Screening and Identification of Metacaspase Inhibitors: Evaluation of Inhibition Mechanism and Trypanocidal Activity.

A common strategy to identify new antiparasitic agents is the targeting of proteases, due to their essential contributions to parasite growth and development. Metacaspases (MCAs) are cysteine proteases present in fungi, protozoa, and plants. These enzymes, which are associated with crucial cellular events in trypanosomes, are absent in the human host, thus arising as attractive drug targets.

Update on relevant trypanosome peptidases: Validated targets and future challenges.

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, and Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, the agents of Sleeping sickness (Human African Trypanosomiasis, HAT), as well as Trypanosoma brucei brucei, the agent of the cattle disease nagana, contain cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes are the cysteine proteases from the Clan CA, the Cathepsin L-like cruzipain and rhodesain, and the Cathepsin B-like enzymes, which have essential roles in the parasites and thus are potential targets for chemotherapy. In addition, several other proteases, present in one or both parasites, have been characterized, and some of them are also promising candidates for the developing of new drugs.

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 (K = 5.

Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti-kinetoplastid chemical boxes.

Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas disease, respectively. These enzymes are absent in humans and several functions have been proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been reported so far for these enzymes.

Target-based Screening of the Chagas Box: Setting Up Enzymatic Assays to Discover Specific Inhibitors Across Bioactive Compounds.

Chagas disease is a neglected tropical illness caused by the protozoan parasite Trypanosoma cruzi. The disease is endemic in Latin America with about 6 million people infected and many more being at risk. Only two drugs are available for treatment, Nifurtimox and Benznidazole, but they have a number of side effects and are not effective in all cases.

Author Correction: Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

DNA-damage inducible protein 1 is a conserved metacaspase substrate that is cleaved and further destabilized in yeast under specific metabolic conditions.

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death in plants and in progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. Here, we report that the DNA-damage inducible protein 1 (Ddi1) represents a conserved protein substrate for metacaspases belonging to divergent unicellular eukaryotes (trypanosomes and yeasts).

Substrate specificity profiling of M32 metallocarboxypeptidases from Trypanosoma cruzi and Trypanosoma brucei.

Metallocarboxypeptidases (MCPs) of the M32 family, while broadly distributed among prokaryotic organisms, have so far been only found in a few eukaryotes including trypanosomatids. Among these organisms are human and animal pathogens of medical relevance such as Trypanosoma brucei and Trypanosoma cruzi, the respective causative agents of sleeping sickness and Chagas disease. The M32 MCP orthologues found in these parasites share 72% protein sequence identity.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome.

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.

Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.

Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets.

Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla.

Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum.

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 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction.

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.

All Trypanosoma cruzi developmental forms present lysosome-related organelles.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T.

The glucose-6-phosphate dehydrogenase from Trypanosoma cruzi: its role in the defense of the parasite against oxidative stress.

The Trypanosoma cruzi glucose-6-phosphate dehydrogenase (G6PDH) is encoded by several genes located in three of the parasite chromosomes. All the sequences present two possible start codons, 111bp apart, also present in its Trypanosoma brucei counterpart. As the 37 residues comprised between the two candidate initiator methionines of T.

Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus.

Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.

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.

The 6-phosphogluconate dehydrogenase from Trypanosoma cruzi: the absence of two inter-subunit salt bridges as a reason for enzyme instability.

The third enzyme of the pentose phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6PGDH), is present in the four major stages of Trypanosoma cruzi, CL Brener clone. The enzyme was too unstable to be purified from epimastigote cell-free extracts. Two genes encoding 6PGDH were cloned and sequenced; the predicted amino acid sequences differ only in five non-essential residues.

Cloning and heterologous expression of a broad specificity aminotransferase of Leishmania mexicana promastigotes1.

We have previously reported that Leishmania mexicana promastigotes possess a broad substrate specificity aminotransferase (BSAT), able to transaminate aspartate, aromatic amino acids, methionine and leucine. We have confirmed now this unusual substrate specificity by cloning its gene and expressing in Escherichia coli the recombinant active protein. The amino acid sequence of BSAT shares over 40% identity with other eukaryotic and prokaryotic aspartate aminotransferases, thus showing that the enzyme belongs to the subfamily Ialpha of aminotransferases, and has only 6% identity with the tyrosine aminotransferase from Trypanosoma cruzi, which has a similar substrate specificity.

The trypanosomiases.

The trypanosomiases consist of a group of important animal and human diseases caused by parasitic protozoa of the genus Trypanosoma. In sub-Saharan Africa, the final decade of the 20th century witnessed an alarming resurgence in sleeping sickness (human African trypanosomiasis). In South and Central America, Chagas' disease (American trypanosomiasis) remains one of the most prevalent infectious diseases.

Specific cleavage sites on human IgG subclasses by cruzipain, the major cysteine proteinase from Trypanosoma cruzi.

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time.

Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi.

Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.

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.

Proteinases of Trypanosoma cruzi: patential targets for the chemotherapy of Changas desease.

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas Disease, contains cysteine, serine, threonine and metallo proteinases. Aspartic proteinases have not been found so far. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a complex mixture of isoforms by the major developmental stages of the parasite, including some membrane-bound isoforms.