Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

Recombinant and native TviCATL from Trypanosoma vivax: Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis.

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.

Production, purification and crystallization of a trans-sialidase from Trypanosoma vivax.

Sialidases and trans-sialidases play important roles in the life cycles of various microorganisms. These enzymes can serve nutritional purposes, act as virulence factors or mediate cellular interactions (cell evasion and invasion). In the case of the protozoan parasite Trypanosoma vivax, trans-sialidase activity has been suggested to be involved in infection-associated anaemia, which is the major pathology in the disease nagana. The physiological role of trypanosomal trans-sialidases in host-parasite interaction as well as their structures remain obscure. Here, the production, purification and crystallization of a recombinant version of T. vivax trans-sialidase 1 (rTvTS1) are described. The obtained rTvTS1 crystals diffracted to a resolution of 2.5 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 57.3, b = 78.4, c = 209.0 Å.

A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.

Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection.

Trypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite.

A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.

QM/MM molecular dynamics study of purine-specific nucleoside hydrolase.

Although various T. vivax purine-specific inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH) crystal structures were determined in recent years, the mechanistic details for the cleavage of N-glycosidic bond and the release of base are still unclear. Herein, the irreversible hydrolysis reaction has been studied by ab initio QM/MM MD simulations, and the results indicate a highly dissociative and concerted mechanism. The protonation of substrate at N7 of inosine is found to strongly facilitate the hydrolysis process, while the hydrolysis reaction is less sensitive to the protonation state of Asp 40 residue. The proton-transfer channel and the dependence of activity on the anti/syn-conformation of substrate are also explored.

Design of new chemotherapeutics against the deadly anthrax disease. Docking and molecular dynamics studies of inhibitors containing pyrrolidine and riboamidrazone rings on nucleoside hydrolase from Bacillus anthracis.

Anthrax is a disease caused by Bacillus anthracis, a dangerous biological warfare agent already used for both military and terrorist purposes. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. Having this in mind we have performed molecular docking studies, aiming to analyze the three-dimensional positioning of six known inhibitors of Trypanosoma vivax NH (TvNH) in the active site of B. anthracis NH (BaNH). We also analyzed the main interactions of these compounds with the active site residues of BaNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compounds as lead for the design of potential inhibitors of BaNH. Most of the docking and MD results obtained corroborated to each other. Additionally, the docking results also suggested a good correlation with experimental data.

Proline racemases are conserved mitogens: characterization of a Trypanosoma vivax proline racemase.

Trypanosoma cruzi proline racemases (TcPRAC) are the only eukaryotic proline racemases described so far. Except their role in the interconversion of free L- and D-proline enantiomers, parasite TcPRACs are involved in major T. cruzi biological pathways. These essential enzymes are implicated in the process of parasite differentiation and the acquisition of virulence during metacyclogenesis and are currently considered as key targets for drug development against Chagas' disease. In this study, we searched for the presence of TcPRAC gene homologues among other trypanosomatid genomes. Despite the high degree of gene synteny observed in Kinetoplastidae genomes, PRAC genes are missing in Trypanosoma brucei, Trypanosoma congolense and Leishmania spp. genomes. Interestingly, we identified a hypothetical PRAC gene in Trypanosoma vivax that is the major hemoparasite responsible for livestock trypanosomiasis, a serious economical impact for most of African and South American countries. We report here that the product of this T. vivax gene is bona fide a proline racemase with an activity comparable to the one we described previously for TcPRAC. Inhibition studies using the pyrrole-2-carboxylic acid confirmed that this compound is a competitive inhibitor for both TcPRAC and TvPRAC enzymes. Similarly to TcPRAC and all members of the racemase family studied so far in other pathogenic and nosocomial bacteria, our results show that TvPRAC is a T-cell-independent B-cell mitogen. Therefore the product of the novel TvPRAC gene identified in T. vivax and reported herein has the potential to be used as a drug target for this parasite-based trypanosomiasis.

