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Journal of Medicinal Chemistry May 2020Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and...
Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.
Topics: Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Crystallography, X-Ray; Drug Design; Mesylates; Neprilysin; Peptidyl-Dipeptidase A; Protein Binding; Protein Structure, Secondary; Pyridines; Thiazepines; Tyrosine
PubMed: 32337993
DOI: 10.1021/acs.jmedchem.0c00441 -
Chemical & Pharmaceutical Bulletin 2017Acid-catalyzed allylating reagent 2,4,6-tris(allyloxy)-1,3,5-triazine (TriAT-allyl) and its substituted derivatives have been developed. The reaction of acid-, and...
Acid-catalyzed allylating reagent 2,4,6-tris(allyloxy)-1,3,5-triazine (TriAT-allyl) and its substituted derivatives have been developed. The reaction of acid-, and alkali-labile alcohols with these reagents in the presence of a catalytic amount of trifluoromethanesulfonic acid (TfOH) afforded the corresponding allyl ethers in good yields. Reactions using these reagents with an unsymmetrically-substituted regioisometric allyl group suggested that a single isometric allylic cation species would be involved.
Topics: Catalysis; Mesylates; Molecular Structure; Stereoisomerism; Triazines
PubMed: 28049907
DOI: 10.1248/cpb.c16-00744 -
The Biochemical Journal Dec 2003The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation,... (Review)
Review
The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) [Edge, Faltynek, Hof, Reichert, and Weber, (1981) Anal. Biochem. 118, 131-137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function.
Topics: Amino Acid Sequence; Bacterial Proteins; Enzymes; Epitopes; Glycoconjugates; Glycoproteins; Glycosylation; Mesylates; Plant Proteins; Protein Transport; Structure-Activity Relationship
PubMed: 12974674
DOI: 10.1042/BJ20030673 -
Journal of Visualized Experiments : JoVE Jan 2011This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4,...
This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.
Topics: Animals; Ethyl Methanesulfonate; Hermaphroditic Organisms; Male; Mutagenesis; Nematoda; Trioxsalen; Ultraviolet Rays
PubMed: 21248706
DOI: 10.3791/2293 -
Journal of Virology Aug 1976After treatment with methyl or ethyl methane sulfonate, T7 amber mutants display a reduced capacity for recombination. Moreover, alkylation reduces recombination...
After treatment with methyl or ethyl methane sulfonate, T7 amber mutants display a reduced capacity for recombination. Moreover, alkylation reduces recombination frequency involving markers on the right-hand side of the genetic map more than it reduces recombination frequency involving markers on the left-hand side. We interpret this to mean that alkylation can stop DNA injection at any point along the DNA molecule, and that T7 phage injects its DNA in a unique fashion starting from the end carrying the genes for early proteins.
Topics: Alkylation; Coliphages; DNA, Viral; Ethyl Methanesulfonate; Mesylates; Methyl Methanesulfonate; Mutation; Recombination, Genetic
PubMed: 183007
DOI: 10.1128/JVI.19.2.318-324.1976 -
Cancer Chemotherapy and Pharmacology Jan 2013Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is an alkylating agent with activity against renal cell carcinoma and is being evaluated clinically. To support... (Comparative Study)
Comparative Study
Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is an alkylating agent with activity against renal cell carcinoma and is being evaluated clinically. To support clinical trials, we developed an LC-MS/MS assay to detect and quantitate BEN and its metabolites/decomposition products. We tested the stability and products of BEN and benzoic acid dimethane sulfonate (BA) in plasma, blood and five renal carcinoma cell lines in vitro. Further, we determined the IC(50) of BEN, BA and four of their products in these cell lines. Low temperature and pH stabilized the analytes, and utilizing this resulted in an accurate, precise and reproducible assay. The half-lives of BEN and BA added to plasma in vitro were 220 and 5 min, while the half-life of BEN in whole blood was 18 min. The generation and degradation of up to 12 analytes were monitored, and structures confirmed with available authentic standards. The IC(50) for BEN was 5- to 500-fold lower than that of any of its products, while the cellular metabolic activity toward BEN correlated with ALDH activity and IC(50) values. We detected six of the in vitro products and their respective glucuronides in murine plasma after dosing BEN. The information gained from these experiments will be instrumental in the evaluation of the pharmacology of BEN in ongoing human trials.
