Identification and characterization of novel inhibitors of Plasmodium falciparum hemozoin formation.

摘要

After hundreds of years of scientific research, drug development and attempts at vector control, malaria still poses an enormous public health burden. One million fatalities were reported in 2006 with 91% of malaria deaths occurring in Africa and primarily in children under five years old. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum , metabolizes hemoglobin and releases toxic heme, which is neutralized by biologically controlled biomineralization (BCM) into a crystal known as hemozoin. Inhibition of this process is thought to be one of the most important drug targets in the malaria parasite, putatively the target of the quinoline antimalarials including chloroquine and amodiaquine.;We've developed a 384-well microtiter plate in vitro high throughput screen (HTS) to detect small molecules that disrupt heme crystallization, the cell free heme crystallization screen (CFHCS). This colorimetric assay requires no parasites or parasite-derived reagents and no radioactive materials. Seventeen compounds were identified from a screen of 16,000 small molecules that both inhibit heme crystallization in the CFHCS and inhibit P. falciparum growth in a separate HTS.;We've conducted a series of experiments to determine if the seventeen CFHCS hits inhibit P. falciparum growth by inhibiting heme crystallization, including heme binding assays, structure activity relationship studies, investigations of drug sensitivity in multidrug resistant parasites and experiments to determine if hemoglobin protease inhibitors antagonize the activity of the CFHCS hits. Additionally, we conducted phenotypic studies to determine if these compounds changed parasite morphology, especially the morphology of the hemozoin crystal and parasite food vacuole. Each of these assays has been previously described to support the mechanism of action of the quinoline antimalarials. Through these experiments we were able to rule out BCM as the mechanism of action of at least one compound and provide strong evidence to support this mechanism for nine compounds. Our finding also have interesting implications for the development of drugs which act by inhibiting BCM, one of the most important drug targets in the malaria parasite.