{"gene":"PF3D7_1011400","run_date":"2026-09-08T12:51:27","timeline":{"discoveries":[{"year":2016,"finding":"The cryo-EM structure of the P. falciparum 20S proteasome at 3.6 Å resolution revealed the active-site architecture of the β5 subunit (PF3D7_1011400) and neighboring subunits. Substrate profiling showed differences in specificity between human and P. falciparum proteasome active sites, and designed inhibitors were found to preferentially inhibit the β2-subunit rather than β5, establishing the structural basis for selectivity.","method":"Cryo-electron microscopy and single-particle analysis; substrate profiling with fluorogenic peptide libraries; inhibitor binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct cryo-EM structure at 3.6 Å with functional inhibitor binding validation and substrate profiling, published in high-impact peer-reviewed journal","pmids":["26863983"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"The cryo-EM structure of the Pf20S in complex with PfPA28 regulatory caps (at 3.9 and 3.8 Å resolution) defined the architecture of the 20S catalytic core, including the β5 subunit (PF3D7_1011400), and showed how PA28 binds asymmetrically to the α-ring face of the 20S complex, engaging subunits on only one side.","method":"Single-particle cryo-electron microscopy; crystal structure of PfPA28; small-angle X-ray scattering; molecular dynamics simulations","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with complementary biophysical methods in a peer-reviewed journal; β5 is a constituent subunit of the resolved Pf20S complex","pmids":["31384003"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"Amino-amide boronate inhibitors (e.g., MPI-5, MPI-13) were shown by cryo-EM structures to bind specifically to the Pf20S β5 active site (PF3D7_1011400), with greater affinity than for human constitutive 20S, establishing the β5 active site as both the functional target and the structural basis for species-selective inhibition and fast-acting antimalarial activity.","method":"Cryo-electron microscopy structure of Pf20S–inhibitor complex; biochemical β5 activity assays; in vitro parasite growth inhibition; in vivo SCID mouse model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with direct biochemical inhibition assay of β5 active site and in vivo validation, peer-reviewed","pmids":["34548400"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""}]},{"year":2023,"finding":"High-resolution cryo-EM structures revealed that the macrocyclic peptide TDI-8304 binds both the β2 and β5 subunits (PF3D7_1011400) of wild-type Pf20S, and that a β6 subunit mutation (A117D) conferring resistance to TDI-8304 also causes conformational changes that enhance inhibitor (WLW-vs) binding to β2 and β5, demonstrating that β5 participates in a dual-subunit binding mode and is involved in resistance/collateral sensitivity mechanisms.","method":"Cryo-electron microscopy structures of Pf20S–inhibitor complexes (wild-type and β6A117D mutant); in vitro parasite killing assays; resistance selection","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple high-resolution cryo-EM structures with mutagenesis and functional resistance data in a peer-reviewed journal","pmids":["38097652"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium cynomolgi","strain":""}]},{"year":2024,"finding":"Resistance selection with piperidine carboxamide SW042 identified point mutations in the Pf β5 active site (PF3D7_1011400), and cryo-EM structure of the Pf20S–SW584 complex showed the inhibitor binds non-covalently at a pocket at the β5/β6/β3 subunit interface, distal from the catalytic threonine. Inhibition of Pfβ5 activity correlated directly with parasite killing without inhibiting human proteasome isoforms.","method":"Resistance selection with sequencing; cryo-EM structure of Pf20S–SW584; biochemical β5 activity assays; in vivo mouse model","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus resistance mutations plus direct biochemical correlation, peer-reviewed","pmids":["39084225"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"Cryo-EM structural studies of optimized carboxypiperidine reversible inhibitors confirmed binding at the Pf20S β5 subunit (PF3D7_1011400), and structure-activity relationship studies established that selective engagement of this site is sufficient for nanomolar potency against P. falciparum blood-stage parasites with selectivity over human proteasome.","method":"Cryo-EM structure of Pf20S–inhibitor complex; SAR studies; in vitro parasite growth inhibition; pharmacokinetic assays","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — single lab cryo-EM study with SAR, no independent replication reported","pmids":["41148577"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"Treatment of P. falciparum with thiostrepton and derivatives caused accumulation of ubiquitinated proteins in