The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Claude F. Bernasconi - One of the best experts on this subject based on the ideXlab platform.
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Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the Allosteric Site.
Biochemistry, 2003Co-Authors: Viola Ruddat, Stephanie Whitman, Theodore R. Holman, Claude F. BernasconiAbstract:Herein, we report on the role of the Allosteric Site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The KD for the activation was determined to be 25.9 ± 2.3 μM and the rate constant for the oxidation of the iron cofactor, k2, to be 182 ± 4 s-1. Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an Allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a Ki of 13.7 ± 1.3 μM for the catalytic Site and a Ki‘of 140 ± 9 μM for the Allosteric Site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a Ki of 17.5 ± 3.8 μM. These results suggest that OS binds to an Allosteric Site that is separate from the catalytic iron Site. We further observed that the Allosteric Site binding selectivity is sensitive to inh...
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Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the Allosteric Site.
Biochemistry, 2003Co-Authors: Viola Ruddat, Stephanie Whitman, Theodore R. Holman, Claude F. BernasconiAbstract:Herein, we report on the role of the Allosteric Site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an Allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic Site and a K(i)' of 140 +/- 9 microM for the Allosteric Site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an Allosteric Site that is separate from the catalytic iron Site. We further observed that the Allosteric Site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.
Viola Ruddat - One of the best experts on this subject based on the ideXlab platform.
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Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the Allosteric Site.
Biochemistry, 2003Co-Authors: Viola Ruddat, Stephanie Whitman, Theodore R. Holman, Claude F. BernasconiAbstract:Herein, we report on the role of the Allosteric Site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The KD for the activation was determined to be 25.9 ± 2.3 μM and the rate constant for the oxidation of the iron cofactor, k2, to be 182 ± 4 s-1. Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an Allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a Ki of 13.7 ± 1.3 μM for the catalytic Site and a Ki‘of 140 ± 9 μM for the Allosteric Site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a Ki of 17.5 ± 3.8 μM. These results suggest that OS binds to an Allosteric Site that is separate from the catalytic iron Site. We further observed that the Allosteric Site binding selectivity is sensitive to inh...
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Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the Allosteric Site.
Biochemistry, 2003Co-Authors: Viola Ruddat, Stephanie Whitman, Theodore R. Holman, Claude F. BernasconiAbstract:Herein, we report on the role of the Allosteric Site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an Allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic Site and a K(i)' of 140 +/- 9 microM for the Allosteric Site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an Allosteric Site that is separate from the catalytic iron Site. We further observed that the Allosteric Site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.
Keith A Wreggett - One of the best experts on this subject based on the ideXlab platform.
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an intracellular Allosteric Site for a specific class of antagonists of the cc chemokine g protein coupled receptors ccr4 and ccr5
Molecular Pharmacology, 2008Co-Authors: Glen Andrews, Carolyn Jones, Keith A WreggettAbstract:A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an Allosteric, intracellular Site on the receptor. The existence of this Site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel Site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding Site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and β-arrestin) suggests that this could be a generic Allosteric Site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a Site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular Site.
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an intracellular Allosteric Site for a specific class of antagonists of the cc chemokine g protein coupled receptors ccr4 and ccr5
Molecular Pharmacology, 2008Co-Authors: Glen Andrews, Carolyn Jones, Keith A WreggettAbstract:A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an Allosteric, intracellular Site on the receptor. The existence of this Site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel Site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding Site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and beta-arrestin) suggests that this could be a generic Allosteric Site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a Site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular Site.
Pedro H. M. Torres - One of the best experts on this subject based on the ideXlab platform.
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Inhibiting Mycobacterium tuberculosis CoaBC by targeting an Allosteric Site.
Nature communications, 2021Co-Authors: Vitor Mendes, Simon Green, Joanna C. Evans, Jeannine Hess, Michal Blaszczyk, Christina Spry, Owain J. Bryant, James Cory-wright, Daniel S-h. Chan, Pedro H. M. TorresAbstract:Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic Allosteric Site within CoaB.
