Development of bis-thiazoles as inhibitors of triosephosphate isomerase from Trypanosoma cruzi. Identification of new non-mutagenic agents that are active in vivo.

The neglected disease American trypanosomiasis is one of the major health problems in Latin America. Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the etiologic agent of this disease, has been proposed as a druggable target. Some bis-benzothiazoles have been described as irreversible inhibitors of this enzyme. On the other hand, new bioactive furane-containing thiazoles have been described as excellent in vivo anti-T. cruzi agents. This encouraged us to design and develop new bis-thiazoles with potential use as drugs for American trypanosomiasis. The bis-thiazol 5, 3,3'-allyl-2,2'-bis[3-(2-furyl)-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, showed the best in vitro anti-T. cruzi profile with a higher selectivity index than the reference drugs Nifurtimox and Benznidazole against amastigote form of the parasite. This derivative displayed marginal activity against TcTIM however the bis-thiazol 14, 3-allyl-2-[3-(2-furyl)-2-propenylidenehydrazono]-3'-phenyl-2'-(3-phenyl-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, was an excellent inhibitor of the enzyme of the parasite. The absence of both in vitro mutagenic and in vivo toxicity effects, together with the activity of bis-thiazol 5in vivo, suggests that this compound is a promising anti-T. cruzi agent surpassing the "hit-to-lead" stage in the drug development process.

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus.

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 µmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers.

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.

Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer.

The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.