Investigation of trypanothione synthetase of Leishmania infantum as a potential target for new anti-parasitic drugs

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Leishmania infantum is a protozoan parasite of the Trypanosomatidae family, responsible for human and canine leishmaniasis in Mediterranean countries. Control of these vector-borne diseases is unsatisfactory and new chemotherapeutics are urgently needed. Trypanothione biosynthesis, owing to its unique and essential character, is regarded as an attractive target for therapeutic intervention. Trypanothione is a bis-(glutathionyl)spermidine conjugate, responsible for redox homeostasis in trypanosomatids. It is synthesized by the sequential addition of two molecules of glutathione to a spermidine molecule. Trypanothione synthetase (TRYS), which catalyzes both conjugation steps, has no counterpart in mammals and is essential to Trypanosoma brucei. This scenario is somewhat different in L. infantum, which harbors one additional enzyme mono(glutathionyl)spermidine synthetase or GSPS capable of driving the first step of trypanothione biosynthesis. Since mono(glutathionyl)spermidine can replace some metabolic functions of trypanothione in vitro, the actual significance of TRYS is still disputed in GSPS-harboring trypanosomatids. This work aimed at clarifying this issue by functionally characterizing both TRYS and GSPS in L. infantum promastigotes (insect stage) and amastigotes (mammalian stage), employing a classical gene replacement strategy. Concerning TRYS, elimination of both alleles in promastigotes was only possible upon complementation with an extrachromosomal copy of the gene. Maintenance of this episome for 6 months in the absence of drug pressure proved that TRYS is crucial and cannot be replaced by GSPS. Work is on going to assess TRYS essentiality in amastigotes. In parallel, we have initiated the chemical validation of TRYS using a N5-substituted paullone (FS-554) that irreversibly inhibits the enzyme in vitro. We observed that the leishmanicidal effect of FS-554 towards promastigotes and intramacrophagic amastigotes correlated with TRYS expression levels, confirming that this enzyme can be targeted by drug-like compounds in the cell context. In what regards GSPS, production of homozygous knockouts is still underway to be used in future work.

Supported by a grant (PTDC/BIA-MIC/10091/2008) from “Fundação para a Ciência e Tecnologia”(FCT)