ABSTRACT
Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen that identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib inhibits SARS-CoV-2 and MERS-CoV Mac1 but not the closest human homologue, MacroD2. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for the screening of large compound libraries to identify improved macrodomain inhibitors and to explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.
Subject(s)
Antiviral Agents/pharmacology , High-Throughput Screening Assays/methods , N-Glycosyl Hydrolases/antagonists & inhibitors , SARS-CoV-2/drug effects , Dasatinib/pharmacology , Protein Domains , SARS-CoV-2/enzymologyABSTRACT
Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.
Subject(s)
Antineoplastic Agents/pharmacology , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Apoptosis , CRISPR-Cas Systems , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Replication , DNA, Neoplasm/metabolism , Deoxycytidine Monophosphate/analogs & derivatives , Deoxycytidine Monophosphate/metabolism , Deoxycytidine Monophosphate/pharmacology , Deoxyuracil Nucleotides/metabolism , Drug Resistance, Neoplasm , Genes, BRCA1 , Humans , Hydrolysis , N-Glycosyl Hydrolases/genetics , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins/genetics , Synthetic Lethal Mutations , Thymidine/analogs & derivatives , Thymidine/antagonists & inhibitors , Thymidine/metabolism , Thymidine/pharmacology , Uracil-DNA Glycosidase/metabolismABSTRACT
Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.
Subject(s)
Biofilms/drug effects , Enzyme Inhibitors/pharmacology , Fluorouracil/pharmacology , High-Throughput Screening Assays/methods , Methicillin-Resistant Staphylococcus aureus/drug effects , Quorum Sensing/drug effects , Animals , Bacterial Proteins/antagonists & inhibitors , Carbon-Sulfur Lyases/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Female , Fluorouracil/therapeutic use , HEK293 Cells , Homoserine/analogs & derivatives , Homoserine/biosynthesis , Humans , Lactones , Methicillin-Resistant Staphylococcus aureus/metabolism , Mice, Inbred C57BL , N-Glycosyl Hydrolases/antagonists & inhibitors , Small Molecule Libraries , Staphylococcal Infections/prevention & controlABSTRACT
The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.
Subject(s)
DNA, Single-Stranded/metabolism , N-Glycosyl Hydrolases/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , DNA/metabolism , DNA Replication , DNA, Single-Stranded/chemistry , Humans , Mice , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Zinc FingersABSTRACT
Nucleoside hydrolases are a strategic target for the development of drugs to treat leishmaniasis, a neglected disease that affects 700 thousand to one million people annually. The present study aimed to identify Leishmania donovani nucleoside hydrolase (LdNH) inhibitors from the leaves of Ormosia arborea, a tree endemic to Brazilian ecosystems, through a strategy based on 1H NMR analyses and chemometrics. The aqueous EtOH extract of O. arborea leaves inhibited LdNH activity by 95%. The extract was fractionated in triplicate (13 in each step, making a total of 39 fractions). Partial least squares discriminant analysis (PLS-DA) was used to correlate the 1H NMR spectra of the fractions with their LdNH inhibitory activity and thus to identify the spectral regions associated with the bioactivity. The strategy aimed at isolating the probable bioactive substances and led to two new A-type proanthocyanidins, linked to a p-coumaroyl unit (1 and 2), which appeared as noncompetitive inhibitors of LdNH (IC50: 28.2 ± 3.0 µM and 25.6 ± 4.1 µM, respectively). This study confirms the usefulness of the NMR-based chemometric methods to accelerate the discovery of drugs from natural products.
Subject(s)
Fabaceae/chemistry , Leishmania donovani/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Brazil , Ecosystem , Fabaceae/metabolism , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/metabolismABSTRACT
BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4⯵g/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Borrelia burgdorferi/enzymology , Lyme Disease/drug therapy , N-Glycosyl Hydrolases/genetics , Borrelia burgdorferi/drug effects , Borrelia burgdorferi/pathogenicity , Deoxyadenosines/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic/drug effects , Humans , Lyme Disease/enzymology , Lyme Disease/microbiology , N-Glycosyl Hydrolases/antagonists & inhibitors , S-Adenosylhomocysteine/metabolism , Thionucleosides/metabolismABSTRACT
NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 µM and 250 µM, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.
