis [3].

Targeting Extracellular Loop Variations

The extracellular loop (ECL) domains of P-type ATPases show considerable sequence and structural variation across species, representing promising targets for selective inhibition. In PfATP4, the ECL domain juts into the parasitophorous vacuole lumen and comprises four long β-sheets connected by long loops and flanked by two small helices [19]. This architecture differs significantly from mammalian P2-type ATPases and contains species-specific epitopes for targeted intervention.

Experimental Data and Comparative Inhibition Profiles

Quantitative Analysis of Inhibitor Selectivity

Experimental data from inhibition studies reveal substantial differences in compound sensitivity across P-type ATPase orthologs. The table below summarizes selectivity profiles for established and emerging inhibitors:

Table 1: Comparative Inhibition Profiles of P-type ATPase Targeting Compounds

Compound	Target ATPase	Species	IC₅₀	Selectivity Against Human Orthologs	Structural Basis of Selectivity
Cipargamin	PfATP4	P. falciparum	<100 nM [19]	>100-fold vs. Na+/K+-ATPase [19]	Binds pocket adjacent to ion site; resistance mutations G358S/A on TM3 [19]
PA21A092	PfATP4	P. falciparum	~100 nM [19]	High (clinical candidate) [19]	A211V resistance mutation in TM2 near ion site [19]
Demethoxycurcumin	PM H+-ATPase	S. cerevisiae	~10 μM [64]	~3-fold vs. SERCA [64]	Non-competitive ATP antagonist; binds conserved allosteric site
P2W18	SERCA Ca²⁺-ATPase	Rabbit	0.6 μM [47]	Limited (similar Na+/K+-ATPase inhibition) [47]	Dawson-type polyoxotungstate; charge density-dependent inhibition
Se2W29	SERCA Ca²⁺-ATPase	Rabbit	0.3 μM [47]	>10-fold vs. Na+/K+-ATPase [47]	Large heteropolytungstate; selective for Ca²⁺-ATPase
Thapsigargin	SERCA	Mammalian	~10 nM [12]	Pan-SERCA inhibitor (low isoform selectivity)	Binds transmembrane region; clinical prodrug (Mipsagargin) [12]

Structural Mapping of Resistance Mutations

Analysis of resistance-conferring mutations provides critical insights into species-specific structural determinants. In PfATP4, resistance mutations against leading antimalarial compounds cluster in specific transmembrane regions:

Table 2: Resistance Mutations in PfATP4 and Their Structural Implications

Mutation	Compound	Structural Location	Proposed Resistance Mechanism
G358S/A	Cipargamin, (+)-SJ733 [19]	TM3, adjacent to Na+ coordination site	Steric hindrance by introduced side chain blocks drug access
A211V	PA21A092 [19]	TM2, near ion-binding site	Alters drug-binding pocket conformation
L263V	Cipargamin [19]	TM4, facing drug-binding pocket	Reduces binding affinity through altered side-chain interactions
E345K	Spiroindolones [19]	Extracellular end of TM6	Disrupts critical drug-protein contacts

These mutations map to regions with significant structural divergence between parasite and human ATPases, highlighting the potential for designing compounds that exploit these differences to overcome resistance.

Experimental Protocols for Selectivity Assessment

Ortholog Replacement and Functional Characterization

A powerful approach for evaluating species-specific inhibitor effects involves ortholog replacement followed by functional characterization:

Protocol:

• Gene Replacement: Replace the endogenous ATPase gene with orthologs from different species using CRISPR-Cas9 [19]
• Membrane Preparation: Isolate plasma membranes via differential centrifugation and sucrose density gradient centrifugation [64]
• ATPase Activity Assay: Measure ATP hydrolysis using spectrophotometric NADH oxidation with an ATP-regenerating system [64] [3]
• Ion Transport Assay: Quantify ion transport using radioisotope fluxes or fluorescent indicators
• Inhibitor Dose-Response: Determine IC₅₀ values for candidate inhibitors across orthologs

This approach was successfully used to demonstrate that drug sensitivity in P. falciparum is a function of PfATP4 primary sequence [19].

Structural Biology Workflow for Comparative Analysis

Determining high-resolution structures of ATPases from different species enables rational design of selective inhibitors:

Key Methodological Considerations:

• Endogenous Purification: Isolate ATPases directly from native sources to preserve native post-translational modifications and protein complexes [19]
• Cryo-EM Conditions: Optimize grid preparation using different lipid environments to stabilize specific conformational states
• Resolution Targets: Aim for ≤4Å resolution to visualize side-chain interactions and identify water molecules in binding sites
• Molecular Dynamics: Supplement structural data with simulations to understand dynamic differences between orthologs

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for Studying P-type ATPase Selectivity

Reagent/Category	Specific Examples	Function/Application	Species-Specific Considerations
Expression Systems	P. pastoris, Sf9 insect cells, HEK293	Heterologous expression for structural studies	PfATP4 requires endogenous system [19]; other ATPases express well heterologously [3]
Activity Assays	Baginski assay, NADH-coupled ATPase assay	Functional characterization of ATP hydrolysis	Ion specificity varies (Na+ for PfATP4, Ca2+ for SERCA, Zn2+ for PIB-4) [3]
Membrane Models	SR vesicles, plasma membrane preparations	Native environment for functional studies	Lipid composition significantly affects activity [47] [64]
Selective Inhibitors	Cipargamin, Demethoxycurcumin, Thapsigargin	Selectivity profiling and validation	Compound sensitivity varies dramatically between orthologs [64] [19]
Structural Tools	Cryo-EM, X-ray crystallography, homology modeling	High-resolution structure determination	Endogenous purification critical for some targets [19]
Engineering Tools	CRISPR-Cas9, ortholog replacement	Functional validation in native context	Essential for establishing species-specific determinants [19]

The strategic exploitation of species-specific structural variations in P-type ATPases represents a powerful approach for developing selective inhibitors with improved therapeutic indices. Key structural features—including accessory protein interactions, ion pathway architectures, extracellular loops, and regulatory domains—provide promising targets for intervention. The experimental frameworks and comparative data presented here offer researchers validated methodologies for identifying and exploiting these differences.

Future directions in this field will likely include:

• Increased use of cryo-EM for determining endogenous structures from pathogenic organisms
• Computational approaches leveraging AlphaFold2 predictions for rapid identification of species-specific variations
• Development of bifunctional compounds targeting both the catalytic core and accessory protein interfaces
• High-throughput screening platforms incorporating ortholog panels to rapidly assess selectivity

As structural biology techniques continue to advance, our ability to identify and exploit subtle structural variations across species will dramatically improve, enabling the rational design of highly selective P-type ATPase inhibitors with minimal off-target effects.

P-type ATPases constitute a large superfamily of membrane transporters that are essential for maintaining cellular ion gradients. Among these, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the Na+/K+-ATPase (NKA) are critical therapeutic targets and well-recognized sites for off-target drug interactions. The structural and evolutionary conservation within the P-type ATPase family means that compounds designed to target one member may inadvertently inhibit others, potentially leading to adverse effects. This comparative profiling examines the mechanisms, chemical space, and experimental approaches for identifying and characterizing off-target interactions with SERCA and NKA, providing a framework for more specific drug development in the context of comparative genomic analysis of P-type ATPase inhibitors.

SERCA: Architecture and Biological Role

SERCA is an intracellular membrane transporter that utilizes energy from ATP hydrolysis to actively pump calcium ions from the cytoplasm into the sarco/endoplasmic reticulum lumen, thereby playing a fundamental role in cellular calcium signaling and homeostasis. SERCA's structure comprises three cytoplasmic domains (actuator [A], nucleotide-binding [N], and phosphorylation [P] domains) and a transmembrane region of 10 helical segments (TM1–TM10) that contain two calcium binding sites [66]. In humans, multiple SERCA isoforms encoded by three different genes (ATP2A1-3) exhibit tissue-specific expression patterns, with SERCA1a predominantly expressed in skeletal muscle, SERCA2a in cardiac muscle, and the more ubiquitous SERCA2b found in various tissues including vascular smooth muscle cells [67] [68].

Na+/K+-ATPase: Structure and Physiological Significance

The Na+/K+-ATPase is a plasma membrane transporter that establishes the essential sodium and potassium gradients across the cell membrane, crucial for maintaining membrane potential, cellular volume, and secondary active transport. Its structure consists of a catalytic α-subunit with 10 transmembrane helices, a glycosylated β-subunit, and a regulatory FXYD subunit [69]. The mammalian Na+/K+-ATPase features multiple isoforms of its α-subunit (α1, α2, α3, and sperm-specific α4) that are approximately 87% identical in sequence but exhibit distinct tissue distributions and physiological roles [69]. The Na+/K+-ATPase is famously the molecular target for cardiotonic steroids like digoxin and ouabain, which have been used therapeutically for heart conditions for centuries [69].

Table 1: Comparative Features of SERCA and Na+/K+-ATPase

Feature	SERCA	Na+/K+-ATPase
Primary Function	Transports Ca2+ from cytoplasm to SR/ER lumen	Exchanges 3Na+ for 2K+ across plasma membrane
Cellular Location	Sarco/Endoplasmic reticulum membrane	Plasma membrane
Transport Stoichiometry	2 Ca2+ per ATP (exchanged for luminal protons)	3 Na+ out, 2 K+ in per ATP
Key Physiological Roles	Muscle relaxation, calcium signaling, cell homeostasis	Membrane potential, cellular volume, secondary transport
Major Isoforms	SERCA1a, SERCA2a, SERCA2b, SERCA3	α1, α2, α3, α4 (catalytic subunits)
Characteristic Inhibitors	Thapsigargin, cyclopiazonic acid, BHQ	Ouabain, digoxin, bufalin

Established Inhibitors and Off-Target Compounds

SERCA-Targeting Compounds

SERCA is inhibited by several well-characterized compounds with diverse chemical structures. Thapsigargin, a sesquiterpene lactone derived from the plant Thapsia garganica, is an exceptionally potent and selective SERCA inhibitor with IC50 values in the sub-nanomolar range [66]. Other specific SERCA inhibitors include cyclopiazonic acid, a fungal metabolite, and the synthetic compound 2,5-di(tert-butyl)hydroquinone (BHQ) [66]. Crystallographic studies have revealed that these compounds bind to distinct but partially overlapping sites within the transmembrane domain of SERCA.

Recent research has identified several FDA-approved drugs with unexpected SERCA inhibitory activity. A novel machine learning-based screening approach combining data augmentation techniques predicted and experimentally validated that seven statins (lipid-lowering medications) act as partial inhibitors of SERCA1a and SERCA2a [67]. Atomistic simulations predicted that these drugs bind to two different allosteric sites on the pump, providing a molecular pathway to explain statin-associated muscular toxicity reported in clinical settings [67].

Na+/K+-ATPase Inhibitors and Their Off-Target Effects

The most well-characterized Na+/K+-ATPase inhibitors are the cardiotonic steroids, which include plant-derived cardenolides (e.g., ouabain, digoxin) and bufadienolides from animal sources (e.g., bufalin, cinobufagin) [69] [70]. These compounds bind to the extracellular side of the pump in the ion exchange pathway, as revealed by crystal structures of inhibitor-pump complexes [69].

