Ki Values (ki + value)

Distribution by Scientific Domains
Distribution within Chemistry

Selected Abstracts

Inhibition of Diamino Pelargonic Acid Aminotransferase, an Enzyme of the Biotin Biosynthetic Pathway, by Amiclenomycin: A Mechanistic Study

Stéphane Mann
The mechanism of action of amiclenomycin (1a), a naturally occuring inhibitor of diaminopelargonic acid aminotransferase, has been established. The enzyme catalyzes the formation of an aromatic adduct between the inhibitor and pyridoxal-5,-phosphate. The structure of the adduct, determined by mass spectrometry, is in agreement with the reported X-ray crystal structure. Kinetic parameters, characteristic of kcat inhibitors, have been observed, with a KI value of 2,,M and a kinact value of 0.4,min,1. The irreversibility of the inactivation observed, in spite of the absence of covalent bond between the inhibitor and the protein, reveals the high affinity of the adduct for the active site. Two other cis -1-amino-4-substituted-cyclohexa-2,5-dienes, 3a and 4a, were also found to efficiently inhibit the enzyme. The trans -isomers were either much less potent (1b) or inactive (3b and 4b). The aminocyclohexadiene moiety, which is, apparently, responsible for the inhibition, could constitute an original pharmacophore for the design of new herbicides. [source]

Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

Nikola Lon
Abstract Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5,9.5) and temperature optimum of 45°C with the KM ratio of 0.065,mM for benzoyl-arginine- p -nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (KI value of 0.012,mM, Ic50 value of 0.204,mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5,kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38,kDa, suggesting that the enzyme is a monomer. © 2010 Wiley Periodicals, Inc. [source]

Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile

FEBS JOURNAL, Issue 8 2009
Markus Pietsch
The hydrolysis of acetylthiocholine by acetylcholinesterase from Electrophorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound 1. The interaction of the enzyme with the substrate and the inhibitors was characterized by the parameters KI, ,,, b or ,, Km and Vmax, which were determined directly and simultaneously from nonlinear Michaelis,Menten plots. Tacrine was shown to act as a mixed-type inhibitor with a strong noncompetitive component (,, , 1) and to completely block deacylation of the acyl-enzyme. In contrast, acetylcholinesterase inhibition by gallamine followed the ,steric blockade hypothesis', i.e. only substrate association to as well as substrate/product dissociation from the active site were reduced in the presence of the inhibitor. The relative efficiency of the acetylcholinesterase,gallamine complex for the catalysis of substrate conversion was determined to be 1.7,25% of that of the free enzyme. Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (KI = 0.25 m). The interaction of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven- to 10-fold increase in the KI values, whereas the principal mode of inhibition was not affected by the organic solvent. The determination of the inhibitory parameters of compound 1 in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase. The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7,9.6% relative efficiency. [source]

Bradyzide, a potent non-peptide B2 bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia

Gillian M Burgess
Bradyzide is from a novel class of rodent-selective non-peptide B2 bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). Bradyzide has high affinity for the rodent B2 receptor, displacing [3H]-bradykinin binding in NG108-15 cells and in Cos-7 cells expressing the rat receptor with KI values of 0.51±0.18 nM (n=3) and 0.89±0.27 nM (n=3), respectively. Bradyzide is a competitive antagonist, inhibiting B2 receptor-induced 45Ca efflux from NG108-15 cells with a pKB of 8.0±0.16 (n=5) and a Schild slope of 1.05. In the rat spinal cord and tail preparation, bradyzide inhibits bradykinin-induced ventral root depolarizations (IC50 value; 1.6±0.05 nM (n=3)). Bradyzide is much less potent at the human than at the rodent B2 receptor, displacing [3H]-bradykinin binding in human fibroblasts and in Cos-7 cells expressing the human B2 receptor with KI values of 393±90 nM (n=3) and 772±144 nM (n=3), respectively. Bradyzide inhibits bradykinin-induced [3H]-inositol trisphosphate (IP3) formation with IC50 values of 11.6±1.4 nM (n=3) at the rat and 2.4±0.3 ,M (n=3) at the human receptor. Bradyzide does not interact with a range of other receptors, including human and rat B1 bradykinin receptors. Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. Bradyzide shows long-lasting oral activity in rodent models of inflammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED50, 0.84 ,mol kg,1; duration of action >4 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal effect exceeding that of the non-steroidal anti-inflammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. In summary, bradyzide is a potent, orally active, antagonist of the B2 bradykinin receptor, with selectivity for the rodent over the human receptor. British Journal of Pharmacology (2000) 129, 77,86; doi:10.1038/sj.bjp.0703012 [source]

