Abstract
Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.
Highlights
Inhibiting PfDHODH kills malaria parasites, but the potential for drug resistance is unknown
We identified compounds that were synergistic with wild-type PfDHODH inhibitors but did not exhibit mutually incompatible resistance
We identified five parasite lines that were resistant to PfDHODH inhibitors without mutation in or copy number variation of the pfdhodh gene (Fig. 4 and Table 9)
Summary
Inhibiting PfDHODH kills malaria parasites, but the potential for drug resistance is unknown. Target gene amplification and unknown mechanisms contributed to resistance, albeit to a lesser extent These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Identifying and combining antimalarial compounds that selectively target the bulk of the wild-type population and the small, emerging resistant population are novel approaches to antimalarial combination therapy We tested this idea, coined “targeting resistance,” with inhibitors of pyrimidine biosynthesis. We carried out additional resistance selections with mutant parasites and these negative cross-resistant, mutant-selective inhibitors These sequential selections gave rise to three more point mutations in the target gene as follows. Selecting parasites with a combination of wild-type and mutant-type selective PfDHODH inhibitors, a test of targeting resistance, maintained a wild-type population and prevented the emergence of resistance far more often than selection with either drug alone. Crystallography of wild-type PfDHODH protein with three inhibitors implied that selectivity is largely due to hydrophobic interactions rather than hydrogen bonding or steric clashes
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