Substrate-dependent modulation of enzyme activity by allosteric effector antibodies.

We investigate the kinetic effects of antibody variable domain fragments derived from heavy chain antibodies (VHH domains) that behave as allosteric effectors of the nucleoside hydrolase from Trypanosoma vivax (TvNH). Strikingly, these antibodies can stimulate or inhibit TvNH steady-state activity, depending on the substrate used. This effect was investigated in greater detail using steady-state and pre-steady-state kinetic experiments. The most potent allosteric effector, VHH 1589, inhibits certain steps on the TvNH catalytic pathway (e.g. N-glycosidic bond cleavage) but increases the rates of others (e.g. substrate and product release). For the natural nucleoside 7-methyl guanosine, where product ribose release is rate determining, the net effect of VHH 1589 binding is to increase k(cat). For the poor substrate pNPR, VHH 1589 causes chemistry (O-glycosidic bond cleavage) to become rate determining and both k(cat)/K(m) and k(cat) to decrease. Thus, the substrate-dependent effects of VHH 1589 binding are caused by differences in the relative rates of chemistry with respect to subsequent steps on the catalytic pathway for these two substrates. We discuss possible mechanisms for these kinetic effects and the implications for allosteric effector drug development.

Crystal structures of T. vivax nucleoside hydrolase in complex with new potent and specific inhibitors.

Diseases caused by parasitic protozoa remain a major health problem, mainly due to old toxic drugs and rising drug resistance. Nucleoside hydrolases are key enzymes of the purine salvage pathway of parasites from the Trypanosomatidae family and are considered as possible drug targets. N-Arylmethyl substituted iminoribitols have been developed as selective nanomolar affinity inhibitors against the purine-specific nucleoside hydrolase of Trypanosoma vivax. The current paper describes the crystal structures of the T. vivax nucleoside hydrolase in complex with two of these inhibitors, to 1.3 and 1.85 A resolution. These high resolution structures provide an accurate picture of the mode of binding of these inhibitors and their mechanism of transition-state mimicry, and are valuable tools to guide further inhibitor design. Comparison of the current structures with previously solved structures of the enzyme in complex with ground-state and transition-state-analogue inhibitors also allows for the elucidation of a detailed molecular mechanism of active-site loop opening/closing. These loop movements can be coupled to the complex kinetic mechanism of the T. vivax nucleoside hydrolase.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America--characterization, relationships and diagnostic implications.

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa.

A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax.

The nucleoside hydrolase of Trypanosoma vivax hydrolyzes the N-glycosidic bond of purine nucleosides. Structural and kinetic studies on this enzyme have suggested a catalytic role for a flexible loop in the vicinity of the active sites. Here we present the analysis of the role of this flexible loop via the combination of a proline scan of the loop, loop deletion mutagenesis, steady state and pre-steady state analysis, and x-ray crystallography. Our analysis reveals that this loop has an important role in leaving group activation and product release. The catalytic role involves the entire loop and could only be perturbed by deletion of the entire loop and not by single site mutagenesis. We present evidence that the loop closes over the active site during catalysis, thereby ordering a water channel that is involved in leaving group activation. Once chemistry has taken place, the loop dynamics determine the rate of product release.

N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase.

A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino-N-(8-quinolinyl)methyl-d-ribitol (UAMC-00115, K(i) 10.8nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.1nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.4nM) being the three most active compounds. Docking studies of the most active inhibitors revealed several important interactions with the enzyme. Among these interactions are aromatic stacking of the nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhibitors and amino acid residues in the active site. During the course of these docking studies we also identified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an interaction which has not previously been considered as being important.

Synthesis and biochemical evaluation of guanidino-alkyl-ribitol derivatives as nucleoside hydrolase inhibitors.