Carbonic Anhydrase Inhibitors: Inhibition of Human Erythrocyte Isozymes I and II with a Series of Phenolic Acids

S. Beyza Öztürk Sar, kaya
The inhibitory effects of some phenolic acids on the cytosolic human carbonic anhydrase (hCA, EC isozymes hCA I and hCA II were investigated. Ellagic acid, gallic acid, ferulic acid, caffeic acid, quercetin, p -coumaric acid, p -hydroxybenzoic acid, and syringic acid showed KI values in the range of 99,1061 ,m for hCA I and of 105,758 ,m against hCA II, respectively. Quercetin (for hCA I), p -coumaric acid (for hCA II), and gallic acid (for hCA II) exhibited competitive inhibitory effects with 4-nitrophenyl acetate as substrate. All of the other phenolic acids were found as non-competitive inhibitors with 4-nitrophenylacetate as substrate for hCA I and hCA II. The phenolic acids investigated here showed thus interesting hCA I and hCA II inhibitory effects and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents. [source]

Structural Basis of ,-Fucosidase Inhibition by Iminocyclitols with Ki Values in the Micro- to Picomolar Range,

Hsing-Ju Wu Dr.
Zwei Schleifen, die sich nach innen zum aktiven Zentrum von ,-Fucosidase bewegen, führen zu einer geschlossenen Konformation des Komplexes mit Inhibitoren mit Ki -Werten vom Mikro- bis zum Nanomolbereich. Bei Inhibitoren mit subnanomolaren Ki -Werten treten zwar keine weiteren Konformationsänderungen in den beiden Schleifen auf, aber die Schleifen werden durch Wasserstoffbrücken und hydrophobe Wechselwirkungen weiter stabilisiert. [source]

Interaction of bisphenol a with human UDP-glucuronosyltransferase 1A6 enzyme

Nobumitsu Hanioka
Abstract The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC50 values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 ,M for liver microsomes, and 84.6 and 80.3 ,M for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with Ki values of 84.9 and 72.3 ,M, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (Ki value, 65.5 ,M), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (Ki value, 42.5 ,M). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source]

Inhibition of the D -alanine:D -alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wall

FEBS JOURNAL, Issue 12 2005
Juergen J. May
The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the d -alanylation of the lipoteichoic acid. The incorporation of d -Ala into lipoteichoic acid requires the d -alanine:d -alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo-DltC is recognized by both endogenous 4,-phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (d -alanylacyl-sulfamoyl-adenosine), which is able to block the d -Ala adenylation by DltA at a Ki value of 232 nmin vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin. [source]

Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase

FEBS JOURNAL, Issue 14 2002
Tadayoshi Kanao
The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source]

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme

Thomas Hansen
Abstract The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP+ followed Michaelis,Menten kinetics with apparent Km values of 0.15 mM and 0.03 mM, respectively; apparent Vmax values were about 20 U mg,1. The enzyme also reduced NAD+ (apparent Km 12 mM, Vmax 12 U mg,1). The 1000-fold higher catalytic activity (kcat/Km) with NADP+ over NAD+ defines the G6PD as NADP+ specific in vivo. G6PD activity was competitively inhibited by NADPH with a Ki value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described. [source]

Synthesis of Monosaccharide-Derived Spirocyclic Cyclopropylamines and Their Evaluation as Glycosidase Inhibitors

