Search

The present paper investigated the electrochemical behaviour of Schiff base and its zinc complex at a working electrode by cyclic voltammetry technique. In the phosphate and Britton-Robinson buffers, the electrochemical study of Schiff base was performed within various pH values 5-9. Cyclic voltammograms of the examined Schiff base exhibited irreversible and diffusion controlled waves at different sweep rates, which is associated with the two electron reduction process of azomethine group. The symmetry transfer coefficient (αn), the diffusion coefficient (D01/2) and rate constant (k°f,h) of the reactant species were calculated. Cyclic voltammetric study of zinc complex showed one electron diffusion controlled, quasi-reversible peaks at 50-250 mV/s sweep rates. Antimicrobial study of zinc complex showed that zinc complex found to be more potent than uncoordinated Schiff base.

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

Protein S-sulphenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulphenic acids. Mapping the specific sites of protein S-sulphenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulphenylation using sulphenic acid-specific chemical probes and mass spectrometry-based proteomics. The major steps in this protocol are (1) optimization of conditions for selective labeling of cysteine S-sulphenic acids in intact cells with the commercially available dimedone-based probe, DYn-2, (2) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(I)-catalyzed azide-alkyne cycloaddition reaction, (3) enrichment of the biotin-tagged tryptic peptides with streptavidin, (4) liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics and (5) computational data analysis. We also outline strategies for quantitative analysis of this modification in cells responding to redox perturbations and discuss special issues pertaining to experimental design of thiol redox studies. Our chemoproteomic platform should be broadly applicable to the investigation of other bioorthogonal-chemically engineered posttranslational modifications. The entire analysis protocol takes about 1 week to complete.

Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

Monoamine oxidase A (MAOA) plays important roles in the pathogenesis of several neurological and cardiovascular disorders. The mechanism of transcriptional regulation of MAOA under basal and pathological conditions, however, remains incompletely understood. Here, we report systematic identification and characterization of cis elements and transcription factors that govern the expression of MAOA gene. Extensive computational analysis of MAOA promoter, followed by 5?-promoter deletion/reporter assays, revealed that the -71/-40 bp domain was sufficient for its basal transcription. Gel-shift and chromatin immunoprecipitation assays provided evidence of interactions of the transcription factors GATA-binding protein 2 (GATA2), Sp1 and TATA-binding protein (TBP) with this proximal promoter region. Consistently, over-expression of GATA2, Sp1 and TBP augmented MAOA promoter activity in a coordinated manner. In corroboration, siRNA-mediated down-regulation of GATA2/Sp1/TBP repressed the endogenous MAOA expression as well as transfected MAOA promoter activity. Tumor necrosis factor-? and forskolin activated MAOA transcription that was reversed by Sp1 siRNA, in support, tumor necrosis factor-?- and forskolin-induced activities were enhanced by ectopic over-expression of Sp1. On the other hand, MAOA transcription was diminished upon exposure of neuroblasts or cardiac myoblasts to ischemia-like conditions because of reduced binding of GATA2/Sp1/TBP with MAOA promoter. In conclusion, this study revealed previously unknown roles of GATA2, Sp1 and TBP in modulating MAOA expression under basal as well as pathophysiological conditions such as inflammation and ischemia, thus providing new insights into the molecular basis of aberrant MAOA expression in neuronal/cardiovascular disease states. � 2015 International Society for Neurochemistry.

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in “druggable” proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand–protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable “redoxome” with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function.

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5?-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions. � 2014 American Chemical Society.