Research has demonstrated that cardiac glycosides, particularly bufadienolides, possess proarrhythmic potential that operates through mechanisms independent of their Na+/K+-ATPase inhibitory activity. Specifically, bufalin and cinobufagin demonstrate concentration-dependent inhibition of L-type calcium channels (ICa,L) and the rapidly activating component of the delayed rectifier K+ current (IKr) even when Na+/K+-ATPase activity is completely blocked by ouabain pre-treatment [70]. These findings reveal off-target ion channel effects that may contribute to the complex cardiotoxicity profile of these compounds.

Table 2: Documented Off-Target Effects of ATPase-Targeting Compounds

Compound	Primary Target	Documented Off-Target Effects	Experimental Evidence
Statins (e.g., Atorvastatin)	HMG-CoA reductase	SERCA1a/2a inhibition (micromolar)	Machine learning prediction + in vitro ATPase assays [67]
Bufadienolides (e.g., Bufalin)	Na+/K+-ATPase	L-type Ca2+ channel inhibition (IC50 ~12.5 µM); hERG channel inhibition	Electrophysiology with Ca2+ chelation and Na+/K+-ATPase blockade [70]
Thapsigargin	SERCA	Non-selective; affects all SERCA isoforms	Functional assays, crystallography [66]
Cardiac Glycosides (e.g., Ouabain)	Na+/K+-ATPase	Limited off-target channel effects at high concentrations	Electrophysiology screening [70]

Experimental Approaches for Profiling Inhibitor Specificity

Machine Learning and Virtual Screening

Modern computational approaches have significantly advanced the prediction of off-target effects. A novel machine learning-based screening method that combines data augmentation successfully identified FDA-approved drugs interacting with SERCA by mapping and probing the chemical space of pharmacological targets [67]. This approach used a conservative data augmentation strategy to expand the original training dataset of SERCA inhibitors, creating robust maps of the pharmacological space and improving classifier performance. The model trained with the augmented dataset showed dramatically improved performance (accuracy: 0.84, Matthews correlation coefficient: 0.60) compared to the model trained on the original dataset alone (accuracy: 0.59, Matthews correlation coefficient: 0.11) [67].

Complementary approaches include virtual screening protocols combining recursive partitioning and computational ligand docking methodologies. One such study screened a compound library of 345,000 entries for novel SERCA inhibitors, identifying 17 inhibitors with potencies below 100 µM from 72 tested compounds [71]. These protocols employed physical property filters (log P between 1.5-4.5, molecular weight 150-350 g/mol) to narrow the chemical space to regions occupied by known SERCA inhibitors [71].

Functional Assays and Electrophysiology

Direct functional assessment of inhibitor effects remains essential for validating off-target interactions. ATPase activity assays provide quantitative measurements of inhibitor potency against specific ATPase targets. For SERCA, these assays typically monitor NADH oxidation coupled to ATP hydrolysis in purified enzyme preparations or membrane fractions [67].

Electrophysiological techniques based on solid supported membranes (SSM) enable pre-steady state measurements of charge displacements within the first transport cycle of ATPases. This method involves adsorbing ATPase-containing vesicles on a hybrid alkanethiol/phospholipid bilayer supported by a gold electrode, then activating the ATPase with rapid substrate concentration jumps while measuring transient current signals [66]. The technique has been successfully employed to characterize drug interactions with SERCA and other P-type ATPases, and automated implementations (e.g., SURFE2R 96SE device) allow high-throughput screening of compound libraries [66].

For assessing Na+/K+-ATPase inhibition and off-target effects on cardiac electrophysiology, patch clamp techniques in heterologous expression systems and stem cell-derived cardiomyocytes provide critical information. Studies examining the NKA inhibition-independent effects of cardiac glycosides typically employ intracellular Ca2+ chelators (EGTA, BAPTA) and complete Na+/K+-ATPase blockade to isolate direct channel effects [70].

Diagram 1: Integrated workflow for profiling off-target effects against SERCA and Na+/K+-ATPase, combining computational and experimental approaches

Pathway Analysis and Physiological Implications

Calcium Signaling Pathways and Cross-Talk

The physiological interplay between SERCA and Na+/K+-ATPase occurs primarily through calcium signaling pathways. In cardiac myocytes, Na+/K+-ATPase inhibition leads to increased intracellular Na+ concentrations, which promotes Ca2+ accumulation via the Na+/Ca2+ exchanger (NCX) operating in reverse mode. This elevated cytoplasmic Ca2+ must then be sequestered by SERCA into the sarcoplasmic reticulum, creating functional coupling between the two transport systems [70] [72].

Off-target inhibition of SERCA by compounds primarily designed to affect other pathways can disrupt calcium homeostasis and contribute to adverse effects. For example, statin-associated muscular toxicity may be partially explained by SERCA inhibition, which would impair calcium handling in skeletal muscle cells [67]. Similarly, the proarrhythmic effects of cardiac glycosides involve both their primary action on Na+/K+-ATPase and secondary effects on calcium cycling, potentially exacerbated by direct off-target inhibition of ion channels [70].

Implications for Therapeutic Development

The structural conservation among P-type ATPases presents both challenges and opportunities for drug development. On one hand, it complicates the achievement of compound specificity; on the other, it enables potential repurposing of existing drugs for new indications. The discovery that statins inhibit SERCA suggests potential applications beyond cholesterol lowering, possibly in conditions where SERCA modulation is therapeutically desirable [67].

Understanding the molecular determinants of inhibitor specificity between SERCA and Na+/K+-ATPase can guide the design of more selective compounds. Structural analyses reveal that while both pumps share similar architecture and catalytic mechanisms, differences in their transmembrane domains and extracellular regions provide opportunities for developing target-specific inhibitors [69] [66].

Diagram 2: Signaling pathways and physiological consequences of off-target inhibition of SERCA and Na+/K+-ATPase, highlighting interconnected calcium handling and emergent toxicity profiles

The Scientist's Toolkit: Essential Research Reagents and Methodologies

Table 3: Key Research Reagents and Experimental Solutions for Off-Target Profiling

Reagent/Solution	Function in Research	Example Applications
Thapsigargin	High-affinity SERCA inhibitor; reference compound	Positive control for SERCA inhibition studies [66]
Ouabain	Specific Na+/K+-ATPase inhibitor; reference compound	Positive control for Na+/K+-ATPase inhibition; tool for isolating NKA-independent effects [70]
ATPase Assay Buffer Systems	Monitor NADH oxidation coupled to ATP hydrolysis	Quantitative measurement of ATPase activity and inhibitor potency [67]
SSM (Solid Supported Membrane) Electrophysiology	Measures pre-steady state charge displacements	Characterization of electrogenic steps in ATPase transport cycle [66]
Ca2+ Chelators (EGTA, BAPTA)	Buffer intracellular Ca2+ concentrations	Isolating direct inhibitor effects from Ca2+-mediated secondary effects [70]
Patch Clamp Solutions	Maintain ionic gradients for electrophysiology	Assessment of off-target ion channel effects [70]
Machine Learning Classifiers	Predict compound-target interactions	Virtual screening for off-target potential [67]

Comparative profiling of compounds against SERCA and Na+/K+-ATPase reveals a complex landscape of on-target and off-target interactions that must be carefully characterized during drug development. The structural and functional similarities between these P-type ATPases, combined with their interconnected roles in cellular ion homeostasis, create challenges for achieving compound specificity. Integrated approaches combining machine learning prediction with functional validation across multiple assay platforms provide the most comprehensive strategy for identifying and understanding these interactions. As structural information for both targets continues to expand and screening technologies advance, the rational design of specific inhibitors with reduced off-target potential becomes increasingly feasible, promising safer therapeutic agents that modulate these critical cellular transporters.

The strategic choice between allosteric and orthosteric inhibition represents a pivotal decision in modern drug discovery, particularly for challenging targets such as P-type ATPases. Orthosteric drugs compete with endogenous substrates by binding at the evolutionarily conserved active site, whereas allosteric modulators bind at topographically distinct sites to fine-tune protein function through conformational changes [73]. This distinction is not merely academic; it fundamentally influences therapeutic outcomes, safety profiles, and resistance mechanisms [74]. Within the context of comparative genomic analysis of P-type ATPase inhibitors, understanding these mechanisms provides a framework for developing more selective and resilient therapeutic agents.

The recent elucidation of P-type ATPase structures, including the Plasmodium falciparum sodium efflux pump PfATP4, has revealed unprecedented opportunities for targeted inhibitor design [19]. Concurrently, advances in computational methodologies—including machine learning, molecular dynamics simulations, and network-based approaches—are transforming our capacity to identify and characterize novel binding sites, especially transient allosteric pockets that evade conventional experimental detection [75]. This guide objectively compares these complementary inhibition strategies, providing researchers with experimental data and protocols to inform future inhibitor development.

Orthosteric vs. Allosteric Inhibition: A Comparative Framework

Table 1: Fundamental Characteristics of Orthosteric and Allosteric Inhibitors

Characteristic	Orthosteric Inhibitors	Allosteric Inhibitors
Binding Site Location	Active site (substrate-binding site) [76]	Topographically distinct regulatory site [76]
Conservation	Highly conserved across protein families [75]	Often less conserved, offering subtype selectivity [76] [75]
Mechanism of Action	Direct competition with endogenous substrate [74]	Modulation of protein conformation and function indirectly [74] [76]
Effect on Physiology	Can "hijack" or block natural receptor physiology [74]	Works with the system, acting as a "tuning knob" [74]
Regulatory Limit	Full inhibition possible	Upper bound to regulation, preserves basal signaling [76] [75]
Selectivity Potential	Lower due to conserved active sites	Higher, can target specific isoforms [76] [75]
Therapeutic Window	Can be narrower due to off-target effects	Often wider due to spatial/temporal selectivity [73]
Resistance Mechanisms	Mutations in the active site that impair drug binding [77]	Mutations that disrupt allosteric communication pathways [77]

Experimental and Computational Protocols for Binding Site Characterization

Protocol 1: Identification of Orthosteric Sites and Resistance Mutations

The identification of orthosteric sites and associated resistance mutations relies heavily on structural biology and mutational analysis, as demonstrated in studies of the antimalarial target PfATP4.

• Protein Engineering and Purification: Use CRISPR-Cas9 to engineer an affinity tag (e.g., 3×FLAG) into the target gene in its native organism (e.g., P. falciparum). Affinity-purify the protein from its native environment to preserve endogenous complex partners [19].
• Structural Determination: Employ single-particle cryo-electron microscopy (cryo-EM) to determine the high-resolution (e.g., 3.7 Å) structure of the purified protein. This allows for de novo modeling of the protein's atomic coordinates [19].
• Active Site Mapping: Analyze the cryo-EM density to identify the canonical active site. For P-type ATPases like PfATP4, this includes the transmembrane domain (TMD) ion-binding site (between TM4, TM5, TM6, and TM8) and the intracellular nucleotide-binding (N) and phosphorylation (P) domains [19].
• Mapping Resistance Mutations: Clinically identified resistance mutations (e.g., G358S and A211V for PfATP4 inhibitors like Cipargamin) can be mapped onto the atomic model. Analyze how these mutations, often located near the ion-binding site, might sterically or electrostatically hinder inhibitor binding without disrupting substrate binding essential for native function [19].

