RESUMEN
CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of small molecular weight compounds with potential use against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified a new selective inhibitor chemotype of TIM from T. cruzi (TcTIM), 1,2,4-thiadiazol-5(4H)-one. OBJECTIVE: Study the mechanism of TcTIM inhibition by a 1,2,4-thiadiazol derivative. METHODS: We performed the biochemical characterization of the interaction of the 1,2,4-thiadiazol derivative with the wild-type and mutant TcTIMs, using DOSY-NMR and MS experiments. Studies of T. cruzi growth inhibition were additionally carried out. RESULTS AND CONCLUSION: At low micromolar concentrations, the compound induces highly selective irreversible inactivation of TcTIM through non-covalent binding. Our studies indicate that it interferes with the association of the two monomers of the dimeric enzyme. We also show that it inhibits T. cruzi growth in culture.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Tiadiazoles/farmacología , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Trypanosoma cruzi/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/química , Triosa-Fosfato Isomerasa/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.