Christian Blüchel
The glucose-, mannose-, and galactose-derived spirocyclic cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d were prepared as potential glycosidase inhibitors. Cyclopropanation of the diazirine 5 with ethyl acrylate led in 71% yield to a 4,:,5,:,1,:,20 mixture of the ethyl cyclopropanecarboxylates 7a,7d, while the Cu-catalysed cycloaddition of ethyl diazoacetate to the exo -glycal 6 afforded 7a,7d (6,:,2,:,5,:,3) in 93,98% yield (Scheme,1). Saponification, Curtius degradation, and subsequent addition of BnOH or t- BuOH led in 60,80% overall yield to the Z- or Boc-carbamates 11a,11d and 12a,12d, respectively. Hydrogenolysis of 11a,11d afforded 1a,1d, while 12a,12d was debenzylated to 13a,13d prior to acidic cleavage of the N -Boc group. The manno - and galacto -isomers 2a,2d and 3a,3d, respectively, were similarly obtained in comparable yields (Schemes,2 and 4). Also prepared were the differentially protected manno- configured esters 24a,24d; they are intermediates for the synthesis of analogous N -acetylglucosamine-derived cyclopropanes (Scheme,3). The cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d are very weak inhibitors of several glycosidases (Tables,1 and 2). Traces of Pd compounds, however, generated upon catalytic debenzylation, proved to be strong inhibitors. PdCl is, indeed, a reversible, micromolar inhibitor for the ,- glucosidases from C. saccharolyticum and sweet almonds (non-competitive), the , -galactosidases from bovine liver and from E. coli (both non-competitive), the , -galactosidase from Aspergillus niger (competitive), and an irreversible inhibitor of the , -glucosidase from yeast and the , -galactosidase from coffee beans. The cyclopropylamines derived from 1a,1d or 3a,3d significantly enhance the inhibition of the ,- glucosidase from C. saccharolyticum by PdCl, lowering the Ki value from 40,,M (PdCl) to 0.5,,M for a 1,:,1 mixture of PdCl and 1d. A similar effect is shown by cyclopropylamine, but not by several other amines. [source]

Effects of oral administration of extracts of Hypericum perforatum (St John's wort) on brain serotonin transporter, serotonin uptake and behaviour in mice

Kazufumi Hirano
The pharmacological effects of extracts of Hypericum perforatum (St John's wort) were characterized in-vitro and ex-vivo, in relation to its behavioural effects. In in-vitro experiments, St John's wort inhibited brain synaptosomal [3H]serotonin uptake in mice with little effect on specific [3H]paroxetine binding. For selective serotonin-reuptake inhibitors (SSRIs), the IC50 value for [3H]serotonin uptake (molar concentration of unlabelled drug necessary to displace 50% of specific uptake) correlated well with the inhibition constant Ki value for [3H]paroxetine binding in mouse brain. Oral administration of St John's wort (900 mg kg,1), paroxetine (1 mg kg,1) and sertraline (10 mg kg,1) brought about significant increases in the Km value for [3H]serotonin uptake into brain synaptosomes 4 h later, and only SSRIs suppressed specific [3H]paroxetine binding in mouse brain. St John's wort and SSRIs significantly inhibited marble-burying behaviour in mice and the time-course of attenuation of this behaviour by St John's wort was similar to that of [3H]serotonin uptake inhibition. In the forced swimming test, St John's wort, but not SSRIs, suppressed the immobility time of mice after oral administration. These results provide the first in-vivo evidence to suggest that the mode of antidepressant action of St John's wort differs from that of SSRIs. Thus, this study may have a significant impact on phytotherapy with St John's wort. [source]

Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids

L Thors
Background and purpose: Recent studies have demonstrated that the naturally occurring isoflavone compounds genistein and daidzein inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the low micromolar concentration range. The purpose of the present study was to determine whether this property is shared by flavonoids. Experimental approach: The hydrolysis of anandamide in homogenates and intact cells was measured using the substrate labelled in the ethanolamine part of the molecule. Key results: Twenty compounds were tested. Among the commonly occurring flavonoids, kaempferol was the most potent, inhibiting FAAH in a competitive manner with a Ki value of 5 ,M. Among flavonoids with a more restricted distribution in nature, the two most active toward FAAH were 7-hydroxyflavone (IC50 value of 0.5,1 ,M depending on the solvent used) and 3,7-dihydroxyflavone (IC50 value 2.2 ,M). All three compounds reduced the FAAH-dependent uptake of anandamide and its metabolism by intact RBL2H3 basophilic leukaemia cells. Conclusions and implications: Inhibition of FAAH is an additional in vitro biochemical property of flavonoids. Kaempferol, 7-hydroxyflavone and 3,7-dihydroxyflavone may be useful as templates for the synthesis of novel compounds, which target several systems that are involved in the control of inflammation and cancer. British Journal of Pharmacology (2008) 155, 244,252; doi:10.1038/bjp.2008.237; published online 16 June 2008 [source]

Oleamide is a selective endogenous agonist of rat and human CB1 cannabinoid receptors

James D Leggett
The ability of the endogenous fatty acid amide, cis -oleamide (ODA), to bind to and activate cannabinoid CB1 and CB2 receptors was investigated. ODA competitively inhibited binding of the nonselective cannabinoid agonist [3H]CP55,940 and the selective CB1 antagonist [3H]SR141716A to rat whole-brain membranes with Ki values of 1.14 ,M (0.52,2.53 ,M, Hill slope=0.80, n=6) and 2.63 ,M (0.62,11.20 ,M, Hill slope=0.92, n=4), respectively. AEA inhibited [3H]CP55,940 binding in rat whole-brain membranes with a Ki of 428 nM (346,510 nM, Hill slope=,1.33, n=3). ODA competitively inhibited [3H]CP55,940 binding in human CB1 (hCB1) cell membranes with a Ki value of 8.13 ,M (4.97,13.32 ,M, n=2). In human CB2 transfected (hCB2) HEK-293T cell membranes, 100 ,M ODA produced only a partial (42.5±7%) inhibition of [3H]CP55,940 binding. ODA stimulated [35S]GTP,S binding in a concentration-dependent manner (EC50=1.64 ,M (0.29,9.32 ,M), R2=0.99, n=4,9), with maximal stimulation of 188±9% of basal at 100 ,M. AEA stimulated [35S]GTP,S binding with an EC50 of 10.43 ,M (4.45,24.42 ,M, R2=1.00, n=3, 195±4% of basal at 300 ,M). Trans -oleamide (trans- ODA) failed to significantly stimulate [35S]GTP,S binding at concentrations up to 100 ,M. ODA (10 ,M)-stimulated [35S]GTP,S binding was reversed by the selective CB1 antagonist SR141716A (IC50=2.11 nM (0.32,13.77 nM), R2=1.00, n=6). The anatomical distribution of ODA-stimulated [35S]GTP,S binding in rat brain sections was indistinguishable from that of HU210. Increases of similar magnitude were observed due to both agonists in the striatum, cortex, hippocampus and cerebellum. ODA (10 ,M) significantly inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in mouse neuroblastoma N1E 115 cells (P=0.02, n=11). ODA-mediated inhibition was completely reversed by 1 ,M SR141716A (P<0.001, n=11) and was also reversed by pretreatment with 300 ng ml,1 pertussis toxin (P<0.001, n=6). These data demonstrate that ODA is a full cannabinoid CB1 receptor agonist. Therefore, in addition to allosteric modulation of other receptors and possible entourage effects due to fatty acid amide hydrolase inhibition, the effects of ODA may be mediated directly via the CB1 receptor. British Journal of Pharmacology (2004) 141, 253,262. doi:10.1038/sj.bjp.0705607 [source]

New Peptolides from the Cyanobacterium Nostoc insulare as Selective and Potent Inhibitors of Human Leukocyte Elastase

CHEMBIOCHEM, Issue 16 2008
Christian Mehner
Abstract Eight new cyanopeptolins (insulapeptolides A,H) were obtained from the cyanobacterium Nostoc insulare. Their isolation was guided by their bioactivity toward the target enzyme human leukocyte elastase, molecular biological investigations, and MALDI-TOF analysis. These peptides are selective inhibitors of human leukocyte elastase with activities in the nanomolar range. Insulapeptolide D (4) was the most potent compound with an IC50 value of 85 nM (Ki value of 36 nM). [source]

