CONICET | Buscador de Institutos y Recursos Humanos

Targeting proteases is a common strategy to identify new antiparasitic agents due totheir essential contribution to parasite growth and development. Metacaspases (MCAs)are cysteine proteases (Clan CD) present in fungi, protozoa and plants. Theseenzymes, which are associated with crucial events in protozoa parasites (i.e. cell deathand cell cycle progression), are absent in the human host, thus arising as attractivedrug targets.To find new MCA inhibitors bearing trypanocidal activity, we adapted a continuousfluorescent enzymatic assay to a medium-throughput format and carried out thescreening of different compounds collections, followed by the construction of dose-response curves for the most promising hits. We used MCA5 from T. brucei (TbMCA5)as a model for the identification of inhibitors from the GlaxoSmithKline HAT andCHAGAS chemical boxes; two collections grouping 404 non-cytotoxic compounds withhigh antiparasitic potency, drug-likeness, structural diversity and scientific novelty. Wealso assessed a third collection of 9 compounds from Maybridge database identifiedby virtual screening as potential inhibitors of the cysteine peptidase falcipain-2 (ClanCA) from Plasmodium falciparum. As a result, 4 hits from the HAT and CHAGAS boxesshowed modest IC 50 values in the range 79-142 µM. Remarkable, HTS01959(Maybridge collection) resulted the most potent inhibitor with IC 50 of 14,39 µM; alsoinhibiting other MCAs from T. brucei and T. cruzi (TbMCA2=4,14 µM, TbMCA3=5,04µM and TcMCA5=151 µM). HTS01959 behaves as a reversible, slow binding andnoncompetitive inhibitor of TbMCA2, where the mechanism of action includes RedOxcomponents.Importantly, HTS01959 displays trypanocidal activity against bloodstream forms of T.brucei and trypomastigotes forms of T. cruzi, with non-cytotoxic effect on VERO cells.Thus, HTS01959 seems to be a promissory starting point to develop more specific andpotent chemical structures to target MCAs from trypanosomatids parasites.