blood stages, indicating inhibition of the parasite proteasome (of which β5/PF3D7_1011400 is a catalytic subunit). Expression profiling showed the proteasome is present in the nucleus and cytoplasm of trophozoites, schizonts, and gametocytes, and compound treatment eliminated gametocytes.","method":"Ubiquitinated protein accumulation assay; expression profiling; stage-specific growth inhibition assays","journal":"Antimicrobial agents and chemotherapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect evidence of proteasome inhibition via ubiquitin accumulation; β5 subunit not individually distinguished from other catalytic subunits in a single-lab study","pmids":["21245445"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_1011400 (Pf20S β5) is a chymotrypsin-like catalytic subunit of the P. falciparum 20S proteasome whose active site, located at the β5/β6/β3 subunit interface and containing a catalytic threonine, has been resolved by cryo-EM and shown to be the primary target of species-selective inhibitors that kill parasites across blood, liver, and sexual stages, with its inhibition correlating directly with parasite killing and point mutations in its active site conferring drug resistance."},"narrative":{"mechanistic_narrative":"PF3D7_1011400 encodes the chymotrypsin-like (β5) catalytic subunit of the Plasmodium falciparum 20S proteasome, whose active-site architecture was resolved by cryo-EM and shown to differ in substrate specificity from the human proteasome, establishing a structural basis for species-selective inhibition [PMID:26863983]. The β5 active site sits at the β5/β6/β3 subunit interface and contains the catalytic threonine that defines its proteolytic activity [PMID:39084225]. This subunit is the primary target of a series of species-selective antimalarial inhibitors—amino-amide boronates, macrocyclic peptides, piperidine carboxamides, and carboxypiperidines—that engage the β5 site (in some cases together with β2) with greater affinity for Pf20S than for human proteasome isoforms, and whose biochemical inhibition of β5 correlates directly with parasite killing across blood, liver, and sexual stages [PMID:34548400, PMID:38097652, PMID:39084225, PMID:41148577]. Point mutations selected in the β5 active site confer resistance to these inhibitors, and a neighboring β6 mutation (A117D) reshapes β5/β2 inhibitor binding, placing β5 at the center of both drug action and resistance/collateral-sensitivity mechanisms [PMID:38097652, PMID:39084225]. Within the 20S core, β5 is a constituent catalytic subunit of the complex that is capped asymmetrically by the PfPA28 regulator [PMID:31384003]. Beyond its role as a catalytic subunit of the 20S proteasome and its function as a validated antimalarial drug target, no additional mechanistic detail specific to PF3D7_1011400 has been characterized in the available corpus.","teleology":[{"year":2016,"claim":"The question of why the parasite proteasome could be targeted selectively over the human enzyme was answered by resolving the β5 active-site architecture and showing its substrate specificity differs from the human proteasome.","evidence":"3.6 Å cryo-EM structure with fluorogenic peptide substrate profiling and inhibitor binding assays in P. falciparum","pmids":["26863983"],"confidence":"High","gaps":["Designed inhibitors in this study preferentially hit β2 rather than β5, leaving β5-selective inhibition unproven at this stage","No in vivo validation of β5 targeting"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"The structural context of β5 within the assembled 20S core and its regulation was defined by resolving the Pf20S in complex with the PfPA28 regulatory cap.","evidence":"Single-particle cryo-EM (3.9/3.8 Å), crystallography of PfPA28, SAXS and molecular dynamics in P. falciparum","pmids":["31384003"],"confidence":"High","gaps":["Functional consequence of asymmetric PA28 capping for β5 catalytic activity not established","Does not address β5 as a drug target"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Whether β5 could be selectively drugged for fast-acting antimalarial effect was answered by showing amino-amide boronates bind the β5 active site with greater affinity than human 20S and kill parasites.","evidence":"Cryo-EM of Pf20S–inhibitor complex, biochemical β5 activity assays, in vitro growth inhibition, and SCID mouse model in P. falciparum and P. vivax","pmids":["34548400"],"confidence":"High","gaps":["Resistance liability of β5-targeting boronates not yet mapped in this study","Covalent boronate mechanism differs from later reversible inhibitors"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""}]},{"year":2023,"claim":"The role of β5 in a dual-subunit binding mode and in resistance was clarified by showing a macrocyclic peptide engages both β2 and β5 and that a β6 A117D mutation reshapes inhibitor binding at these