New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasynodium vivax have been cloned and expressed for a varie ty of structural and enzymatic studies. Recombinant plasmepsins possess kin etic similarity to the native enzymes, indicating their suitability for tar get-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen comp ounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) d iphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K -i of 1 to 6 muM. This compound appears to be selective for plasmepsin, sin ce it is a poor inhibitor of the human aspartic protease cathepsin D (K-i g reater than 280 muM). NVR268961 inhibited the growth of P. falciparum strai ns W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 mug/ml, but was much less toxic to mammalian cells. The Walter Reed chemica l database contains over 1,500 compounds with a diphenylurea core structure , 9 of which inhibit the plasmepsins, with K-i values ranging from 0.05 to 0.68 muM. These nine compounds show specificity for the plasmepsins over hu man cathepsin D, but they are poor inhibitors of P. falciparum growth in vi tro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.