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PubMed2003

Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor.

A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.

Allosteric Regulation↗
PubMed2000

Zinc inhibits miniature GABAergic currents by allosteric modulation of GABAA receptor gating.

Zinc is abundantly present in the CNS, and after nerve stimulation is thought to be released in sufficient quantity to modulate the synaptic transmission. Although it is known that this divalent cation inhibits the GABAergic synaptic currents, the underlying mechanisms were not fully elucidated. Here we report that zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of GABA(A) receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding (k(on)) and the transition rate from closed to open state (beta(2)). Moreover, slower onset and recovery from desensitization as well as an increased unbinding rate (k(off)) were shown to underlie the accelerated deactivation kinetics in the presence of zinc. The nonequilibrium conditions of GABA(A) receptor activation were found to strongly affect zinc modulation of this receptor. In particular, an extremely fast clearance of synaptic GABA is implicated to be responsible for a stronger zinc effect on mIPSCs than on current responses to exogenous GABA. Finally, the analysis of currents evoked by GABA coapplied with zinc indicated that the interaction between zinc and GABA(A) receptors was too slow to explain zinc effects in terms of competitive antagonism. In conclusion, our results provide evidence that inhibition of mIPSCs by zinc is attributable to the allosteric modulation of GABA(A) receptor gating.

Allosteric Regulation↗
PubMed2005

The Na+ binding channel of human coagulation proteases: novel insights on the structure and allosteric modulation revealed by molecular surface analysis.

Thrombovascular diseases result from imbalanced haemostasis and comprise important health problems in the aging population worldwide. The activity of enzymes pertaining to the coagulation cascade of mammalians exhibit several control mechanisms in order to maintain a proper balance between bleeding and thrombosis. For instance, human coagulation serine proteases carrying a F225 or Y225 are allosteric modulated by the binding of Na+ in a water-filled channel connected to the primary specificity pocket (S1 subsite) of these enzymes. We have characterized the structure, topography and lipophilicity of this channel in the ligand-free fast (sodium-bound) and slow (sodium-free) forms of thrombin, in the sole available structure of activated protein C and in several structures of the coagulation factors VIIa, IXa and Xa, differing in the nature of the bound inhibitor and in the occupancy of exosite-I as well as the Ca2+ and Na+ binding sites. Opposite to thrombin, the aqueous channels in all other coagulation enzymes sheltering a Na+ binding site do not have an aperture on the enzyme surface opposite to the S1 subsite entrance. In these enzymes, the lack of the three-residue insertion in loop 1 (183-189) as found in thrombin allied to compensatory mutations in the positions 187-185 and 222 effects a constriction in the water-filled channel that ends up by segregating the ion binding site from the S1 subsite. We also disclosed major topographical changes on the thrombin's surface upon sodium release and transition to the slow form that culminate in the narrowing of the S1 subsite entrance and, strikingly, in the loss of communication between the primary specificity pocket and the exosite-I. Such observation is in accordance with existing experimental data demonstrating thermodynamic linkage between these distant regions on the thrombin surface. Conformational changes in F34, L40, R73 and T74 were the main responsible for this effect. A path by which these changes in the vicinity of exosite-I could be transmitted to the S1 subsite and, consequently, to the sodium binding site is proposed.

Allosteric Regulation↗
PubMed2001

Allosteric modulation in spontaneously active mutant gamma-aminobutyric acidA receptors.

Tryptophan substitutions were made in the second transmembrane domain of the gamma-aminobutyric acid(A) (GABAA) receptor alpha and beta subunits and the resulting mutant receptors, containing alpha2(S270W) and/or beta1 (S265W), were expressed in Xenopus oocytes. Mutation of either or both subunits resulted in receptors that exhibited enhanced sensitivity to agonist and were spontaneously active in the absence of GABA. The spontaneous activity was blocked by picrotoxin or bicuculline. The enhancement of GABA-induced currents by pentobarbital, by the neurosteroid 5alpha-pregnan-3alpha-ol-20-one, and by the benzodiazepine flunitrazepam was dramatically reduced in the mutant receptors. These results are consistent with the idea that a mutation that promotes gating behavior in a ligand-gated ion channel will also show reduced effects of all positive allosteric modulators in a generalized manner, even when these modulators act at distinct sites on the receptor.

Allosteric Regulation↗
PubMed2005

Molecular pharmacology and therapeutic prospects of metabotropic glutamate receptor allosteric modulators.

The metabotropic glutamate receptors (mGluR) consist of a family of eight G-protein-coupled receptors that differ in their function, distribution and physiological roles within the central nervous system. In recent years substantial efforts have been made towards developing selective agonists and antagonists which have proven useful for elucidating their potential as novel targets for the treatment of psychiatric and neurological diseases. In the present review we will provide an update of the recent developments of functional allosteric modulators of the mGluR family and explore their therapeutic potential for anxiety/depression, schizophrenia, epilepsy/stroke, pain and Alzheimer's, Parkinson's and Huntington's diseases.