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Inhibiting Mycobacterium tuberculosis CoaBC by targeting an Allosteric Site
Nature Communications, 2021Co-Authors: Vitor Mendes, Joanna C. Evans, Jeannine Hess, Michal Blaszczyk, Christina Spry, James Cory-wright, Daniel S-h. Chan, Simon R. Green, Owain Bryant, Pedro H. M. TorresAbstract:The bifunctional enzyme CoaBC catalyses the second and third step in the Coenzyme A (CoA) biosynthesis pathway and is of interest as a M. tuberculosis drug target. Here, the authors present the full-length crystal structure of Mycobacterium smegmatis CoaBC, which is regulated by CoA and CoA thioesters and forms a dodecamer and by performing a high-throughput screen they identify selective inhibitors of M. tuberculosis CoaB that bind to an Allosteric Site within CoaB. Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis . The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis , by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis . Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis , describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic Allosteric Site within CoaB.
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Inhibiting Mycobacterium tuberculosis CoaBC by targeting a new Allosteric Site
2019Co-Authors: Vitor Mendes, Simon Green, Joanna C. Evans, Jeannine Hess, Michal Blaszczyk, Christina Spry, Owain J. Bryant, James Cory-wright, Daniel S-h. Chan, Pedro H. M. TorresAbstract:Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps. However, in the vast majority of prokaryotes including M. tuberculosis, a single bifunctional protein, CoaBC, catalyses the second and third steps. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent inhibitors of M. tuberculosis CoaB. Crucially we further show that these compounds bind to a novel cryptic Allosteric Site within CoaB.
Klaus Mohr - One of the best experts on this subject based on the ideXlab platform.
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Allosteric Site in M_2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist
Naunyn-Schmiedeberg's Archives of Pharmacology, 2006Co-Authors: Maren Großmüller, Ulrike Holzgrabe, Christian Trankle, Johannes Antony, Klaus MohrAbstract:Muscarinic acetylcholine receptors contain two distinct ligand binding Sites, i.e. the orthosteric Site for acetylcholine and other conventional ligands, and an Allosteric Site located at the entrance of the ligand binding pocket. We used a set of Allosteric agents to probe whether muscarinic M_2 receptors whose orthosteric Site is occupied by an agonist still reveal the common Allosteric Site that has been identified in M_2 receptors being occupied by an orthosteric antagonist ( N -methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [^3H]oxotremorine M ([^3H]OxoM) or the antagonist [^3H]NMS. The affinities of the Allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M_2 receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [^3H]OxoM-liganded receptors compared with [^3H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [^3H]OxoM- and [^3H]NMS-occupied receptors. The Allosteric antagonistic tool obidoxime inhibited WDuo3 action in [^3H]OxoM-liganded receptors with the same potency as in [^3H]NMS-liganded receptors. We conclude that the common Allosteric Site is still present in OxoM-liganded M_2 receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.
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Allosteric Site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist
Naunyn-Schmiedeberg's archives of pharmacology, 2005Co-Authors: Ulrike Holzgrabe, Christian Trankle, Maren Großmüller, Johannes Antony, Klaus MohrAbstract:Muscarinic acetylcholine receptors contain two distinct ligand binding Sites, i.e. the orthosteric Site for acetylcholine and other conventional ligands, and an Allosteric Site located at the entrance of the ligand binding pocket. We used a set of Allosteric agents to probe whether muscarinic M2 receptors whose orthosteric Site is occupied by an agonist still reveal the common Allosteric Site that has been identified in M2 receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [3H]oxotremorine M ([3H]OxoM) or the antagonist [3H]NMS. The affinities of the Allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M2 receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [3H]OxoM-liganded receptors compared with [3H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [3H]OxoM- and [3H]NMS-occupied receptors. The Allosteric antagonistic tool obidoxime inhibited WDuo3 action in [3H]OxoM-liganded receptors with the same potency as in [3H]NMS-liganded receptors. We conclude that the common Allosteric Site is still present in OxoM-liganded M2 receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.