Protocol 2: Computational Prediction of Allosteric Sites

Computational tools have become indispensable for discovering allosteric sites, which are often cryptic and not evident in static structures.

• Input Preparation: Obtain a high-quality 3D structure of the target protein (resolution <2.5 Å) from the PDB or via homology modeling. Ensure there are no missing loops in the main chain. Remove small molecules, ions, and solvents from the file [76].
• Job Submission on Allosite Server: Access the Allosite web server (http://mdl.shsmu.edu.cn/AST). Submit the job either by "PDB ID" or by uploading a "PDB File". Specify a unique "Job Name" and select the relevant protein chain(s) to initiate the calculation. A typical job for a 400-residue protein requires approximately 15 seconds [76].
• Result Retrieval and Analysis: The server returns 0–4 predicted allosteric pockets. The results page displays the sites with properties like "Pocket Volume", "Pocket Total SASA", "Pocket Polar SASA", and a "Pocket Druggability Score". The predicted site can be visualized directly on the web platform or downloaded for offline analysis [76].
• Offline Visualization with PyMOL: Download the result file, which contains a PyMOL script (pml). Open this script in PyMOL to visualize the query protein structure with the predicted allosteric site residues shown as stick models and the pocket represented as white spheres [76].

The following workflow diagram illustrates the core steps and decision points in the computational protocol for allosteric site prediction.

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 2: Key Research Reagent Solutions for Inhibitor Discovery

Reagent / Solution	Function in Research
CRISPR-Cas9 Engineering System	Enables endogenous tagging and purification of target proteins (e.g., PfATP4) directly from pathogenic organisms, preserving native complexes [19].
Allosite Web Server	A computational tool that uses a support vector machine (SVM) model to identify potential allosteric sites on protein structures based on pocket topology and physiochemical properties [76].
Polyoxotungstates (POTs)	A diverse class of transition metal complexes (e.g., P2W18, Se2W29) used as experimental P-type ATPase inhibitors to study structure-activity relationships and selectivity [47].
FPocket Software	An open-source package for detecting binding pockets in protein structures, which forms the foundation for feature extraction in tools like Allosite [76].
PyMOL Molecular Graphics System	Industry-standard software for visualizing protein structures, binding sites, and molecular docking results, essential for analyzing computational predictions [76].
Sarcoplasmic Reticulum (SR) Vesicles	A well-established in vitro model system, rich in Ca2+-ATPase, used to study the inhibitory effects of compounds on P-type ATPase activity and kinetics [47].

Signaling Pathways and Resistance Landscapes

Allosteric Modulation of Key Drug Targets

Allosteric modulators provide nuanced control over protein function across diverse target classes. The following diagram generalizes the mechanisms by which allosteric modulators exert their effects on different protein types, highlighting their therapeutic advantages.

Comparative Resistance Mechanisms

Resistance poses a significant threat to both orthosteric and allosteric inhibitors, though the underlying mechanisms differ.

Table 3: Comparative Analysis of Resistance Mechanisms

Resistance Aspect	Orthosteric Inhibitors	Allosteric Inhibitors
Primary Mechanism	Mutations in the active site that directly impair drug binding but may retain substrate affinity [77].	Mutations that disrupt the allosteric communication pathway or the modulator's binding site, diminishing regulatory efficacy [77].
Impact on Function	Can be deleterious if they disrupt native substrate binding, limiting evolutionary pathways.	May have less impact on native function, as the orthosteric site remains intact, potentially facilitating resistance.
Overcoming Strategy	Bitopic Inhibitors: Molecules that simultaneously engage both the orthosteric and an allosteric site can mitigate the impact of single resistance mutations [77].	Combination Therapy: Using an allosteric inhibitor in concert with an orthosteric inhibitor can create a high genetic barrier to resistance [77].
Clinical Example	PfATP4 mutations (e.g., G358S) confer resistance to orthosteric inhibitors like Cipargamin by altering the ion-binding pocket [19].	Resistance to allosteric inhibitors in kinases and other enzymes can arise from mutations that alter the dynamics of the alloster network [77].

The strategic choice between orthosteric and allosteric inhibition is central to the future of targeting P-type ATPases and other challenging drug targets. Orthosteric inhibitors provide potent blockade but face challenges of selectivity and susceptibility to resistance mutations in the conserved active site. In contrast, allosteric modulators offer a sophisticated means to fine-tune biological activity with greater potential for selectivity and preservation of physiological signaling, though their discovery is hampered by difficulties in identifying often transient binding sites.

The integration of advanced computational methods—including machine learning platforms like Allosite, molecular dynamics simulations, and network analysis—with high-resolution experimental structures is rapidly overcoming these hurdles [76] [75]. The recent discovery of an apicomplexan-specific binding partner for PfATP4, PfABP, further underscores that endogenous regulatory complexes represent a rich, unexplored avenue for allosteric drug discovery [19]. As the computational toolkit continues to evolve, the rational design of bitopic inhibitors and combination therapies that leverage both orthosteric and allosteric principles presents a promising path to overcome resistance and develop more effective and durable therapeutics.

The efficacy of pharmaceutical inhibitors targeting membrane-bound proteins is not solely determined by the drug's chemical structure or its affinity for the protein's active site. A critical, yet often overlooked, factor is the lipid membrane environment in which the protein is embedded. The lipid bilayer is not merely a passive scaffold but an active participant that modulates protein conformation, dynamics, and ultimately, function [78] [79]. This is particularly relevant for P-type ATPases, a family of ion pumps that are crucial drug targets in various diseases, including malaria [21] [44]. The lipid environment can influence how these proteins respond to inhibitory compounds, a phenomenon with profound implications for drug development and resistance. This guide explores the evidence for this lipid-protein interplay, comparing how different membrane properties can alter the inhibitory landscape for this important class of targets, with a specific focus on insights from comparative genomic analyses.

Lipid Modulation of P-Type ATPases: A Comparative Framework

P-type ATPases, despite shared structural motifs, exhibit diverse responses to their lipid environment and to inhibitors, depending on their specific type and cellular location. The table below summarizes key comparative findings.

Table 1: Comparative Influence of Lipid Environment on P-Type ATPase Function and Inhibition

P-Type ATPase & Source	Native Membrane Lipid Composition	Impact of Lipid Environment on Activity	Relevance to Inhibitor Efficacy
PfATP4(P. falciparum plasma membrane) [21]	Not fully characterized; discovery of specific binding partner PfABP suggests complex lipid-protein network.	Purified protein shows Na+-dependent ATPase activity inhibitable by compounds like Cipargamin. Lipid dependencies under investigation.	Resistance mutations (e.g., G358S, A211V) cluster near ion-binding site, likely altering inhibitor binding pocket [21].
Na+,K+-ATPase (NKA)(Animal plasma membrane) [78]	~51% Phospholipids, ~36% Cholesterol, ~5% Acylglycerols [78].	Cholesterol and phospholipids crucial for stability and conformational transitions of the α-subunit [78].	Lipid-dependent conformational shifts (E1-E2 states) can occlude or expose inhibitor binding sites, modulating drug access.
Gastric H+,K+-ATPase (HKA)(Mammalian gastric membrane) [78]	~58% Phospholipids, ~38% Cholesterol, ~4% Glycosphingolipids; Phosphatidylethanolamine is dominant phospholipid [78].	Requires specific lipid composition for optimal activity and structural stability mediated by its β-subunit [78].	Membrane thickness and charge may influence the binding and potency of proton pump inhibitors.
SERCA(Sarco/Endoplasmic Reticulum) [78]	Predominantly phospholipids (~89%) with low cholesterol content (~7%) [78].	Activity is highly sensitive to membrane thickness and fluidity due to large conformational changes during its cycle [78] [79].	Lipid packing can either facilitate or hinder the structural transitions that inhibitors often target.
Plant PM H+-ATPase(Plant plasma membrane) [29]	Rich in sterols (~30%), anionic phospholipids, and sphingolipids; forms potential nanodomains.	Anionic phospholipids and sterols fine-tune pump activity; electrostatic interactions with lipid headgroups modulate function [29].	Not directly studied for inhibitors, but lipid-mediated spatial organization with regulatory kinases could affect therapeutic targeting.

The functional impact of the membrane can be broken down into specific, measurable physical properties. A systematic study on the bacterial ABC transporter BmrA, while not a P-type ATPase, provides a clear framework for understanding these parameters. The study identified that BmrA's ATPase activity is controlled by the membrane's hydrophobic thickness, its negative surface charge, and the packing of lipids in both the acyl-chain and headgroup regions [80]. These principles are directly applicable to P-type ATPases, as their activity also involves major conformational shifts that must be accommodated by the surrounding lipid bilayer [78] [79].

Experimental Protocols for Studying Lipid-Protein-Inhibitor Interplay

To generate comparative data on inhibitor efficacy across different membrane environments, robust and reproducible experimental protocols are essential. The following methodologies are commonly employed in the field.

Protein Purification and Functional Reconstitution

• Endogenous Purification: For proteins like PfATP4 that are difficult to express heterologously, CRISPR-Cas9 can be used to tag the endogenous gene (e.g., with a 3×FLAG epitope). The protein is then affinity-purified directly from its native cellular context (e.g., from P. falciparum-infected red blood cells) to preserve native interactions, as demonstrated with the discovery of the PfABP binding partner [21].
• Reconstitution into Proteoliposomes: The purified protein is incorporated into pre-formed liposomes of defined lipid composition. This process involves solubilizing the protein in detergent, mixing it with lipids, and then removing the detergent to form sealed vesicles. This allows for precise control over the lipid environment during functional assays [80] [79].

ATPase Activity Assays

• Protocol: The activity of P-type ATPases is measured by quantifying the release of inorganic phosphate (Pi) from hydrolyzed ATP. A classic method is the colorimetric Fiske-Subbarow assay, where Pi reacts with molybdate to form a phosphomolybdate complex, which is then reduced to a blue compound measurable by spectrophotometry.
• Inhibitor Testing: The assay is run in the presence of necessary co-factors (e.g., Na+ for PfATP4, Ca2+ for SERCA) with and without the inhibitor compound. The reaction is linear over time, and the difference in Pi liberation rates quantifies the degree of inhibition [21] [44].
• Membrane Variation: To test lipid dependence, the assay is repeated using protein reconstituted into liposomes with systematically varying lipid compositions—for instance, different concentrations of cholesterol, anionic lipids like phosphatidylserine, or lipids with acyl chains of different lengths to modulate thickness [80].

Structural Analysis of Binding

• Cryo-Electron Microscopy (Cryo-EM): This is a powerful technique for visualizing protein-inhibitor and protein-lipid interactions. Proteins can be purified in nanodiscs or detergents and frozen in vitreous ice. High-resolution structures (e.g., the 3.7 Å structure of PfATP4) can reveal the binding pose of an inhibitor and identify resistance mutations that alter the binding site [21] [79].
• Molecular Dynamics (MD) Simulations: Simulations are used to model the dynamic interactions between the protein, lipid bilayer, and inhibitor. After building a system with the protein embedded in a realistic lipid bilayer, simulations can run for microseconds to observe how lipids influence protein conformation and the stability of inhibitor binding, providing atomic-level insights into the mechanisms suggested by experimental data [44] [79].

