{"gene":"PF3D7_1211900","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2001,"finding":"PfATP4 (PF3D7_1211900) expressed in Xenopus laevis oocytes exhibits Ca2+-dependent ATPase activity stimulated over a broad [Ca2+]free range (3.2–320 µM), is resistant to inhibition by ouabain (200 µM) or thapsigargin (0.8 µM), but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 µM). mRNA expression is variable across asexual stages with a maximal ~5-fold increase in meronts compared with rings.","method":"Xenopus oocyte expression, ATPase activity assays with pharmacological inhibitors, quantitative PCR on synchronized asexual stages","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional expression in Xenopus oocytes with multiple inhibitor assays and stage-specific mRNA quantification, single lab","pmids":["11145964"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1996,"finding":"The PfATP4 (PF3D7_1211900) gene encodes a ~190 kDa P-type ATPase protein that is detectable immunologically throughout the asexual erythrocytic stages and in free merozoites. Immunofluorescence microscopy shows the protein is concentrated in discrete compartments at the periphery of the parasite. An 8 kb transcript appears shortly after erythrocyte invasion but is not detected in later stages, while the protein persists.","method":"Gene cloning, immunological detection (Western blot), immunofluorescence microscopy","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — immunofluorescence localization and Western blot, single lab, two orthogonal methods","pmids":["8813672"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"Spiroindolone antimalarials (including NITD609) rapidly inhibit protein synthesis in P. falciparum, and this effect is ablated in parasites bearing nonsynonymous mutations in the gene encoding PfATP4 (PF3D7_1211900), establishing PfATP4 as the target of spiroindolones.","method":"Resistance selection with whole-genome sequencing identifying mutations in PfATP4; protein synthesis inhibition assay","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — resistance-conferring mutations in PfATP4 ablate the spiroindolone effect, replicated across multiple resistant lines","pmids":["20813948"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PfATP4 (PF3D7_1211900) localizes to the parasite plasma membrane and functions as a Na+ efflux ATPase, actively extruding Na+ against an inward gradient to maintain low cytosolic [Na+]. Spiroindolones cause a profound disruption of Na+ homeostasis in P. falciparum that is attenuated in parasites bearing resistance-conferring mutations in PfATP4; those mutant parasites also show some impairment of Na+ regulation.","method":"Fluorescent Na+ indicator measurements in live parasites; genetic resistance-conferring mutations in PfATP4 characterized by whole-genome sequencing; ion homeostasis assays","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct functional measurement of Na+ regulation combined with genetic epistasis via resistance mutations, replicated across multiple compound classes","pmids":["23414762"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"Treatment of P. falciparum-infected erythrocytes with (+)-SJ733, which acts on PfATP4 (PF3D7_1211900), causes rapid perturbation of parasite Na+ homeostasis followed by increased membrane rigidity and externalization of phosphatidylserine in infected cells, consistent with eryptosis/senescence. Resistance-conferring mutations in pfatp4 carry a high fitness cost.","method":"Ion homeostasis assays (fluorescent Na+ indicators), membrane rigidity measurements, phosphatidylserine externalization assay, in vivo mouse model, resistance selection with whole-genome sequencing","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ion assays, membrane biophysics, in vivo efficacy, genetic resistance mapping) in a single study","pmids":["25453091"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"Pyrazoleamide antimalarials disrupt Na+ regulation in blood-stage P. falciparum in a manner dependent on PfATP4 (PF3D7_1211900); mutations in PfATP4 (in combination with mutations in PfCDPK5) are necessary to confer full pyrazoleamide resistance, establishing PfATP4 as part of their mechanism of action.","method":"Resistance selection, whole-genome sequencing, gene transfer (complementation), Na+ homeostasis assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — resistance mapping via whole-genome sequencing, gene transfer validation, and direct ion homeostasis measurement","pmids":["25422853"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"Aminopyrazole antimalarials (e.g. GNF-Pf4492) target PfATP4 (PF3D7_1211900): three independently derived resistant lines each acquired independent mutations in PfATP4. The aminopyrazoles disrupt intracellular Na+ homeostasis and block parasite transmission to mosquitoes, phenocopying spiroindolone effects.","method":"Resistance selection, whole-genome sequencing of three independent resistant lines, Na+ homeostasis assay, gametocyte/transmission assay","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent resistance lines with PfATP4 mutations, multiple orthogonal functional assays, single lab","pmids":["25322084"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"}]},{"year":2014,"finding":"Twenty-eight compounds from the Malaria Box affect P. falciparum Na+ and pH regulation consistent with PfATP4 (PF3D7_1211900) inhibition; six chemically diverse compounds showed reduced antiplasmodial activity against spiroindolone-resistant