| Malaria, one of the most deadly infectious diseases in the world, is caused by a protozoan parasite, Plasmodium. The malaria parasite has a very unique way of disposing of toxic free heme. Heme detoxification is achieved through heme polymerization, generating hemozoin (or malaria pigment). In vivo heme polymerization has been demonstrated in the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a 30 kDa protein composed of 35% histidines. In addition, chloroquine, a 4-aminoquinoline antimalarial drug, has been shown to inhibit PfHRP2-mediated heme polymerization.; Investigation of PfHRP2 and its roles in heme detoxification has demonstrated PfHRP2 to be capable of binding 50 hemes per polypeptide of PfHRP2; furthermore, spectrosopic studies have revealed that 95% of the bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. We propose that heme binding has the immediate effect of lowering the free heme concentration within the parasite thereby protecting the parasite against free heme-mediated toxicity; moreover, heme binding is the first step to PfRP2-mediated heme polymerization.; Probing the mechanism of chloroquine inhibition of PfHRP2-mediated heme polymerization demonstrated that (i) chloroquine binds to the PfHRP2-heme complex only in the presence of heme, (ii) chloroquine does neither displace the heme bound to PfHRP2 nor inhibit heme binding to PfHRP2. Chloroquine binding brings about spectroscopic changes to the PfHRP2-heme complex, suggesting a close interaction between PfHRP2 and chloroquine. Based on our findings, we have proposed new models for the mechanism of action of chloroquine.; As heme detoxification is both required for parasite survival and uniquely distinct from that of the human host, it makes for an attractive drug target. We have developed a quick and efficient microplate-based assay to screen for compounds that inhibit PfHRP2-mediated heme detoxification (by inhibiting heme binding to PfHRP2). Using this novel microplate heme-binding inhibition assay, we screened a library of potential antimalarial compounds. Our results reveal a good correlation between heme-binding inhibition and parasite growth inhibition. Hence, we have not only established the effectiveness of this novel screening method but have identified compounds with potent antiparasitic activity. |
