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.

Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/ß TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.

Glucose metabolism in Trypanosoma cruzi.

The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major pathways: glycolysis and the pentose phosphate pathway. Glucose is taken up via one facilitated transporter and its catabolism by the glycolytic pathway leads to the excretion of reduced products, succinate and l-alanine, even in the presence of oxygen; the first six enzymes are located in a peroxisome-like organelle, the glycosome, and the lack of regulatory controls in hexokinase and phosphofructokinase results in the lack of the Pasteur effect. All of the enzymes of the pentose phosphate pathway are present in the four major stages of the parasite's life cycle, and some of them are possible targets for chemotherapy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase are present, but there is no reserve polysaccharide.

Functional characterization of NADP-dependent isocitrate dehydrogenase isozymes from Trypanosoma cruzi.

Trypanosoma cruzi exhibits two putative isocitrate dehydrogenases (IDHs). Both idh genes were cloned and the recombinant enzymes expressed in Escherichia coli. Our results showed that T. cruzi IDHs are strictly dependent on NADP(+) and display apparent affinities towards isocitrate and the coenzyme in the low micromolar range. In T. cruzi, IDHs are cytosolic and mitochondrial enzymes, and there is no evidence for the typical Krebs cycle-related NAD-dependent IDH. Hence, like in Trypanosoma brucei, the Krebs cycle is not a canonical route in T. cruzi. However, the citrate produced in the mitochondrion could be isomerized into isocitrate in the cytosol and the mitochondrion by means of the putative aconitase, which would provide the substrate for both IDHs. The cytosolic IDH is significantly more abundant in amastigotes, cell-derived and metacyclic trypomastigotes than in epimastigotes. This observation fits in well with the expected oxidative burst this pathogen has to face when infecting the mammalian host.

Comparative studies on the biochemical properties of the malic enzymes from Trypanosoma cruzi and Trypanosoma brucei.

Comparative studies showed that, like Trypanosoma cruzi, Trypanosoma brucei exhibits functional cytosolic and mitochondrial malic enzymes (MEs), which are specifically linked to NADP. Kinetic studies provided evidence that T. cruzi and T. brucei MEs display similarly high affinities towards NADP(+) and are also almost equally efficient in catalyzing the production of NADPH. Nevertheless, in contrast to the cytosolic ME from T. cruzi, which is highly activated by l-aspartate (over 10-fold), the T. brucei homologue is slightly more active (50%) in the presence of this amino acid. In T. brucei, both isozymes appear to be clearly more abundant in the insect stage, although they can be immunodetected in the bloodstream forms. By contrast, in T. cruzi the expression of the mitochondrial ME seems to be clearly upregulated in amastigotes, whereas the cytosolic isoform appears to be more abundant in the insect stages of the parasite. It might be hypothesized that in those environments where glucose is very low or absent, these pathogens depend on NADP-linked dehydrogenases such as the MEs for NADPH production, as in those conditions the pentose phosphate pathway cannot serve as a source of essential reducing power.

Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxido-reductase activity.

Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas' disease, have shown side effects and variable efficiency. With the aim to describe parasite enzymes involved in the mechanisms of action of trypanocidal drugs and since it has been reported that reductases are crucial in their metabolism, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi. The percolation of a soluble fraction of epimastigote lysates through a Cibacron Blue-Sepharose column followed by elution by NADPH yielded a predominant protein with an apparent molecular weight of 32 kDa. This protein was identified by MALDI-TOF as an aldo-keto reductase (AKR) and hence denominated TcAKR. TcAKR was mainly localized in the cytosol and was also present in trypomastigote and amastigote lysates. The recombinant TcAKR (recTcAKR) showed NADPH-dependent reductase activity with the AKR substrates 4-nitrobenzaldehyde and 2-dihydroxyacetone. The saturation curves for both substrates were consistent with the Michaelis-Menten model. We also tested whether recTcAKR may reduce naphthoquinones (NQ), since many of these compounds have displayed important trypanocidal activity. recTcAKR reduced o-NQ (1,2-naphthoquinone, 9,10-phenanthrenquinone and beta-lapachone) with concomitant generation of free radicals but did not exhibit affinity for p-NQ (5-hydroxy-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, alpha-lapachone and menadione). The substrate saturation curve with o-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In addition, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. TcAKR is the first member of the AKR family described in T. cruzi. Our results indicate that this enzyme may participate in the mechanisms of action of trypanocidal drugs.

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 Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle.

BACKGROUND: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages. RESULTS: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure. CONCLUSIONS: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei.

Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.

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.

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.

Functional characterization and subcellular localization of the three malate dehydrogenase isozymes in Leishmania spp.

As part of a study on the malate dehydrogenase isozymes (MDHs) from Trypanosomatids, three different fractions with MDH activity were obtained from crude extracts of Leishmania mexicana promastigotes combining two different chromatographic steps. Gel filtration chromatography in native conditions showed that most of the MDH activity present in the crude extracts eluted in a single peak, which corresponded to a lower apparent molecular mass ( congruent with 57kDa) than the value expected for typical MDHs. To further characterize the leishmanial isozymes, three putative MDH genes, presumably corresponding to the mitochondrial, glycosomal and cytosolic isoforms were amplified by PCR, cloned into bacterial expression vectors, and the recombinant enzymes purified. Digitonin extraction of intact L. mexicana promastigotes and immunofluorescence microscopy of L. major promastigotes confirmed the subcellular compartmentation of each of the three isozymes. Western blot analysis showed that the three MDHs are developmentally regulated. At the protein level, these isozymes are remarkably more abundant in amastigotes than in promastigotes of L. mexicana. Altogether our results demonstrate the presence of three MDH isoforms with slightly distinct biochemical properties and different subcellular localization in Leishmania spp. Presumably the functional and biochemical features of these isozymes reflect the metabolic adaptation to the different nutrient sources these parasites have to face along their life cycle. These results also emphasize the differences among Trypanosomatids in this area of metabolism, since in the case of Trypanosoma brucei the cMDH is the only isoform expressed in bloodstream trypomastigotes, whereas in Trypanosoma cruzi cMDH is absent.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.

Transketolase from Leishmania mexicana has a dual subcellular localization.

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.

The pentose phosphate pathway in Trypanosoma cruzi.

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).

Pentose phosphate metabolism in Leishmania mexicana.

The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.