Design, synthesis, and evaluation of substrate - analogue inhibitors of Trypanosoma cruzi ribose 5-phosphate isomerase type B.

Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (Ki = 5.5 ± 0.1 µM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC50 = 17.40 ± 1.055 µM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.

Functional characterization of stage-specific aminotransferases from trypanosomatids.

As part of a study on aminotransferases, genes coding for putative enzymes from Trypanosoma brucei and Leishmania major (alanine aminotransferases: ALATs, Tb927.1.3950 and LmjF12.0630; kynurenine aminotransferase: KAT, Tb10.389.1810; and tyrosine aminotransferase: TAT, LmjF36.2360) were cloned and functionally expressed in Escherichia coli. The putative T. brucei KAT, in fact coded for a glutamine aminotransferase (GlnAT), which exhibited a notably high affinity (in the micromolar range) towards glutamine and cysteine; in addition, like bacterial GlnATs and mammalian KATs, it was able to utilize different 2-oxoacids as amino acceptors. L. major TAT resembled T. cruzi TAT in substrate specificity, although the leishmanial enzyme did not exhibit ALAT activity. On the other hand, T. brucei ALAT, shortened by the first 65 amino acids assigned in the data bases, was functional and actively transaminated the substrate pair l-alanine and 2-oxoglutarate. Moreover in Western blots, the molecular size of the protein detected in crude extracts of T. brucei procyclics was identical to the value of the recombinant enzyme. Like T. brucei and T. cruzi orthologues, L. major ALAT displayed narrow substrate specificity. The leishmanial ALAT, like the T. cruzi enzyme, exhibited a dual subcellular localization, in the cytosol and in the mitochondrion. In line with the findings of comparative proteomic analyses of insect and mammalian stages of T. brucei and Leishmania parasites, our results also showed that T. cruzi ALAT is constitutively expressed, with remarkably higher levels being detected in amastigotes than in epimastigotes. ALATs are expressed in the clinically important stages of TriTryps, probably fulfilling an essential role, which deserves further studies.

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.