sites.","evidence":"Cryo-EM of wild-type and β6A117D mutant Pf20S–inhibitor complexes, parasite killing assays, and resistance selection in P. falciparum and P. cynomolgi","pmids":["38097652"],"confidence":"High","gaps":["Relative contribution of β5 versus β2 engagement to parasite killing not deconvolved","Mechanism of collateral sensitivity not fully resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium cynomolgi","strain":""}]},{"year":2024,"claim":"Direct causal linkage between β5 inhibition and parasite death was established by mapping resistance mutations to the β5 active site and showing β5 activity inhibition correlates with killing without hitting human proteasome.","evidence":"Resistance selection with sequencing, cryo-EM of Pf20S–SW584 revealing a non-covalent pocket at the β5/β6/β3 interface, biochemical β5 assays and mouse model in P. falciparum","pmids":["39084225"],"confidence":"High","gaps":["The SW584 binding pocket is distal to the catalytic threonine, so allosteric mechanism of inhibition is not fully defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Whether selective engagement of β5 alone suffices for potency was addressed by showing optimized carboxypiperidine reversible inhibitors bind β5 and achieve nanomolar blood-stage potency with human selectivity.","evidence":"Cryo-EM of Pf20S–inhibitor complex, SAR, in vitro growth inhibition and PK assays in P. falciparum","pmids":["41148577"],"confidence":"Medium","gaps":["Single-lab study without independent replication","In vivo efficacy and resistance profile of this chemotype not reported here"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"Early evidence that inhibiting the parasite proteasome disrupts protein turnover across stages came from thiostrepton-induced ubiquitinated protein accumulation and gametocyte elimination.","evidence":"Ubiquitinated protein accumulation assay, expression profiling, and stage-specific growth inhibition in P. falciparum","pmids":["21245445"],"confidence":"Low","gaps":["β5 subunit not individually distinguished from other catalytic subunits","Indirect readout of proteasome inhibition rather than direct β5 activity measurement"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How β5 catalytic activity is regulated within the assembled 20S core and how its inhibition selectively disrupts parasite proteostasis across the life cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No mapping of endogenous β5 substrate repertoire in vivo","Functional link between PA28 capping and β5 activity not established","Mechanism connecting distal non-covalent inhibitor binding to catalytic inhibition unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["20S proteasome catalytic core (Pf20S)","Pf20S–PfPA28 regulatory complex"],"partners":["Β2 PROTEASOME SUBUNIT (CO-ENGAGED BY DUAL-SUBUNIT INHIBITORS)","Β6 PROTEASOME SUBUNIT (A117D RESISTANCE MUTATION, Β5/Β6/Β3 INTERFACE)","Β3 PROTEASOME SUBUNIT (Β5/Β6/Β3 ACTIVE-SITE INTERFACE)","PFPA28 REGULATORY CAP"],"other_free_text":["Chymotrypsin-like catalytic subunit with active-site catalytic threonine at the β5/β6/β3 subunit interface [#4]","Primary target of species-selective antimalarial proteasome inhibitors active across blood, liver, and sexual stages [#2, #4]","Active-site point mutations confer inhibitor resistance [#3, #4]","Substrate specificity differs from the human proteasome, providing basis for selectivity [#0]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"26863983","id":"PMC_26863983","title":"Structure- and function-based design of Plasmodium-selective proteasome inhibitors.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26863983","citation_count":196,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21245445","id":"PMC_21245445","title":"Thiostrepton and derivatives exhibit antimalarial and gametocytocidal activity by dually targeting parasite proteasome and apicoplast.","date":"2011","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/21245445","citation_count":98,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31384003","id":"PMC_31384003","title":"The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.","date":"2019","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/31384003","citation_count":37,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"34548400","id":"PMC_34548400","title":"Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.","date":"2021","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/34548400","citation_count":33,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38097652","id":"PMC_38097652","title":"Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38097652","citation_count":21,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"39084225","id":"PMC_39084225","title":"Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria.","date":"2024","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/39084225","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41148577","id":"PMC_41148577","title":"Optimization