Hemozoin: A Target-Based Approach to Antimalarial Drug Discovery

Abstract

The emergence of drug resistant strains of Plasmodium spp. creates a critical need for the development of novel antimalarials. Hemozoin, a crystalline heme detoxification biomineral vital to parasite survival, serves as an important drug target. The quinoline antimalarials including chloroquine and amodiaquine owe their antimalarial activity to inhibition of hemozoin formation. Although hemozoin has been studied since the 18th century, scientists have only begun to obtain a detailed understanding of the structure and formation of this biomineral in the last 25 years. The goal of this dissertation was to first elucidate the details of hemozoin formation in the malaria parasite and second to mimic this process in a high-throughput screening (HTS) assay to identify inhibitors of beta-hematin (synthetic hemozoin) formation. Neutral lipid droplets (NLDs) were investigated as the location of hemozoin formation in the malaria parasite. Free heme was shown to rapidly partition into synthetic NLDs under physiological pH and temperature conditions. These synthetic NLDs were also shown to facilitate the rapid formation of beta-hematin. Using this information, the low cost, lipophilic detergent, NP-40, was identified as a surrogate for synthetic NLDs. Consequently, the NP-40 beta-hematin formation assay was utilized to screen 144,330 compounds in an HTS campaign. This effort resulted in the identification of 530 hits. To establish the effectiveness of these target-based beta-hematin inhibitors against P. falciparum, each hit was further tested in cultures of parasitized red blood cells. This effort revealed that 171 of the beta-hematin inhibitors are also active against the parasite. Dose-response data identified 73 of these beta-hematin inhibitors have IC50 values < 5 ìM, including 25 compounds with nanomolar activity against P. falicparum. Of these hits, four scaffolds were selected for a preliminary structure-activity relationship analysis. The results of this screening effort were also used as a knowledge-base to establish quantitative structure-activity relationship (QSAR) models. These models were applied to virtually screen the publically available GlaxoSmithKline (GSK) and Novartis databases of in vitro antimalarial compounds. The GSK in vitro antimalarials were obtained (13,229 compounds) to experimentally validate the QSAR models.