Topics: Aldehyde Dehydrogenase; Animals; Antineoplastic Agents, Alkylating; Benzaldehydes; Carcinoma, Renal Cell; Cell Line, Tumor; Chromatography, Liquid; Female; Glucuronides; Half-Life; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Inhibitory Concentration 50; Kidney Neoplasms; Mesylates; Mice; Reproducibility of Results; Tandem Mass Spectrometry; Temperature; Time Factors; para-Aminobenzoates
PubMed: 23053264
DOI: 10.1007/s00280-012-1980-1 -
PloS One 2022Acid-sensing ion channels (ASICs) are neuronal, proton-gated, Na+-selective ion channels. They are involved in various physiological and pathological processes such as...
Acid-sensing ion channels (ASICs) are neuronal, proton-gated, Na+-selective ion channels. They are involved in various physiological and pathological processes such as neurodegeneration after stroke, pain sensation, fear behavior and learning. To obtain information on the activation mechanism of ASIC1a, we attempted in this study to impose distance constraints between paired residues in different channel domains by using cross-linkers reacting with engineered Cys residues, and we measured how this affected channel function. First, the optical tweezer 4'-Bis(maleimido)azobenzene (BMA) was used, whose conformation changes depending on the wavelength of applied light. After exposure of channel mutants to BMA, an activation of the channel by light was only observed with a mutant containing a Cys mutation in the extracellular pore entry, I428C. Western blot analysis indicated that BMA did not cross-link Cys428 residues. Extracellular application of methanethiosulfonate (MTS) cross-linkers of different lengths changed the properties of several Cys mutants, in many cases likely without cross-linking two Cys residues. Our observations suggest that intersubunit cross-linking occurred in the wrist mutant A425C and intrasubunit cross-linking in the acidic pocket mutant D237C/I312C. In these mutants, exposure to cross-linkers favored a non-conducting channel conformation and induced an acidic shift of the pH dependence and a decrease of the maximal current amplitude. Overall, the cross-linking approaches appeared to be inefficient, possibly due to the geometrical requirements for successful reactions of the two ends of the cross-linking compound.
Topics: Acid Sensing Ion Channels; Hydrogen-Ion Concentration; Mesylates; Optical Tweezers; Protein Structure, Tertiary
PubMed: 35802631
DOI: 10.1371/journal.pone.0270762 -
PloS One 2015Quantitatively, methanesulfonate (MSA) is a very relevant compound in the global biogeochemical sulfur cycle. Its utilization by bacteria as a source of carbon and...
Quantitatively, methanesulfonate (MSA) is a very relevant compound in the global biogeochemical sulfur cycle. Its utilization by bacteria as a source of carbon and energy has been described and a specific enzyme, methanesulfonate monooxygenase (MSAMO), has been found to perform the first catabolic step of its oxidation. Other proteins seemingly involved in the import of MSA into bacterial cells have been reported. In this study, we obtained novel sequences of genes msmA and msmE from marine, estuary and soil MSA-degraders (encoding the large subunit of the MSAMO enzyme and the periplasmic component of the import system, respectively). We also obtained whole-genome sequences of two novel marine Filomicrobium strains, Y and W, and annotated two full msm operons in these genomes. Furthermore, msmA and msmE sequences were amplified from North Atlantic seawater and analyzed. Good conservation of the MsmA deduced protein sequence was observed in both cultured strains and metagenomic clones. A long spacer sequence in the Rieske-type [2Fe-2S] cluster-binding motif within MsmA was found to be conserved in all instances, supporting the hypothesis that this feature is specific to the large (α) subunit of the MSAMO enzyme. The msmE gene was more difficult to amplify, from both cultivated isolates and marine metagenomic DNA. However, 3 novel msmE sequences were obtained from isolated strains and one directly from seawater. With both genes, our results combined with previous metagenomic analyses seem to imply that moderate to high-GC strains are somehow favored during enrichment and isolation of MSA-utilizing bacteria, while the majority of msm genes obtained by cultivation-independent methods have low levels of GC%, which is a clear example of the misrepresentation of natural populations that culturing, more often than not, entails. Nevertheless, the data obtained in this work show that MSA-degrading bacteria are abundant in surface seawater, which suggests ecological relevance for this metabolic group of bacteria.