Conceptual Workflow: From Membrane Properties to Inhibitor Efficacy

The relationship between membrane properties, protein function, and inhibitor binding can be conceptualized as a logical pathway. The following diagram illustrates this interconnected workflow, from the initial composition of the lipid bilayer to the ultimate functional outcome for a potential drug.

The Scientist's Toolkit: Essential Research Reagents and Solutions

Research in this field relies on a suite of specialized reagents and tools to dissect the complex relationships between lipids, proteins, and inhibitors.

Table 2: Key Research Reagent Solutions for Lipid-Protein Interaction Studies

Reagent / Solution	Function / Application	Specific Examples / Notes
Defined Lipid Liposomes	Creating a controlled membrane environment for protein reconstitution and in vitro assays. Allows systematic variation of cholesterol %, anionic lipids, and acyl chain length.	Available from vendors like Avanti Polar Lipids. Crucial for probing general vs. specific lipid effects [78] [80].
Detergents	Solubilizing membrane proteins from their native environment for purification while attempting to maintain function.	Choice of detergent (e.g., DDM, LMNG) is critical for stability and can influence which lipids remain bound [79].
CRISPR-Cas9 Tools	For endogenous tagging and purification of difficult-to-express proteins from native hosts, preserving authentic protein complexes.	Used to tag PfATP4 with 3×FLAG in P. falciparum, enabling its purification and the discovery of PfABP [21].
Cryo-EM Reagents	For high-resolution structural determination of membrane proteins in near-native states, often revealing bound lipids and inhibitors.	Grids (e.g., gold or copper), vitrification devices, and advanced detectors are key for structures like the 3.7 Å PfATP4 map [21] [79].
MD Simulation Software	Modeling atomistic dynamics of protein-lipid-inhibitor systems to generate testable hypotheses and explain experimental data.	Packages like GROMACS, NAMD, and CHARMM are widely used with force fields (e.g., Martini) adapted for lipids [44] [79].
P-Type ATPase Inhibitors	Tool compounds for probing pump function and resistance mechanisms in different lipid contexts.	Cipargamin, PA21A092 (for PfATP4) [21]; Thapsigargin (for SERCA); Phenolic compounds (for PfATP6) [44].

The membrane environment is a decisive factor in the functional efficacy of inhibitors targeting P-type ATPases. Evidence from comparative genomics and structural biology shows that resistance mutations can directly alter inhibitor binding sites, while biophysical studies demonstrate that lipid composition can control the conformational equilibrium of these pumps, thereby modulating drug access and potency. Future drug discovery efforts must move beyond a purely protein-centric view. Incorporating the lipid dimension—through techniques like native membrane reconstitution, cryo-EM, and molecular dynamics—will be crucial for designing next-generation therapeutics that are less susceptible to resistance and more effective within the complex lipid milieu of the native cell membrane. Understanding the specific lipid dependencies of pathogenic targets like PfATP4, in contrast to human P-type ATPases, could open avenues for developing highly selective antimalarials and other infectious disease treatments.

Comparative Inhibition Profiling and Therapeutic Validation Across Systems

The fungal plasma membrane H+-ATPase (Pma1) represents a prime target for the development of novel antifungal agents due to its essential role in fungal cell physiology and its absence in mammals [81]. As a P-type ATPase that creates the electrochemical gradient necessary for nutrient uptake, Pma1 belongs to the type III family of P-type ATPases, while related human ATPases (Na+,K+-ATPase, SERCA Ca2+-ATPase, and H+,K+-ATPase) belong to the type II family [82]. This evolutionary divergence is significant for drug discovery, as mammalian ATPases share less than 30% amino acid sequence identity with Pma1, theoretically enabling selective inhibition [82]. Within the context of comparative genomic analysis of P-type ATPase inhibitors, this review systematically evaluates the selectivity profiles of documented Pma1 inhibitors against mammalian ATPase counterparts, providing a structural and functional framework for understanding cross-species inhibition.

Quantitative Comparison of Inhibitor Selectivity

Selectivity Profiling of Pma1 Inhibitory Compounds

A high-throughput screening initiative of approximately 191,000 compounds identified numerous Pma1 inhibitors, which were subsequently counterscreened against mammalian Na+,K+-ATPase and SERCA to determine selectivity [82]. The following table summarizes the half-maximal inhibitory concentration (IC50) values for the most promising hit compounds and reference inhibitors, illustrating the varying degrees of selectivity achieved.

Table 1: Inhibition potency (IC50) and selectivity of Pma1 inhibitors against mammalian ATPases

Compound	Pma1 IC50 (μM)	Na+,K+-ATPase IC50 (μM)	SERCA IC50 (μM)	Selectivity Profile
Compound 1	0.040 ± 0.035	13.2 ± 0.91	17.5 ± 4.7	Selective for Pma1
Compound 2	2.1 ± 1.3	>105	>105	Selective for Pma1
Compound 3	2.5 ± 2.5	>105	>105	Selective for Pma1
Compound 4	3.7 ± 0.7	15.0 ± 7.2	4.1 ± 0.2	Moderate selectivity
Compound 5	4.4 ± 0.6	1.3 ± 0.3	0.46 ± 0.04	More potent vs. mammalian ATPases
Compound 14	9.3 ± 1.2	20.3 ± 0.9	20.6 ± 2.6	Selective for Pma1
Ebselen	~0.1 (Pma1)	~0.5 (Na,K)	~1.0 (SERCA)	Non-selective
BM2	0.5 (Pma1)	Active (Na,K)	N/D	Non-selective

Compound 14, a pyrido-thieno-pyrimidine, emerged as the most promising hit from the screen, being the only compound more potent against Pma1 than against the mammalian ATPases while also displaying a broad spectrum of antifungal activity [82]. In contrast, several compounds with potent Pma1 inhibition (e.g., Compounds 5, 7, 10, and 13) were generally more potent against the mammalian ATPases, rendering them non-selective and likely unsuitable for development as antifungal agents due to potential host toxicity [82].

Antifungal Efficacy of Selective Inhibitors

The translation of enzymatic inhibition to fungal growth suppression is a critical step in validating Pma1 as a drug target. The following table correlates the inhibitory potency (IC50) of selected compounds with their minimum inhibitory concentration (MIC) against various Candida species.

Table 2: Correlation between Pma1 inhibition and antifungal activity for selected compounds

Compound	Pma1 IC50 (μM)	C. albicans MIC (μM)	C. glabrata MIC (μM)	Antifungal Efficacy
Compound 1	0.040 ± 0.035	>150	>150	Poor
Compound 4	3.7 ± 0.7	13	>38	Moderate (C. albicans)
Compound 5	4.4 ± 0.6	6.5	5.8	Good
Compound 7	5.5 ± 0.3	7.3	4.7	Good
Compound 14	9.3 ± 1.2	7.8	3.1	Good
BM2 Peptide	0.5	0.63 (S. cerevisiae)	N/D	Good, but non-selective

A key observation is that potent enzymatic inhibition does not always guarantee effective fungal growth suppression in vitro. For instance, Compound 1 displayed sub-micromolar potency against Pma1 (IC50 = 40 nM) yet failed to inhibit the growth of Candida albicans or Candida glabrata at concentrations up to 150 μM [82]. This disconnect can arise from factors such as poor compound solubility, inadequate cellular penetration, or efflux by membrane transporters.

Experimental Protocols for Cross-Species Inhibition Analysis

Primary Screening Assay: Pma1 ATP Hydrolysis Inhibition

The foundational protocol for identifying Pma1 inhibitors involves measuring compound effects on ATPase activity in purified fungal plasma membranes [82].

• Membrane Preparation: Plasma membranes containing Pma1 are isolated from Saccharomyces cerevisiae via differential centrifugation.
• Reaction Conditions: The assay measures the liberation of inorganic phosphate (Pi) from ATP hydrolysis. A standard reaction mixture includes membrane samples, test compound (typically at 20 μM for screening), and MgATP in an appropriate buffer.
• Detection: The formed Pi is quantified colorimetrically, often using a malachite green-based reagent.
• Hit Criteria: In the primary screen, compounds causing >50% inhibition of enzymatic activity at 20 μM are classified as hits for further characterization [82].

Counterscreening Assay: Selectivity Profiling

To establish selectivity, confirmed hits are evaluated against mammalian P-type ATPases.

• Mammalian ATPase Sources: Na+,K+-ATPase is purified from porcine cerebral cortex, and SERCA (Ca2+-ATPase) is purified from rabbit skeletal muscle.
• Standardized Testing: Compounds are tested in concentration-response format (e.g., 0.1-100 μM) against all three ATPases (Pma1, Na+,K+-ATPase, SERCA) under parallel conditions.
• Data Analysis: IC50 values are determined for each ATPase. The selectivity index is calculated as the ratio of the IC50 for the mammalian ATPase to the IC50 for Pma1 [82].

Functional Cellular Assays

Compounds exhibiting desirable potency and selectivity are advanced to cellular assays.

• Membrane Depolarization: Inhibitor-induced depolarization of the fungal membrane potential is measured using fluorescent dyes like DiBAC4(3).
• Intracellular ATP Levels: Pma1 inhibition blocks a major ATP-consuming process, often leading to a measurable increase in intracellular ATP concentration, which is quantified using a luciferase-based assay.
• Extracellular Acidification: The pump's proton-pumping activity is monitored as the rate of extracellular medium acidification using a pH-sensitive electrode; inhibition slows acidification [82].

The following diagram illustrates the key steps in the screening and validation workflow for identifying selective Pma1 inhibitors.

Figure 1: Experimental workflow for identifying selective Pma1 inhibitors.

Structural and Mechanistic Basis for Selective Inhibition

Unique Hexameric Architecture of Fungal Pma1

A key structural feature that differentiates fungal Pma1 from mammalian P-type ATPases is its hexameric organization. While mammalian P-type ATPases typically function as monomers or heterodimers, cryo-EM structures have confirmed that Pma1 assembles into a stable hexameric ring [18] [81]. This oligomerization is mediated by:

• Transmembrane Domain Interfaces: TMH7 and TMH10 of one subunit interact with TMH3 and TMH4 of an adjacent subunit.
• Endogenous Lipids: A central ring of 115 well-defined fatty acid acyl chains within the hexamer stabilizes the assembly.
• N-terminal Extension (NTE): The NTE of one subunit binds to the nucleotide-binding domain (N domain) of its neighbor, creating a domain-swapped interaction crucial for cooperative function [18] [83].

This cooperativity mechanism means that conformational changes in one subunit are coupled to adjacent subunits, enabling a more efficient and regulated proton-pumping activity [83]. The unique NTE interface and the hexameric state present apicomplexan-specific targeting opportunities absent in mammalian ATPases.

Divergent Allosteric Regulation and Inhibitor Binding Sites

The regulatory mechanisms of Pma1 have diverged significantly from those of mammalian pumps.

