ABSTRACT
Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).
Subject(s)
Enzyme Inhibitors/pharmacology , Leishmania infantum/enzymology , Multienzyme Complexes/antagonists & inhibitors , Oxidoreductases/antagonists & inhibitors , Thymidylate Synthase/antagonists & inhibitors , Catalysis , Chromatography, Affinity , Cloning, Molecular , Drug Evaluation, Preclinical , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Inhibitory Concentration 50 , Multienzyme Complexes/genetics , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Oxidoreductases/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/isolation & purification , Tetrahydrofolate Dehydrogenase/metabolism , Thymidylate Synthase/genetics , Thymidylate Synthase/isolation & purification , Thymidylate Synthase/metabolismABSTRACT
Among the neglected tropical diseases, leishmaniasis stands out for its worldwide distribution and diversity of symptoms. Cutaneous leishmaniasis (CL), for instance, is endemic in 18 countries, but the available drugs to fight it have high toxicity and low patient adherence. In order to overcome this, dilemma drugs that target enzymes which are absent in the human host, such as Leishmania braziliensis sterol C24-methyltransferase (SMT-C24, EC 2.1.1.41), are needed. However, medicinal chemistry efforts toward this goal have been hampered by the low yield of soluble recombinant SMT-C24 afforded by currently available expression systems. Herein, we show that a combination of molecular biology and chromatographic strategies may increase the yield of LbSMT-C24 in up to fivefold. These results lay the ground for future investigation of this enzyme as a drug target.