of Species-Selective Reversible Proteasome Inhibitors for the Treatment of Malaria.","date":"2025","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41148577","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6288,"output_tokens":2207,"usd":0.025985,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7560,"output_tokens":3389,"usd":0.061262,"stage2_stop_reason":"end_turn"},"total_usd":0.087247,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"The cryo-EM structure of the P. falciparum 20S proteasome at 3.6 Å resolution revealed the active-site architecture of the β5 subunit (PF3D7_1011400) and neighboring subunits. Substrate profiling showed differences in specificity between human and P. falciparum proteasome active sites, and designed inhibitors were found to preferentially inhibit the β2-subunit rather than β5, establishing the structural basis for selectivity.\",\n      \"method\": \"Cryo-electron microscopy and single-particle analysis; substrate profiling with fluorogenic peptide libraries; inhibitor binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct cryo-EM structure at 3.6 Å with functional inhibitor binding validation and substrate profiling, published in high-impact peer-reviewed journal\",\n      \"pmids\": [\"26863983\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The cryo-EM structure of the Pf20S in complex with PfPA28 regulatory caps (at 3.9 and 3.8 Å resolution) defined the architecture of the 20S catalytic core, including the β5 subunit (PF3D7_1011400), and showed how PA28 binds asymmetrically to the α-ring face of the 20S complex, engaging subunits on only one side.\",\n      \"method\": \"Single-particle cryo-electron microscopy; crystal structure of PfPA28; small-angle X-ray scattering; molecular dynamics simulations\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with complementary biophysical methods in a peer-reviewed journal; β5 is a constituent subunit of the resolved Pf20S complex\",\n      \"pmids\": [\"31384003\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Amino-amide boronate inhibitors (e.g., MPI-5, MPI-13) were shown by cryo-EM structures to bind specifically to the Pf20S β5 active site (PF3D7_1011400), with greater affinity than for human constitutive 20S, establishing the β5 active site as both the functional target and the structural basis for species-selective inhibition and fast-acting antimalarial activity.\",\n      \"method\": \"Cryo-electron microscopy structure of Pf20S–inhibitor complex; biochemical β5 activity assays; in vitro parasite growth inhibition; in vivo SCID mouse model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with direct biochemical inhibition assay of β5 active site and in vivo validation, peer-reviewed\",\n      \"pmids\": [\"34548400\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"High-resolution cryo-EM structures revealed that the macrocyclic peptide TDI-8304 binds both the β2 and β5 subunits (PF3D7_1011400) of wild-type Pf20S, and that a β6 subunit mutation (A117D) conferring resistance to TDI-8304 also causes conformational changes that enhance inhibitor (WLW-vs) binding to β2 and β5, demonstrating that β5 participates in a dual-subunit binding mode and is involved in resistance/collateral sensitivity mechanisms.\",\n      \"method\": \"Cryo-electron microscopy structures of Pf20S–inhibitor complexes (wild-type and β6A117D mutant); in vitro parasite killing assays; resistance selection\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple high-resolution cryo-EM structures with mutagenesis and functional resistance data in a peer-reviewed journal\",\n      \"pmids\": [\"38097652\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Resistance selection with piperidine carboxamide SW042 identified point mutations in the Pf β5 active site (PF3D7_1011400), and cryo-EM structure of the Pf20S–SW584 complex showed the inhibitor binds non-covalently at a pocket at the β5/β6/β3 subunit interface, distal from the catalytic threonine. Inhibition of Pfβ5 activity correlated directly with parasite killing without inhibiting human proteasome isoforms.\",\n      \"method\": \"Resistance selection with sequencing; cryo-EM structure of Pf20S–SW584; biochemical β5 activity assays; in vivo mouse model\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus resistance mutations plus direct biochemical correlation, peer-reviewed\",\n      \"pmids\": [\"39084225\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structural studies of optimized carboxypiperidine reversible inhibitors confirmed binding at the Pf20S β5 subunit (PF3D7_1011400), and structure-activity relationship studies established that selective engagement of this site is sufficient for nanomolar potency against P. falciparum blood-stage parasites with selectivity over human proteasome.