{ "abstract": "Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX‑selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis–Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks. Author summary: Malaria treatment relies heavily on combination therapies, but drug resistance continues to undermine their effectiveness. We investigated whether Plasmodium falciparum could develop resistance to two classes of experimental inhibitors that target parasite aspartic proteases: compounds that selectively block plasmepsin X (PMX) and compounds that simultaneously inhibit plasmepsin IX and X (PMIX/X). PMX-selective inhibitors could, with prolonged laboratory exposure, select parasites with reduced sensitivity driven by amplification of the pmx gene and by specific point mutations (S315P, D245N, I363L, S359P) that decrease drug binding yet preserve enzyme function. By contrast, dual PMIX/X inhibitors resisted resistance selection even after extended pressure; no resistant parasites were recovered, and no pre-existing resistance was detected in a diverse panel of resistant lines. Importantly, dual inhibitors retained activity against PMX-altered parasites. Resistance-associated pmx changes imposed measurable fitness costs on parasites, reducing their competitiveness without drug pressure. These results indicate that dual-target inhibition markedly raises the barrier to resistance and support prioritising PMIX/X dual inhibitors for antimalarial development to prolong drug utility.", "abstract_shown": true, "affiliations": [ "The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia", "Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia", "Merck & Co., Inc., West Point, Pennsylvania, United States of America", "Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom", "Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom" ], "article_type": "Research Article", "arxiv": null, "author_total": 18, "authors": [ { "affs": [ 0 ], "name": "Paola Favuzza" }, { "affs": [ 1 ], "name": "Madeline G. Dans" }, { "affs": [ 2 ], "name": "Wenyin Su" }, { "affs": [ 3 ], "name": "Jennifer K. Thompson" }, { "affs": [ 4 ], "name": "Anthony N. Hodder" }, { "name": "Anna Ngo" }, { "name": "Jocelyn Sietsma Penington" }, { "name": "Danushka S. Marapana" }, { "name": "Jerzy M. Dziekan" }, { "name": "Anthony T. Papenfuss" }, { "name": "Manuel de Lera Ruiz" }, { "name": "Rachael Coyle" }, { "name": "Marcus C. S. Lee" }, { "name": "John A. McCauley" }, { "name": "Kym Lowes" }, { "name": "David B. Olsen" }, { "name": "Brad E. Sleebs" }, { "name": "Alan F. Cowman" } ], "bibtex": "https://research.draconitic.com/paper/fm21as3fnb02s.bib", "cited_by": 2, "doi": "10.1371/journal.ppat.1014068", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014068", "id": "fm21as3fnb02s", "issns": [ "1553-7374" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014068", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2739|Malaria Research and Control" ], [ "plos", "/Biology and life sciences/Biochemistry/Enzymology/Enzymes/Proteases" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Enzymes/Proteases" ], [ "plos", "/Biology and life sciences/Genetics/Gene amplification" ], [ "plos", "/Biology and life sciences/Genetics/Genomics" ], [ "plos", "/Biology and life sciences/Genetics/Mutation/Point mutation" ], [ "plos", "/Biology and life sciences/Microbiology/Microbial control/Antimicrobial resistance" ], [ "plos", "/Biology and life sciences/Parasitology/Parasite groups/Apicomplexa/Plasmodium" ], [ "plos", "/Biology and life sciences/Parasitology/Parasite replication" ], [ "plos", "/Medicine and health sciences/Pharmacology/Antimicrobial resistance" ], [ "plos", "/Medicine and health sciences/Pharmacology/Drugs/Antimalarials" ] ], "openalex": "W7134988195", "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014068&type=printable", "pmcid": null, "pmid": null, "published": "2026-09-08", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/fm21as3fnb02s.ris", "sources": [ { "first_seen": "2026-10-30T12:17:56Z", "native_id": "10.1371/journal.ppat.1014068", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014068" } ], "subjects": [ { "field": "biology", "origin": "plos:genetics", "subfield": "genetics-genomics" }, { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" }, { "field": "medicine", "origin": "plos:pharmacology", "subfield": "pharmacology-toxicology" } ], "title": "Dual plasmepsin IX and X inhibitors are refractory to development of resistance", "title_text": "Dual plasmepsin IX and X inhibitors are refractory to development of resistance", "type": "peer-reviewed", "updated_at": "2026-10-30T12:17:56Z", "url": "https://research.draconitic.com/paper/fm21as3fnb02s", "venue": "PLOS Pathogens", "versions": [] }