parasites carrying pfatp4 mutations, and resistance selection with two compounds yielded parasites with new mutations in pfatp4. This establishes PfATP4 as a target for diverse chemotypes and indicates it co-regulates both Na+ and cytosolic pH (alkalinization).","method":"Ion homeostasis screening (fluorescent Na+ and pH indicators), cross-resistance testing against pfatp4 mutant parasites, in vitro resistance selection with whole-genome sequencing","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple chemotypes, genetic validation via resistance mutations, ion homeostasis assays; independent confirmation of PfATP4 as pH and Na+ regulator","pmids":["25145582"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"CRISPR/Cas9-mediated introduction of specific point mutations into pfatp4 (PF3D7_1211900) using a plasmid-free ribonucleoprotein + ssODN approach confirmed that those mutations confer resistance to the antimalarial SJ733, validating the genetic link between PfATP4 and SJ733 action.","method":"Plasmid-free CRISPR/Cas9 genome editing, ssODN repair template, whole-genome sequencing, growth inhibition assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct genetic engineering (CRISPR) introducing specific mutations confirms resistance phenotype, single lab","pmids":["28542423"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"A G358S mutation in PfATP4 (PF3D7_1211900) confers clinically relevant resistance to cipargamin and (+)-SJ733, allowing parasites to withstand micromolar concentrations while remaining susceptible to non-PfATP4-targeting antimalarials. G358S reduces the affinity of PfATP4 for Na+ and is associated with increased resting cytosolic [Na+], but causes no detectable defect in parasite growth or transmissibility. The equivalent G358S mutation in Toxoplasma gondii ATP4 also decreases sensitivity to cipargamin and SJ733.","method":"Clinical trial resistance marker sequencing; functional ion homeostasis assays; genetic introduction of G358S into Toxoplasma gondii ATP4; parasite growth and transmissibility assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — clinical validation plus multiple orthogonal functional assays (ion homeostasis, cross-species genetic confirmation), single primary paper","pmids":["36180431"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"A 3.7 Å cryo-EM structure of PfATP4 (PF3D7_1211900) purified from CRISPR-engineered P. falciparum parasites reveals a previously unknown apicomplexan-specific binding partner, PfABP, which forms a conserved interaction with PfATP4 and likely plays a modulatory role.","method":"CRISPR engineering, native protein purification, cryo-EM structure determination at 3.7 Å","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution cryo-EM structure of endogenous protein from CRISPR-engineered parasites with binding partner identification; single study","pmids":["41115914"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"The isolated soluble catalytic domain of PfATP4 (PF3D7_1211900) exhibits Mg2+-dependent ATPase activity in vitro at rates comparable to native cellular PfATP4. Mutation of key nucleotide-binding residues (Lys619, Lys652, Arg703 → Met) decreases enzymatic activity to ~one quarter. Mutation of Mg2+-coordinating residues Asp865+Asp869 together also reduces activity to ~one quarter. Mutation of the putative γ-phosphate-receiving Asp451 does not prevent Pi release, indicating ATP hydrolysis by this domain proceeds independently of phosphorylation at Asp451.","method":"Recombinant protein expression and purification, in vitro phosphate colorimetric ATPase assay, site-directed mutagenesis, AlphaFold 3 structural modelling","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis of key catalytic residues; single lab, no full-length protein structural validation","pmids":["40091859"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"Homology modelling of PfATP4 (PF3D7_1211900) combined with a merodiploid conditional expression genetic system identified residues critical for Na+ coordination and the phosphorylation cycle. Phenotypic characterization of mutants confirmed that PfATP4 is localized to the parasite plasma membrane, that mutation of these residues impairs parasite growth and Na+ regulation, and that resistance-associated mutations reduce inhibitor sensitivity with some conferring a fitness cost consistent with model predictions.","method":"Homology modelling (SERCA-based), molecular dynamics simulation, CRISPR-mediated merodiploid conditional allele system, parasite growth assays, subcellular localization (imaging), Na+ homeostasis assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic engineering, localization, ion homeostasis, growth phenotyping, computational modelling) in a single rigorous study confirming plasma membrane localization and Na+ pump function","pmids":["39773028"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"Solvent-induced proteome profiling (SPP) identified PfATP4 (PF3D7_1211900; annotated as non-SERCA-type Ca2+-transporting P-ATPase) as the protein target of cipargamin in P. falciparum lysates, demonstrating ligand-induced stabilization of PfATP4 consistent with direct drug-protein interaction.","method":"Solvent proteome profiling (SPP), mass spectrometry-based proteomics","journal":"International journal for parasitology. Drugs and drug resistance","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — proteomics-based target engagement assay identifies PfATP4 stabilization by cipargamin, single lab, single