Synthesis and SAR Study of Opioid Receptor Ligands: Mono- and Bis-Indolomorphinans

Fuying Li
Mono- and bis-indolomorphinans were synthesized through a multi-step synthetic approach from the alkaloid, thebaine, to further explore the C-ring SAR (structure-activity relationship) of morphinan scaffold. Both mono-indoles displayed good binding affinity and selectivity for the , receptor, with compound 6b possessed the highest Ki value of 1.45 nm at this receptor. Bisindolomorphinans 7a,b did not have appreciable affinity for both , and , receptors, but moderate binding at the , receptor was observed. Functional assays indicated that the newly synthesized mono-indole 6b was ,-agonist, opposite to the ,-antagonist profile of naltrindole. Bisindoles 7a,b were ,-agonists. This work further confirms that the phenol component in opioids is essential for higher binding to the opioid receptors. The different binding ability, receptor selectivity, and the functional activity profiles of naltrindole 2, monoindole 6b, and bisindole 7b clearly indicated that they interact with the opioid receptors in different modes. [source]

Inhibition of Histidine Ammonia Lyase by Heteroaryl-alanines and Acrylates

Adrian Katona
Abstract Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the , -position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with Ki values between 18 and 139,,M. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L -histidine can be dissected into 4-methyl-1H -imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a Ki value of 1.7±0.09,mM which is to be compared with the Km value of 15.6,mM for the normal reaction. We conclude that 5-methyl-1H -imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation. [source]

Highly Potent and Selective Substrate Analogue Factor Xa Inhibitors Containing D -Homophenylalanine Analogues as P3 Residue: Part 2

CHEMMEDCHEM, Issue 7 2007
Anne Stürzebecher Dr.
Abstract A series of highly potent substrate-analogue factor Xa inhibitors containing D -homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl- d- hPhe-Gly-4-amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0,nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl- D -homo-2-pyridylalanyl(N-oxide)-Gly-4-amidinobenzylamide, inhibits fXa with a Ki value of 0.32,nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280,nM and 170,nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC50 value of 5,nM and is approximately 50 times more potent than the reference inhibitor DX-9065a in this assay. [source]

Nanomolar Competitive Inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor Type,II Dehydroquinase

CHEMMEDCHEM, Issue 2 2007
Verónica F.
Abstract Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid have been synthesized and tested as inhibitors of M.,tuberculosis and S.,coelicolor type,II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross-coupling and copper(I)-catalyzed 2,3-dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para -nitrophenyl derivative is almost 20-fold more potent as a competitive inhibitor against the S.,coelicolor enzyme than that of M.,tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S.,coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4,30,nM). These derivatives are also competitive inhibitors of the M.,tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S.,coelicolor enzyme, a 6-benzothiophenyl derivative, has a Ki value of 4,nM,over 2000-fold more potent than the best previously known inhibitor, (1R,4R,5R)-1,5-dihydroxy-4-(2-nitrophenyl)cyclohex-2-en-1-carboxylic acid (8,,M), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S.,coelicolor enzyme (GOLD,3.0.1), suggest a key , -stacking interaction between the aromatic rings and Tyr,28, a residue that has been identified as essential for enzyme activity. [source]

Synthesis and Biological Evaluation of Bicyclic Nucleosides as Inhibitors of M.,tuberculosis Thymidylate Kinase

CHEMMEDCHEM, Issue 10 2006
Daele, Ineke Van
Abstract Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a Ki value of 2.3,,m, 1-[3-aminomethyl-3,5-dideoxy-2- O,6- N -(thiocarbonyl)-,- D -ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC99 value of 100,,g,mL,1, thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure,activity relationship (SAR) involving molecular modelling and conformational analysis are described. [source]

