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In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.

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.