method","pmids":["41308470"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"N-acetamide indole compounds inhibit PfATP4 (PF3D7_1211900): resistance selection and whole-genome sequencing revealed mutations in PfATP4; analogs showed reduced potency against PfATP4 resistance-mutant parasites; metabolomic signature matched that of the PfATP4 inhibitor KAE609; and in vitro inhibition of Na+-dependent ATPase activity was demonstrated. The compounds also inhibit gamete development and block transmission to mosquitoes.","method":"Resistance selection, whole-genome sequencing, cross-resistance assays, metabolomics, Na+-dependent ATPase inhibition assay in vitro, gametocyte/transmission assay, P. berghei mouse model","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal validation methods including direct in vitro ATPase assay, genetic resistance mapping, and metabolomics; single lab","pmids":["40228810"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PfATP4 (PF3D7_1211900) is an essential P-type cation ATPase localized to the parasite plasma membrane that functions as a Na+ efflux pump (and likely H+ importer) to maintain low cytosolic [Na+] in intraerythrocytic P. falciparum; its inhibition by multiple structurally distinct antimalarial classes (spiroindolones, pyrazoleamides, aminopyrazoles, dihydroisoquinolones, and others) causes rapid disruption of Na+ homeostasis, cytosolic alkalinization, and downstream eryptotic changes leading to parasite death, while a 3.7 Å cryo-EM structure reveals it associates with a novel apicomplexan-specific binding partner PfABP, and mutagenesis studies have defined key residues for Na+ coordination, the phosphorylation cycle, and inhibitor binding."},"narrative":{"mechanistic_narrative":"PF3D7_1211900 (ATP4) is an essential P-type cation ATPase of the intraerythrocytic P. falciparum plasma membrane that maintains parasite ion homeostasis by extruding Na+ against an inward gradient to keep cytosolic [Na+] low, and it co-regulates cytosolic pH [PMID:23414762, PMID:25145582, PMID:39773028]. The protein is a ~190 kDa P-type ATPase expressed throughout the asexual erythrocytic stages and in free merozoites, concentrated at the parasite periphery/plasma membrane [PMID:8813672, PMID:39773028]. Its catalytic mechanism has been dissected biochemically: the isolated soluble catalytic domain shows Mg2+-dependent ATPase activity that depends on nucleotide-binding residues (Lys619, Lys652, Arg703) and Mg2+-coordinating residues (Asp865/Asp869), and homology modelling plus a conditional-allele genetic system have defined residues critical for Na+ coordination and the phosphorylation cycle whose mutation impairs both growth and Na+ regulation [PMID:40091859, PMID:39773028]. A 3.7 Å cryo-EM structure of the endogenous protein revealed an apicomplexan-specific binding partner, PfABP [PMID:41115914]. ATP4 is the molecular target of multiple structurally distinct antimalarial chemotypes — spiroindolones/cipargamin, pyrazoleamides, aminopyrazoles, dihydroisoquinolones such as (+)-SJ733, and others — whose inhibition disrupts Na+ homeostasis and triggers downstream eryptotic changes (increased membrane rigidity, phosphatidylserine externalization) leading to parasite death, and several of these compounds also block transmission to mosquitoes [PMID:20813948, PMID:23414762, PMID:25453091, PMID:25422853, PMID:25322084, PMID:25145582, PMID:40228810]. Resistance-conferring mutations in the pfatp4 gene, including the clinically relevant G358S allele that lowers Na+ affinity, reduce inhibitor sensitivity, confirming ATP4 as the on-target of these compounds [PMID:28542423, PMID:36180431, PMID:41308470].","teleology":[{"year":1996,"claim":"Establishing that the gene product is a large P-type ATPase present at the parasite periphery throughout asexual development set the stage for a membrane-transport function.","evidence":"Gene cloning with Western blot and immunofluorescence in P. falciparum asexual stages and merozoites","pmids":["8813672"],"confidence":"Medium","gaps":["Localization described as peripheral compartments, not yet resolved as plasma membrane","No transported ion identified","No functional transport assay"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2001,"claim":"Heterologous expression provided the first direct enzymatic readout, showing a vanadate- and cyclopiazonic-acid-sensitive but ouabain-/thapsigargin-resistant ATPase, distinguishing it pharmacologically from SERCA and Na/K-ATPases.","evidence":"Xenopus oocyte expression with pharmacological ATPase assays and stage-specific qPCR in P. falciparum","pmids":["11145964"],"confidence":"Medium","gaps":["Ca2+-stimulation reported here was later reframed by Na+-pump studies","Single-lab heterologous system","Physiological transported ion not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Resistance selection against spiroindolones mapped mutations to pfatp4, identifying the gene as an antimalarial drug target and motivating direct interrogation of its function.","evidence":"Resistance selection with whole-genome sequencing and protein-synthesis inhibition assays in P. falciparum","pmids":["20813948"],"confidence":"High","gaps":["Physiological pump activity not yet defined","Downstream mechanism linking inhibition to death unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Live-cell ion measurements resolved the transported species, defining the protein as a plasma-membrane Na+ efflux ATPase and explaining how its inhibition perturbs cytosolic Na+.","evidence":"Fluorescent Na+ indicator