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 (TcRPIB) 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-i = 5.5 +/- 0.1 mu M), 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 NIH3T3 murine host cells (IC50 = 17.40 +/- 1.055 mu M). 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.

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. Recently, new inhibitors, with good selectivity for the parasite proteasomes, have been described and are very promising as lead compounds for the development of new therapies for these neglected diseases. This article is part of a Special Issue entitled: "Play and interplay of proteases in health and disease".

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.

Metacaspases and paracaspases are proteases that were first identified as containing a caspase-like structural fold (Uren et al., 2000Uren A.G. O’Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). Like caspases, metacaspases and paracaspases are multifunctional proteins regulating diverse biological phenomena, such as aging, immunity, proteostasis, and programmed cell death. The broad phylogenetic distribution of metacaspases and paracaspases across all kingdoms of life and large variation of their biochemical and structural features complicate classification and annotation of the rapidly growing number of identified homologs. Establishment of an adequate classification and unified nomenclature of metacaspases and paracaspases is especially important to avoid frequent confusion of these proteases with caspases—a tenacious misnomer that unfortunately does not appear to decline with time. This Letter represents a consensus opinion of researchers studying different aspects of caspases, metacaspases, and paracaspases in various organisms, ranging from microbes to plants and animals. The current classification of proteases provided by the MEROPS database clusters caspases, metacaspases, and paracaspases to the same family, C14, within the CD clan (https://www.ebi.ac.uk/merops/). All members of the C14 family are annotated to possess aspartate P1 cleavage specificity, and the family is further split into two subfamilies: C14A (caspases) and C14B (metacaspases and paracaspases). Importantly, the MEROPS approach of grouping proteases into families or subfamilies is based on statistically significant similarities of the amino acid sequence within the peptidase domain or part thereof, without considering their biochemical properties (Rawlings et al., 2018Rawlings N.D. Barrett A.J. Thomas P.D. Huang X. Bateman A. Finn R.D. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database.Nucleic Acids Res. 2018; 46: D624-D632Crossref PubMed Scopus (756) Google Scholar). Being valuable for high-throughput protease classification, this approach, however, has substantial drawbacks if implemented without further adjustment. Indeed, in contradiction with the MEROPS description, none of the metacaspases or paracaspases characterized so far cleave after an aspartate residue. Instead, paracaspases are arginine specific (Coornaert et al., 2008Coornaert B. Baens M. Heyninck K. Bekaert T. Haegman M. Staal J. Sun L. Chen Z.J. Marynen P. Beyaert R. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.Nat. Immunol. 2008; 9: 263-271Crossref PubMed Scopus (342) Google Scholar, Hachmann et al., 2012Hachmann J. Snipas S.J. van Raam B.J. Cancino E.M. Houlihan E.J. Poreba M. Kasperkiewicz P. Drag M. Salvesen G.S. Mechanism and specificity of the human paracaspase MALT1.Biochem. J. 2012; 443: 287-295Crossref PubMed Scopus (66) Google Scholar, Rebeaud et al., 2008Rebeaud F. Hailfinger S. Posevitz-Fejfar A. Tapernoux M. Moser R. Rueda D. Gaide O. Guzzardi M. Iancu E.M. Rufer N. et al.The proteolytic activity of the paracaspase MALT1 is key in T cell activation.Nat. Immunol. 2008; 9: 272-281Crossref PubMed Scopus (238) Google Scholar), whereas metacaspases can cleave after either arginine or lysine (Figure S1A; Sundström et al., 2009Sundström J.F. Vaculova A. Smertenko A.P. Savenkov E.I. Golovko A. Minina E. Tiwari B.S. Rodriguez-Nieto S. Zamyatnin Jr., A.A. Välineva T. et al.Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.Nat. Cell Biol. 2009; 11: 1347-1354Crossref PubMed Scopus (164) Google Scholar, Vercammen et al., 2004Vercammen D. van de Cotte B. De Jaeger G. Eeckhout D. Casteels P. Vandepoele K. Vandenberghe I. Van Beeumen J. Inzé D. Van Breusegem F. Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine.J. Biol. Chem. 2004; 279: 45329-45336Crossref PubMed Scopus (263) Google Scholar). Such fundamental differences in the proteolytic specificity between caspases, metacaspases, and paracaspases imply distinct repertoires of new proteoforms that they generate and point to the complex diversification and coevolution of their substrates and downstream pathways. One unfortunate consequence of the current classification is the misuse of caspase-specific probes for studying metacaspases and paracaspases that is commonly found in the literature and leads to false conclusions. Apart from substrate specificity, caspases, metacaspases, and paracaspases feature other fundamental differences (Figure S1A). For example, active metacaspases are monomers and their activation usually requires millimolar concentrations of calcium (Hander et al., 2019Hander T. Fernández-Fernández Á.D. Kumpf R.P. Willems P. Schatowitz H. Rombaut D. Staes A. Nolf J. Pottie R. Yao P. et al.Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides.Science. 2019; 363: 1-10Crossref Scopus (113) Google Scholar, McLuskey et al., 2012McLuskey K. Rudolf J. Proto W.R. Isaacs N.W. Coombs G.H. Moss C.X. Mottram J.C. Crystal structure of a Trypanosoma brucei metacaspase.Proc. Natl. Acad. Sci. USA. 2012; 109: 7469-7474Crossref PubMed Scopus (65) Google Scholar, Wong et al., 2012Wong A.H.H. Yan C. Shi Y. Crystal structure of the yeast metacaspase Yca1.J. Biol. Chem. 2012; 287: 29251-29259Crossref PubMed Scopus (70) Google Scholar). In contrast, active caspases and paracaspases are calcium-independent dimers (Hachmann et al., 2012Hachmann J. Snipas S.J. van Raam B.J. Cancino E.M. Houlihan E.J. Poreba M. Kasperkiewicz P. Drag M. Salvesen G.S. Mechanism and specificity of the human paracaspase MALT1.Biochem. J. 2012; 443: 287-295Crossref PubMed Scopus (66) Google Scholar, Wiesmann et al., 2012Wiesmann C. Leder L. Blank J. Bernardi A. Melkko S. Decock A. D’Arcy A. Villard F. Erbel P. Hughes N. et al.Structural determinants of MALT1 protease activity.J. Mol. Biol. 2012; 419: 4-21Crossref PubMed Scopus (64) Google Scholar, Yu et al., 2011Yu J.W. Jeffrey P.D. Ha J.Y. Yang X. Shi Y. Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region.Proc. Natl. Acad. Sci. USA. 2011; 108: 21004-21009Crossref PubMed Scopus (65) Google Scholar). This indicates that upstream pathways regulating activation of caspases, metacaspases, and paracaspases are likewise different. In the past two decades we have learned about important differences between caspases, metacaspases, and paracaspases. Thus, simple extrapolation of features typical for caspases to all other members of the C14 family is not justified anymore. Instead caspases, metacaspases, and paracaspases should be separated into three corresponding groups within the family and each group should be properly annotated by having its key biochemical and structural characteristics provided. We kindly request curators of the MEROPS database to make corresponding changes. Since the structure and substrate specificity of prokaryotic caspase-like proteases named “orthocaspases” remain largely unknown (Klemenčič et al., 2015Klemenčič M. Novinec M. Dolinar M. Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa.Mol. Microbiol. 2015; 98: 142-150Crossref PubMed Scopus (34) Google Scholar), we leave their classification and nomenclature open until their structural and biochemical properties have been clarified. The name “caspase” stands for “cysteine-dependent aspartate-specific protease.” Thus, the names “metacaspase” and “paracaspase” imply the wrong substrate specificity for these proteases. However, since these names have been used for two decades, we propose to keep them, provided that caspases, metacaspases, and paracaspases are recognized as three separate groups within the C14 family. Based on domain composition and arrangement, metacaspases and paracaspases are further subdivided into three and two types, respectively (Figure S1A). For the sake of consistency, we propose to maintain a common nomenclature for the different types of metacaspases and paracaspases using Latin numerals (e.g., type I metacaspases). As for the conserved protein structures, they will be referred to as the p20-like region, the p10-like region, the linker region, and the N-terminal pro-domain, matching the nomenclature of caspases (Figure S1A; Alnemri et al., 1996Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Human ICE/CED-3 protease nomenclature.Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2139) Google Scholar). The p20, p10, and linker regions have been previously defined for the caspase group of the C14 family (Fuentes-Prior and Salvesen, 2004Fuentes-Prior P. Salvesen G.S. The protein structures that shape caspase activity, specificity, activation and inhibition.Biochem. J. 2004; 384: 201-232Crossref PubMed Scopus (687) Google Scholar) and can be easily identified in metacaspases and paracaspase homologs based on a hidden Markov model (HMM) alignment with the C14 peptidase domain (Figure S1B). Notably, although not always clearly stated in the literature, most known members of the C14 family contain the linker region. Furthermore, type II metacaspases are distinguished by a long linker between the p20 and p10 regions and an additional linker within the p10 region (Figure S1A), which are frequently referred to as a single linker. We suggest the consideration of the active form of metacaspases or paracaspases as a monomer if it is a cleaved or intact polypeptide chain derived from a single translational event, and a dimer if it comprises uncut or processed products of two translational events. We propose to establish a unified nomenclature of metacaspases and paracaspases in order to (i) facilitate the comparison of orthologs from different organisms and (ii) make it suitable for annotating homologs of species with partially sequenced genomes. Thus, we suggest using simple root symbols such as MCA for metacaspases and PCA for paracaspases. When naming individual family members, these root symbols will be preceded by the abbreviated Latin name of the species and followed by a hyphen, a Latin number representing the type, and then a small alpha character indicating in alphabetical order the number of the homolog of this type in a given genome (Figure S1C). Proenzymes that require proteolytic processing for activation could be annotated with the prefix “pro-”, e.g. pro-AtMCA-Ia for the metacaspase 1 of type I from A. thaliana. Spliceoforms should be indicated by a decimal number (e.g. AtMCA-Ia.1). Please note that these conventions do not consider the letter case, which should conform to gene and protein nomenclature established for a given model organism or taxonomic group. Importantly, this nomenclature should be used synonymously for metacaspases and paracaspase homologs with well-established names, e.g., human MALT1/HsPCA-Ia and A. thaliana AtMC1/AtMCA-Ia or AtMC4/AtMCA-IIa. We encourage all researchers to adopt these recommendations. The new classification and unified nomenclature of metacaspases and paracaspases will facilitate a more comprehensive exchange of relevant findings within the scientific community and help to bridge already existing knowledge with newly discovered homologs, thus promoting mechanistic understanding of these ancient, evolutionarily conserved proteases. This work was supported by the Knut and Alice Wallenberg Foundation. We apologize to colleagues whose work has not been cited due to space limitations. 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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. This makes urgently necessary the development of new drugs, more efficient, less toxic and affordable to the poor people, who are most of the infected population. In this review we will summarize the current strategies used for drug discovery considering drug repositioning, phenotyping screenings and target-based approaches. In addition, we will describe in detail the considerations for setting up robust enzymatic assays aimed at identifying and validating small molecule inhibitors in high throughput screenings.

