Diseño y evaluación de inhibidores peptídicos dirigidos a la interfaz de dimerización de la tripanotión reductasa de Leishmania Infantum

Abstract

The pharmacological treatment of Leishmaniasis still leaves much to be desired in terms of efficacy and effort devoted to the development of new agents. In the search for novel molecular entities, it is always preferable to focus on targets that are absent in the human host. Leishmania parasites lack the ubiquitous glutathione/glutathione reductase system (GSH/GluR) that is used in most prokaryotic and eukaryotic cells to detoxify their intracellular milieu from the damaging oxidizing agents produced during metabolism. Instead, they make use of the rather unique trypanothione/trypanothione reductase (T(SH)2/TryR) system, the blockage of which can be used to impair parasite survival. T(SH)2 is formed by two glutathione molecules bound together by a spermidine bridge. This molecule is so effective (more than glutathione) as an antioxidant that Leishmania relies solely on this system to withstand the harsh oxidant environment found inside of the phagolysosome. TryR is a homodimeric enzyme with C2 symmetry and two independent catalytic sites, each of which is made up of residues belonging to both monomers. Therefore, the dimeric configuration of this enzyme is an absolute requirement for its activity. At the core of the interaction between TryR subunits lays the dimerization interface, a hydrophobic patch that keeps both monomers tightly joined. Right in that hydrophobic patch we have identified a couple of amino acids that are important for maintaining a proper interaction between the monomers. These amino acids, glutamic acid 492 and glutamine 495, were incorporated into a 13-mer a-helix that we designed as an inhibitory peptide targeted to the dimerization interface of TryR. To assess this peptide and its follow-up derivatives, we implemented an easy scalable activity assay and developed a new ELISA that allowed us to quantify their impact on TryR dimerization and activity. The results obtained in this work allow us to conclude that TRL35 interacts with the Leishmania infantum TryR dimerization interface destabilizing its dimeric conformation and triggering the irreversible loss of activity because of enzyme precipitation.