Investigating the antimalarial properties of small-molecule compounds and exploring Plasmodium falciparum hexokinase as a potential therapeutic target

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2020-05

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The Ohio State University

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Malaria, a deadly tropical disease transmitted by infected mosquitos, is caused by Plasmodium parasites. Plasmodium falciparum is the most pathogenic form, responsible for >95% of mortality. Continual development of therapeutic drug resistance necessitates the search for novel antimalarial therapeutics. In an aim to find novel anti-malarial therapeutics, the five small-molecule compounds were screened for their ability to kill P. falciparum parasites. The compounds were purified from Cinnamosma fragnans, a plant endemic to Madagascar commonly used as an antimalaria treatment. A 72-hour dose response assay was performed with the asexual stages of the parasite, and the percent inhibition of each drug was determined relative to the negative DMSO-control. CMOS was the most potent with an IC50 of 0.4148 micromolar, followed by CM18 with an IC50 of 0.9858 micromolar. In a target-based approach, this second part of the project focuses on characterizing the biochemical properties of P. falciparum hexokinase (PfHk) and studying how its properties change through the intra-erythrocytic life stages of P. falciparum. The parasite's single hexokinase enzyme is solely responsible for the conversion of glucose to glucose-6-phosphate, the necessary substrate for production of ATP via glycolysis or generation of reducing equivalents (NADPH) and ribose-5-phosphate via the pentose phosphate pathway. Glycolytic flux in the parasite has been shown to be regulated during the parasite's pathogenic, erythrocytic stages, with PfHK activity being suggested as the rate-limiting step. We are exploring the idea that regulated post-translational modification of PfHK and/or turnover is responsible for its catalytic activity. To test this hypothesis, polyclonal antisera was generated, which specifically recognizes PfHK. Western blotting of P. falciparum whole-cell lysate, under reducing conditions, recognizes a single band of ~55 kDa as is predicted. However, under native conditions, a single band of ~220 kDa is detected, suggestive of PfHK forming a homotetramer in vivo, which has not been described for other eukaryotic HKs. Recently published structural studies of recombinant PvHK in the Morris lab supports this observation (1). Furthermore, using our PfHK antisera we have successfully immunopurified the protein from the parasite and are in process of identifying post-translational modifications and possible binding partners. Immunofluorescence assays (IFA) were also performed to determine the location and expression of PfHk in the different life stages. Results from the IFA support previous hypotheses that the enzyme is cytosolic and is expressed in all stages. Additionally, SeaHorse XF and kinetic assays were performed to determine how the glycolytic flux and the activity of PfHk changes through the life cycle of the parasite, respectively. The results from the kinetic experiments show that trophozoites have the highest HK content with the lowest turnover rate; whereas the gametocytes had unmeasurable PfHk activity. The SeaHorse XF assays also showed that the asexual stages had measurable glycolytic flux relative to the sexual stages, which had a very low glycolytic flux. Thus, even though HK is expressed in the matured sexual stages, it is not active. Therefore, unraveling the mechanism by which PfHk activity is regulated in sexual stages is a promising next step to understanding how PfHk could be targeted in novel antimalarial therapeutics.

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malaria, drug-discovery, biochemistry, infectious disease

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