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Jamshed Iqbal
Abstract Fast and convenient CE assays were developed for the screening of adenosine kinase,(AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260,nm. An MEKC method using borate buffer (pH,9.5) containing 100,mM SDS (method,A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH,7.5 or 8.5) was used and a constant current (95,,A) was applied (method,B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10,min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method,C). After hydrodynamic injection of a plug of reaction buffer (20,mM Tris-HCl, 0.2,mM MgCl2, pH,7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1,mM,ATP, 100,,M adenosine, and 20,,M,UMP as an internal standard,(I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5,kV separation voltage (negative polarity) for 0.20,min to let the plugs interpenetrate. The voltage was turned off for 5,min (zero-potential amplification) and again turned on at a constant current of ,60,,A to elute the products within 7,min. The method employing a polyacrylamide-coated capillary of 20,cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose,response curves and calculated Ki values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay. [source]

Interaction of bisphenol a with human UDP-glucuronosyltransferase 1A6 enzyme

Nobumitsu Hanioka
Abstract The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC50 values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 ,M for liver microsomes, and 84.6 and 80.3 ,M for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with Ki values of 84.9 and 72.3 ,M, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (Ki value, 65.5 ,M), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (Ki value, 42.5 ,M). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source]

In the Search of Glycogen Phosphorylase Inhibitors: Synthesis of C- D -Glycopyranosylbenzo(hydro)quinones , Inhibition of and Binding to Glycogen Phosphorylase in the Crystal,

Li He
Abstract Penta- O -acetyl-,- D -glycopyranoses and 1,4-dimethoxybenzene led selectively by electrophilic substitution to C-,- D -glycopyranosyl-1,4-dimethoxybenzenes which were converted by simple and efficient reactions (oxidation, reduction and deacetylation) to the corresponding C-glycosylhydro- and C-glycosylbenzoquinones, with either an acetylated or deprotected sugar moiety. C-,- D -Glucosylbenzoquinone 19 and C-,- D -Glucosylhydroquinone 23 were found to be competitive inhibitors of rabbit muscle glycogen phosphorylase b (GPb), with respect to the substrate ,- D -glucose-1-phosphate, with Ki values of 1.3 and 0.9 mM, respectively, whereas C-,- D -glucosylhydroquinone 17 was not effective up to a concentration of 8 mM. In order to elucidate the structural basis of inhibition, we determined the crystal structures of 19 and 23 in complex with GPb at a 2.03,2.05 Å resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as ,- D -glucose and stabilise the transition state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source]

Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain

FEBS JOURNAL, Issue 6 2009
Potential substrates and/or inhibitors of Ap4A hydrolases
Dinucleoside polyphosphates (NpnN,s; where N and N, are nucleosides and n = 3,6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of NpnN, analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap4A hydrolase [bis(5,-nucleosyl)-tetraphosphatase (symmetrical); EC] from E. coli and of two asymmetrically acting Ap4A hydrolases [bis(5,-nucleosyl)-tetraphosphatase (asymmetrical); EC] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH2OpOCH2pA (1), ApOCH2pCH2OpA (2), ApOpCH2OpOpA (3), ApCH2OpOpOCH2pA (4), ApOCH2pOpCH2OpA (5) and ApOpOCH2pCH2OpOpA (6). The eukaryotic asymmetrical Ap4A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and ,,,-methyleneoxy-ATP (pOCH2pOpA), whereas hydrolysis of analog 5 gave two molecules of ,,,-oxymethylene ADP (pCH2OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having Ki values within the range of the Km for Ap4A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap4A hydrolase. [source]

A Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1

Romain Siegrist
Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source]

1-(Arylalkyl)quinolizidine Derivatives and Thio-Isosteric Analogues as Ligands for Sigma Receptors

Anna Sparatore
A set of 1-(arylalkyl)quinolizidines, isosteric thioanalogues, and variously functionalized congeners were synthesized (see 1,25) and tested for affinity to sigma 1 and sigma 2 receptor subtypes, by displacing [3H]- (+)-pentazocine and [3H]DTG from guinea pig brain and rat brain preparations, respectively. All compounds exhibited a good affinity for the ,1 subtype, with subnanomolar Ki values for the best of them, while only modest or poor affinity for the ,2 subtype was observed (Tables,1 and 2). Some structureactivity relationships were put forward. [source]