• Autoinhibitory C-terminal Tail: Pma1 contains a C-terminal regulatory domain (residues 880–918 in yeast) that folds into two helices in the autoinhibited state, stabilizing a compact conformation [81]. This regulatory element is not found in mammalian ATPases.
• Allosteric Inhibitor Binding: The discovery of an apicomplexan-specific P-type ATPase modulator (PfABP) in Plasmodium falciparum highlights the potential for parasite-specific regulatory complexes that can be targeted for inhibition [19].

The following diagram contrasts the structural and functional features of fungal Pma1 with mammalian P-type ATPases.

Figure 2: Structural and functional comparison between fungal and mammalian P-type ATPases.

The Scientist's Toolkit: Key Research Reagents

Table 3: Essential research reagents for studying Pma1 inhibition and selectivity

Reagent / Tool	Type	Primary Function in Research	Example/Source
Purified S. cerevisiae Plasma Membranes	Biological Material	Source of native Pma1 for primary ATPase inhibition assays	Isolated from S. cerevisiae cultures [82]
Porcine Na+,K+-ATPase	Biological Material	Mammalian ATPase for counterscreening and selectivity profiling	Purified from porcine cerebral cortex [82]
Rabbit SERCA	Biological Material	Mammalian Ca2+-ATPase for counterscreening and selectivity profiling	Purified from rabbit skeletal muscle [82]
DiBAC4(3) Fluorescent Dye	Chemical Probe	Measurement of membrane potential changes in fungal cells following Pma1 inhibition	Commercial suppliers [82]
Luciferase-Based ATP Assay Kit	Assay Kit	Quantification of intracellular ATP levels upon Pma1 inhibition	Commercial suppliers [82]
Anti-H,K-ATPase α subunit mAb (1H9)	Antibody	Detection and immunoprecipitation of mammalian gastric H+,K+-ATPase	MBL (Code No. D031-3) [84]
Compound 14 (Pyrido-thieno-pyrimidine)	Chemical Inhibitor	Selective Pma1 inhibitor; lead compound for mechanism-of-action studies	Custom synthesis [82]

This cross-species inhibition analysis demonstrates that while achieving selective inhibition of fungal Pma1 over mammalian ATPases is challenging, it is feasible, as evidenced by compounds like Compound 14. The structural and mechanistic uniqueness of Pma1—specifically its hexameric organization, cooperative function mediated by the NTE, and distinct regulatory domains—provides a solid foundation for designing inhibitors that exploit these fungal-specific features. Future antifungal drug discovery efforts should leverage the detailed structural insights from recent cryo-EM studies and the robust experimental frameworks for selectivity screening outlined here to develop novel agents that overcome the limitations of current antifungal therapies.

P-type ATPases constitute a large, ancient superfamily of primary active pumps that transport diverse substrates across biological membranes, from ions like H⁺, Na⁺, K⁺, and Ca²⁺ to phospholipids [58] [2]. The significance of these enzymes in biology cannot be overstated, as they are involved in critical physiological processes across all domains of life [58] [2]. These membrane transporters share a common catalytic mechanism that involves alternation between high- and low-affinity conformations induced by autophosphorylation and dephosphorylation of a conserved aspartate residue—a characteristic that gives the "P-type" its name [58] [2] [12]. This phosphorylation event triggers conformational changes between E1 and E2 states, enabling vectorial transport of substrates against concentration gradients [2].

From a therapeutic perspective, many P-type ATPase family members are implicated in pathophysiological conditions or provide critical functions for pathogens, making them promising targets for drug development [12]. The Na⁺,K⁺-ATPase, H⁺,K⁺-ATPase, and Ca²⁺-ATPases are well-established targets for cardiovascular, gastrointestinal, and other disorders, while fungal H⁺-ATPases and parasite-specific ATPases like PfATP4 represent valuable targets for antimicrobial development [12] [85] [19]. The recent structural characterization of various P-type ATPase family members reveals that all share a similar basic structure and ion transport mechanism, featuring two access channels leading from either side of the membrane to ion binding sites at a central cavity [12]. This structural conservation, along with pathogen-specific adaptations, provides a foundation for targeted therapeutic intervention across multiple disease contexts.

Comparative Analysis of P-type ATPase Inhibitors

The therapeutic potential of P-type ATPase inhibitors spans anticancer, antimalarial, and antifungal applications. The table below provides a structured comparison of key inhibitors, their targets, and experimental evidence supporting their applications.

Table 1: Comparative Analysis of P-type ATPase Inhibitors Across Therapeutic Areas

Therapeutic Area	Inhibitor/Compound	Target P-type ATPase	Key Experimental Findings	Resistance Mechanisms
Antimalarial	KAE609 (Cipargamin) [86] [85]	PfATP4 (P. falciparum) [86] [85]	Direct inhibition of ATPase activity; increases cytoplasmic [H⁺] in yeast; induces rapid parasite clearance in vivo [86] [85]	Mutations in PfATP4 (e.g., G358S/A, A211V) [19]
Antimalarial	(+)-SJ733 [85]	PfATP4 (P. falciparum) [85]	Acts through ATP4 to induce host-mediated parasite clearance [85]	Cross-resistance with Cipargamin via G358S mutation [19]
Antimalarial	PA21A092 [19]	PfATP4 (P. falciparum) [19]	Inhibits Na⁺-dependent ATPase activity of PfATP4 [19]	A211V mutation confers resistance [19]
Antifungal	Edelfosine [86]	ScPma1p (S. cerevisiae) [86]	Displaces ScPma1p from plasma membrane; shows cross-sensitivity in KAE609-resistant yeast [86]	Not specified
Anticancer	Edelfosine (LPC analogue) [86]	Not fully specified (affects lipid rafts) [86]	Modifies lipid raft composition, induces internalization of plasma membrane transporters [86]	Not specified

Target Validation and Resistance Profiling

The validation of PfATP4 as a high-value antimalarial target exemplifies the rigorous approach required for translational success. Multiple chemical classes, including spiroindolones (KAE609/Cipargamin) and dihydroisoquinolones (+)-SJ733, converge on this target, as evidenced by resistance mutations that map directly to the PfATP4 gene [85] [19]. The recent 3.7 Å cryoEM structure of endogenously purified PfATP4 has revolutionized our understanding of this target, revealing an apicomplexan-specific binding partner, PfABP, which forms a conserved, likely modulatory interaction with PfATP4 [19]. This discovery presents an unexplored avenue for designing next-generation PfATP4 inhibitors that could potentially overcome existing resistance mechanisms.

The structural analysis shows that resistance-conferring mutations (e.g., G358S, A211V) mainly localize around the Na⁺ binding site within the transmembrane domain [19]. The G358S mutation, found in recrudescent parasites from Cipargamin clinical trials, likely blocks drug binding by introducing a serine sidechain into the proposed binding pocket [19]. Conversely, the A211V mutation that confers resistance to pyrazoleamide PA21A092 surprisingly increases susceptibility to Cipargamin, illustrating the complex structure-activity relationships that can be exploited for combination therapies [19].

Experimental Protocols for Target Validation

In Vitro ATPase Activity Assay

Objective: To measure the direct inhibitory effect of compounds on P-type ATPase activity in a cell-free system [86] [85].

Methodology:

• Membrane Preparation: Isolate plasma membrane fractions containing the target P-type ATPase (e.g., from Saccharomyces cerevisiae expressing ScPma1p or P. falciparum parasites expressing PfATP4) using differential centrifugation [86] [85].
• Reaction Setup: Prepare assay mixtures containing assay buffer (e.g., 50 mM HEPES, pH 7.4), 5 mM MgCl₂, and varying concentrations of the test compound [86].
• ATP Hydrolysis Measurement: Initiate the reaction by adding ATP (typically 2-5 mM). Incubate at 37°C for 30-60 minutes [86].
• Detection: Terminate the reaction and quantify inorganic phosphate (Pi) release using colorimetric methods (e.g., malachite green assay) [86].
• Data Analysis: Calculate ATPase activity as μmol Pi released/mg protein/hour. Determine IC₅₀ values using non-linear regression analysis of inhibition curves [86].

Chemical Genomics and Resistance Selection

Objective: To identify the cellular target of compounds and validate target engagement through resistance mutation mapping [86] [85].

Methodology:

• Resistance Selection: Expose organisms (e.g., S. cerevisiae or P. falciparum) to sublethal concentrations of the test compound, gradually increasing concentration over multiple generations [86] [85].
• Clone Isolation: Isplicate individual resistant clones and sequence candidate target genes (e.g., PfATP4 for malaria parasites or ScPMA1 for yeast) based on prior knowledge or whole-genome sequencing [86] [85].
• Genetic Validation: Introduce identified mutations into naive strains via genetic engineering (e.g., CRISPR-Cas9) to confirm sufficiency for resistance [19].
• Cross-Sensitivity Testing: Evaluate resistant strains against unrelated compounds to determine specificity of the resistance mechanism [86].

Intracellular pH Measurement

Objective: To assess the functional consequence of P-type ATPase inhibition on cellular ion homeostasis [86].

Methodology:

• Dye Loading: Incubate cells with a pH-sensitive fluorescent dye (e.g., BCECF-AM) for 30-45 minutes [86].
• Compound Treatment: Expose dye-loaded cells to the test inhibitor at various concentrations.
• Fluorescence Measurement: Monitor fluorescence intensity at excitation/emission wavelengths specific for the protonated and deprotonated forms of the dye using a plate reader or fluorescence microscopy [86].
• Calibration: Perform a calibration curve using buffers of known pH with ionophores to relate fluorescence ratios to actual intracellular pH values [86].
• Data Interpretation: Compare intracellular acidification rates between treated and untreated cells as evidence of H⁺-ATPase inhibition [86].

Visualization of P-type ATPase Structure, Function, and Inhibition

The following diagrams illustrate the structural organization of P-type ATPases, their transport mechanism, and the molecular basis of inhibitor action.

P-type ATPase Domain Architecture and Transport Cycle

Molecular Mechanism of PfATP4 Inhibition

The Scientist's Toolkit: Essential Research Reagents

The following table catalogues key reagents and their applications in P-type ATPase research, providing scientists with essential tools for experimental design.

Table 2: Essential Research Reagents for P-type ATPase Studies

Reagent/Cell Line	Specifications	Research Application	Therapeutic Relevance
S. cerevisiae (ScPMA1)	Wild-type and PMA1 mutant strains [86]	Heterologous expression system for P-type ATPase studies; resistance selection [86]	Antifungal target validation; chemical genomics [86]
P. falciparum (PfATP4)	Dd2 parasite line with endogenously tagged PfATP4 [19]	Native target validation; inhibitor susceptibility testing; structural studies [19]	Primary antimalarial target; resistance mechanism studies [19]
KAE609 (Cipargamin)	Spiroindolone compound [86] [85]	Positive control inhibitor for PfATP4/ScPma1p studies; resistance selection [86] [85]	Clinical antimalarial; prototype ATPase inhibitor [86] [85]
PA21A092	Pyrazoleamide compound [19]	PfATP4 inhibitor with distinct resistance profile; combination studies [19]	Antimalarial candidate; demonstrates target vulnerability [19]
Edelfosine	Alkyl-lysophospholipid [86]	Membrane-disrupting agent; causes Pma1p displacement in yeast [86]	Anticancer agent; demonstrates pleiotropic effects on membranes [86]
CRISPR-Cas9 System	For parasite genetic engineering [19]	Introduction of resistance mutations; epitope tagging for purification [19]	Validation of target engagement; structural biology [19]
ATPase Assay Kit	Colorimetric (e.g., malachite green) [86]	Direct measurement of ATP hydrolysis inhibition [86]	Quantification of inhibitor potency (IC₅₀) [86]
pH-Sensitive Dyes	BCECF-AM or similar [86]	Measurement of intracellular acidification upon inhibition [86]	Functional consequence of H⁺-ATPase inhibition [86]

The comparative analysis of P-type ATPase inhibitors reveals both the promise and challenges of targeting this enzyme family for therapeutic applications. The validation of PfATP4 as a antimalarial target exemplifies a successful trajectory from phenotypic screening to target identification and mechanistic understanding [86] [85] [19]. The recent structural elucidation of PfATP4 in complex with its apicomplexan-specific binding partner PfABP opens new avenues for drug design that could overcome existing resistance mechanisms [19].

