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PubMed2004

Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain.

A three-dimensional model of the human extracellular Ca(2+)-sensing receptor (CaSR) has been used to identify specific residues implicated in the recognition of two negative allosteric CaSR modulators of different chemical structure, NPS 2143 and Calhex 231. To demonstrate the involvement of these residues, we have analyzed dose-inhibition response curves for the effect of these calcilytics on Ca(2+)-induced [(3)H]inositol phosphate accumulation for the selected CaSR mutants transiently expressed in HEK293 cells. These mutants were further used for investigating the binding pocket of two chemically unrelated positive allosteric CaSR modulators, NPS R-568 and (R)-2-[1-(1-naphthyl)ethylaminomethyl]-1H-indole (Calindol), a novel potent calcimimetic that stimulates (EC(50) = 0.31 microM) increases in [(3)H]inositol phosphate levels elicited by activating the wild-type CaSR by 2 mM Ca(2+). Our data validate the involvement of Trp-818(6.48), Phe-821(6.51), Glu-837(7.39), and Ile-841(7.43) located in transmembranes (TM) 6 and TM7, in the binding pocket for both calcimimetics and calcilytics, despite important differences observed between each family of compounds. The TMs involved in the recognition of both calcilytics include residues located in TM3 (Arg-680(3.28), Phe-684(3.32), and Phe-688(3.36)). However, our study indicates subtle differences between the binding of these two compounds. Importantly, the observation that some mutations that have no effect on calcimimetics recognition but which affect the binding of calcilytics in TM3 and TM5, suggests that the binding pocket of positive and negative allosteric modulators is partially overlapping but not identical. Our CaSR model should facilitate the development of novel drugs of this important therapeutic target and the identification of the molecular determinants involved in the binding of allosteric modulators of class 3 G-protein-coupled receptors.

Allosteric Regulation↗

PubMed1993

Allosteric modulation by ATP, calcium and magnesium ions of rat osseous plate alkaline phosphatase.

Alkaline phosphatase from rat osseous plate is allosterically modulated by ATP, calcium and magnesium at pH 7.5. At pH 9.4, the hydrolysis of ATP and PNPP follows Michaelis-Menten kinetics with K0.5 values of 154 microM and 42 microM, respectively. However, at pH 7.5 both substrates exhibit more complex saturation curves, while only ATP exhibited site-site interactions. Ca(2+)-ATP and Mg(2+)-ATP were effective substrates for the enzyme, while the specific activity of the enzyme for the hydrolysis of ATP at pH 7.5 was 800-900 U/mg and was independent of the ion species. ATP, but not PNPP, was hydrolyzed slowly in the absence of metal ions with a specific activity of 140 U/mg. These data demonstrate that in vitro and at pH 7.5 rat osseous plate alkaline phosphatase is an active calcium or magnesium-activated ATPase.

4-Nitrophenylphosphatase↗

PubMed2004

QSAR study of anticonvulsant negative allosteric modulators of the AMPA receptor.

A quantitative structure-activity relationship (QSAR) study was performed on a set of 49 negative allosteric modulators of AMPA receptor, acting as anticonvulsant agents, using multiple linear regression. The predictive ability of the resulting model was evaluated against a set of 12 compounds; the results showed good statistics in regression and revealed high correlation between anticonvulsant activity and some electrotopological descriptors.

Allosteric Regulation↗

PubMed2005

A close structural analog of 2-methyl-6-(phenylethynyl)-pyridine acts as a neutral allosteric site ligand on metabotropic glutamate receptor subtype 5 and blocks the effects of multiple allosteric modulators.

The metabotropic glutamate receptor subtype 5 (mGlu5) activates calcium mobilization via binding of glutamate, the major excitatory neurotransmitter in the central nervous system. Allosteric modulation of the receptor has recently emerged as a promising alternative method of regulation to traditional regulation through orthosteric ligands. We now report three novel compounds that bind to the allosteric 2-methyl-6-(phenylethynyl)-pyridine (MPEP) site on mGlu5 but have only partial inhibition or no functional effects on the mGlu5 response. Two of these compounds, 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP) and 2-(2-(5-bromopyridin-3-yl)ethynyl)-5-methylpyridine (Br-5MPEPy), act as partial antagonists of mGlu5 in that they only partially inhibit the response of this receptor to glutamate. The third compound, 5-methyl-6-(phenylethynyl)-pyridine (5MPEP), acts as a neutral allosteric site ligand that binds to the MPEP site and has no effects alone. However, 5MPEP blocks the effects of both the allosteric antagonist MPEP and potentiators 3,3'-difluorobenzaldazine and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB). This compound also blocks depolarization effects of both MPEP and CDPPB in neurons in the subthalamic nucleus. These novel compounds provide valuable new insight into the pharmacology of allosteric sites on G protein-coupled receptors and provide valuable new tools for determining the effects of allosteric site ligands in native systems.

