Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation
Aboge, Gabriel O
Terkawi, Mohamad A
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Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolatesonits enzymeactivity,aswellasoninvitroparasitegrowth.Thefull-lengthgene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identiﬁed from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K m values of 4.70 0.059 (mean standard error of the mean) and 9.75 1.64M for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC50] 68.6 5.20 nM) than pyrimethamine (IC50 55.0 2.08M) and trimethoprim (IC50 50 12.5M). Moreover, the antifolates’ inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, theB.gibsoni DHFR-TS is a molecular antifolate drug target.