For anticancer applications, the journey is less advanced. While compounds like edelfosine show pleiotropic effects on membrane organization and P-type ATPase localization [86], more specific inhibitors targeting cancer-specific ATPase isoforms or their modulators are needed. The demonstrated role of heavy metal ATPases (HMAs) in plant copper homeostasis suggests similar mechanisms might be exploitable in cancer cells exposed to heavy metal stressors [87].

Future directions should focus on leveraging structural information for rational drug design, exploring combination therapies that target multiple conformational states of P-type ATPases, and investigating pathogen-specific adaptations like PfABP that offer opportunities for selective inhibition without host toxicity. The integration of comparative genomics, chemical genomics, and structural biology will continue to drive innovation in this field, potentially yielding novel therapeutic agents across the infectious disease, oncology, and antifungal domains.

This guide provides a comparative analysis of the half-maximal inhibitory concentration (IC50) values for three distinct classes of P-type ATPase inhibitors: Tetrahydrocarbazoles, Pyrazoleamides, and Polyoxometalates. IC50 serves as a crucial quantitative measure of a compound's potency, indicating the concentration required to inhibit a specific biological process by 50% [88]. The data presented focuses on the inhibition of calcium-transporting P-type ATPases, key regulators of cellular calcium homeostasis. By compiling experimental data and detailed methodologies, this guide aims to support research in comparative genomic analysis and the development of novel therapeutic agents targeting these enzymes.

P-type ATPases constitute a large superfamily of primary active pumps that transport various ions across membranes against concentration gradients, utilizing energy from ATP hydrolysis. They are characterized by the formation of a phosphorylated intermediate (hence "P-type") during their catalytic cycle [58]. This review focuses on calcium-transporting P-type ATPases, specifically the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and plasma membrane Ca²⁺-ATPase (PMCA).

The PMCA, a crucial regulator of intracellular calcium, extrudes Ca²⁺ from the cytosol to the extracellular space to terminate calcium signals. Its ultrafast transport rates, which can exceed 5,000 cycles per second, are distinct from other P-type ATPases and are essential for processes like neuronal signal transmission [27].

The IC50 value is the most widely used and informative measure of a drug's efficacy, indicating how much of a compound is needed to inhibit a biological process by half [89]. It is a key metric in pharmacological research for evaluating the therapeutic potential of inhibitory compounds [88]. For ATP-dependent enzymes like P-type ATPases, the IC50 value can be interdependent with the concentration of ATP, especially if the inhibition is competitive [88].

Comparative IC50 Data

The following table summarizes the available experimental IC50 data for the three compound classes against their respective P-type ATPase targets. This compilation allows for a direct comparison of inhibitory potency.

Table 1: Experimentally Determined IC50 Values for Different Compound Classes

Compound Class	Specific Compound / Type	Target Enzyme	IC50 Value	Experimental Model
Polyoxometalates	Dawson-type (C₂H₈N)₆[V₂Mo₁₈O₆₂]·3H₂O [90]	Sarcoplasmic Reticulum Ca²⁺-ATPase [90]	3.4 µM [90]	Sarcoplasmic Reticulum Vesicles, Coupled Enzyme Assay [90]
Polyoxometalates	Lindqvist-type (C₄H₁₆N₃)₄[V₂W₄O₁₉]₃·12H₂O [90]	Sarcoplasmic Reticulum Ca²⁺-ATPase [90]	45.1 µM [90]	Sarcoplasmic Reticulum Vesicles, Coupled Enzyme Assay [90]
Tetrahydrocarbazoles	Data not located in search results	Target not specified	Data not located	Data not located
Pyrazoleamides	Data not located in search results	Target not specified	Data not located	Data not located

As illustrated, among the Polyoxometalates, the Dawson-type structure demonstrates significantly higher potency (lower IC50) against Ca²⁺-ATPase compared to the Lindqvist-type structure. A structure-activity relationship study correlates this enhanced potency with the compound's higher net charge and charge density [90]. A comprehensive search did not yield specific IC50 data for Tetrahydrocarbazoles or Pyrazoleamides in the context of P-type ATPase inhibition.

Experimental Protocols for IC50 Determination

Coupled Enzyme Assay for Ca²⁺-ATPase Activity

This spectrophotometric method is a standard functional assay for determining the IC50 of P-type ATPase inhibitors like Polyoxometalates [90].

• Primary Objective: To measure the ATP hydrolytic activity of Ca²⁺-ATPase in the presence of varying concentrations of an inhibitory compound.
• Workflow Overview:
  • Sample Preparation: Sarcoplasmic reticulum (SR) vesicles, which are rich in Ca²⁺-ATPase, are isolated and used as the enzyme source [90].
  • Inhibition Reaction: The SR vesicles are pre-incubated with a range of concentrations of the compound under investigation (e.g., Dawson- or Lindqvist-type POMs).
  • ATP Hydrolysis Measurement: The assay is initiated by adding ATP to the reaction mixture. The ATPase activity is coupled to the oxidation of NADH, which is monitored by a decrease in absorbance at 340 nm. The coupled enzyme system includes pyruvate kinase and lactate dehydrogenase [90].
  • Data Analysis: The rate of ATP hydrolysis is calculated for each inhibitor concentration. The IC50 value is determined as the concentration of the inhibitor that reduces the ATPase activity by 50% compared to the uninhibited control.

Surface Plasmon Resonance (SPR) for Interaction-Specific IC50

SPR can be used as an alternative, interaction-specific method to determine IC50 values, providing molecular resolution for inhibitor screening [89].

• Primary Objective: To determine the concentration of an inhibitor required to block 50% of a specific ligand-receptor interaction (e.g., between Ca²⁺-ATPase and a regulatory molecule).
• Workflow Overview:
  • Immobilization: An antibody against the Fc tag is immobilized on a CM5 sensor chip using amine-coupling chemistry [89].
  • Capture: The target receptor (e.g., a Ca²⁺-ATPase extracellular domain fused to an Fc tag) is captured onto the sensor chip surface via the immobilized antibody [89].
  • Injection with Inhibitor: A fixed concentration of the ligand is pre-incubated with a range of concentrations of the inhibitor. This mixture is then injected over the sensor chip surface [89].
  • Data Analysis: The reduction in the SPR response signal (indicating blocked binding) is measured. The IC50 is calculated by fitting the dose-response data of signal inhibition versus inhibitor concentration using software like GraphPad Prism [89].

The Scientist's Toolkit: Research Reagent Solutions

The following table details key reagents and materials essential for conducting the experiments described in this guide.

Table 2: Essential Research Reagents and Materials

Reagent/Material	Function/Application	Key Details / Examples
Sarcoplasmic Reticulum (SR) Vesicles [90]	In vitro model system rich in Ca²⁺-ATPase for functional inhibition studies.	Isolated from skeletal muscle; provides a high concentration of the target enzyme for spectrophotometric assays [90].
Coupled Enzyme Assay System [90]	To indirectly measure ATP hydrolysis activity by Ca²⁺-ATPase.	Includes pyruvate kinase and lactate dehydrogenase; monitors NADH oxidation spectrophotometrically at 340 nm [90].
Surface Plasmon Resonance (SPR) System [89]	Label-free, real-time analysis of biomolecular interactions for determining interaction-specific IC50.	Utilizes a sensor chip (e.g., CM5) with immobilized capture antibody (e.g., anti-Fc) to study ligand-receptor binding kinetics [89].
Fc-Tagged Recombinant Proteins [89]	Provides a universal capture method for SPR and other assay formats.	Receptors (e.g., ATPase domains) are expressed as fusion proteins with an IgG1-Fc tag for easy purification and immobilization [89].
In-Cell Western Assay Kits [91]	Cell-based, high-throughput method for IC50 determination within intact cells.	Combines immunoassay and Western blot principles to assess protein expression and phosphorylation in a physiological context [91].

This comparison guide highlights the current state of experimental IC50 data for several compound classes targeting P-type ATPases. Among the compounds surveyed, Polyoxometalates, particularly the Dawson-type architecture, show promising nanomolar-range potency against Ca²⁺-ATPase [90]. The available data underscores the importance of structural features, such as net charge and charge density, in determining inhibitor potency [90]. The provided experimental protocols and toolkit offer a foundation for standardizing the evaluation of existing and novel inhibitors, such as Tetrahydrocarbazoles and Pyrazoleamides, for which public IC50 data on these targets is currently limited. This structured approach to IC50 comparison is vital for advancing the comparative genomic analysis of P-type ATPase inhibitors and for rational drug design.

In the discovery of P-type ATPase inhibitors, a critical step in developing new therapeutic agents, researchers face the fundamental challenge of translating findings from simple, controlled laboratory systems to more complex, biologically relevant environments. The journey from in vitro (test tube) experiments to ex vivo (tissue-based) systems represents a crucial bridge in validating biochemical activity within a cellular context, ultimately informing the potential for future in vivo (whole organism) success. This transition is particularly significant in the study of P-type ATPases—a large superfamily of ion pumps that are structurally related and traverse a catalytic cycle involving phosphorylation and dephosphorylation of a conserved aspartate residue [58]. These enzymes, including Ca²⁺-ATPase and Na⁺/K⁺-ATPase, are essential for cellular functions ranging from muscle contraction and neurotransmission to calcium homeostasis, making them important pharmacological targets [47] [13].

The correlation between in vitro and ex vivo models enables researchers to assess not only the potency of potential inhibitors but also their selectivity, cellular penetration, and activity in more physiologically relevant systems. As these ATPases represent pharmacologically important targets due to their role in health and disease, understanding this correlation is paramount for developing effective metallodrugs and therapeutic agents [47]. This guide objectively compares the performance of various experimental systems for evaluating P-type ATPase inhibitors, providing researchers with a framework for selecting appropriate assays and interpreting translational data.

Comparative Analysis of Experimental Systems for P-type ATPase Inhibition

Fundamental Differences Between In Vitro and Ex Vivo Approaches

In vitro assays typically involve isolated enzymes or membrane preparations under controlled conditions, allowing for precise measurement of biochemical activity without the complexity of cellular systems. For P-type ATPase research, this often utilizes vesicle preparations from specific membrane sources, such as sarcoplasmic reticulum vesicles for Ca²⁺-ATPase studies [47] [48]. These systems offer advantages in screening efficiency, mechanistic insight, and the ability to precisely control experimental conditions such as ATP concentration, calcium levels, and pH.