Subject(s)
Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , N-Glycosyl Hydrolases/antagonists & inhibitors , Biological Assay , Drug Discovery , Enzyme Inhibitors , Escherichia coli/metabolism , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , N-Glycosyl Hydrolases/metabolismABSTRACT
NH36 is a vital enzyme of the DNA metabolism and a specific target for anti-Leishmania chemotherapy. We developed second-generation vaccines composed of the FML complex or its main native antigen, the NH36 nucleoside hydrolase of Leishmania (L.) donovani and saponin, and a DNA vaccine containing the NH36 gene. All these vaccines were effective in prophylaxis and treatment of mice and dog visceral leishmaniasis (VL). The FML-saponin vaccine became the first licensed veterinary vaccine against leishmaniasis (Leishmune®) which reduced the incidence of human and canine VL in endemic areas. The NH36, DNA or recombinant protein vaccines induced a Th1 CD4+IFN-γ+ mediated protection in mice. Efficacy against VL was mediated by a CD4+TNF-α T lymphocyte response against the NH36-F3 domain, while against tegumentary leishmaniasis (TL) a CD8+ T lymphocyte response to F1 was also required. These domains were 36-41 % more protective than NH36, and a recombinant F1F3 chimera was 21% stronger than the domains, promoting a 99.8% reduction of the parasite load. We also identified the most immunogenic NH36 domains and epitopes for PBMC of active human VL, cured or asymptomatic and DTH+ patients. Currently, the NH36 subunit recombinant vaccine is turning into a multi-epitope T cell synthetic vaccine against VL and TL.
Subject(s)
Epitopes, T-Lymphocyte/immunology , Leishmania/enzymology , Leishmaniasis Vaccines/immunology , Leishmaniasis/immunology , N-Glycosyl Hydrolases/immunology , Animals , Antiprotozoal Agents/pharmacology , Dog Diseases/immunology , Dog Diseases/parasitology , Dog Diseases/prevention & control , Dogs , Humans , Leishmania/immunology , Leishmaniasis Vaccines/genetics , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/prevention & control , Leishmaniasis, Visceral/veterinary , Mice , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/geneticsABSTRACT
Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis and is the most prevalent, nonviral sexually transmitted disease. The parasite has shown increasing resistance to the current 5-nitroimidazole therapies indicating the need for new therapies with different mechanisms. T. vaginalis is an obligate parasite that scavenges nucleosides from host cells and then uses salvage pathway enzymes to obtain the nucleobases. The adenosine/guanosine preferring nucleoside ribohydrolase was screened against a 2000-compound diversity fragment library using a 1H NMR-based activity assay. Three classes of inhibitors with more than five representatives were identified: bis-aryl phenols, amino bicyclic pyrimidines, and aryl acetamides. Among the active fragments were 10 compounds with ligand efficiency values greater than 0.5, including five with IC50 values <10 µM. Jump-dilution and detergent counter screens validated reversible, target-specific activity. The data reveals an emerging SAR that is guiding our medicinal chemistry efforts aimed at discovering compounds with nanomolar potency.
Subject(s)
Antiprotozoal Agents/chemistry , Enzyme Inhibitors/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Trichomonas vaginalis/enzymology , Antiprotozoal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Female , Humans , Ligands , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trichomonas Vaginitis/parasitology , Trichomonas vaginalis/chemistry , Trichomonas vaginalis/drug effects , Trichomonas vaginalis/geneticsABSTRACT
It is reasonable to consider that Type I-ribosomal inactivation proteins (RIP) retain some specific affinity to harmful pathogens to complete the role as a bio-defense relating protein. In the present studies, it was shown that two Type I-RIPs, α- and ß-momorcharins, maintained the abilities to bind with N-acetylglucosamine (NAG) to change the conformation around the active sites and to regulate their N-glycosidase activities. By the binding of NAG, the freedom of internal motion of Trp192 in α-momorcharin was increased 1.5 times near the active site and, on the other hand, the corresponding motion of Trp190 was limited 50% in ß-momorcharin. The results in the fluorescence resonance excitation energy transfer experiments demonstrated that Trp-190 of ß-momorcharin was kept away from Tyr-70 but Trp192 contrarily approached closer to the nearest neighboring Tyr residue consisting of the active center of α-momorcharin by the binding with NAG. These conformational changes near the active site close correlated with promotion and/or suppression of the N-glucosidase activities of ß- and α-momorcharins.
