Functional interaction of diphenols with polyphenol oxidase - Molecular determinants of substrate/inhibitor specificity SO FEBS JOURNAL

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on o-diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His 109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants Of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.

Exploring the interaction energies for the binding of hydroxydiphenyl ethers to enoyl-acyl carrier protein reductases

It is now well established that the potent anti-microbial compound, triclosan, interrupts the type II fatty acid synthesis by inhibiting the enzyme enoyl-ACP reductase in a number of organisms. Existence of a high degree of similarity between the recently discovered enoyl-ACP reductase from R falciparum and B. napus enzyme permitted building of a satisfactory model for the former enzyme that explained some of the key aspects of the enzyme such as its specificity for binding to the cofactor and the inhibitor. We now report the interaction energies between triclosan and other hydroxydiphenyl ethers with the enzymes from B. napus, E. coli and R falciparum. Examination of the triclosan-enzyme interactions revealed that subtle differences exist in the ligand binding sites of the enzymes from different sources i.e., B. napus, E. coli and P falciparum. A comparison of their binding propensities thus determined should aid in the design of effective inhibitors for the respective enzymes.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Activation of succinate dehydrogenase by 2,4-dinitrophenol-a competitive inhibitor

1.The reported inhibition of the succinate oxidase system at high concentrations of dinitrophenol, considered to be at the primary dehydrogenase level, is now confirmed by measuring the activity of succinate dehydrogenase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1) in the presence of dinitrophenol, using the dye reduction method. 2. 2. The results indicate that the inhibition of substrate-activated succinate dehydrogenase by dinitrophenol is competitive. 3. 3. Low concentrations of dinitrophenol inhibited the basal activity, while at higher concentrations the kinetics were complicated by an apparent activation. 4. 4. Preincubation of mitochondria with dinitrophenol stimulated the enzyme activity, a phenomenon shown by succinate and competitive inhibitors. This activation was very rapid at 37°, compared to that by succinate; activation by dinitrophenol was observed even at 25°, under conditions where succinate had no effect. 5. 5. Repeated washing of the activated mitochondrial samples with the sucrose homogenizing medium reduced the succinate-stimulated activity to the basal level, but only partially reversed the dinitrophenol activation. 6. 6. The relevance of this activation phenomenon to the physiological modulation of this enzyme system is discussed.

X-ray Crystallographic Analysis of the Complexes of Enoyl Acyl Carrier Protein Reductase of Plasmodium falciparum with Triclosan Variants to Elucidate the Importance of Different Functional Groups in Enzyme Inhibition

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. (C) 2010 IUBMB IUBMB Life, 62(6): 467-476.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE-32 at near neutral pH, followed by a cation exchange chromatography on SPC-50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI-4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80000. Subjecting 125I labelled GI-4 to chromatography on Sephadex G-100 helped obtain a homogeneous material, Gl-5. The 125I labelled GI-5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand-receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.

An Eventful Synthetic Approach Towards the Biologically Potent Natural Product Ottelione A: Enantio-, Regio- and Stereoselective Construction of the Bicyclic Core

A novel synthetic approach towards the recently reported anti-tumor and anti-tuberculor natural product ottelione A from the readily available Diels-Alder adduct of cyclopentadiene and p-benzoquinone is delineated. Our short strategy, besides being enantio-, regio- and stereoselective, charts an eventful course and is inherently well-suited for adaptation towards diverse synthetic analogues of this biologically potent natural product.

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98-degrees-C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.

Studies on simultaneous nhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences.The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.