Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61+/-2% of the iron within parasitized erythrocytes, of which 92+/-6% is located within the food vacuole. Of this, 88+/-9% is in the form of haemozoin. (57)Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.

Using cell fractionation and measurement of Fe(III)heme-pyridine, the antimalarial chloroquine (CQ) has been shown to cause a dose-dependent decrease in hemozoin and concomitant increase in toxic “free” heme in cultured Plasmodium falciparum that is directly correlated with parasite survival. Transmission electron microscopy techniques have further shown that heme is redistributed from the parasite digestive vacuole to the cytoplasm and that CQ disrupts hemozoin crystal growth, resulting in mosaic boundaries in the crystals formed in the parasite. Extension of the cell fractionation study to other drugs has shown that artesunate, amodiaquine, lumefantrine, mefloquine and quinine, all clinically important antimalarials, also inhibit hemozoin formation in the parasite cell, while the antifolate pyrimethamine and its combination with sulfadoxine do not. This study finally provides direct evidence in support of the hemozoin inhibition hypothesis for the mechanism of action of CQ and shows that other quinoline and related antimalarials inhibit cellular hemozoin formation.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

A novel class of imidazopyridazines identified from whole cell screening of a SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant strain) and NF54 (sensitive strain). Structure-activity relationship studies led to the identification of highly potent compounds against both strains. Compound 35 was highly active (IC50: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35 exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 × 50 mg/kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35 displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or higher metabolic clearance in mice.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.