measurements with genetic resistance-mutation epistasis in P. falciparum","pmids":["23414762"],"confidence":"High","gaps":["Mechanism of H+ counter-transport not directly demonstrated","Structural basis of Na+ coordination unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Multiple chemotypes and phenotypic studies established a unified mechanism-of-action: ATP4 inhibition disrupts Na+ (and pH) homeostasis, drives eryptotic changes and death, and blocks transmission, while defining the fitness cost of resistance.","evidence":"Resistance mapping, ion/pH homeostasis and membrane-biophysics assays, transmission assays, and in vivo models for pyrazoleamides, aminopyrazoles, SJ733, and Malaria Box compounds in P. falciparum","pmids":["25453091","25422853","25322084","25145582"],"confidence":"High","gaps":["Whether pH dysregulation is a direct pump activity or downstream consequence not resolved","Molecular details of inhibitor binding site not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"claim":"CRISPR introduction of specific point mutations proved the causal genetic link between pfatp4 alleles and SJ733 resistance, moving beyond correlative resistance mapping.","evidence":"Plasmid-free CRISPR/Cas9 with ssODN editing and growth-inhibition assays in P. falciparum","pmids":["28542423"],"confidence":"High","gaps":["Structural interpretation of the mutated residues not provided"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"The clinically relevant G358S mutation linked field/clinical resistance to a measurable reduction in Na+ affinity without a growth or transmission cost, and its conservation in Toxoplasma gondii extended the mechanism across apicomplexans.","evidence":"Clinical resistance marker sequencing, ion homeostasis assays, and cross-species genetic introduction into T. gondii ATP4","pmids":["36180431"],"confidence":"High","gaps":["Structural basis of how G358S alters Na+ and inhibitor binding not shown here"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Structure, biochemistry, and residue-level genetics converged to define the catalytic machinery, plasma-membrane localization, Na+-coordination and phosphorylation-cycle residues, a novel binding partner PfABP, and direct drug engagement.","evidence":"3.7 Å cryo-EM of endogenous protein, recombinant catalytic-domain ATPase assays with mutagenesis, homology modelling plus conditional-allele genetics, SPP target engagement, and N-acetamide indole in vitro Na+-ATPase inhibition in P. falciparum","pmids":["41115914","40091859","39773028","41308470","40228810"],"confidence":"High","gaps":["Functional role of PfABP not experimentally defined","Catalytic-domain assay lacks full-length structural validation","Asp451-independent Pi release complicates a simple phosphoenzyme model"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The molecular role of the PfABP binding partner, the precise inhibitor-binding site, and the mechanistic coupling of Na+ efflux to cytosolic pH regulation remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["No functional assay defining what PfABP contributes to pump activity","Inhibitor binding pocket not resolved structurally in a drug-bound complex","Direct demonstration of counter-ion (H+) transport still lacking"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["PfATP4–PfABP complex (cryo-EM, apicomplexan-specific binding partner) [#10]"],"partners":["PFABP (APICOMPLEXAN-SPECIFIC BINDING PARTNER) [#10]"],"other_free_text":["Essential Na+ efflux P-type ATPase of the intraerythrocytic parasite plasma membrane [#3, #12]","Validated molecular target of multiple antimalarial chemotypes: spiroindolones/cipargamin (NITD609/KAE609), pyrazoleamides, aminopyrazoles, dihydroisoquinolones (SJ733), Malaria Box compounds, and N-acetamide indoles [#2, #4, #5, #6, #7, #13, #14]","Resistance-conferring point mutations in pfatp4, including clinically relevant G358S which lowers Na+ affinity without growth/transmission cost [#8, #9]","Catalytic residues defined by mutagenesis: nucleotide-binding Lys619/Lys652/Arg703 and Mg2+-coordinating Asp865/Asp869; ATP hydrolysis by the isolated domain proceeds independently of phosphorylation at Asp451 [#11]","Pharmacologically vanadate- and cyclopiazonic-acid-sensitive but ouabain- and thapsigargin-resistant [#0]","Inhibition also blocks parasite transmission to mosquitoes [#6, #14]","Equivalent G358S mutation in Toxoplasma gondii ATP4 reduces cipargamin/SJ733 sensitivity, indicating a conserved apicomplexan mechanism [#9]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"19641202","id":"PMC_19641202","title":"Artemisinin resistance in Plasmodium falciparum malaria.","date":"2009","source":"The New England journal of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/19641202","citation_count":2627,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfSERCA"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20813948","id":"PMC_20813948","title":"Spiroindolones, a potent compound class for the treatment of malaria.","date":"2010","source":"Science (New York, 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stimulated over a broad [Ca2+]free range (3.2–320 µM), is resistant to inhibition by ouabain (200 µM) or thapsigargin (0.8 µM), but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 µM). mRNA expression is variable across asexual stages with a maximal ~5-fold increase in meronts compared with rings.