Alkynes↗

PubMed2006

Challenges in the development of mGluR5 positive allosteric modulators: the discovery of CPPHA.

This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.

Allosteric Regulation↗

PubMed2002

Synthesis of 3,4-dihydro-2H-1,2,4-benzo-thiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/kainate receptors.

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

Allosteric Regulation↗

PubMed1987

Kinetic mechanism of Ascaris suum phosphofructokinase desensitized to allosteric modulation by diethylpyrocarbonate modification.

The kinetic mechanism of phosphofructokinase has been determined at pH 8 for native enzyme and pH 6.8 for an enzyme desensitized to allosteric modulation by diethylpyrocarbonate modification. In both cases, the mechanism is predominantly steady state ordered with MgATP binding first in the direction of fructose 6-phosphate (F6P) phosphorylation and rapid equilibrium random in the direction of MgADP phosphorylation. This is a unique kinetic mechanism for a phosphofructokinase. Product inhibition by MgADP is competitive versus MgATP and noncompetitive versus F6P while fructose 1,6-bisphosphate (FBP) is competitive versus fructose 6-phosphate and uncompetitive versus MgATP. The uncompetitive pattern obtained versus F6P is indicative of a dead-end E.MgATP.FBP complex. Fructose 6-phosphate is noncompetitive versus either FBP or MgADP. Dead-end inhibition by arabinose 5-phosphate or 2,5-anhydro-D-mannitol 6-phosphate is uncompetitive versus MgATP corroborating the ordered addition of MgATP prior to F6P. In the direction of MgADP phosphorylation, inhibition by anhydromannitol 1,6-bisphosphate is noncompetitive versus MgADP, while Mg-adenosine 5'(beta, gamma-methylene)triphosphate is noncompetitive versus FBP. Anhydromannitol 6-phosphate is a slow substrate, while anhydroglucitol 6-phosphate is not. This suggests that the enzyme exhibits beta-anomeric specificity.

Adenosine Triphosphate↗

PubMed2003

Investigation of the interaction of a putative allosteric modulator, N-(2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide (SCH-202676), with M1 muscarinic acetylcholine receptors.

The interaction between a novel G protein-coupled receptor modulator, N-(2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide (SCH-202676), and the M(1) muscarinic acetylcholine receptor (mAChR) was investigated. In contrast to the prototypical mAChR allosteric modulator, heptane 1,7-bis-(dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3-phth), SCH-202676 had no effect on the dissociation kinetics of [(3)H]N-methylscopolamine ([(3)H]NMS) at M(1) mAChRs stably expressed in Chinese hamster ovary (CHO) cell membranes. However, SCH-202676 completely inhibited the binding of [(3)H]NMS in membrane preparations, with a Hill slope significantly greater than unity, indicative of positive cooperativity in the binding of the inhibitor. Moreover, SCH-202676 caused dextral shifts of the [(3)H]NMS saturation binding curve that were greater than expected for a competitive interaction. The addition of C(7)/3-phth (100 microM) had no significant effect on the inhibitory potency of SCH-202676. In contrast to the findings in cell membranes, the interaction between SCH-202676 and [(3)H]NMS in intact M(1) CHO cells yielded saturation and inhibition isotherms that were compatible with the predictions for a competitive interaction. Intact cell assays of acetylcholine-mediated phosphoinositide hydrolysis in the absence or presence of SCH-202676 revealed a mixed competitive/noncompetitive mode of interaction that was dependent on the concentration of SCH-202676. These data reveal that the nature of the interaction between SCH-202676 and the M(1) mAChR is dependent on whether it is studied using intact versus broken cell preparations. It is proposed that SCH-202676 uses a dual mode of ligand-receptor interaction involving both extra- and intracellular attachment points on the M(1) mAChR that are distinct from the allosteric binding site recognized by prototypical mAChR modulators such as C(7)/3-phth.

Allosteric Regulation↗

PubMed2006

Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors.

It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejçí and Tuçek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.

Allosteric Regulation↗

PubMed1987

Co-localization and co-release of GABA and putative allosteric modulators of GABA receptor.

