APPLYING ACTIVITY-BASED PROTEIN PROFILING TO IDENTIFY POTENTIALLY DRUGGABLE SERINE HYDROLASE ENZYMES IN LEISHMANIA SPP

Abstract

Leishmaniasis is a poverty related and one of the most important tropical diseases in the world with more than 12 million infected people, 0.9 to 1.6 million new cases each year, between 20000 and 30000 deaths per year, and 350 million people at risk of infection. Available drugs face problems with toxicity, administration and resistance which challenge their effectiveness. Consequently, the identification of new drug targets and the development of new treatments are imperative. Although serine hydrolases have been demonstrated to participate in crucial roles in the life cycle of the parasite and its virulence, these have not been yet characterized and the Leishmania serinome remains surprisingly neglected. This project attempts map and explore therapeutic targets within SHs present in the Leishmania proteome using an activity-based protein profiling (ABPP) strategy in the quest to find new protein drug targets for drug discovery. Initial experiments using commercial fluorophosphonate (FPs) probes revealed significant differences between the SH expression levels throughout their life cycles and between different Leishmania spp. As these probes are only effective for in vitro labelling, a suite of cell permeable probes has been synthesized and applied to study the Leishmania serinome in whole cells. Following proteome labelling and enrichment, the mass spectrometry-based tagging method, iTRAQ, led to the identification of two serine proteases: Carboxypeptidase LmxM.18.0450 and prolyl oligopeptidase (POP) LmxM.36.6750. Using a competitive ABPP approach, we were able to identify small molecule inhibitors for these enzymes which did showed activity against both L. mexicana promastigotes and axenic amastigotes. Collectively, these findings suggest that the serinome is a valuable source of new drug targets and that ABPP is a reliable approach for target discovery.