Pharmacological characterization of cis -nitromethylene neonicotinoids in relation to imidacloprid binding sites in the brown planthopper, Nilaparvata lugens

X. Xu
Abstract Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) and extensively used in areas of crop protection and animal health to control a variety of insect pest species. Here we describe that two cis -nitromethylene neonicotinoids (IPPA152002 and IPPA152004), recently synthesized in our laboratory, discriminated between the high and low affinity imidacloprid binding sites in the brown planthopper, Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. [3H]imidacloprid has two binding sites with different affinities (Kd value of 0.0035 ± 0.0006 nM for the high-affinity site and 1.47 ± 0.22 nM for the low-affinity site). Although the cis -nitromethylene neonicotinoids showed low displacement ability (Ki values of 0.15 ± 0.03 µM and 0.42 ± 0.07 µM for IPPA152002 and IPPA152004, respectively) against [3H]imidacloprid binding, low concentrations (0.01 µM) of IPPA152002 completely inhibited [3H]imidacloprid binding at its high-affinity site. In Xenopus oocytes co-injected with cRNA encoding Nl,1 and rat ,2 subunits, obvious inward currents were detected in response to applications of IPPA152002 and IPPA152004, although the agonist potency is reduced to that of imidacloprid. The previously identified Y151S mutation in Nl,1 showed significant effects on the agonist potency of IPPA152002 and IPPA152004, such as a 75.8% and 70.6% reduction in Imax, and a 2.4- and 2.1-fold increase in EC50. This data clearly shows that the two newly described cis -nitromethylene neonicotinoids act on insect nAChRs and like imidacloprid, discriminated between high and low affinity binding sites in N. lugens native nAChRs. These compounds may be useful tools to further elucidate the pharmacology and nature of neonicotinoid binding sites. [source]

Extrapolating in vitro metabolic interactions to isolated perfused liver: Predictions of metabolic interactions between R -bufuralol, bunitrolol, and debrisoquine

Sami Haddad
Abstract Drug,drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (Ki) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R -bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF,BUN, BUF,DBQ, BUN,DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the Ki values estimated were similar to their Km values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration,time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF,DBQ and BUN,DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF,BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4406,4426, 2010 [source]

Tocolytic Effects of a Long-acting ,2 -Adrenoceptor Agonist, Formoterol, in Rats

We have assessed the tocolytic activity of formoterol, a novel long-acting and potent ,2 -adrenoceptor agonist, through its production of cyclic adenosine monophosphate, in comparison with ritodrine, a ,2 -adrenoceptor agonist used clinically to counter premature delivery. Formoterol and ritodrine inhibited the amplitude and frequency of rat uterine contraction, with IC50 values of 3.8 times 10,10 and 4.7 times 10,7 M, respectively. Intravenous administration of formoterol or ritodrine caused inhibition of uterine motility and increased heart rate in a dose-dependent manner. Inhibition of uterine motility by oral administration of formoterol (0.3 and 1 mg kg,1) continued for at least 60 min, whereas that with ritodrine (100 mg kg,1) persisted for 15 min with rapid recovery thereafter in pregnant rats. The ,-adrenoceptor binding of [125I]iodopindolol to the myometrium of pregnant rats was competitive with formoterol and ritodrine, with Ki values of 0.04 and 6.10 nM, respectively. Formoterol (10,6 , 10,4 M) and ritodrine (10,6 , 10,4 M) increased the level of cyclic adenosine monophosphate in lymphocytes in a dose-dependent manner. The results suggested that formoterol caused relaxation of uterine motility through production of cyclic adenosine monophosphate. Thus, formoterol may be useful as a treatment to counter premature delivery. [source]

Inhibitor design for ribonuclease A: the binding of two 5,-phosphate uridine analogues

Vicky G. Tsirkone
In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5,-phosphate (U5P) and uridine 5,-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with Ki values of 4.0 and 0.65,mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily. [source]