Diazepam binding inhibitor (DBI) belongs to a family of newly discovered neuropeptides that, when acting on the benzodiazepine/beta-carboline recognition site, provide an allosteric modulation of the function of GABAA receptor. The molecular size of DBI (10K Da) and its amino acid sequence characteristics are compatible with the view that this polypeptide can function as a precursor of smaller biologically active neuropeptides. In neurons of the cerebral cortex of the neonatal rat, in primary culture, DBI coexists with at least 4 different processing products. These peptides immunoreact with an antiserum directed against a biologically active octadecaneuropeptide (ODN) amino acid sequence of which (QATVGDVNTDRPGLLDLK) is included in the middle portion of the amino acid sequence of DBI. One of the immunoreactive peptides extracted from neurons has a retention time in high pressure liquid chromatography (HPLC) identical to that of synthetic ODN. Double immunofluorescence staining of the cultured neurons with glutamic acid decarboxylase (GAD) and antibodies for ODN indicates that ODN and ODN-like peptides are localized with GABA in 58% of the GAD-positive neurons. Moreover, the proportion of the neuronal stores of GABA, ODN, DBI-like peptides and DBI that are released together following depolarization with veratridine is similar. These experiments provide evidence to suggest that ODN, ODN-like peptides derived from DBI, might participate as putative neuromodulators of physiological significance in changing the probability that a quantum of GABA opens specific chloride (Cl-) channels located on post-synaptic cell membranes.

Amino Acid Sequence↗

PubMed2002

Allosteric modulators of group I metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic perspectives.

Recent drug discovery programs aimed at identifying selective metabotropic mGlu receptor ligands by high-throughput functional screening efforts have revealed subtype-selective allosteric modulators of mGlu1 and mGlu5 receptors that are structurally unrelated to glutamate. In contrast to competitive ligands, which bind to the glutamate binding site located in the large N-terminal extracellular domain, these modulators act as non-competitive antagonists, inverse agonists or positive modulators by binding to specific residues in the seven-transmembrane domain. More recent studies to assess the potential of these compounds in in vivo models of nervous system disorders have implicated the mGlu5 receptor subtype as a potentially important therapeutic target for inflammatory pain, anxiety, Parkinson's disease and drug abuse, and mGlu1 and mGlu5 receptors as potential targets for anticonvulsant and neuroprotective therapies. Very recent findings indicate an important regulatory role for intracellular proteins interacting with metabotropic glutamate receptors, which might constitute novel drug targets for modulating metabotropic glutamate receptor activity.

Allosteric Regulation↗

PubMed1997

Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

Animals↗

PubMed2006

Substituent effects of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides on positive allosteric modulation of the metabotropic glutamate-5 receptor in rat cortical astrocytes.

CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR5 subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an EC50 value of 77 +/- 15 nM in potentiating mGluR5-mediated responses in cortical astrocytes and a Ki value of 3760 +/- 430 nM in displacing [3H]methoxyPEPy binding in membranes of cultured HEK-293 cells expressing rat mGluR5. The structure-activity relationships showed that electronegative aromatic substituents in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing Ki = 156 +/- 29 nM and EC50 = 9.6 +/- 1.9 nM in the binding and functional assays, respectively.

Allosteric Regulation↗

PubMed1997

Three allosteric modulators act at a common site, distinct from that of competitive antagonists, at muscarinic acetylcholine M2 receptors.

Functional studies were conducted on guinea pig atrial muscarinic acetylcholine M2 receptors with the allosteric modulators heptane-1,7-bis(dimethyl-3'-phthalimidopropyl)ammonium bromide (C7/3'-phth), gallamine and alcuronium to determine whether these ligands are able to recognize a common accessory site. The three modulators inhibited the negative inotropic response to carbachol in this tissue. When used in combination, C7/3'-phth and gallamine or C7/3'-phth and alcuronium gave dose ratios that were either additive or underadditive. In contrast, the combinations of C7/3'-phth or alcuronium with the competitive antagonists, N-methylscopolamine or atropine, yielded supra-additive dose ratios. The data could be reconciled with a model involving a ternary complex between (1) the receptor, (2) carbachol, N-methylscopolamine or atropine acting at the orthosteric binding site and (3) C7/3'-phth, alcuronium or gallamine acting at a common, allosteric site with varying degrees of heterotropic cooperativity.

Alcuronium↗

PubMed2006

A novel class of AMPA receptor allosteric modulators. Part 1: design, synthesis, and SAR of 3-aryl-4-cyano-5-substituted-heteroaryl-2-carboxylic acid derivatives.

The synthesis and initial SAR studies of novel, highly potent positive allosteric modulators of AMPA receptors based on 3-(4-tert-butylphenyl)-4-cyano-5-methylsulfanyl-thiophene-2-carboxylic acid (6a) are described. SAR studies at the thioether moiety indicated that substitution at this position was mandatory and better potency was achieved with small groups.

