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

NIEMIROWICZ, GABRIELA TERESA; SALAS SURDUY, EMIR; CAZZULO, JUAN JOSÉ; AGUERO, FERNAN; ALVAREZ, VANINA EDER

Santa Fe

Encuentro; XXVIII Reunión Anual de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias; 2016

Members of the Trypanosomatidae family comprise a large number of species that are causative agents of several highly disabling and often fatal diseases such as sleeping sickness, Chagas? disease and Leishmaniasis. The current chemotherapy used in the treatment of these infections presents serious side-effects, and in some cases have low effectiveness, which makesimperative the development of new chemotherapeutic compounds. The M32 family ofmetallopeptidases (MCPs) contains a group of hydrolases, which although being broadly distributed among prokaryotic organisms, can only be found in a few eukaryotes including some green algae and trypanosomatids. The absence of M32 MCPs in metazoans constitutes an attractive trait due to the high specificity/selectivity potential of this family for drug development. In particular, Trypanosoma cruzi and T. brucei contain conserved M32 MCPs (at 71% identity) whose structure and biochemistry are well characterized. Nonetheless, no inhibitors or biological functions for these two proteins have been reported to date. On the basis of their biochemical properties and stage-specific expression, they have been implicated in the catabolism of peptides and proteins to single amino acids required for protein synthesis, the regulation of small peptide metabolism, and more recently, the replication cycle of the kinetoplast in the case of T. brucei MCP-1 . In this work, we present the identification of new M32 inhibitory scaffolds within the GlaxoSmithKline HAT and CHAGAS chemicalboxes (two compound collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty). To this end, we developed and optimized a continuous enzymatic assay to a 384-multiwell format and carried out a primary screening of both compound collections, followed by construction and analysis of dose-response curves for the most promising hits. Using this strategy we identified several inhibitors which have differential potency and selectivity towards both T. cruzi and T. brucei MCP-1 peptidases. These results provide a testable target and hypothesis on the mechanism of action of these compounds. Moreover these inhibitors represent new useful tools to understand the action mode of M32 MCPs and also enable further studies towards the functional understanding of role of these enzymes in trypanosomatid biology.