\",\n      \"method\": \"Xenopus oocyte expression, ATPase activity assays with pharmacological inhibitors, quantitative PCR on synchronized asexual stages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional expression in Xenopus oocytes with multiple inhibitor assays and stage-specific mRNA quantification, single lab\",\n      \"pmids\": [\"11145964\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The PfATP4 (PF3D7_1211900) gene encodes a ~190 kDa P-type ATPase protein that is detectable immunologically throughout the asexual erythrocytic stages and in free merozoites. Immunofluorescence microscopy shows the protein is concentrated in discrete compartments at the periphery of the parasite. An 8 kb transcript appears shortly after erythrocyte invasion but is not detected in later stages, while the protein persists.\",\n      \"method\": \"Gene cloning, immunological detection (Western blot), immunofluorescence microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — immunofluorescence localization and Western blot, single lab, two orthogonal methods\",\n      \"pmids\": [\"8813672\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Spiroindolone antimalarials (including NITD609) rapidly inhibit protein synthesis in P. falciparum, and this effect is ablated in parasites bearing nonsynonymous mutations in the gene encoding PfATP4 (PF3D7_1211900), establishing PfATP4 as the target of spiroindolones.\",\n      \"method\": \"Resistance selection with whole-genome sequencing identifying mutations in PfATP4; protein synthesis inhibition assay\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — resistance-conferring mutations in PfATP4 ablate the spiroindolone effect, replicated across multiple resistant lines\",\n      \"pmids\": [\"20813948\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PfATP4 (PF3D7_1211900) localizes to the parasite plasma membrane and functions as a Na+ efflux ATPase, actively extruding Na+ against an inward gradient to maintain low cytosolic [Na+]. Spiroindolones cause a profound disruption of Na+ homeostasis in P. falciparum that is attenuated in parasites bearing resistance-conferring mutations in PfATP4; those mutant parasites also show some impairment of Na+ regulation.\",\n      \"method\": \"Fluorescent Na+ indicator measurements in live parasites; genetic resistance-conferring mutations in PfATP4 characterized by whole-genome sequencing; ion homeostasis assays\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct functional measurement of Na+ regulation combined with genetic epistasis via resistance mutations, replicated across multiple compound classes\",\n      \"pmids\": [\"23414762\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Treatment of P. falciparum-infected erythrocytes with (+)-SJ733, which acts on PfATP4 (PF3D7_1211900), causes rapid perturbation of parasite Na+ homeostasis followed by increased membrane rigidity and externalization of phosphatidylserine in infected cells, consistent with eryptosis/senescence. Resistance-conferring mutations in pfatp4 carry a high fitness cost.\",\n      \"method\": \"Ion homeostasis assays (fluorescent Na+ indicators), membrane rigidity measurements, phosphatidylserine externalization assay, in vivo mouse model, resistance selection with whole-genome sequencing\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ion assays, membrane biophysics, in vivo efficacy, genetic resistance mapping) in a single study\",\n      \"pmids\": [\"25453091\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Pyrazoleamide antimalarials disrupt Na+ regulation in blood-stage P. falciparum in a manner dependent on PfATP4 (PF3D7_1211900); mutations in PfATP4 (in combination with mutations in PfCDPK5) are necessary to confer full pyrazoleamide resistance, establishing PfATP4 as part of their mechanism of action.\",\n      \"method\": \"Resistance selection, whole-genome sequencing, gene transfer (complementation), Na+ homeostasis assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — resistance mapping via whole-genome sequencing, gene transfer validation, and direct ion homeostasis measurement\",\n      \"pmids\": [\"25422853\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Aminopyrazole antimalarials (e.g. GNF-Pf4492) target PfATP4 (PF3D7_1211900): three independently derived resistant lines each acquired independent mutations in PfATP4. The aminopyrazoles disrupt intracellular Na+ homeostasis and block parasite transmission to mosquitoes, phenocopying spiroindolone effects.\",\n      \"method\": \"Resistance selection, whole-genome sequencing of three independent resistant lines, Na+ homeostasis assay, gametocyte/transmission assay\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent resistance lines with PfATP4 mutations, multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"25322084\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Twenty-eight compounds from the Malaria Box affect P. falciparum Na+ and pH regulation consistent with PfATP4 (PF3D7_1211900) inhibition; six chemically diverse compounds showed reduced antiplasmodial activity against spiroindolone-resistant parasites carrying pfatp4 mutations, and resistance selection with two compounds yielded parasites with new mutations in pfatp4. This establishes PfATP4 as a target for diverse chemotypes and indicates it co-regulates both Na+ and cytosolic pH (alkalinization).