Allosteric Regulation↗
PubMed1996

Negative allosteric modulation of wild-type and mutant AMPA receptors by GYKI 53655.

Benzothiadiazides such as cyclothiazide potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor responses, whereas 2,3-benzodiazepines such as 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4 -dihydro- 5H-2,3-benzodiazepine (GYKI 53655) act as noncompetitive antagonists; both drugs act through allosteric modulation. Controversy exists as to whether cyclothiazide and GYKI 53655 act at a common site. Recent mutational analysis has led to the identification of a serine residue in flip splice variants that is critical for directing the interaction of cyclothiazide with AMPA receptors. We tested whether the mutation of this residue to glutamine, which abolishes potentiation by cyclothiazide, can in addition block antagonism by 2,3-benzodiazepines, as would be predicted for action at a common site. We found that the S to Q mutation does not alter antagonism by 2,3-benzodiazepines, suggesting that the molecular determinants directing the interaction between GYKI 53655 and AMPA receptors are not identical to those controlling sensitivity to cyclothiazide. Additional support for this was obtained from analysis of the responses of AMPA receptor flip/flop splice variants, which, despite differences in equilibrium desensitization and sensitivity to cyclothiazide, show only small differences in sensitivity to 2,3-benzodiazepines. Furthermore, introduction of the flip exon from GluRA into GluR6, conferred sensitivity to cyclothiazide but did not increase sensitivity to 2,3-benzodiazepines. Of interest, experiments with native AMPA receptors generated from hippocampal and forebrain poly(A)+ mRNA revealed greater sensitivity to 2,3-benzodiazepines than receptors generated by expression of recombinant AMPA receptors, possibly indicating the existence of an unidentified accessory protein or novel receptor subunit.

Allosteric Regulation↗
PubMed2006

Allosteric modulation of the effect of escitalopram, paroxetine and fluoxetine: in-vitro and in-vivo studies.

Clinical and preclinical studies have shown that the effect of citalopram on serotonin (5-HT) reuptake inhibition and its antidepressant activity resides in the S-enantiomer. In addition, using a variety of in-vivo and in-vitro paradigms, it was shown that R-citalopram counteracts the effect of escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 microg/kg i.v.) blocked the suppressant effect on neuronal firing activity of both escitalopram (100 microg/kg i.v.) and paroxetine (500 microg/kg i.v.), but not fluoxetine (10 mg/kg i.v.). Interestingly, administration of R-citalopram (8 mg/kg i.p.) attenuated the increase of extracellular levels of 5-HT ([5-HT]ext) in the ventral hippocampus induced by both escitalopram (0.28 microM) and paroxetine (0.75 microM), but not fluoxetine (10 microM). In conclusion, the present in-vitro and in-vivo studies show that R-citalopram counteracts the activity of escitalopram and paroxetine, but not fluoxetine, by acting at the allosteric binding site of the 5-HT transporter, either located in the dorsal raphe nucleus or post-synaptically in the ventral hippocampus. This conclusion is strengthened by the observation that the inhibitory effect of fluoxetine, which has no stabilizing effect on the radioligand/hSERT complex, was not blocked by co-administration of R-citalopram.

Action Potentials↗
PubMed2000

Allosteric modulation in spontaneously active mutant gamma-aminobutyric acid(A) receptors [corrected].

Tryptophan substitutions were made in the second transmembrane domain of the gamma-aminobutyric acid(A) (GABA(A)) receptor alpha and beta subunits and the resulting mutant receptors, containing alpha(2)(S270W) and/or beta(1)(S265W), were expressed in Xenopus oocytes. Mutation of either or both subunits resulted in receptors that exhibited enhanced sensitivity to agonist and were spontaneously active in the absence of GABA. The spontaneous activity was blocked by picrotoxin or bicuculline. The enhancement of GABA-induced currents by pentobarbital, by the neurosteroid 5alpha-pregnan-3alpha-ol-20-one, and by the benzodiazepine flunitrazepam was dramatically reduced in the mutant receptors. These results are consistent with the idea that a mutation that promotes gating behavior in a ligand-gated ion channel will also show reduced effects of all positive allosteric modulators in a generalized manner, even when these modulators act at distinct sites on the receptor.

Allosteric Regulation↗
PubMed2005

A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization.

Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the alpha7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human alpha7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by alpha4beta2, alpha3beta4, and alpha9alpha10 nAChRs. PNU-120596 increased the channel mean open time of alpha7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of alpha7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances alpha7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Acetylcholine↗
PubMed1999

Allosteric modulation and accelerated resensitization of human P2X(3) receptors by cibacron blue.

