Mutations in a Putative Esterase Confer Resistance to Pestatin n-butyl ester. Plasmodium falciparum

We investigated the reported order-of-magnitude variance in potency of the aspartic protease inhibitor pepstatin against asexual Plasmodium falciparum. From a more potent preparation, we isolated the fraction that had strong anti-parasitic activity and identified the active component as butyl ester-modified pepstatin. We synthesized a number of derivatives and characterized an analog (pepstatin hexyl ester) with low nanomolar activity. Compared to the ester derivatives, charged pepstatin is poorly taken up by parasites. Pepstatin ester-resistant parasites were readily obtained. Full-genome sequencing revealed SNPs in a putative esterase, PF_0709700. Resistance-conferring mutations were in the C-terminal helix or close to the active site; others had nonsense mutations resulting in enzyme truncation. Knock-in of esterase-inactivating mutations did not affect parasite growth and made pepstatin esters inert. Parasites with esterase mutations were also resistant to a compound from the Medicines for Malaria Venture (MMV) collection of antimalarials, MMV11438. Recombinant esterase hydrolyzed pepstatin esters and de-esterified MMV11438. We conclude that 1) pepstatin is a potent, but poorly bioavailable anti-malarial; 2) PF_0709700 is an active esterase with moderate substrate specificity that is capable of activating prodrugs; 3) Mutations in this esterase constitute a new mechanism of antimalarial resistance.