\",\n      \"method\": \"Ion homeostasis screening (fluorescent Na+ and pH indicators), cross-resistance testing against pfatp4 mutant parasites, in vitro resistance selection with whole-genome sequencing\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple chemotypes, genetic validation via resistance mutations, ion homeostasis assays; independent confirmation of PfATP4 as pH and Na+ regulator\",\n      \"pmids\": [\"25145582\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CRISPR/Cas9-mediated introduction of specific point mutations into pfatp4 (PF3D7_1211900) using a plasmid-free ribonucleoprotein + ssODN approach confirmed that those mutations confer resistance to the antimalarial SJ733, validating the genetic link between PfATP4 and SJ733 action.\",\n      \"method\": \"Plasmid-free CRISPR/Cas9 genome editing, ssODN repair template, whole-genome sequencing, growth inhibition assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct genetic engineering (CRISPR) introducing specific mutations confirms resistance phenotype, single lab\",\n      \"pmids\": [\"28542423\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A G358S mutation in PfATP4 (PF3D7_1211900) confers clinically relevant resistance to cipargamin and (+)-SJ733, allowing parasites to withstand micromolar concentrations while remaining susceptible to non-PfATP4-targeting antimalarials. G358S reduces the affinity of PfATP4 for Na+ and is associated with increased resting cytosolic [Na+], but causes no detectable defect in parasite growth or transmissibility. The equivalent G358S mutation in Toxoplasma gondii ATP4 also decreases sensitivity to cipargamin and SJ733.\",\n      \"method\": \"Clinical trial resistance marker sequencing; functional ion homeostasis assays; genetic introduction of G358S into Toxoplasma gondii ATP4; parasite growth and transmissibility assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clinical validation plus multiple orthogonal functional assays (ion homeostasis, cross-species genetic confirmation), single primary paper\",\n      \"pmids\": [\"36180431\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A 3.7 Å cryo-EM structure of PfATP4 (PF3D7_1211900) purified from CRISPR-engineered P. falciparum parasites reveals a previously unknown apicomplexan-specific binding partner, PfABP, which forms a conserved interaction with PfATP4 and likely plays a modulatory role.\",\n      \"method\": \"CRISPR engineering, native protein purification, cryo-EM structure determination at 3.7 Å\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution cryo-EM structure of endogenous protein from CRISPR-engineered parasites with binding partner identification; single study\",\n      \"pmids\": [\"41115914\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The isolated soluble catalytic domain of PfATP4 (PF3D7_1211900) exhibits Mg2+-dependent ATPase activity in vitro at rates comparable to native cellular PfATP4. Mutation of key nucleotide-binding residues (Lys619, Lys652, Arg703 → Met) decreases enzymatic activity to ~one quarter. Mutation of Mg2+-coordinating residues Asp865+Asp869 together also reduces activity to ~one quarter. Mutation of the putative γ-phosphate-receiving Asp451 does not prevent Pi release, indicating ATP hydrolysis by this domain proceeds independently of phosphorylation at Asp451.\",\n      \"method\": \"Recombinant protein expression and purification, in vitro phosphate colorimetric ATPase assay, site-directed mutagenesis, AlphaFold 3 structural modelling\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis of key catalytic residues; single lab, no full-length protein structural validation\",\n      \"pmids\": [\"40091859\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Homology modelling of PfATP4 (PF3D7_1211900) combined with a merodiploid conditional expression genetic system identified residues critical for Na+ coordination and the phosphorylation cycle. Phenotypic characterization of mutants confirmed that PfATP4 is localized to the parasite plasma membrane, that mutation of these residues impairs parasite growth and Na+ regulation, and that resistance-associated mutations reduce inhibitor sensitivity with some conferring a fitness cost consistent with model predictions.\",\n      \"method\": \"Homology modelling (SERCA-based), molecular dynamics simulation, CRISPR-mediated merodiploid conditional allele system, parasite growth assays, subcellular localization (imaging), Na+ homeostasis assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic engineering, localization, ion homeostasis, growth phenotyping, computational modelling) in a single rigorous study confirming plasma membrane localization and Na+ pump function\",\n      \"pmids\": [\"39773028\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Solvent-induced proteome profiling (SPP) identified PfATP4 (PF3D7_1211900; annotated as non-SERCA-type Ca2+-transporting P-ATPase) as the protein target of cipargamin in P. falciparum lysates, demonstrating ligand-induced stabilization of PfATP4 consistent with direct drug-protein interaction.\",\n      \"method\": \"Solvent proteome profiling (SPP), mass spectrometry-based proteomics\",\n      \"journal\": \"International journal for parasitology. Drugs and drug resistance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — proteomics-based target engagement assay identifies PfATP4 stabilization by cipargamin, single lab, single method\",\n      \"pmids\": [\"41308470\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"N-acetamide indole compounds inhibit PfATP4 (PF3D7_1211900): resistance selection and whole-genome sequencing revealed mutations in PfATP4; analogs showed reduced potency against PfATP4 resistance-mutant parasites; metabolomic signature matched that of the PfATP4 inhibitor KAE609; and in vitro inhibition of Na+-dependent ATPase activity was demonstrated. The compounds also inhibit gamete development and block transmission to mosquitoes.