Identification of M32 peptidase inhibitors in new compound sets with anti-kinetoplastid activity

GABRIELA NIEMIROWICZ,; CAZZULO JJ; AGÜERO F; ALVAREZ, V. E.

Santa Fe

Congreso; XXVIII Reunión Anual de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias y SIMPOSIO Internacional de Biología Celular y Molecular de la Enfermedad de Chagas; 2016

Sociedad Argentina de Protozoología y Enfermedades Parasitarias (SAP)

Members of the Trypanosomatidae familycomprise a large number of species that arecausative agents of several highly disabling andoften fatal diseases such as sleeping sickness,Chagas? disease and Leishmaniasis. The currentchemotherapy used in the treatment of theseinfections presents serious side-effects, and in some cases have low effectiveness, which makesimperativethedevelopmentofnewchemotherapeutic compounds. The M32 family ofmetallopeptidases (MCPs) contains a group ofhydrolases, which although being broadlydistributed among prokaryotic organisms, can onlybe found in a few eukaryotes including somegreen algae and trypanosomatids. The absence ofM32 MCPs in metazoans constitutes an attractivetrait due to the high specificity/selectivity potentialof this family for drug development. In particular,Trypanosoma cruzi and T. brucei containconserved M32 MCPs (at 71% identity) whosestructure and biochemistry are well characterized.Nonetheless, no inhibitors or biological functionsfor these two proteins have been reported to date.On the basis of their biochemical properties andstage-specific expression, they have beenimplicated in the catabolism of peptides andproteins to single amino acids required for proteinsynthesis, the regulation of small peptidemetabolism, and more recently, the replicationcycle of the kinetoplast in the case of T. bruceiMCP-1 . In this work, we present the identificationof new M32 inhibitory scaffolds within theGlaxoSmithKline HAT and CHAGAS chemicalboxes (two compound collections grouping 404non-cytotoxic compounds with high antiparasiticpotency, drug-likeness, structural diversity andscientific novelty). To this end, we developed andoptimized a continuous enzymatic assay to a 384-multiwell format and carried out a primaryscreening of both compound collections, followedby construction and analysis of dose-responsecurves for the most promising hits. Using thisstrategy we identified several inhibitors whichhave differential potency and selectivity towardsboth T. cruzi and T. brucei MCP-1 peptidases.These results provide a testable target andhypothesis on the mechanism of action of thesecompounds. Moreover these inhibitors representnew useful tools to understand the action mode ofM32 MCPs and also enable further studiestowards the functional understanding of role ofthese enzymes in trypanosomatid biology.