A Diverse Range of Hemozoin Inhibiting Scaffolds Act on Plasmodium falciparum as Heme Complexes.

A diverse series of hemozoin-inhibiting quinolines, benzamides, triarylimidazoles, quinazolines, benzimidazoles, benzoxazoles, and benzothiazoles have been found to lead to exchangeable heme levels in cultured Plasmodium falciparum (NF54) that ranged over an order of magnitude at the IC50. Surprisingly, less active compounds often exhibited higher levels of exchangeable heme than more active ones. Quantities of intracellular inhibitor measured using the inoculum effect exhibited a linear correlation with exchangeable heme, suggesting formation of heme-inhibitor complexes in the parasite. In an effort to confirm this, the presence of a Br atom in one of the benzimidazole derivatives was exploited to image its distribution in the parasite using electron spectroscopic imaging of Br, an element not naturally abundant in cells. This showed that the compound colocalized with iron, consistent with its presence as a heme complex. Direct evidence for this complex was then obtained using confocal Raman microscopy. Exchangeable heme and inhibitor were found to increase with decreased rate of killing, suggesting that slow-acting compounds have more time to build up exchangeable heme complexes. Lastly, some but not all compounds evidently cause pro-oxidant effects because their activity could be attenuated with N-acetylcysteine and potentiated with t-butyl hydroperoxide. Collectively, these findings suggest that hemozoin inhibitors act as complexes with free heme, each with its own unique activity.

Hemozoin inhibiting 2-phenylbenzimidazoles active against malaria parasites.

The 2-phenylbenzimidazole scaffold has recently been discovered to inhibit ß-hematin (synthetic hemozoin) formation by high throughput screening. Here, a library of 325,728 N-4-(1H-benzo[d]imidazol-2-yl)aryl)benzamides was enumerated, and Bayesian statistics used to predict ß-hematin and Plasmodium falciparum growth inhibition. Filtering predicted inactives and compounds with negligible aqueous solubility reduced the library to 35,124. Further narrowing to compounds with terminal aryl ring substituents only, reduced the library to 18, 83% of which were found to inhibit ß-hematin formation <100 µM and 50% parasite growth <2 µM. Four compounds showed nanomolar parasite growth inhibition activities, no cross-resistance in a chloroquine resistant strain and low cytotoxicity. QSAR analysis showed a strong association of parasite growth inhibition with inhibition of ß-hematin formation and the most active compound inhibited hemozoin formation in P. falciparum, with consequent increasing exchangeable heme. Pioneering use of molecular docking for this system demonstrated predictive ability and could rationalize observed structure activity trends.