Dual plasmepsin IX and X inhibitors are refractory to development of resistance
5 affiliations
- 1The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- 2Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia
- 3Merck & Co., Inc., West Point, Pennsylvania, United States of America
- 4Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom
- 5Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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.
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BibTeX
@article{favuzza2026dual,
title = {{Dual plasmepsin IX and X inhibitors are refractory to development of resistance}},
author = {Paola Favuzza and Madeline G. Dans and Wenyin Su and Jennifer K. Thompson and Anthony N. Hodder and Anna Ngo and Jocelyn Sietsma Penington and Danushka S. Marapana and Jerzy M. Dziekan and Anthony T. Papenfuss and Manuel de Lera Ruiz and Rachael Coyle and Marcus C. S. Lee and John A. McCauley and Kym Lowes and David B. Olsen and Brad E. Sleebs and Alan F. Cowman},
journal = {PLOS Pathogens},
year = {2026},
month = sep,
doi = {10.1371/journal.ppat.1014068},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014068},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Dual plasmepsin IX and X inhibitors are refractory to development of resistance
AU - Paola Favuzza
AU - Madeline G. Dans
AU - Wenyin Su
AU - Jennifer K. Thompson
AU - Anthony N. Hodder
AU - Anna Ngo
AU - Jocelyn Sietsma Penington
AU - Danushka S. Marapana
AU - Jerzy M. Dziekan
AU - Anthony T. Papenfuss
AU - Manuel de Lera Ruiz
AU - Rachael Coyle
AU - Marcus C. S. Lee
AU - John A. McCauley
AU - Kym Lowes
AU - David B. Olsen
AU - Brad E. Sleebs
AU - Alan F. Cowman
PY - 2026
DA - 2026/09/08
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014068
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014068
AB - 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.
SN - 1553-7374
ER -