\",\n      \"method\": \"Resistance selection, whole-genome sequencing, cross-resistance assays, metabolomics, Na+-dependent ATPase inhibition assay in vitro, gametocyte/transmission assay, P. berghei mouse model\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal validation methods including direct in vitro ATPase assay, genetic resistance mapping, and metabolomics; single lab\",\n      \"pmids\": [\"40228810\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PfATP4 (PF3D7_1211900) is an essential P-type cation ATPase localized to the parasite plasma membrane that functions as a Na+ efflux pump (and likely H+ importer) to maintain low cytosolic [Na+] in intraerythrocytic P. falciparum; its inhibition by multiple structurally distinct antimalarial classes (spiroindolones, pyrazoleamides, aminopyrazoles, dihydroisoquinolones, and others) causes rapid disruption of Na+ homeostasis, cytosolic alkalinization, and downstream eryptotic changes leading to parasite death, while a 3.7 Å cryo-EM structure reveals it associates with a novel apicomplexan-specific binding partner PfABP, and mutagenesis studies have defined key residues for Na+ coordination, the phosphorylation cycle, and inhibitor binding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1211900 (ATP4) is an essential P-type cation ATPase of the intraerythrocytic P. falciparum plasma membrane that maintains parasite ion homeostasis by extruding Na+ against an inward gradient to keep cytosolic [Na+] low, and it co-regulates cytosolic pH [#3, #7, #12]. The protein is a ~190 kDa P-type ATPase expressed throughout the asexual erythrocytic stages and in free merozoites, concentrated at the parasite periphery/plasma membrane [#1, #12]. Its catalytic mechanism has been dissected biochemically: the isolated soluble catalytic domain shows Mg2+-dependent ATPase activity that depends on nucleotide-binding residues (Lys619, Lys652, Arg703) and Mg2+-coordinating residues (Asp865/Asp869), and homology modelling plus a conditional-allele genetic system have defined residues critical for Na+ coordination and the phosphorylation cycle whose mutation impairs both growth and Na+ regulation [#11, #12]. A 3.7 Å cryo-EM structure of the endogenous protein revealed an apicomplexan-specific binding partner, PfABP [#10]. ATP4 is the molecular target of multiple structurally distinct antimalarial chemotypes — spiroindolones/cipargamin, pyrazoleamides, aminopyrazoles, dihydroisoquinolones such as (+)-SJ733, and others — whose inhibition disrupts Na+ homeostasis and triggers downstream eryptotic changes (increased membrane rigidity, phosphatidylserine externalization) leading to parasite death, and several of these compounds also block transmission to mosquitoes [#2, #3, #4, #5, #6, #7, #14]. Resistance-conferring mutations in the pfatp4 gene, including the clinically relevant G358S allele that lowers Na+ affinity, reduce inhibitor sensitivity, confirming ATP4 as the on-target of these compounds [#8, #9, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing that the gene product is a large P-type ATPase present at the parasite periphery throughout asexual development set the stage for a membrane-transport function.\",\n      \"evidence\": \"Gene cloning with Western blot and immunofluorescence in P. falciparum asexual stages and merozoites\",\n      \"pmids\": [\"8813672\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Localization described as peripheral compartments, not yet resolved as plasma membrane\", \"No transported ion identified\", \"No functional transport assay\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Heterologous expression provided the first direct enzymatic readout, showing a vanadate- and cyclopiazonic-acid-sensitive but ouabain-/thapsigargin-resistant ATPase, distinguishing it pharmacologically from SERCA and Na/K-ATPases.\",\n      \"evidence\": \"Xenopus oocyte expression with pharmacological ATPase assays and stage-specific qPCR in P. falciparum\",\n      \"pmids\": [\"11145964\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ca2+-stimulation reported here was later reframed by Na+-pump studies\", \"Single-lab heterologous system\", \"Physiological transported ion not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resistance selection against spiroindolones mapped mutations to pfatp4, identifying the gene as an antimalarial drug target and motivating direct interrogation of its function.\",\n      \"evidence\": \"Resistance selection with whole-genome sequencing and protein-synthesis inhibition assays in P. falciparum\",\n      \"pmids\": [\"20813948\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Physiological pump activity not yet defined\", \"Downstream mechanism linking inhibition to death unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Live-cell ion measurements resolved the transported species, defining the protein as a plasma-membrane Na+ efflux ATPase and explaining how its inhibition perturbs cytosolic Na+.\",\n      \"evidence\": \"Fluorescent Na+ indicator measurements with genetic resistance-mutation epistasis in P. falciparum\",\n      \"pmids\": [\"23414762\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism of H+ counter-transport not directly demonstrated\", \"Structural basis of Na+ coordination unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Multiple chemotypes and phenotypic studies established a unified mechanism-of-action: ATP4 inhibition disrupts Na+ (and pH) homeostasis, drives eryptotic changes and death, and blocks transmission, while defining the fitness cost of resistance.