\",\n      \"method\": \"Cryo-EM structure of Pf20S–inhibitor complex; SAR studies; in vitro parasite growth inhibition; pharmacokinetic assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — single lab cryo-EM study with SAR, no independent replication reported\",\n      \"pmids\": [\"41148577\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Treatment of P. falciparum with thiostrepton and derivatives caused accumulation of ubiquitinated proteins in blood stages, indicating inhibition of the parasite proteasome (of which β5/PF3D7_1011400 is a catalytic subunit). Expression profiling showed the proteasome is present in the nucleus and cytoplasm of trophozoites, schizonts, and gametocytes, and compound treatment eliminated gametocytes.\",\n      \"method\": \"Ubiquitinated protein accumulation assay; expression profiling; stage-specific growth inhibition assays\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect evidence of proteasome inhibition via ubiquitin accumulation; β5 subunit not individually distinguished from other catalytic subunits in a single-lab study\",\n      \"pmids\": [\"21245445\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_1011400 (Pf20S β5) is a chymotrypsin-like catalytic subunit of the P. falciparum 20S proteasome whose active site, located at the β5/β6/β3 subunit interface and containing a catalytic threonine, has been resolved by cryo-EM and shown to be the primary target of species-selective inhibitors that kill parasites across blood, liver, and sexual stages, with its inhibition correlating directly with parasite killing and point mutations in its active site conferring drug resistance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PF3D7_1011400 encodes the chymotrypsin-like (β5) catalytic subunit of the Plasmodium falciparum 20S proteasome, whose active-site architecture was resolved by cryo-EM and shown to differ in substrate specificity from the human proteasome, establishing a structural basis for species-selective inhibition [#0]. The β5 active site sits at the β5/β6/β3 subunit interface and contains the catalytic threonine that defines its proteolytic activity [#4]. This subunit is the primary target of a series of species-selective antimalarial inhibitors—amino-amide boronates, macrocyclic peptides, piperidine carboxamides, and carboxypiperidines—that engage the β5 site (in some cases together with β2) with greater affinity for Pf20S than for human proteasome isoforms, and whose biochemical inhibition of β5 correlates directly with parasite killing across blood, liver, and sexual stages [#2, #3, #4, #5]. Point mutations selected in the β5 active site confer resistance to these inhibitors, and a neighboring β6 mutation (A117D) reshapes β5/β2 inhibitor binding, placing β5 at the center of both drug action and resistance/collateral-sensitivity mechanisms [#3, #4]. Within the 20S core, β5 is a constituent catalytic subunit of the complex that is capped asymmetrically by the PfPA28 regulator [#1]. Beyond its role as a catalytic subunit of the 20S proteasome and its function as a validated antimalarial drug target, no additional mechanistic detail specific to PF3D7_1011400 has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"The question of why the parasite proteasome could be targeted selectively over the human enzyme was answered by resolving the β5 active-site architecture and showing its substrate specificity differs from the human proteasome.\",\n      \"evidence\": \"3.6 Å cryo-EM structure with fluorogenic peptide substrate profiling and inhibitor binding assays in P. falciparum\",\n      \"pmids\": [\"26863983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Designed inhibitors in this study preferentially hit β2 rather than β5, leaving β5-selective inhibition unproven at this stage\", \"No in vivo validation of β5 targeting\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The structural context of β5 within the assembled 20S core and its regulation was defined by resolving the Pf20S in complex with the PfPA28 regulatory cap.\",\n      \"evidence\": \"Single-particle cryo-EM (3.9/3.8 Å), crystallography of PfPA28, SAXS and molecular dynamics in P. falciparum\",\n      \"pmids\": [\"31384003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of asymmetric PA28 capping for β5 catalytic activity not established\", \"Does not address β5 as a drug target\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Whether β5 could be selectively drugged for fast-acting antimalarial effect was answered by showing amino-amide boronates bind the β5 active site with greater affinity than human 20S and kill parasites.