Nucleoside hydrolase (NH) is a key enzyme in the purine salvage pathway. The purine specificity of the IAG-NH from Trypanosoma vivax is at least in part due to cation-pi-stacking interactions. Guanidinium ions can be involved in cation-pi-stacking interactions, therefore a series of guanidino-alkyl-ribitol derivatives were synthesized in order to examine the binding affinity of these compounds towards the target enzyme. The compounds show moderate to good inhibiting activity towards the IAG-NH from T. vivax.

Examination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from Trypanosoma vivax.

The mechanism and energetics of the purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) are examined by stopped-flow at low temperatures. TvNH is shown to follow an ordered uni-bi kinetic mechanism and high forward commitment with inosine as substrate (C(f) = 1.9 +/- 0.6). Measurement of partitioning of the Michaelis complex, which exists at negligible concentrations in the steady state, is achieved using a novel sequential-mixing stopped-flow method. A product burst is observed with p-nitrophenyl riboside (pNPR) in the pre-steady state, indicating that a step after chemistry rate determines k(cat). Comparison of the kinetics of inosine and pNPR turnover shows that the dominant energetic contribution towards catalysis in TvNH comes from ribosyl and water activation (11 kcal/mol); however, leaving group activation still makes a considerable (8 kcal/mol) contribution. A solvent isotope effect ((D2O)k = 1.7) on the chemistry transient tau1 with guanosine as substrate was observed. Therefore, the leaving group is unlikely to be protonated prior to N-glycosidic bond cleavage. We propose that leaving group protonation is, by itself, unlikely to account for the large energetic contribution of leaving group activation. Instead, we postulate that active site binding interactions to the purine leaving group are required for efficient ribosyl and/or water activation.

Multiple transients in the pre-steady-state of nucleoside hydrolase reveal complex substrate binding, product base release, and two apparent rates of chemistry.

We have investigated the transient kinetics of the nucleoside hydrolase from Trypanosoma vivax (TvNH) at low temperatures (5 degrees C). Three novel absorbance transients (termed tau1, tau3, and tau4) were detected during multiple-guanosine turnover stopped-flow absorbance spectroscopy, in addition to a transient (tau2) that had been observed previously at 35 degrees C. At 5 degrees C, TvNH displays full-sites activity and not half-of-the-sites activity as is apparent at 35 degrees C. Both tau1 and tau2 are assigned to chemistry based on rapid-quench results. For tau1, the rate of chemistry is ca. 3000-fold faster than kcat (1-2 orders of magnitude greater than previous estimates). The pH dependencies of the burst amplitudes for tau1 and tau2 indicate that these transients arise from the formation of two different dimeric TvNH.substrate complexes and not from TvNH that contains kinetically asymmetric monomers. The saturating burst rates for tau1 and tau2 are surprisingly pH-independent, given the prominent role of acid-base chemistry in the proposed mechanism for TvNH. tau3 and tau4 are assigned to the substrate binding and base release processes, respectively, and shown to be equivalent to two fluorescence transients (tau3 and tau4, respectively) observed previously by stopped-flow methods at 35 degrees C. The rate of base release is shown to be an apparent rate. Together with steady-state product inhibition results, the data indicate that TvNH follows an ordered uni-bi kinetic mechanism with a TvNH.base dead-end complex, and not the rapid equilibrium random uni-bi mechanism proposed for other NHs. Two applicable kinetic models are presented and their implications for future mechanistic studies discussed.

Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis.

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity.

Characterization of sialidase from bloodstream forms of Trypanosoma vivax.

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.

Therapeutic nanoreactors: combining chemistry and biology in a novel triblock copolymer drug delivery system.

Triblock copolymeric nanoreactors are introduced as an alternative for liposomes as encapsulating carrier for prodrug activating enzymes. Inosine-adenosine-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in nanometer-sized vesicles constructed of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-(2-methyloxazoline) triblock copolymers. The nanoreactor is functionalized by incorporation of bacterial porins, OmpF or Tsx, in the reactor wall. Efficient cleavage of three natural substrates and one prodrug, 2-fluoroadenosine, by the nanoreactors was demonstrated.