In contrast, ex vivo models maintain tissue architecture and more closely approximate the in vivo environment while remaining outside the living organism. For Na⁺/K⁺-ATPase studies, researchers have successfully used the opercular epithelium of killifish to investigate effects on epithelial chloride secretion coupled with ATPase activity [47] [48]. These systems preserve native cellular environments, including membrane polarity, accessory proteins, and intracellular signaling networks that may influence inhibitor efficacy.

Quantitative Comparison of P-type ATPase Inhibitors Across Systems

Table 1: Comparison of P-type ATPase Inhibitor Potency Across Experimental Systems

Inhibitor	P-type ATPase Target	In vitro IC₅₀ (μM)	Ex vivo Inhibition	Experimental Model
K₉(C₂H₈N)₅[H₁₀Se₂W₂₉O₁₀₃] (Se₂W₂₉)	Ca²⁺-ATPase	0.3 μM [47]	N/R	SR vesicles from skeletal muscle [47]
K₆[α-P₂W₁₈O₆₂] (P₂W₁₈)	Ca²⁺-ATPase	0.6 μM [47]	99% at 1 μM [47]	SR vesicles (in vitro); killifish opercular epithelium (ex vivo) [47]
K₆[α-P₂W₁₈O₆₂] (P₂W₁₈)	Na⁺/K⁺-ATPase	N/R	99% at 1 μM [47]	Killifish opercular epithelium [47]
K₉(C₂H₈N)₅[H₁₀Se₂W₂₉O₁₀₃] (Se₂W₂₉)	Na⁺/K⁺-ATPase	N/R	14% at 1 μM [47]	Killifish opercular epithelium [47]
Cs₅.₆H₃.₄PV₁₄O₄₂ (PV₁₄)	Ca²⁺-ATPase	5 μM [48]	N/R	SR vesicles from skeletal muscle [48]
Cs₅.₆H₃.₄PV₁₄O₄₂ (PV₁₄)	Na⁺/K⁺-ATPase	N/R	78% at 10 μM [48]	Killifish opercular epithelium [48]
Decavanadate (V₁₀)	Ca²⁺-ATPase	15 μM [48]	N/R	SR vesicles from skeletal muscle [48]
Decavanadate (V₁₀)	Na⁺/K⁺-ATPase	N/R	66% at 10 μM [48]	Killifish opercular epithelium [48]
Monovanadate (V₁)	Ca²⁺-ATPase	80 μM [48]	N/R	SR vesicles from skeletal muscle [48]
Monovanadate (V₁)	Na⁺/K⁺-ATPase	N/R	33% at 10 μM [48]	Killifish opercular epithelium [48]
[Cl@Vᵛ⁷Vᴵⱽ⁸O₃₆]⁶⁻ (V₁₅)	Ca²⁺-ATPase	14.2 μM [10]	N/R	SR vesicles from skeletal muscle [10]

Abbreviations: N/R = Not reported; SR = Sarcoplasmic reticulum

Key Insights from Comparative Data Analysis

The comparative data reveals several critical patterns in P-type ATPase inhibitor evaluation:

• Potency Translation Variability: Some compounds maintain strong inhibitory activity across systems, such as P₂W₁₈ which showed exceptional potency in both in vitro (IC₅₀ = 0.6 μM) and ex vivo (99% inhibition) models for Ca²⁺-ATPase [47].

• Selectivity Profiles: Significant differences in target selectivity emerge between systems. Se₂W₂₉ demonstrated remarkable selectivity for Ca²⁺-ATPase over Na⁺/K⁺-ATPase (0.3 μM IC₅₀ vs. only 14% inhibition at 1 μM) [47], highlighting how ex vivo models can reveal specificity that might inform therapeutic windows.

• Structural-Activity Relationships: Polyoxometalates with higher charge densities generally exhibit greater inhibitory potency, with the most potent compounds (IC₅₀ < 16 μM) including Se₂W₂₉, P₂W₁₈, CoW₁₁Ti, SiW₉, and P₂W₁₂ [47].

Experimental Protocols for P-type ATPase Inhibition Studies

Standardized In Vitro Ca²⁺-ATPase Activity Assay

Principle: This assay measures ATP hydrolysis activity of Ca²⁺-ATPase in sarcoplasmic reticulum (SR) vesicles by quantifying inorganic phosphate release [47].

Protocol:

• Vesicle Preparation: SR vesicles are isolated from rabbit skeletal muscle through differential centrifugation and homogenization in sucrose-EDTA buffers [47].
• Reaction Setup: Prepare assay mixture containing 20 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl₂, 5 mM ATP, 0.1 mM CaCl₂, and 5-20 μg of SR vesicle protein.
• Inhibitor Incubation: Pre-incubate vesicles with polyoxometalate inhibitors (0.1-200 μM) for 5 minutes at 37°C.
• Reaction Initiation: Start ATP hydrolysis by adding ATP, then incubate for 30 minutes at 37°C.
• Reaction Termination: Stop the reaction by adding ice-cold trichloroacetic acid (5% final concentration).
• Phosphate Detection: Quantify inorganic phosphate using the colorimetric method of Fiske and Subbarow or a malachite green-based assay.
• Data Analysis: Calculate IC₅₀ values by fitting inhibition data to a sigmoidal dose-response curve using appropriate statistical software.

Key Controls: Include samples without calcium (EGTA-added), without ATP, and without enzyme to account for non-specific phosphatase activity and background phosphate [47].

Ex Vivo Na⁺/K⁺-ATPase Activity in Killifish Opercular Epithelium

Principle: This assay evaluates Na⁺/K⁺-ATPase function by measuring its role in powering chloride secretion across an intact epithelial membrane [47] [48].

Protocol:

• Tissue Preparation: Isolve the opercular epithelium from Fundulus heteroclitus (killifish) and mount in specialized Ussing chambers [47] [48].
• Physiological Solution: Bathe both sides of the epithelium with oxygenated Ringer's solution (pH 7.8) containing (in mM): 140 NaCl, 2.5 KCl, 1.5 CaCl₂, 1.0 MgCl₂, 5.0 glucose, and 10 HEPES.
• Electrophysiological Measurements: Monitor short-circuit current (Iₛc) as a direct measure of net chloride ion transport, which is energetically coupled to Na⁺/K⁺-ATPase activity.
• Inhibitor Application: Add polyoxometalate inhibitors (typically 1-10 μM) to the serosal side and measure changes in Iₛc.
• Validation: Confirm Na⁺/K⁺-ATPase specificity using ouabain (a specific Na⁺/K⁺-ATPase inhibitor) as a positive control.
• Data Quantification: Express inhibition as percentage reduction in Iₛc compared to baseline levels [48].

Technical Considerations: This model is particularly valuable because the opercular epithelium contains rich mitochondria-rich cells with abundant Na⁺/K⁺-ATPase, making it highly sensitive for detecting ATPase inhibition [48].

Visualization of Experimental Workflows and Signaling Pathways

P-type ATPase Inhibition Assay Workflow

Calcium Signaling and ATPase Interplay

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Research Reagent Solutions for P-type ATPase Studies

Reagent/Material	Function/Application	Specific Examples	Experimental Considerations
Sarcoplasmic Reticulum Vesicles	Source of Ca²⁺-ATPase for in vitro assays	Rabbit skeletal muscle preparations [47]	Maintain at -80°C; avoid freeze-thaw cycles; confirm activity with positive controls
Polyoxometalate Inhibitors	P-type ATPase inhibition studies	Dawson-type POTs (P₂W₁₈), Keggin-type structures (CoW₁₁Ti) [47]	Prepare fresh stock solutions; keep on ice to prevent decomposition; characterize by IR spectroscopy [47]
Killifish Opercular Epithelium	Ex vivo Na⁺/K⁺-ATPase model	Fundulus heteroclitus tissue [47] [48]	Mount immediately in Using chambers; oxygenate Ringer's solution; monitor tissue viability
Using Chamber System	Electrophysiological measurement of ion transport	Commercial Using chambers with voltage/current clamp [48]	Calibrate electrodes daily; maintain temperature at 25-28°C; bubble with air/O₂ mixture
ATP Regeneration System	Maintain constant ATP levels in prolonged assays	Pyruvate kinase/phosphoenolpyruvate system [47]	Include in assays >30 minutes; optimize concentration for specific ATPase activity
Phosphate Detection Reagents	Quantify ATP hydrolysis activity	Malachite green assay; Fiske-Subbarow method [47]	Prepare fresh standards; account for non-enzymatic ATP hydrolysis in controls
Calcium Buffers	Control free Ca²⁺ concentration in assays	EGTA/Ca²⁺ buffering systems [13]	Calculate using binding constants; verify with calcium electrodes; consider pH dependence
Protease Inhibitor Cocktails	Prevent protein degradation during preparations	Commercial mixes or custom combinations	Add fresh to isolation buffers; consider target-specific inhibitors (e.g., PMSF for serine proteases)

The correlation between in vitro and ex vivo systems for evaluating P-type ATPase inhibitors provides an essential framework for drug discovery, particularly in the development of polyoxometalate-based therapeutics. The data demonstrates that while in vitro systems excel in rapid screening and mechanistic studies, ex vivo models reveal critical information about cellular penetration, tissue-specific activity, and selectivity that may predict in vivo performance.

Successful implementation of this correlative approach requires careful consideration of model systems, experimental design, and data interpretation. The killifish opercular epithelium model has proven particularly valuable for Na⁺/K⁺-ATPase studies, while SR vesicle preparations remain the gold standard for initial Ca²⁺-ATPase inhibitor screening. As research advances, integrating these correlative models with structural biology and genomic approaches will further enhance our ability to develop targeted P-type ATPase inhibitors with therapeutic potential.

Researchers should prioritize compounds that demonstrate consistent activity across both in vitro and ex vivo systems, while paying close attention to selectivity profiles that emerge in more complex biological environments. This integrated approach maximizes the translational potential of preclinical findings and supports the rational design of next-generation P-type ATPase-targeting therapeutics.

Conclusion

Comparative genomic analysis of P-type ATPase inhibitors reveals both significant challenges and promising avenues for therapeutic development. The integration of high-resolution structural data with genomic insights enables precise mapping of resistance mutations and identification of species-specific features for selective targeting. Emerging methodologies, including computational design and high-throughput screening, are accelerating inhibitor discovery, while polyoxometalates and tetrahydrocarbazoles represent promising chemotypes with broad activity spectra. Future directions should focus on exploiting newly discovered binding partners like PfABP, engineering enhanced selectivity through structural insights, and developing combination therapies to circumvent resistance. The continued convergence of genomic, structural, and chemical approaches will ultimately yield next-generation ATPase inhibitors with improved clinical efficacy across infectious diseases, cancer, and other pathological conditions.

