Abstract
Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance.
Highlights
Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and has parasiticidal activity
By selecting parasite mutants resistant to pepstatin esters, we found that a parasite alpha/beta hydrolase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase), is required for activation of the esterified compound
We have clarified the differences in potency of different pepstatin preparations reported previously and found that antimalarial activity is due not to pepstatin, but to low levels of a highly potent pepstatin ester
Summary
Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and has parasiticidal activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance. Pepstatin is a natural product isolated nearly 50 years ago from Streptomyces argenteolus and related actinomycetes[4] It is a wellcharacterized aspartic protease inhibitor and derivatives of this scaffold are in clinical use as antiretroviral therapies. By selecting parasite mutants resistant to pepstatin esters, we found that a parasite alpha/beta hydrolase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase), is required for activation of the esterified compound.
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