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Angewandte Chemie (International Ed. in... Aug 2016The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the...
The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.
Topics: Animals; Enzyme Assays; Enzyme Inhibitors; Glutathione; Isoxazoles; Molecular Probes; Nuclear Magnetic Resonance, Biomolecular; Rats; gamma-Glutamyltransferase
PubMed: 27483206
DOI: 10.1002/anie.201603731 -
Structure (London, England : 1993) Jul 2016The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy...
The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26- to 4,900-fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Furthermore, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity.
Topics: Allosteric Regulation; Allosteric Site; Aminohydrolases; Aminoimidazole Carboxamide; Isoxazoles; Ligands; Molecular Docking Simulation; Ribonucleotides
PubMed: 27238967
DOI: 10.1016/j.str.2016.04.010 -
Biosensors & Bioelectronics Jul 2016The first near-infrared fluorescent probe with excellent water-solubility for γ-glutamyl transpeptidase (GGT) has been developed by combining glutathione (GSH) as a...
The first near-infrared fluorescent probe with excellent water-solubility for γ-glutamyl transpeptidase (GGT) has been developed by combining glutathione (GSH) as a recognition unit with a near-infrared hemicyanine fluorophore through an acrylyl linker. The probe exhibits a highly selective and sensitive fluorescent off-on response to GGT with a detection limit of 0.50U/L, and the response mechanism is based on the enzyme-catalyzed cleavage of the γ-glutamyl bond of GSH, followed by the spontaneous intramolecular cyclization and the release of the fluorophore. Notably, the probe has been used to image GGT in zebrafish and evaluate the inhibition ability of three common inhibitors of GGT both in vitro and in vivo, revealing that their inhibition efficiencies are acivicin >6-diazo-5-oxo-L-norleucine >L-serine-borate complex, and their corresponding IC50 values are 0.11±0.01mM, 0.34±0.04mM and 2.06±0.24mM, respectively. The proposed probe is simple, and may have great potential for screening GGT inhibitors.
Topics: Animals; Enzyme Assays; Enzyme Inhibitors; Fluorescent Dyes; Humans; Infrared Rays; Optical Imaging; Solubility; Spectrometry, Fluorescence; Water; Zebrafish; Zebrafish Proteins; gamma-Glutamyltransferase
PubMed: 26995285
DOI: 10.1016/j.bios.2016.03.021 -
ChemMedChem Jan 2016Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re-emphasized, the selective,...
Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re-emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3-chloro-4,5-dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3-halo-4,5-dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3-halo-4,5-dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.
Topics: Carbon-Nitrogen Ligases; Cysteine Proteases; Drug Design; Enzyme Inhibitors; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Isoxazoles; Molecular Conformation
PubMed: 26607551
DOI: 10.1002/cmdc.201500496 -
JAMA Dermatology May 2015The risk for herpes zoster (HZ) in patients with psoriasis treated with biologic medications or other systemic treatments has been given little attention to date.
IMPORTANCE
The risk for herpes zoster (HZ) in patients with psoriasis treated with biologic medications or other systemic treatments has been given little attention to date.
OBJECTIVE
To describe the risk for HZ in patients with psoriasis and its relation to treatment.
DESIGN, SETTING, AND PARTICIPANTS
A cohort study was performed using the administrative database of Clalit Health Services, the largest public health care provider organization in Israel, in the setting of general community clinics, primary care and referral centers, and ambulatory and hospitalized care. We extracted information for all patients who received a psoriasis diagnosis from January 2002 to June 2013. Follow-up was conducted until the end of July 2013. The study included 95,941 patients with psoriasis in the analysis, with 522,616 person-years of follow-up. Incidence of HZ events was calculated for each systemic antipsoriatic medication provided, during a follow-up period of 11 years and 7 months. We used a generalized estimating equation Poisson regression model to examine the effect of each systemic treatment for psoriasis on HZ incidence, adjusting for age, sex, psoriasis severity, Charlson comorbidity index, steroid treatment, and socioeconomic status.
MAIN OUTCOMES AND MEASURES
Incidence of HZ associated with systemic therapies.
RESULTS
In a multivariate analysis, it was observed that treatment with phototherapy (rate ratio [RR], 1.09 [95% CI, 0.62-1.93]; P = .99), methotrexate (RR, 0.98 [95% CI, 0.78-1.23]; P = .83), cyclosporine (RR, 1.16 [95% CI, 0.48-2.80]; P = .49), and biologic medications as a single agent (RR, 2.67 [95% CI, 0.69-10.3]; P = .14) was not associated with HZ. The use of combination treatment with biologic medications and methotrexate was significantly associated with an increased incidence of HZ (RR, 1.66 [95% CI, 1.08-2.57]; P = .02). The use of acitritin was associated with decreased incidence of HZ (RR, 0.69 [95% CI, 0.49-0.97]; P = .004).
CONCLUSIONS AND RELEVANCE
Physicians may need to consider offering an HZ preventive vaccine to patients receiving combination treatment with biologic medications and methotrexate, particularly if they have additional risk factors for HZ.