References

• 1. P-type ATPase [https://en.wikipedia.org/wiki/P-type_ATPase]
• 2. P-type ATPases: Many more enigmas left to solve - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC10654040/]
• 3. Structure and ion-release mechanism of PIB-4-type ATPases [https://elifesciences.org/articles/73124]
• 4. Structure and ion-release mechanism of PIB-4-type ATPases [https://pmc.ncbi.nlm.nih.gov/articles/PMC8880997/]
• 5. Structure and transport mechanism of P5B-ATPases [https://www.nature.com/articles/s41467-021-24148-y]
• 6. Comparative genomics uncovers adaptive and ... [https://www.nature.com/articles/s41598-025-25044-x]
• 7. Integrative Genomics and Metabolomics Analyses Provide ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12472927/]
• 8. The mechanism of copper transporters in ovarian cancer ... [https://www.sciencedirect.com/science/article/abs/pii/S0162013423002064]
• 9. Fungal Plasma Membrane H+-ATPase: Structure, Mechanism ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11051447/]
• 10. ATPase Inhibition Potential and Anti-Breast Cancer Activity [https://www.mdpi.com/2304-6740/13/9/306]
• 11. Recent advances in understanding the P-type IIIA ATPase ... [https://www.sciencedirect.com/science/article/abs/pii/S0981942825008666]
• 12. Review P-type ATPases as drug targets: Tools for medicine ... [https://www.sciencedirect.com/science/article/pii/S0005272808007639]
• 13. Crucial roles of calcium ATPases and phosphoinositides [https://pmc.ncbi.nlm.nih.gov/articles/PMC12329087/]
• 14. Structural basis of the P4B ATPase lipid flippase activity [https://www.nature.com/articles/s41467-021-26273-0]
• 15. Structural analysis of full-length human transmembrane ... [https://www.nature.com/articles/s41421-025-00806-z]
• 16. Structure, function and biogenesis of the fungal proton ... [https://www.sciencedirect.com/science/article/pii/S0167488923001738]
• 17. Structure and activation mechanism of the hexameric ... [https://www.nature.com/articles/s41467-021-26782-y]
• 18. Assembly and cooperative mechanism of the hexameric ... [https://www.sciencedirect.com/science/article/pii/S2211124725005248]
• 19. Endogenous structure of antimalarial target PfATP4 reveals ... [https://www.nature.com/articles/s41467-025-64815-y]
• 20. ATPase Pma1 inhibitors that extend chronological lifespan ... [https://pubmed.ncbi.nlm.nih.gov/40484900/]
• 21. Endogenous structure of antimalarial target PfATP4 reveals ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12537908/]
• 22. College of Medicine [https://drexel.edu/medicine/news-events/news-archive/2025/October/Drexel-Researchers-Gain-New-Insights-From-Malaria-Causing-Parasite/]
• 23. New Insights into Malaria Could Reshape Treatment [https://www.cuimc.columbia.edu/news/new-insights-malaria-could-reshape-treatment]
• 24. 9n10 - P. falciparum P-type ATPase, PfATP4 - Summary [https://pdbj.org/mine/summary/9n10]
• 25. Catalytic dwell oscillations complete the F1-ATPase ... [https://www.nature.com/articles/s42004-025-01443-z]
• 26. Molecular choreography of E1 enzymes in ubiquitin-like ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12355090/]
• 27. Molecular mechanism of ultrafast transport by plasma ... [https://www.nature.com/articles/s41586-025-09402-3]
• 28. The role of Na,K‑ATPase in lung diseases (Review) [https://www.spandidos-publications.com/10.3892/mmr.2025.13665]
• 29. Lipid-Protein Interplay: How the Plant Plasma Membrane H ... [https://premierscience.com/pjpb-25-967/]
• 30. Conduction pathway for potassium through the Escherichia ... [https://elifesciences.org/articles/107397]
• 31. Measuring In Vitro ATPase Activity for Enzymatic ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5091952/]
• 32. Identification and expression analysis of P-type ATPase IIIA ... [https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-025-11219-3]
• 33. ATPase Assay Kits [https://bellbrooklabs.com/atpase-assay-kits/?srsltid=AfmBOoooefsNdZiJtj3-rXaO85BXGH5aQvbv9OCF56DfPIKIs6EkUv16]
• 34. A High-Throughput TNP-ATP Displacement Assay for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3065726/]
• 35. ATP Assays | What is an ATP Assay? [https://www.promega.com/resources/guides/cell-biology/atp-assays/]
• 36. Targeting protein-protein interactions for therapeutic ... [https://www.nature.com/articles/s41598-018-29685-z]
• 37. Structure-Based Virtual Screening for Drug Discovery [https://pmc.ncbi.nlm.nih.gov/articles/PMC3282008/]
• 38. A Comparative Study of Modern Homology Modeling ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6068592/]
• 39. Comparative analysis of the P-type ATPase gene family in ... [https://www.sciencedirect.com/science/article/pii/S0888754319305099]
• 40. A Genome-Wide Identification and Comparative Analysis of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10606463/]
• 41. Structure-based virtual screening discovers potent and ... [https://www.sciencedirect.com/science/article/pii/S0223523423003859]
• 42. Identification of novel VPS4 inhibitors using multi-tiered ... [https://link.springer.com/article/10.1007/s11030-025-11412-2]
• 43. Homology Modeling of the Human P-glycoprotein (ABCB1) ... [https://www.mdpi.com/1422-0067/21/11/4058]
• 44. Small phenolic inhibitors of PfATP6, a Plasmodium ... [https://www.sciencedirect.com/science/article/pii/S0968089625002834]
• 45. In Silico Screening for Inhibitors of P-Glycoprotein That ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4244591/]
• 46. Revisiting Plasmodium falciparum P - type 4 in malarial... ATPase [https://pubmed.ncbi.nlm.nih.gov/40512986/]
• 47. The P-type ATPase inhibiting potential of polyoxotungstates [https://pmc.ncbi.nlm.nih.gov/articles/PMC5824666/]
• 48. Inhibition of Na+/K+- and Ca2+-ATPase activities by ... [https://www.sciencedirect.com/science/article/pii/S0162013419300224]
• 49. Dawson- and Lindqvist-Type Hybrid Polyoxometalates ... [https://www.mdpi.com/1420-3049/30/22/4334]
• 50. The P-type ATPase inhibiting potential of polyoxotungstates. [https://www.lifescience.net/publications/1678473/the-p-type-atpase-inhibiting-potential-of-polyoxot/]
• 51. Properties of FDA-approved small molecule protein kinase ... [https://pubmed.ncbi.nlm.nih.gov/40252783/]
• 52. ATP-competitive inhibitors for cancer treatment – kinases and the... [https://pubs.rsc.org/en/content/articlehtml/2025/md/d5md00235d]
• 53. Tetrahydrocarbazoles are a novel class of potent P-type ... [https://pubmed.ncbi.nlm.nih.gov/29293507/]
• 54. Tetrahydrocarbazoles are a novel class of potent P-type ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5749684/]
• 55. Comparative genomic analysis of the DUF71/COG2102 family ... [https://biologydirect.biomedcentral.com/articles/10.1186/1745-6150-7-32]
• 56. Comparative genomic analysis reveals metabolic flexibility ... [https://www.nature.com/articles/s41467-021-25565-9]
• 57. Structure-based identification of salvianolic acid B as an ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12538069/]
• 58. P-type ATPases: Many more enigmas left to solve [https://www.sciencedirect.com/science/article/pii/S0021925823023803]
• 59. Recent advances in AI-driven protein-ligand interaction ... [https://pubmed.ncbi.nlm.nih.gov/39999605/]
• 60. AI Designs Binders with High Affinity and Specificity to ... [https://www.genengnews.com/topics/artificial-intelligence/ai-designs-binders-with-high-affinity-and-specificity-to-proteins/]
• 61. Recent Advances on P-Glycoprotein (ABCB1) Transporter ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9740644/]
• 62. Mutations in the P-Type Cation-Transporter ATPase 4 ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4340351/]
• 63. A G358S mutation in the Plasmodium falciparum Na+ ... [https://www.nature.com/articles/s41467-022-33403-9]
• 64. Demethoxycurcumin Is A Potent Inhibitor of P-Type ATPases ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5028038/]
• 65. P-Type ATPase - an overview [https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/p-type-atpase]
• 66. ATPase From Sarco(Endo)Plasmic Reticulum (SERCA) [https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2018.00036/full]
• 67. A novel machine learning-based screening identifies statins ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10193016/]
• 68. The role of SERCA in vascular diseases, a potential ... [https://www.sciencedirect.com/science/article/abs/pii/S014341602500048X]
• 69. The Structure and Function of the Na,K-ATPase Isoforms in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5459889/]
• 70. Identification of Na+/K+-ATPase inhibition-independent ... [https://www.nature.com/articles/s41598-017-02496-4]
• 71. Discovery of novel SERCA inhibitors by virtual screening of a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3065555/]
• 72. Pharmacological or genetic inhibition of LTCC promotes ... [https://www.nature.com/articles/s41536-025-00389-z]
• 73. Allosterism vs. Orthosterism: Recent Findings and Future ... [https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2021.652121/full]
• 74. Orthosteric vs. Allosteric Interactions: The Silent Decider of ... [https://www.ecosystem.drgpcr.com/post/orthosteric-vs-allosteric-the-silent-decider-of-safety-and-success]
• 75. Recent advances in computational strategies for allosteric ... [https://www.sciencedirect.com/science/article/abs/pii/S1359644625001795]
• 76. Computational Tools for Allosteric Drug Discovery: Site ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7920515/]
• 77. Resistance to Allosteric Inhibitors [https://www.sciencedirect.com/science/article/abs/pii/S0022283625001998]
• 78. General and specific interactions of the phospholipid bilayer ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6557964/]
• 79. Emerging Diversity in Lipid–Protein Interactions - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC6509647/]
• 80. Membrane properties control the ATPase activity of ... [https://www.sciencedirect.com/science/article/pii/S0005273625000240]
• 81. Fungal Plasma Membrane H+-ATPase: Structure, ... [https://www.mdpi.com/2309-608X/10/4/273]
• 82. Identification of Antifungal H+-ATPase Inhibitors with Effect ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5487681/]
• 83. Assembly and cooperative mechanism of the hexameric ... [https://pubmed.ncbi.nlm.nih.gov/40413744/]
• 84. Anti-Proton Pump (H,K-ATPase α subunit) mAb [https://www.mblbio.com/bio/g/dtl/A/?pcd=D031-3]
• 85. chemical genomics reveal that the spiroindolone... Comparative [https://pubmed.ncbi.nlm.nih.gov/27291296/]
• 86. (PDF) Comparative chemical genomics reveal that the spiroindolone... [https://www.academia.edu/53111970/Comparative_chemical_genomics_reveal_that_the_spiroindolone_antimalarial_KAE609_Cipargamin_is_a_P_type_ATPase_inhibitor]
• 87. Genome-wide analysis of heavy metal ATPases (HMAs) in ... [https://www.nature.com/articles/s41598-023-32023-7]
• 88. IC50 [https://en.wikipedia.org/wiki/IC50]
• 89. Determination of half-maximal inhibitory concentration ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4955526/]
• 90. Dawson- and Lindqvist-Type Hybrid Polyoxometalates [https://pmc.ncbi.nlm.nih.gov/articles/PMC12654597/]
• 91. In-cell Western Assays for IC50 Determination [https://azurebiosystems.com/blog/in-cell-western-assays-for-ic50-determination/]