\",\n      \"evidence\": \"Cryo-EM of Pf20S–inhibitor complex, biochemical β5 activity assays, in vitro growth inhibition, and SCID mouse model in P. falciparum and P. vivax\",\n      \"pmids\": [\"34548400\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resistance liability of β5-targeting boronates not yet mapped in this study\", \"Covalent boronate mechanism differs from later reversible inhibitors\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The role of β5 in a dual-subunit binding mode and in resistance was clarified by showing a macrocyclic peptide engages both β2 and β5 and that a β6 A117D mutation reshapes inhibitor binding at these sites.\",\n      \"evidence\": \"Cryo-EM of wild-type and β6A117D mutant Pf20S–inhibitor complexes, parasite killing assays, and resistance selection in P. falciparum and P. cynomolgi\",\n      \"pmids\": [\"38097652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of β5 versus β2 engagement to parasite killing not deconvolved\", \"Mechanism of collateral sensitivity not fully resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium cynomolgi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Direct causal linkage between β5 inhibition and parasite death was established by mapping resistance mutations to the β5 active site and showing β5 activity inhibition correlates with killing without hitting human proteasome.\",\n      \"evidence\": \"Resistance selection with sequencing, cryo-EM of Pf20S–SW584 revealing a non-covalent pocket at the β5/β6/β3 interface, biochemical β5 assays and mouse model in P. falciparum\",\n      \"pmids\": [\"39084225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The SW584 binding pocket is distal to the catalytic threonine, so allosteric mechanism of inhibition is not fully defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Whether selective engagement of β5 alone suffices for potency was addressed by showing optimized carboxypiperidine reversible inhibitors bind β5 and achieve nanomolar blood-stage potency with human selectivity.\",\n      \"evidence\": \"Cryo-EM of Pf20S–inhibitor complex, SAR, in vitro growth inhibition and PK assays in P. falciparum\",\n      \"pmids\": [\"41148577\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without independent replication\", \"In vivo efficacy and resistance profile of this chemotype not reported here\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Early evidence that inhibiting the parasite proteasome disrupts protein turnover across stages came from thiostrepton-induced ubiquitinated protein accumulation and gametocyte elimination.\",\n      \"evidence\": \"Ubiquitinated protein accumulation assay, expression profiling, and stage-specific growth inhibition in P. falciparum\",\n      \"pmids\": [\"21245445\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"β5 subunit not individually distinguished from other catalytic subunits\", \"Indirect readout of proteasome inhibition rather than direct β5 activity measurement\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How β5 catalytic activity is regulated within the assembled 20S core and how its inhibition selectively disrupts parasite proteostasis across the life cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mapping of endogenous β5 substrate repertoire in vivo\", \"Functional link between PA28 capping and β5 activity not established\", \"Mechanism connecting distal non-covalent inhibitor binding to catalytic inhibition unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004298\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0004175\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0043161\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0010498\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [\n      \"20S proteasome catalytic core (Pf20S)\",\n      \"Pf20S–PfPA28 regulatory complex\"\n    ],\n    \"partners\": [\n      \"β2 proteasome subunit (co-engaged by dual-subunit inhibitors)\",\n      \"β6 proteasome subunit (A117D resistance mutation, β5/β6/β3 interface)\",\n      \"β3 proteasome subunit (β5/β6/β3 active-site interface)\",\n      \"PfPA28 regulatory cap\"\n    ],\n    \"other_free_text\": [\n      \"Chymotrypsin-like catalytic subunit with active-site catalytic threonine at the β5/β6/β3 subunit interface [#4]\",\n      \"Primary target of species-selective antimalarial proteasome inhibitors active across blood, liver, and sexual stages [#2, #4]\",\n      \"Active-site point mutations confer inhibitor resistance [#3, #4]\",\n      \"Substrate specificity differs from the human proteasome, providing basis for selectivity [#0]\"\n    ]\n  }\n}\n```","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1011400","name":null,"product":"proteasome subunit beta type-5","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1209800","name":null,"product":"proteasome subunit beta type-5, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0811200","name":null,"product":"proteasome subunit beta type-5, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}