Subject(s)
Momordica charantia/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Ribosome Inactivating Proteins/pharmacology , Seeds/chemistry , Catalytic Domain/drug effects , Ligands , Models, Molecular , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/metabolism , Protein Conformation , Ribosome Inactivating Proteins/chemistryABSTRACT
Campylobacter jejuni is a Gram-negative bacterium responsible for food-borne gastroenteritis and associated with Guillain-Barré, Reiter, and irritable bowel syndromes. Antibiotic resistance in C. jejuni is common, creating a need for antibiotics with novel mechanisms of action. Menaquinone biosynthesis in C. jejuni uses the rare futalosine pathway, where 5'-methylthioadenosine nucleosidase ( CjMTAN) is proposed to catalyze the essential hydrolysis of adenine from 6-amino-6-deoxyfutalosine to form dehypoxanthinylfutalosine, a menaquinone precursor. The substrate specificity of CjMTAN is demonstrated to include 6-amino-6-deoxyfutalosine, 5'-methylthioadenosine, S-adenosylhomocysteine, adenosine, and 5'-deoxyadenosine. These activities span the catalytic specificities for the role of bacterial MTANs in menaquinone synthesis, quorum sensing, and S-adenosylmethionine recycling. We determined inhibition constants for potential transition-state analogues of CjMTAN. The best of these compounds have picomolar dissociation constants and were slow-onset tight-binding inhibitors. The most potent CjMTAN transition-state analogue inhibitors inhibited C. jejuni growth in culture at low micromolar concentrations, similar to gentamicin. The crystal structure of apoenzyme C. jejuni MTAN was solved at 1.25 Å, and five CjMTAN complexes with transition-state analogues were solved at 1.42 to 1.95 Å resolution. Inhibitor binding induces a loop movement to create a closed catalytic site with Asp196 and Ile152 providing purine leaving group activation and Arg192 and Glu12 activating the water nucleophile. With inhibitors bound, the interactions of the 4'-alkylthio or 4'-alkyl groups of this inhibitor family differ from the Escherichia coli MTAN structure by altered protein interactions near the hydrophobic pocket that stabilizes 4'-substituents of transition-state analogues. These CjMTAN inhibitors have potential as specific antibiotic candidates against C. jejuni.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , N-Glycosyl Hydrolases/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Campylobacter jejuni/enzymology , Catalytic Domain , Enzyme Inhibitors/chemistry , Kinetics , Molecular Structure , N-Glycosyl Hydrolases/chemistry , Pyrimidines/chemistry , Pyrroles/chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
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.
Subject(s)
N-Glycosyl Hydrolases/metabolism , Protozoan Proteins/metabolism , Trichomonas vaginalis/enzymology , Adenosine/analogs & derivatives , Adenosine/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Quercetin/analogs & derivatives , Quercetin/chemistry , Quercetin/metabolism , Ribavirin/chemistry , Ribavirin/metabolism , Structure-Activity RelationshipABSTRACT
The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 5'-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations.
Subject(s)
Adenosine/pharmacology , Enzyme Inhibitors/pharmacology , N-Glycosyl Hydrolases/antagonists & inhibitors , Phosphates/pharmacology , Adenosine/chemical synthesis , Adenosine/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , N-Glycosyl Hydrolases/metabolism , Phosphates/chemistry , Protein Processing, Post-Translational/drug effects , Structure-Activity RelationshipABSTRACT
N-Acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors both as tools for studying the chemical biology of autolysins and also as antibacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (21) and fgka (5), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 µM, respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrates a propensity for cells to form linked chains, suggesting impaired cell growth and division.