Allosteric Regulation↗

PubMed1997

Zinc allosterically modulates antagonist binding to cloned D1 and D2 dopamine receptors.

Cations of various size and charge were used as atomic scale probes of D1 and D2 dopamine receptors. Those cations that perturbed the binding of D1- and D2-selective dopamine receptor antagonists were identified by screening at 5 mM cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [3H]SCH-23390 and [3H]methylspiperone to cloned D1A and D2L dopamine receptors in either the presence or absence of Zn2+. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant (K(D)) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D1- and D2-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.

Allosteric Regulation↗

PubMed1999

Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.

In this study, thrombin interaction with the basic pancreatic trypsin inhibitor (BPTI) was investigated in the presence of different allosteric modulators of thrombin, that is the C-terminal hirudin peptide 54-65 (Hir54-65), a recombinant thrombomodulin form (TMEGF4-6) and Na+. BPTI binding to alpha-thrombin is positively linked to Na+. Under low sodium concentration (5 mM Na+) the BPTI affinity for alpha-thrombin was roughly threefold lower than in the presence of 150 mM sodium (Ki = 320 microM vs. 100 microM). The hirudin fragment, which binds to the fibrinogen recognition site (FRS) of thrombin, induced a progressive and saturable decrease (3.6-fold) of alpha-thrombin affinity for BPTI, whereas the thrombomodulin peptide, which binds to a more extended region of FRS, caused a 5.5-fold increase of the enzyme affinity for the inhibitor. The opposite effect exerted by Hir54-65 and TMEGF4-6 was also observed for BPTI interaction with zeta-thrombin, in which the amidic bond between W148 and T149 is cleaved. However, in this case the effect by Hir54-65 and TMEGF4-6, although qualitatively similar to that observed with alpha-thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54-65 and TMEGF4-6 peptides. While the latter enhanced the Protein C activation, the former caused a reduction of both alpha- and zeta-thrombin kcat/K(m)' for Protein C cleavage. These results showed that (a) Na+ facilitates BPTI interaction with thrombin; (b) Hir54-65 and TMEGF4-6, though sharing in part the same binding site at the thrombin FRS, can affect in opposite way thrombin's interaction with BPTI and Protein C; (c) such findings along with the results obtained with zeta-thrombin might be explained by admitting that the thermodynamic linkage between FRS and the critical W60-loop is also controlled by ligation and/or conformational state of the W148 insertion loop.

Allosteric Regulation↗

PubMed2005

Novel plant substances acting as beta subunit isoform-selective positive allosteric modulators of GABAA receptors.

GABAA receptors are modulated by a large variety of compounds. A common chemical characteristic of most of these modulators is that they contain a cyclic entity. Three linear molecules of a polyacetylene structure were isolated from the East African medicinal plant Cussonia zimmermannii Harms and shown to allosterically stimulate GABAA receptors. Stimulation was not abolished by the absence of the gamma2 subunit, the benzodiazepine antagonist Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester), or the point mutation beta2N265S that abolishes effects by loreclezole. At a concentration of 30 microM, the substances by themselves elicited only tiny currents. Maximal stimulation at alpha1beta2gamma2 amounted to 110 to 450% for the three substances, and half-maximal stimulation was observed at concentrations of 1 to 2 muM. Stimulation was subunit composition-dependent and was for the substance MS-1, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha3beta2gamma2 > alpha2beta2gamma2 > alpha5beta2gamma2 approximately alpha1beta3gamma2 approximately alpha6beta2gamma2 > alpha1beta1gamma2, for MS-2 alpha1beta2gamma2 approximately alpha3beta2gamma2 approximately alpha1beta2 > alpha2beta2gamma2 approximately alpha6beta2gamma2 approximately alpha5beta2gamma2 > alpha1beta1gamma2, and for MS-4, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha5beta2gamma2 approximately alpha3beta2gamma2 approximately alpha2beta2gamma2 > alpha6beta2gamma2 >> alpha1beta1gamma2. Maximal stimulation by MS-1 was 450% at alpha1beta2gamma2, 80% at alpha1beta1gamma2, and 150% at alpha1beta3gamma2. MS-1 was thus specific for receptors containing the beta2 subunit. The reversal potential was unaffected by 10 microM MS-1, whereas apparent picrotoxin affinity for current inhibition was increased approximately 3-fold. In summary, these positive allosteric modulators of GABAA receptors of plant origin have a novel unusual chemical structure and act at a site independent of that of benzodiazepines and loreclezole.

Allosteric Regulation↗