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. Here, we present the identification of a set of inhibitors for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two collections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzymatic assays to a medium-throughput format and carried out the screening of both collections, followed by the construction of dose-response curves for the most promising hits. As a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE), a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor reported for this family of MCPs and considering its potency and specificity, TCMDC-143620 seems to be a promissory starting point to develop more specific and potent chemical tools targeting M32 MCPs from trypanosomatid parasites.

Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem “head-to-tail” configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including α-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.

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). We show that although the recognized cleavage sequence is not identical among the different model organisms tested, in all of them the proteolysis consequence is the removal of the ubiquitin-associated domain (UBA) present in the protein. We also demonstrate that Ddi1 cleavage is tightly regulated in vivo as it only takes place in yeast when calcium increases but under specific metabolic conditions. Finally, we show that metacaspase-mediated Ddi1 cleavage reduces the stability of this protein which can certainly impact on the many functions ascribed for it, including shuttle to the proteasome, cell cycle control, late secretory pathway regulation, among others.

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/β 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.

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. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

\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. They also present a cytosolic localization, a similar pattern of expression and a marked preference for Arg/Lys residues at P1'. To further explore MCPs substrate specificity beyond the subsite, we employed four positional scanning synthetic combinatorial libraries (PS-SC) of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that the T. brucei enzyme has a restricted selectivity for Phe in P1 position compared to T. cruzi MCP-1, which presented a wider range of substrate acceptance. The S2, S3 and S4 subsites, on the other hand, could accommodate a broad range of residues. On the basis of these results, we synthesized for each enzyme a series of FRET substrates which contained the most favourable residues in every position. In particular, for both MCPs acting on FRET pentapeptide substrates, catalytic efficiencies were similar to 100 times higher compared with previously described chromogenic substrates. In fact, the fluorogenic peptide Abz-LLKFK(Dnp)-OH (Abz = ortho-aminobenzoic acid; Dnp = 2, 4-dinitrophenyl) described here can be used to monitor accurately TbMCP-1 activity in parasite cell-free extracts. These results provide valuable insights to develop selective substrates and inhibitors, to further understand the mechanisms and functions of M32 MCPs.

