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

Abstract

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K(i) of 1 to 6 microM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K(i) greater than 280 microM). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 microg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical 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 microM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.

FIG. 1

Molecular structure of Walter Reed compound WR268961.

FIG. 2

Micrographs of Giemsa-stained schizont-stage parasites. The top panel shows abnormally enlarged food vacuoles in inhibitor-treated parasites. The lower panel shows even more pronounced morphological abnormalities during erythrocytic rupture.

FIG. 3

Parasite food vacuole contents visualized by Coomassie-stained SDS-PAGE. Intact hemoglobin accumulates in parasites treated with the falcipain inhibitor E-64, but not in parasites treated with WR268961.

FIG. 4

Structures of active ortho-phenoxyl diphenylurea compounds.

FIG. 5

Structures of active para-phenoxyl diphenylurea compounds.

FIG. 6

Crystal structure of pepstatin bound to plasmepsin (A) compared with the model of diphenylurea bound to plasmepsin (B). The compounds are colored by atom type (carbon is gray, oxygen is red, and nitrogen is blue), and the plasmepsin enzyme is represented by a molecular surface that is colored based on the surface potential (red is negative and blue is positive). Diphenylurea mimics the core region (the statine residue) of pepstatin.

FIG. 7

Crystal structure of pepstatin bound to plasmepsin (A) compared with the model of WR268961 bound to plasmepsin (B). The compounds are colored by atom type, and the main chain of the plasmepsin enzyme is represented by a semitransparent yellow worm. The side chains of four plasmepsin residues involved in hydrogen bonds (purple dashed lines) are colored by atom type.