New therapeutic targets in leishmania spp. Study and characterization of lmjpes brct

Resum

Leishmaniasis is a group of vector-borne neglected tropical diseases caused by a protozoan parasite from the genus Leishmania and transmitted by hematophagous phlebotomine sandflies. Depending on clinical manifestations, it can be fatal without an adequate treatment. It causes a significant economic and human burden, as this disease currently puts at risk more than 1 billion people. The few drugs that are currently available for the treatment of leishmaniasis, display some kind of deleterious effects such as strong toxicity and drug resistance. In this context, the development of new antileishmanial treatments remains crucial. Rational approaches have been used to develop new specific therapies for leishmaniases. The identification of new targets that are essential for parasite viability or virulence continues as an attractive approach for the development of specific nontoxic antileishmanial agents. In fact, said studies can also be exploited for the identification of optimized inhibitor scaffolds. Consequently, to describe a novel putative drug target in Leishmania spp., the aim of this project was the characterization of a recently discovered leishmanial BRCT domain (present in LmjPES). The results derived from this thesis work would be a valuable contribution to the scientific community in the future development of new drugs to combat leishmaniasis. To achieve the aims of this research project, this thesis was structured as follows: Chapter 1 aimed to characterize LmjPES BRCT as a novel putative therapeutic target in leishmaniasis. By using specific plasmids, gene overexpression was performed to explore the implications of this domain in LmjPES subcellular localization, Leishmania infectivity, and treatment resistance processes. In addition, we used its gene and protein sequences for the phylogenetic and structural studies of this domain. In fact, structures constructed in Chapter 1 would be later used for some of the analyses performed in the following chapters. In Chapter 2, novel compounds able to inhibit this domain have been identified. To this end, previously generated structures were used. In addition, we employed in silico docking, molecular dynamics and ADME predictors to detect the best suited candidates which were then tested by using L. major promastigote cultures and in vitro infections. In Chapter 3, through drug repurposing and structural similarity to our best drug candidate from previous Chapter 2, we identified some antitubercular drugs as potential compounds against leishmaniasis. Candidates were in silico selected and in vitro tested in both, the promastigote and amastigote forms of the parasite. Lastly, the possible mechanism of action of the compound identified in Chapter 2 was explored via RT-qPCR. Chapter 4 described the involvement of studied protein domain in Leishmania infectivity. To achieve this goal, in vivo infection assays using plasmid tools were carried out and in silico protein-protein docking was later performed to analyze the possible proteins interactions responsible for the observed effects. In summary, our work shows that LmjPES BRCT is implicated in key L. major biological processes, it is a novel therapeutic target, and it is susceptible to be exploited for the development of innovative compounds for the treatment of leishmaniasis.