Antiapicoplast and Gametocytocidal Screening To Identify the Mechanisms of Action of Compounds within the Malaria Box

Abstract

cMalaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparum and low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of <1 M.

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

“…Hoechst 33342 at 0.25 g/ml was added to each sample right before imaging. A Zeiss Axioimager M1 microscope with a 100ϫ/1.4 numerical aperture (NA) oil immersion objective and an Axiocam MRm digital camera was used to obtain fluorescent images as reported previously (24). Adobe Photoshop CS2 was used to prepare final figures.…”

“…alamarBlue is a reporter of metabolic activity, and the active ingredient (resazurin) is thought to be reduced by mitochondrial enzymes (26,35,36). Both MitoTracker and alamarBlue have been successfully developed for gametocytocidal assays (24,26,37,38). After fluorescence was read, all wells were smeared and stained with Giemsa for morphological evaluation (Fig.…”

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities

“…Primary hit rates (>80% inhibition at 5 μM) of 4.5-24% [20,23,27,29] were achieved when screened against stage IV/V gametocytes; this increased to 33% against stage I-III gametocytes [23,26]. Furthermore, although the rank-order of hits were similar, there was not complete overlap between MMV Malaria Box screens conducted with different assay platforms or between laboratories using the same assay platform [24,25].…”

“…The Medicines for Malaria Venture (MMV) Malaria Box of 400 compounds (selectively active against asexual parasites [22]) has been screened worldwide for activity against stage IV/V gametocytes [20,[23][24][25][26][27][28][29][30][31]. Primary hit rates (>80% inhibition at 5 μM) of 4.5-24% [20,23,27,29] were achieved when screened against stage IV/V gametocytes; this increased to 33% against stage I-III gametocytes [23,26].…”

New Opportunities in the Structure-based Design of Anti-Protozoan Agents

“…A recent publication [30] relating to the theme of this article is the identification of MMV008138 from the Malaria Box which targets isoprenoid biosynthesis via the non-mevalonate (MEP) pathway. This biosynthetic pathway is inhibited by the antibiotic fosmidomycin (1, Fig.…”