Topics: Adrenal Cortex Hormones; Antibodies, Monoclonal, Humanized; Biological Factors; Causality; Cohort Studies; Comorbidity; Cyclosporine; Dermatologic Agents; Drug Therapy, Combination; Female; Follow-Up Studies; Herpes Zoster; Herpes Zoster Vaccine; Humans; Incidence; Isoxazoles; Male; Methotrexate; Middle Aged; Multivariate Analysis; Phototherapy; Psoriasis; Risk Factors; Sex Distribution; Ustekinumab
PubMed: 25797026
DOI: 10.1001/jamadermatol.2014.4956 -
Chemical Science Dec 2014Acivicin is a natural product with diverse biological activities. Several decades ago its clinical application in cancer treatment was explored but failed due to...
Target discovery of acivicin in cancer cells elucidates its mechanism of growth inhibition†Electronic supplementary information (ESI) available: Synthesis, cloning, protein expression, purification and biochemical assays. See DOI: 10.1039/c4sc02339k.
Acivicin is a natural product with diverse biological activities. Several decades ago its clinical application in cancer treatment was explored but failed due to unacceptable toxicity. The causes behind the desired and undesired biological effects have never been elucidated and only limited information about acivicin-specific targets is available. In order to elucidate the target spectrum of acivicin in more detail we prepared functionalized derivatives and applied them for activity based proteomic profiling (ABPP) in intact cancer cells. Target deconvolution by quantitative mass spectrometry (MS) revealed a preference for specific aldehyde dehydrogenases. Further in depth target validation confirmed that acivicin inhibits ALDH4A1 activity by binding to the catalytic site. In accordance with this, downregulation of ALDH4A1 by siRNA resulted in a severe inhibition of cell growth and might thus provide an explanation for the cytotoxic effects of acivicin.
PubMed: 25580214
DOI: 10.1039/c4sc02339k -
Chemical Research in Toxicology Dec 2014Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible...
Electrophilicity of pyridazine-3-carbonitrile, pyrimidine-2-carbonitrile, and pyridine-carbonitrile derivatives: a chemical model to describe the formation of thiazoline derivatives in human liver microsomes.
Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure-property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile], and pyridines [5-p-tolylpicolinonitrile and 5-(4-fluorophenyl)picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.
Topics: Chromatography, Liquid; Humans; Magnetic Resonance Spectroscopy; Microsomes, Liver; Models, Chemical; Nitriles; Pyridazines; Pyridines; Pyrimidines; Spectrophotometry, Ultraviolet; Tandem Mass Spectrometry; Thiazoles
PubMed: 25372409
DOI: 10.1021/tx500256j -
Frontiers in Pharmacology 2014Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of...
Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of lung lining fluid (LLF) which contains an abundance of the antioxidant glutathione. LLF glutathione metabolism is regulated by γ-glutamyl transferase (GGT). Loss of LLF GGT activity in the mutant GGT(enu1) mouse causes an increase in baseline LLF glutathione content which is magnified in an IL-13 model of allergic airway inflammation and protective against asthma. Normal mice are susceptible to asthma in this model but can be protected with acivicin, a GGT inhibitor. GGT is a target to treat asthma but acivicin toxicity limits clinical use. GGsTop is a novel GGT inhibitor. GGsTop inhibits LLF GGT activity only when delivered through the airway. In the IL-13 model, mice treated with IL-13 and GGsTop exhibit a lung inflammatory response similar to that of mice treated with IL-13 alone. But mice treated with IL-13 and GGsTop show attenuation of methacholine-stimulated airway hyper-reactivity, inhibition of Muc5ac and Muc5b gene induction, decreased airway epithelial cell mucous accumulation and a fourfold increase in LLF glutathione content compared to mice treated with IL-13 alone. Mice treated with GGsTop alone are no different from that of mice treated with saline alone, and show no signs of toxicity. GGsTop could represent a valuable pharmacological tool to inhibit LLF GGT activity in pulmonary disease models. The associated increase in LLF glutathione can protect lung airway epithelial cells against oxidant injury associated with inflammation in asthma.
PubMed: 25132819
DOI: 10.3389/fphar.2014.00179 -
The Biochemical Journal Oct 2014Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid...
Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.
Topics: Amino Acid Transport Systems, Neutral; Amino Acids; Animals; Biological Transport, Active; Down-Regulation; Drug Resistance; Eflornithine; Enzyme Inhibitors; Hydrogen-Ion Concentration; Isoxazoles; Membrane Potentials; Protozoan Proteins; Trypanocidal Agents; Trypanosoma brucei brucei; Xenopus
PubMed: 24988048
DOI: 10.1042/BJ20140719 -
Letters in Applied Microbiology Oct 2014Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the...
Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 μmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.
Topics: Fusarium; Isoxazoles; Magnaporthe; Mycotoxins; Oryza; Plant Diseases; Spores, Fungal; Triticum; Virulence
PubMed: 24863673
DOI: 10.1111/lam.12289