Topics: Aquatic Organisms; Bacteria; Base Composition; DNA, Bacterial; Genome, Bacterial; Hyphomicrobiaceae; Mesylates; Metagenome; Mixed Function Oxygenases; Phylogeny; Seawater; Sequence Analysis, DNA
PubMed: 25978049
DOI: 10.1371/journal.pone.0125735 -
Poultry Science Feb 2022Hematology and serum biochemistry study may provide antique knowledge about the physical status of individuals, making them a valuable tool to differentiate healthy...
Hematology and serum biochemistry study may provide antique knowledge about the physical status of individuals, making them a valuable tool to differentiate healthy animals from affected animals. We aimed to investigate Steroid safety levels in birds through ex-situ studies at regular therapeutic doses. A total of 100 birds were used for hematology and serum biochemistry. This study was designed into 2 trials over the summer and winter, each comprised 5, 10, 15, and 20 d. Each study group was based on 5 control group birds and 20 experimental group birds. A sum of 2 groups representing 2 different steroids trial groups was treated with therapeutic doses to the stretch of 5, 10, 15, and 20 d each season. A therapeutic dose of each of the steroids was given at the rate of 3 drops 2 times a day to each bird. Analysis of data reveals that steroids had severe effects on bird's (Coturnix coturnix) hematological parameters. In most trials, the hematological effects of bromocriptine as mesylate showed an increase in red blood cell count and white blood cell count. On the other hand, steroid estradiol valerate showed a decrease in these parameters. Effect of steroids on serum biochemistry profile indicate acute damage to vital organs, especially to liver and kidney, indicating an increase in cholesterol, total protein, albumin, urea, and uric acid. The overall effect of steroids on the bird's serum and biochemistry of quails were nearly similar but different only in their intensity.
Topics: Animals; Bromocriptine; Chickens; Coturnix; Estradiol; Mesylates; Quail
PubMed: 34942520
DOI: 10.1016/j.psj.2021.101552 -
Journal of the American Veterinary... Feb 2022Doses of buffered tricaine methanesulfonate (MS-222) up to 1000 mg/L for 15 minutes are reported inefficient to produce euthanasia in goldfish. The goal of this study...
OBJECTIVE
Doses of buffered tricaine methanesulfonate (MS-222) up to 1000 mg/L for 15 minutes are reported inefficient to produce euthanasia in goldfish. The goal of this study was to determine if goldfish can be euthanized by more prolonged immersion in MS-222.
ANIMALS
24 healthy goldfish (weight range: 1 to 10 g) were randomly assigned to 4 groups of 6 fish.
PROCEDURES
The first group (G1) was exposed to 500 mg/L buffered MS-222 for 15 minutes then placed in freshwater for 3 hours. The second (G2) and third groups (G3) were exposed to 1000 mg/L of buffered MS-222 for 15 minutes then placed in freshwater for 3 hours and 18 hours respectively. The fourth group (G4) was exposed to 1000 mg/L of buffered MS-222 for 60 minutes then placed in freshwater for 3 hours. Time to cessation and return of operculation were recorded. If the goldfish did not resume operculation, heart rate was evaluated by Doppler ultrasonic flow detector.
RESULTS
Median times to apnea were 35 seconds at 1000 mg/L and 65 seconds at 500 mg/L. Re-operculation occurred only in G1 in 5 out of 6 individuals. All fish from G1, 3 fish from G2, 0 fish from G3, 1 fish from G4 had remaining heartbeats at the end of the observation period.
CLINICAL RELEVANCE
Overall, a dose of 1000 mg/L of buffered MS-222 for 15 minutes was efficient to euthanize juvenile goldfish at 20 °C. Different fish body mass and water quality parameters might explain different results compared to previous studies.
Topics: Aminobenzoates; Anesthetics, Local; Animals; Euthanasia, Animal; Goldfish; Immersion; Mesylates
PubMed: 35143411
DOI: 10.2460/javma.21.09.0416