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critical amino acid residues of the common Allosteric Site on the m2 muscarinic acetylcholine receptor more similarities than differences between the structurally divergent agents gallamine and bis ammonio alkane type hexamethylene bis dimethyl 3 pht
Molecular Pharmacology, 2005Co-Authors: Xi Ping Huang, Stefanie Prilla, John Ellis, Klaus MohrAbstract:The structurally divergent agents gallamine and hexamethylene- bis -[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) are known to interact competitively at a common Allosteric Site on muscarinic receptors. Previous studies reported that the M2 selectivity of gallamine depended largely on the EDGE (172-175) sequence in the second outer loop (o2) and on 419Asn near the junction of o3 and the seventh transmembrane domain (TM7), whereas the selectivity of W84 depended on nearby residues 177Tyr and 423Thr. However, it has so far proven difficult to confer the high sensitivity for Allosteric modulation of the M2 subtype onto the weakly sensitive M5 subtype by substituting these key residues. We now have found that M2 423Thr, not 419Asn, is the dominant residue in the o3/TM7 region for gallamine's high potency, although 419Asn can substitute for 423Thr in some contexts; in contrast, the presence of 419Asn reduces the potency of W84 in every context we have studied. In addition, the orientation of 177Tyr is crucial to high sensitivity toward W84, and it seems that the proline residue at position 179 in M5 (corresponding to M2 172Glu) may interfere with that orientation. Consistent with these observations, a mutant M5 receptor with these three key mutations, M5P179E, Q184Y, and H478T, showed dramatically increased sensitivity for W84 (>100-fold), compared with the wild-type M5 receptor. This same mutant receptor approached M2 sensitivity toward gallamine. Thus, gallamine and W84 derive high potency from the same receptor domains (epitopes in o2 and near the junction between o3 and TM7), even though these Allosteric agents have quite different structures.
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Interactions of Orthosteric and Allosteric Ligands with [3H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic M2 Receptors
Molecular pharmacology, 2003Co-Authors: Christian Trankle, Oliver Weyand, S. Lazareno, Nigel J.m. Birdsall, Uta Voigtländer, Anita Mynett, Klaus MohrAbstract:An optimized assay for the binding of [3H]dimethyl-W84 to its Allosteric Site on M2 muscarinic receptors has been used to directly measure the affinities of Allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric Site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the Allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the Allosteric Site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding Site that is buried within the transmembrane helices. The atypical Allosteric ligands tacrine and 4,4′-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1′-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding Sites. It is suggested that these atypical ligands either bind to the two known spatially separated Allosteric Sites on muscarinic receptors with positive cooperativity or their binding to the common Allosteric Site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated.
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Probing the size of a hydrophobic binding pocket within the Allosteric Site of muscarinic acetylcholine M2-receptors.
Life sciences, 2000Co-Authors: Wiebke Bender, Klaus Mohr, Christian Trankle, Markus Staudt, Ulrike HolzgrabeAbstract:Abstract Hexane-bisammonium-type compounds containing lateral phthalimide moieties are known to have a rather high affinity for the Allosteric Site of muscarinic M2 receptors. In order to get more insight into the contribution of the lateral substituents for alloster binding affinity, a series of compounds with unilaterally varying imide substituents were synthesized and tested for their ability to retard Allosterically the dissociation of [3H]N-methylscopolamine from the receptor protein (control t 1 2 = 2 min; 3 mM MgHCO4, 50 mM Tris, pH 7.3, 37 °C). Among the test compounds, the naphthalimide containing agent (half maximum effect at EC50,diss = 60 nM) revealed the highest potency. Apparently, its affinity for the Allosteric Site in NMS-occupied receptors is 20fold higher compared with the phthalimide containing parent compound W 84. Analysis of quantitative structure-activity relationships yielded a parabolic correlation between the volume of the lateral substituents and the Allosteric potency. The maximal volume was determined to be approximately 600 A3 suggesting that the Allosteric binding Site contains a binding pocket of a defined size for the imide moiety.