Owing to its labile nature, a new role for cysteine sulfenic acid (-SOH) modification has emerged. This oxidative modification modulates protein function by acting as a redox switch during cellular signaling. The identification of proteins that undergo this modification represents a methodological challenge, and its resolution remains a matter of current interest. The development of strategies to chemically modify cysteinyl-containing peptides for LC-MS/MS analysis has increased significantly within the past decade. The method of choice to selectively label sulfenic acid is based on the use of dimedone or its derivatives. For these chemical probes to be effective on a proteome-wide level, their reactivity toward -SOH must be high to ensure reaction completion. In addition, the presence of an adduct should not interfere with electrospray ionization, the efficiency of induced dissociation in MS/MS experiments, or with identification of Cys-modified peptides by automated database searching algorithms. Herein, we employ a targeted proteomics approach to study the electrospray ionization and fragmentation effects of different –SOH specific probes, and compared them to commonly used alkylating agents. We then extend our study to a whole proteome extract using shotgun proteomic approaches. These experiments enable us to demonstrate that dimedone adducts do not interfere with electrospray by suppressing the ionization nor impedes product ion assignment by automated search engines, which detect a + 138 Da increase from unmodified peptides. Collectively, these results suggest dimedone can be a powerful tool to identify sulfenic acid modifications by high-throughput shotgun proteomics of a whole proteome.

Background Reactive oxygen species-mediated cysteine sulfenic acid modification has emerged as an important regulatory mechanism in cell signaling. The stability of sulfenic acid in proteins is dictated by the local microenvironment and ability of antioxidants to reduce this modification. Several techniques for detecting this cysteine modification have been developed, including direct and in situ methods. Scope of review This review presents a historical discussion of sulfenic acid chemistry and highlights key examples of this modification in proteins. A comprehensive survey of available detection techniques with advantages and limitations is discussed. Finally, issues pertaining to rates of sulfenic acid formation, reduction, and chemical trapping methods are also covered. Major conclusions Early chemical models of sulfenic acid yielded important insights into the unique reactivity of this species. Subsequent pioneering studies led to the characterization of sulfenic acid formation in proteins. In parallel, the discovery of oxidant-mediated cell signaling pathways and pathological oxidative stress has led to significant interest in methods to detect these modifications. Advanced methods allow for direct chemical trapping of protein sulfenic acids directly in cells and tissues. At the same time, many sulfenic acids are short-lived and the reactivity of current probes must be improved to sample these species, while at the same time, preserving their chemical selectivity. Inhibitors with binding scaffolds can be rationally designed to target sulfenic acid modifications in specific proteins. General significance Ever increasing roles for protein sulfenic acids have been uncovered in physiology and pathology. A more complete understanding of sulfenic acid-mediated regulatory mechanisms will continue to require rigorous and new chemical insights. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

2,3-Diarylbenzofurans are efficiently generated by the cyclization of ortho-benzyloxybenzophenones using the hindered phosphazene base P 4 - t -Bu.

A sensitive probe that detects protein sulfenylation in cells reveals that sulfenylation of the active site cysteine in EGFR enhances its kinase activity. Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor–mediated signaling results in H2O2 production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H2O2 at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.

The reaction of phenols with 7a led to the synthesis of aurones, while the reaction of phenols with 7b led to the synthesis of flavones.

The effects of substituents on the aryl ring were studied by the preparation and testing of several PD173955 analogs. Inserting a single carbon atom into the C-N bond in the aniline subunit (PDC) reduced the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955, PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs with amino groups in place of the methylthio group are 10 times more inhibitory than the parent molecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for Imatinib.

Dihydroindolo[2,1-a]isoquinolines were synthesized from tetrahydroisoquinolines and alpha-fluoroaldehydes by a novel two-step procedure. These compounds exhibited significant immunosuppressive activity against IL-2, IL-10 and IFN-gamma.

Cysteine S-sulphenylation provides redox regulation of protein functions, but the global cellular impact of this transient post-translational modification remains unexplored. We describe a chemoproteomic workflow to map and quantify over 1,000 S-sulphenylation sites on more than 700 proteins in intact cells. Quantitative analysis of human cells stimulated with hydrogen peroxide or epidermal growth factor measured hundreds of site selective redox changes. Different cysteines in the same proteins displayed dramatic differences in susceptibility to S-sulphenylation. Newly discovered S-sulphenylations provided mechanistic support for proposed cysteine redox reactions and suggested novel redox mechanisms, including S-sulphenyl-mediated redox regulation of the transcription factor HIF1A by SIRT6. S-sulphenylation is favored at solvent-exposed protein surfaces and is associated with sequence motifs that are distinct from those for other thiol modifications. S-sulphenylations affect regulators of phosphorylation, acetylation and ubiquitylation, which suggest regulatory crosstalk between redox control and signalling pathways.