Dottorato in BIOCHIMICA

Titolo della tesi: Inhibition Studies of Trypanothione Reductase & Structural Studies on Ornithine Decarboxylase from Leishmania infantum

ABSTRACT Leishmaniasis is a neglected disease caused by the parasite Leishmania which has a unique redox metabolism involving trypanothione. Trypanothione delivers reducing equivalents which in turn saves the parasite from oxidative damage and is also utilized in the formation of deoxyribonucleotides. Current treatment involved toxic drugs such as pentavalent antimonials targeting the trypanothione metabolism. These therapeutics are unsatisfactory in terms of safety, high cost and low efficacy, due to the emergence of drug resistance against them. Therefore, there is serious need to discover and develop new drugs to treat this life-endangering disease. In the present project, we aimed at studying the inhibition mechanisms of Trypanothione Reductase (TR) and structurally studying the Ornithine Decarboxylase (ODC) from Leishmania infantum involved in trypanothione and polyamine metabolism respectively. Trypanothione reductase has been proved to be a specific enzyme for parasites survival and proposed to be an effective target for the treatment of trypanosomiasis and leishmaniasis. The biochemical basis for the selection of this enzyme as a target and the structural aspects of its inhibition are presented. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities for TR and their prospects as future chemotherapeutic agents has been highlighted. Recently, the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine) has been solved, which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466′, Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777–TR complex, we synthesized, in collaboration with the group of Prof. Di Santo (Sapienza University of Rome), structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase. Moreover, the structural studies on Ornithine decarboxylase (ODC) from Leishmania infantum have been carried out. ODC catalyzes the decarboxylation of ornithine to form putrescine, a key step in the pathway of spermidine biosynthesis, one of the component of trypanothione molecule. Thus, targeting ornithine decarboxylase would inhibit formation of spermidine and subsequently trypanothione, affecting the growth and survival of Leishmania. The structure of ODC from Leishmania species is not yet determined, whereas human, mouse, and Trypanosoma brucei ODC structures are reported in Protein Data Bank. The knowledge of the Leishmania ODC will furnishes new insight to understand at molecular level the polyamine metabolism pathway in Leishmania and will allow the design of new series of compounds against Leishmania parasite targeting this key enzyme. In this thesis I described the cloning, expression, purification, of this protein and the preliminary X-ray data analysis that allow me to solve its low resolution structure.