Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies.

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(ß-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(ß-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Quercetin-enriched Lactobacillus aviarius alleviates hyperuricemia by hydrolase-mediated degradation of purine nucleosides.

The development of hyperuricemia (HUA) and gout is associated with dysbiosis of the gut microbiota. Quercetin can reduce serum uric acid levels and thus alleviate HUA by modulating the gut microbiota. However, the detailed mechanisms involved in this process are not fully understood. Here, we showed that quercetin significantly reduced the serum uric acid level in a chicken HUA model by altering the chicken cecal microbiota structure and function and increasing the abundance of Lactobacillus aviarius. An L. aviarius strain, CML180, was isolated from the quercetin-treated chicken gut microbiota. Strain characterization indicated that quercetin promoted the growth of L. aviarius CML180 and increased its adhesion, hydrophobicity, and co-aggregation abilities. Gavage of live L. aviarius CML180 to a mouse model of HUA-established by adenosine and potassium oxonate-reduced the serum uric acid level and alleviated HUA. The ability of L. aviarius CML180 to decrease the level of uric acid was due to its degradation of purine nucleosides, which are the precursors for uric acid production. A nucleoside hydrolase gene, nhy69, was identified from the genome of L. aviarius CML180, and the resulting protein, Nhy69, exhibited strong purine nucleoside-hydrolyzing activity at mesophilic temperature and neutral pH conditions. These findings provide mechanistic insights into the potential of quercetin to treat HUA or gout diseases via a specific gut microbe.

Purification of a non-specific nucleoside hydrolase from Alaska pea seeds.

A non-specific nucleoside hydrolase has been isolated from germinated Alaska pea seeds. The enzyme catalyzes the hydrolysis of both purines and pyrimidines along with ribo- and deoxyribonucleosides. A purification scheme utilized ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography, resulted in 103-fold purification with a recovery of 2.8%. The purified protein has a specific activity of 0.308 µmol/min•mg. The subunit molecular weight was 26103 Da and the enzyme exists as a dimer. The enzyme retains a significant amount of activity over a wide pH range with the maximum activity occurring at a pH of 6.0. The maximum activity was observed with adenosine as the substrate followed by inosine and guanosine, respectively. The Km for adenosine was 184 ±â€¯34 µM and for inosine 283 ±â€¯88 µM. In addition to the nucleoside hydrolase activity, adenosine deaminase activity was seen in the initial extract. Using adenosine as the substrate with the initial extract from the germinated seeds, the products adenine, inosine, and hypoxanthine were identified based on their retention times during reverse phase HPLC.

Expression, purification and spectrophotometric analysis of nucleoside hydrolase from Leishmania chagasi (LcNH).

Leishmaniasis represents an important public health problem in several countries. The main target in this study is the nucleoside hydrolase Leishmania chagasi (LcNH) that is responsible for causing visceral leishmaniasis, principally in Brazil. Nucleoside hydrolase enzymes are members of this pathway, hydrolyzing the N-glycosidic bond of ribonucleosides for the synthesis of nucleic acids. We present here for the first time, the expression and purification protocols to obtain the enzymes LcNH1 and LcNH2 that can be employed to explore novel strategies to produce nucleoside hydrolase inhibitors for use in chemotherapy. Protein integrity was also confirmed by SDS-PAGE gel, mass spectrometry and enzymatic activity.

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations.

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine-uridine nucleoside hydrolase (CU-NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU-NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU-NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU-NH are identified. Regardless of the wild-type or mutant CU-NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU-NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Identification of a 2'-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries.

Ribose methylation is among the most ubiquitous modifications found in RNA. 2'-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2'-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2'-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Combined virtual screening, MMPBSA, molecular docking and dynamics studies against deadly anthrax: An in silico effort to inhibit Bacillus anthracis nucleoside hydrolase.

Anthrax is a deadly disease caused by Bacillus anthracis, a dangerous biological warfare agent employed for both military and terrorist purposes. A critical selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. In the current study, we have performed molecular docking and dynamics studies, aiming to propose the new potent inhibitors of B. anthracis NH among National Cancer Institute (NCI) Diversity Set. We also analyzed the principal interactions of proposed compounds with the active site residues of NH and the relevant factors to biological activity. Additionally, the physic-chemical properties of free and inhibitor bound NH were evaluated and discussed. Our data showed that compound NSC79887 is a good candidate to inhibit NH and also for biological tests and further development. Also, ADMET prediction revealed that all physic-chemical parameters are within the acceptable range defined for human use.

A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2'-O-methylribonucleosides.

2'-O-Methylribonucleosides (2'-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2'-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2'-O-methyluridine (2'-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2'-OMe-UR to 2'-O-methylribose and uracil, and the kinetic constants were Km of 0.040 mM, kcat of 0.49 s(-1), and kcat/Km of 12 mM(-1) s(-1). In a substrate specificity analysis, LbNH preferred ribonucleosides and 2'-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2'-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes.

Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli.