Subject(s)
Acetylglucosaminidase/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Azo Compounds/chemical synthesis , Bacillus subtilis/drug effects , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , N-Glycosyl Hydrolases/antagonists & inhibitors , Acetylglucosaminidase/genetics , Acetylglucosaminidase/metabolism , Anti-Bacterial Agents/pharmacology , Azo Compounds/pharmacology , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Division/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression , Hydrolysis , Microbial Sensitivity Tests , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Peptidoglycan/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Anthrax/prevention & control , Bacillus anthracis/enzymology , Computer Simulation , Models, Molecular , N-Glycosyl Hydrolases/antagonists & inhibitors , Anthrax/drug therapy , Bacillus anthracis/drug effects , Bacterial Proteins/antagonists & inhibitors , Catalytic Domain , Drug Design , Humans , Molecular Docking Simulation , Molecular Dynamics SimulationABSTRACT
Leishmania is a genus of trypanosomes, which are responsible for leishmaniasis disease, a major trypanosome infection in humans. In recent years, published studies have shown that the search for new drugs for Leishmania treatments has intensified. Through technique modeling it has been possible to develop new compounds, which act as nucleoside hydrolase (NH) inhibitors. The effect of these enzymes is the hydrolysis of certain RNA nucleotides, which include uridine and inosine, necessary for the protozoa to transform certain nucleosides obtained from infected individuals into nucleobases for the preparation of their DNA. The obtention of NH inhibitors is very important to eliminate leishmaniasis disease in infected individuals. The aim of this study is to discuss the research and development of new agents for the treatment of Leishmania, and to stimulate the formulation of new NH inhibitors.
Subject(s)
Leishmania/enzymology , N-Glycosyl Hydrolases/metabolism , Protozoan Proteins/metabolism , Animals , Catalytic Domain , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Leishmaniasis/drug therapy , Leishmaniasis/parasitology , Molecular Conformation , Molecular Docking Simulation , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Ribitol/analogs & derivatives , Ribitol/chemistry , Ribitol/metabolismABSTRACT
Nucleotide metabolism is the target of a large number of anticancer drugs including antimetabolites and specific enzyme inhibitors. We review scientific findings that over the last 10-15 years have allowed the identification of several intracellular nucleotide-degrading enzymes as cancer drug targets, and discuss further potential therapeutic applications for Rcl, SAMHD1, MTH1 and cN-II. We believe that enzymes involved in nucleotide metabolism represent potent alternatives to conventional cancer chemotherapy targets.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Neoplasms , Nucleotides/metabolism , 5'-Nucleotidase/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , DNA Repair Enzymes/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Humans , Molecular Targeted Therapy , Monomeric GTP-Binding Proteins/antagonists & inhibitors , N-Glycosyl Hydrolases/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/enzymology , Nuclear Proteins/antagonists & inhibitors , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , SAM Domain and HD Domain-Containing Protein 1ABSTRACT
Nucleoside salvage pathway enzymes used by Trichomonas vaginalis are distinct from the pathway involved in activation of existing 5-nitroimidazole drugs. They thus represent excellent targets for developing novel, mechanism-based antitrichomonal agents. The purine-specific adenosine/guanosine preferring ribohydrolase (AGNH) was screened against the NIH Clinical Collection to assess its druggability. Eight compounds, including five flavonoids, were identified with IC50 values ⩽10 µM and confirmed in counter screens run in the presence of detergent. The inhibitors are structurally distinct from inhibitors of the pyrimidine-specific uridine ribohydrolase (UNH) thus indicating that AGNH is a distinct, druggable target from UNH.
Subject(s)
Antitrichomonal Agents/chemistry , Flavonoids/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/chemistry , High-Throughput Screening Assays , Quercetin/analogs & derivatives , Quercetin/chemistry , Small Molecule Libraries/chemistry , Stereoisomerism , Trichomonas vaginalisABSTRACT
Transition-state analysis based on kinetic isotope effects and computational chemistry provides electrostatic potential maps to serve as blueprints for the design and chemical synthesis of transition-state analogues. The utility of these molecules is exemplified by potential clinical applications toward leukemia, autoimmune disorders, gout, solid tumors, bacterial quorum sensing and bacterial antibiotics. In some cases, transition-state analogues have chemical features that have allowed them to be repurposed for new indications, including potential antiviral use. Three compounds from this family have entered clinical trials. The transition-state analogues bind to their target proteins with high affinity and specificity. The physical and structural properties of binding teach valuable and often surprising lessons about the nature of tight-binding inhibitors.
Subject(s)
Adenine/analogs & derivatives , Enzyme Inhibitors/chemistry , Pyrrolidines/chemistry , Adenine/chemistry , Adenine/metabolism , Adenosine/analogs & derivatives , Animals , Enzyme Inhibitors/metabolism , Humans , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/metabolism , Protein Binding , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/metabolism , Pyrrolidines/metabolism , Substrate Specificity , ThermodynamicsABSTRACT
Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 µM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 µM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 µM for IA and 0.019 µM for IH). IA, IH and SMIH at 10 µM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.