\",\n      \"evidence\": \"Resistance mapping, ion/pH homeostasis and membrane-biophysics assays, transmission assays, and in vivo models for pyrazoleamides, aminopyrazoles, SJ733, and Malaria Box compounds in P. falciparum\",\n      \"pmids\": [\"25453091\", \"25422853\", \"25322084\", \"25145582\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether pH dysregulation is a direct pump activity or downstream consequence not resolved\", \"Molecular details of inhibitor binding site not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"CRISPR introduction of specific point mutations proved the causal genetic link between pfatp4 alleles and SJ733 resistance, moving beyond correlative resistance mapping.\",\n      \"evidence\": \"Plasmid-free CRISPR/Cas9 with ssODN editing and growth-inhibition assays in P. falciparum\",\n      \"pmids\": [\"28542423\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural interpretation of the mutated residues not provided\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The clinically relevant G358S mutation linked field/clinical resistance to a measurable reduction in Na+ affinity without a growth or transmission cost, and its conservation in Toxoplasma gondii extended the mechanism across apicomplexans.\",\n      \"evidence\": \"Clinical resistance marker sequencing, ion homeostasis assays, and cross-species genetic introduction into T. gondii ATP4\",\n      \"pmids\": [\"36180431\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of how G358S alters Na+ and inhibitor binding not shown here\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structure, biochemistry, and residue-level genetics converged to define the catalytic machinery, plasma-membrane localization, Na+-coordination and phosphorylation-cycle residues, a novel binding partner PfABP, and direct drug engagement.\",\n      \"evidence\": \"3.7 Å cryo-EM of endogenous protein, recombinant catalytic-domain ATPase assays with mutagenesis, homology modelling plus conditional-allele genetics, SPP target engagement, and N-acetamide indole in vitro Na+-ATPase inhibition in P. falciparum\",\n      \"pmids\": [\"41115914\", \"40091859\", \"39773028\", \"41308470\", \"40228810\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional role of PfABP not experimentally defined\", \"Catalytic-domain assay lacks full-length structural validation\", \"Asp451-independent Pi release complicates a simple phosphoenzyme model\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular role of the PfABP binding partner, the precise inhibitor-binding site, and the mechanistic coupling of Na+ efflux to cytosolic pH regulation remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No functional assay defining what PfABP contributes to pump activity\", \"Inhibitor binding pocket not resolved structurally in a drug-bound complex\", \"Direct demonstration of counter-ion (H+) transport still lacking\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016887\", \"supporting_discovery_ids\": [0, 11]},\n      {\"term_id\": \"GO:0008554\", \"supporting_discovery_ids\": [3, 12, 14]},\n      {\"term_id\": \"GO:0005388\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0000287\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 12]},\n      {\"term_id\": \"GO:0071944\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006814\", \"supporting_discovery_ids\": [3, 4, 12]},\n      {\"term_id\": \"GO:0055078\", \"supporting_discovery_ids\": [3, 9]},\n      {\"term_id\": \"GO:0051453\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\n      \"PfATP4–PfABP complex (cryo-EM, apicomplexan-specific binding partner) [#10]\"\n    ],\n    \"partners\": [\n      \"PfABP (apicomplexan-specific binding partner) [#10]\"\n    ],\n    \"other_free_text\": [\n      \"Essential Na+ efflux P-type ATPase of the intraerythrocytic parasite plasma membrane [#3, #12]\",\n      \"Validated molecular target of multiple antimalarial chemotypes: spiroindolones/cipargamin (NITD609/KAE609), pyrazoleamides, aminopyrazoles, dihydroisoquinolones (SJ733), Malaria Box compounds, and N-acetamide indoles [#2, #4, #5, #6, #7, #13, #14]\",\n      \"Resistance-conferring point mutations in pfatp4, including clinically relevant G358S which lowers Na+ affinity without growth/transmission cost [#8, #9]\",\n      \"Catalytic residues defined by mutagenesis: nucleotide-binding Lys619/Lys652/Arg703 and Mg2+-coordinating Asp865/Asp869; ATP hydrolysis by the isolated domain proceeds independently of phosphorylation at Asp451 [#11]\",\n      \"Pharmacologically vanadate- and cyclopiazonic-acid-sensitive but ouabain- and thapsigargin-resistant [#0]\",\n      \"Inhibition also blocks parasite transmission to mosquitoes [#6, #14]\",\n      \"Equivalent G358S mutation in Toxoplasma gondii ATP4 reduces cipargamin/SJ733 sensitivity, indicating a conserved apicomplexan mechanism [#9]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1211900","name":null,"product":"non-SERCA-type Ca2+ -transporting P-ATPase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0610400","name":null,"product":"non-SERCA-type Ca2+ -transporting P-ATPase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1312000","name":null,"product":"non-SERCA-type Ca2+ -transporting P-ATPase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"ATP4","evaluation":null}