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480µM. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5'-OH group or with the 2'-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose.

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram-negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ(Xoo)) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ(Xoo) does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca(2+) coordination shell of a ribose-binding site and the spacious active site in XopQ(Xoo), we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ(Xoo). Here, we report the crystal structure of XopQ(Xoo) in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca(2+) concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ(Xoo) with its ribosyl end tethered to the Ca(2+) coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand-induced conformational changes. These data showed that XopQ(Xoo) is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ(Xoo).

Crystal structure of the effector protein XOO4466 from Xanthomonas oryzae.

Many Gram-negative bacteria deliver their virulence factors into host cells through a secretion system. Those factors, called effector proteins, are involved in the pathogenicity in host cells by interfering with various cellular events. The phytopathogen Xanthomonas oryzae pv. oryzae uses a type III secretion system to inject its effectors, but the functional roles of these proteins remain largely uncharacterized. Here, we determined a crystal structure of XOO4466, an effector from X. oryzae pv. oryzae, and performed a functional analysis. We determined that XOO4466 is similar in sequence to Xanthomonas outer protein Q, a putative nucleoside hydrolase (NH). The overall structure of XOO4466 is homologous to that of NHs, including a metal-binding site, but differs in its oligomeric state and active site topology. Further analysis indicated that antiparallel ß-strands commonly found in NHs adjacent to the active site loop are replaced in XOO4466 with a short loop, causing the active site loop to adopt a conformation distinct from that of NHs. Thus, the catalytic residues emanating from the respective active site loop of NHs are absent in the putative active site of XOO4466. Consistent with these structural features, a functional assay indicated that XOO4466 does not exhibit NH activity and possibly catalyzes yet unknown reactions.

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

Process study of ceramic membrane-coupled mixed-cell fermentation for the production of adenine.

In order to solve the problems of high complexity, many by-products, high pollution and difficult extraction of the existing adenine production process, in this study, ceramic membrane-coupled mixed cell fermentation was used to produce adenine while reducing the synthesis of by-products and simplifying the production process of adenine. Nucleoside hydrolase (encoded by the rihC gene) was used to produce adenine by coordinated fermentation with the adenosine-producing bacterium Bacillus Subtilis XGL. The adenosine hydrolase (AdHy)-expressing strain Escherichia coli BL21-AdHy was successfully employed and the highest activity of the crude enzyme solution was found by orthogonal experiments at 170 W power, 42% duty cycle, and 8 min of sonication. The highest AdHy activity was found after 18 h of induction incubation. E. coli BL21-AdHy was induced for 18 h and sonicated under the above ultrasonic conditions and the resulting crude enzyme solution was used for co-fermentation of the strain and enzyme. Moreover, 15% (v/v) of the AdHy crude enzyme solution was added to fermentation of B. subtilis XGL after 35 h. Finally, the whole fermentation system was dialyzed using coupled ceramic membranes for 45 and 75 h, followed by the addition of fresh medium. In contrast, the AdHy crude enzyme solution was added after 35, 65, and 90 h of B. subtilis fermentation, with three additions of 15, 15, and 10% of the B. subtilis XGL fermentation system. The process was validated in a 5 L fermenter and 14 ± 0.25 g/L of adenine was obtained, with no accumulation of adenosine and d-ribose as by-products. The enzymatic activity of the AdHy crude solution treated with ultrasound was greatly improved. It also reduced the cellular activity of E. coli BL21-AdHy and reduced effects on bacterial co-fermentation. Membrane-coupled dialysis solved the problem of decreased yield due to poor bacterial survival and decreased viability, and eliminated inhibition of the product synthesis pathway by adenosine. The batch addition of crude enzyme broth allowed the continuous conversion of adenosine to adenine. This production method provides the highest yield of biologically produced adenine reported to date, reduces the cost of adenine production, and has positive implications for the industrial production of adenine by fermentation. And it provides a reference for producing other high-value-added products made by fermentation.

High-resolution inhibition profiling and ligand fishing for screening of nucleoside hydrolase ligands in Moringa oleifera Lamarck.

In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.

Nucleoside Metabolism Is Induced in Common Bean During Early Seedling Development.

Nucleoside hydrolases (NSH; nucleosidases) catalyze the cleavage of nucleosides into ribose and free nucleobases. These enzymes have been postulated as key elements controlling the ratio between nucleotide salvage and degradation. Moreover, they play a pivotal role in ureidic legumes by providing the substrate for the synthesis of ureides. Furthermore, nucleotide metabolism has a crucial role during germination and early seedling development, since the developing seedlings require high amount of nucleotide simultaneously to the mobilization of nutrient in cotyledons. In this study, we have cloned two nucleosidases genes from Phaseolus vulgaris, PvNSH1 and PvNSH2, expressed them as recombinant proteins, and characterized their catalytic activities. Both enzymes showed a broad range of substrate affinity; however, PvNSH1 exhibited the highest activity with uridine, followed by xanthosine, whereas PvNSH2 hydrolyses preferentially xanthosine and shows low activity with uridine. The study of the regulation of nucleosidases during germination and early postgerminative development indicated that nucleosidases are induced in cotyledons and embryonic axes just after the radicle emergence, coincident with the induction of nucleases activity and the synthesis of ureides in the embryonic axes, with no remarkable differences in the level of expression of both nucleosidase genes. In addition, nucleosides and nucleobase levels were determined as well in cotyledons and embryonic axes. Our results suggest that PvNSH1 and PvNSH2 play an important role in the mobilization of nutrients during this crucial stage of plant development.