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. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.

Trypanosomatids utilize glucose to sustain critical cellular functions. A key metabolic pathway that relies on glucose is the pentose phosphate pathway (PPP), which comprises reactions oxidizing substrates (oxidative phase) and interconversions to phosphorylated saccharides (non-oxidative phase). The products (ribose-5- phosphate), intermediates (glyceraldehyde-3-phosphate, fructose-6-phosphate), and cofactor (NADPH) of this metabolism are used in the synthesis of nucleic acids and lipids, and for the maintenance of redox homeostasis. Enzymes from the oxidative branch (i.e., glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH)) are phylogenetically related to their cyanobacterial and plant counterparts, and play an essential house-keeping role in the parasites. The components of the non-oxidative branch (i.e., ribose-phosphate isomerase (RPI), ribose-phosphate epimerase (RPE), transketolase (TKT), and transaldolase (TAL) are more heterogeneous, with a member that has no orthologous sequence in mammals (RPI) and others (RPE and TKT) that are developmentally regulated and species-specific dispensable. Except for 6PGDH, no systematic drug discovery studies have been performed on PPP enzymes. Only few chemical entities have been identified as inhibitors of G6PDH, and there are no investigations addressing this issue for 6PGL, RPI, RPE, TKT, and TAL. Thus, the search for inhibitors against PPP enzymes from trypanosomatids does not keep pace with the substantial information available on their biochemical and structural properties. The aim of this chapter is to attract attention towards PPP enzymes that qualify as trypanosomatid drug targets.

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. They also present a cytosolic localization, a similar pattern of expression and a marked preference for Arg/Lys residues at P1′. To further explore MCPs substrate specificity beyond the S1′ subsite, we employed four positional scanning synthetic combinatorial libraries (PS-SC) of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that the T. brucei enzyme has a restricted selectivity for Phe in P1 position compared to T. cruzi MCP-1, which presented a wider range of substrate acceptance. The S2, S3 and S4 subsites, on the other hand, could accommodate a broad range of residues. On the basis of these results, we synthesized for each enzyme a series of FRET substrates which contained the most favourable residues in every position. In particular, for both MCPs acting on FRET pentapeptide substrates, catalytic efficiencies were ∼100 times higher compared with previously described chromogenic substrates. In fact, the fluorogenic peptide Abz-LLKFK(Dnp)-OH (Abz = ortho-aminobenzoic acid; Dnp = 2, 4-dinitrophenyl) described here can be used to monitor accurately TbMCP-1 activity in parasite cell-free extracts. These results provide valuable insights to develop selective substrates and inhibitors, to further understand the mechanisms and functions of M32 MCPs.

During the last decade, de novo drug discovery approaches have come into focus due to the increased number of parasite pathogen genomes sequenced and the subsequent availability of genome-scale functional datasets. In order to prioritize target proteins, these approaches consider traits commonly thought to be desirable in a drug target, including essentiality, druggability (whether drug-like molecules are likely to interact with the target), assayability, importance in lifecycle stages of the pathogen relevant to human health, and specificity (i.e. the target is absent from, or substantially different in, the host). Proteases from protozoan parasites have become popular drug targets since these enzymes accomplish both housekeeping tasks common to many eukaryotes as well as functions highly specific to the parasite life style. Trypanosoma cruzi, the parasitic flagellate, agent of Chagas Disease, contains several cysteine, serine, threonine and metallo proteinases. This review will deal with peculiar families described in this parasite. Among them, two eukaryote homologues of the carboxypeptidases Taq are promising targets due to their particular phylogenetic distribution. Also absent in metazoans, metacaspases are essential peptidases playing important roles in cell growth, death and differentiation of trypanosomatids. Finally, autophagins are involved in the regulation of a conserved degradative pathway, the autophagy pathway, and result important for parasite survival under nutritional stress conditions and differentiation. Although so far there are no specific inhibitors for these families, the increasing knowledge of their biochemical properties, including substrate specificity, crystal structure, and biological functions, is an essential step towards the development of inhibitors.