Evolution of chromone-like compounds as potential antileishmanial agents, through the 21st century.

INTRODUCTION: Leishmaniasis is one of the most neglected diseases of modern times that mainly affects people from developing countries, with approximately 350 million people considered at risk of developing leishmaniasis. Therefore, the development of novel antileishmanial treatments is becoming the focus of numerous research groups, with the support of the World Health Organization, which hopes to eradicate this disease in the near future. AREAS COVERED: This review focuses on the interest of chromones for the development of future treatments against leishmaniasis. In addition to plant-based chromone derivatives, structure-activity relationship studies that aim to identify the optimal structural features of the chromones' antileishmanial activity are also described and discussed. EXPERT OPINION: The numerous examples of chromones depicted in this paper, allied with the SAR studies presented herein, suggest that the chromone scaffold is a privileged core for the design and development of novel antileishmanial agents. However, some concerns have been raised concerning the considerable variability observed in the results throughout the scientific bibliography. These inconsistencies may explain the absence of pharmacodynamic and pharmacokinetic studies as well as clinical trials.

Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut.

Although the gut microbiome is generally symbiotic or commensal, some microbiome members become pathogenic under certain circumstances. However, the factors driving this pathogenic switch are largely unknown. Pathogenic bacteria can generate uracil that triggers host dual oxidase (DUOX) to produce antimicrobial reactive oxygen species (ROS). We show that pathogens generate uracil and ribose upon nucleoside catabolism of gut luminal uridine, which triggers not only host defenses but also inter-bacterial communication and pathogenesis in Drosophila. Uridine-derived uracil triggers DUOX-dependent ROS generation, whereas ribose induces bacterial quorum sensing (QS) and virulence gene expression. Genes implicated in nucleotide metabolism are found in pathogens but not commensal bacteria, and their genetic ablation blocks QS and the commensal-to-pathogen transition in vivo. Furthermore, commensal bacteria lack functional nucleoside catabolism, which is required to achieve gut-microbe symbiosis, but can become pathogenic by enabling nucleotide catabolism. These findings reveal molecular mechanisms governing the commensal-to-pathogen transition in different contexts of host-microbe interactions.

The F1F3 Recombinant Chimera of Leishmania donovani-Nucleoside Hydrolase (NH36) and Its Epitopes Induce Cross-Protection Against Leishmania (V.) braziliensis Infection in Mice.

Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ/IL-10 and TNF-α/IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis.

New nucleoside hydrolase with transribosylation activity from Agromyces sp. MM-1 and its application for enzymatic synthesis of 2'-O-methylribonucleosides.

Microorganisms were screened for transribosylation activity between 2'-O-methyluridine (2'-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2'-O-methylribonucleosides (2'-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2'-O-methyladenosine (2'-OMe-AR) when whole cells were used in a reaction mixture containing 2'-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed AgNH. AgNH was a bi-functional enzyme catalyzing both hydrolysis towards 2'-OMe-NRs and transribosylation between 2'-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, AgNH preferred guanosine analogues as its substrates. In the transribosylation reaction, AgNH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2'-OMe-UR and 6-chloroguanine was determined to 2'-O-methyl-6-chloroguanosine (2'-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2'-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L.

Druggability of the guanosine/adenosine/cytidine nucleoside hydrolase from Trichomonas vaginalis.

Trichomonas vaginalis infects approximately 300 million people worldwide annually. Infected individuals have a higher susceptibility to more serious conditions such as cervical and prostate cancer. The parasite has developed increasing resistance to current drug therapies, with an estimated 5% of clinical cases resulting from resistant strains, creating the need for new therapeutic strategies with novel mechanisms of action. Nucleoside salvage pathway enzymes represent novel drug targets as these pathways are essential for the parasite's survival. The guanosine/adenosine/cytidine nucleoside hydrolase (GACNH) may be particularly important as its expression is upregulated under glucose-limiting conditions mimicking those that occur during infection establishment. GACNH was screened against the NIH Clinical Collection to explore its druggability. Seven compounds were identified with IC50 values <20 µM. Extensive overlap was found between inhibitors of GACNH and the adenosine/guanosine nucleoside hydrolase (AGNH), but no overlap was found with inhibitors of the uridine nucleoside hydrolase. The guanosine analog ribavirin was the only compound found to be specific for GACNH. Compounds that inhibit both AGNH and GACNH purine salvage pathway enzymes may prove critical given the role that GACNH appears to play in the early stages of infection.