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X(3) (hP2X(3)) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X(2), as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X(3) receptors. In 1321N1 cells expressing the hP2X(3) receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca(2+) influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC(50) = 1.4 microM) was independent of the agonist used to activate the hP2X(3) receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X(3) receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X(3) receptor recovery from desensitization (from T(1/2) = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X(3) receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.

Adenosine Triphosphate↗
PubMed1988

Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes.

Chick brain mRNA was isolated and injected into Xenopus oocytes. This led to the expression in the surface membrane of functional GABA-activated channels with properties reminiscent of vertebrate GABAA channels. The GABA-induced current was analyzed quantitatively under voltage-clamp conditions. Picrotoxin inhibited this current in a concentration-dependent manner with IC50 = 0.6 microM. The allosteric modulation of GABA currents by a number of drugs acting at the benzodiazepine binding site was characterized quantitatively. In the presence of the benzodiazepine receptor ligands diazepam and clorazepate, GABA responses were enhanced, and in the presence of the convulsant beta-carboline compound methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), they were depressed. Maximal stimulation of the response elicited by 10 microM GABA was 160% with diazepam and 90% with clorazepate, and maximal inhibition was 42% with DMCM, 30% with methyl beta-carboline-3-carboxylate (beta-CCM), 15% with ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate (Ro 15-1788), and 12% with ethyl beta-carboline-3-carboxylate (beta-CCE). Half-maximal stimulation was observed with 20 nM diazepam and 390 nM clorazepate, respectively, and half-maximal inhibition with 6 nM DMCM. beta-CCM had a similar effect to DMCM, whereas beta-CCE and Ro 15-1788 showed only small inhibition at low concentrations (less than 1 microM). All the tested carboline compounds and Ro 15-1788 showed a biphasic action and stimulated GABA current at concentrations higher than 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Calindol, a positive allosteric modulator of the human Ca(2+) receptor, activates an extracellular ligand-binding domain-deleted rhodopsin-like seven-transmembrane structure in the absence of Ca(2+).

The extracellular calcium-sensing human Ca(2+) receptor (hCaR),2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethyl-aminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca(2+) activation without independently activating the wild type receptor. Because agonists activate rhodopsin-like family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca(2+) receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsin-like 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca(2+) for the ECD-containing wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca(2+) alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that activation of Ca(2+) T903-Rhoc by Calindol truly the is independent using in vitro reconstitution with purified G(q). These findings demonstrate distinct allosteric linkages between Ca(2+) site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics.

Allosteric Regulation↗
PubMed2001

Alpha-amino acid phenolic ester derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABA(A) receptors.

In the search for a novel water-soluble general anesthetic agent the activity of an alpha-amino acid phenolic ester lead, identified from patent literature, was markedly improved. In addition to improving in vivo activity in mice, good in vitro activity at GABA(A) receptors was also conferred. Within the series of compounds good enantioselectivity for both in vitro and in vivo activity was found, supporting a protein-mediated mechanism of action for anesthesia involving allosteric modulation of GABA(A) receptors. alpha-Amino acid phenolic ester 19, as the hydrobromide salt Org 25435, was selected for clinical evaluation since it retained the best overall anesthetic profile coupled with improved stability and water solubility. In the clinic it proved to be an effective intravenous anesthetic in man with rapid onset of and recovery from anesthesia at doses of 3 and 4 mg/kg.

Allosteric Regulation↗
PubMed1995

Allosteric modulation of oxygen binding to the three human embryonic haemoglobins.

Plasmid based yeast expression systems have been developed for the high-level expression of the three human embryonic haemoglobins Gower I (zeta 2 epsilon 2), Gower II (alpha 2 epsilon 2) and Portland (zeta 2 gamma 2). Physiochemical characterization of the three product haemoglobins show them to be in the 'native' state. Oxygen-binding studies show that, under what are usually considered physiological conditions, each of the embryonic haemoglobins shows a high oxygen affinity, coupled to a high degree of co-operativity. Allosteric modulation of the oxygen-binding properties of the three haemoglobins in response to organic phosphates and protons has been investigated. The various responses exhibited by the three haemoglobins are rationalized in terms of their amino acid sequences.

2,3-Diphosphoglycerate↗
PubMed1987

Phenylquinolines PK 8165 and PK 9084 allosterically modulate [35S]t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5 4864 binding site.

The binding of the cage convulsant, [35S]t-butylbicyclophosphorothionate ([35S]TBPS), to a picrotoxin-sensitive site in rat cerebral cortical homogenates was used to identify and characterize the site of action of the phenylquinolines PK 8165 and PK 9084, the isoquinoline PK 11195 and the atypical benzodiazepine (BZ) Ro5 4864. These agents were found to allosterically modulate the binding of 2 nM [35S]TBPS in a pharmacologically relevant fashion. Evidence is presented to suggest that these compounds share a common site of action as modulators of [35S]TBPS binding. The relative potencies of these compounds in vitro are in the submicromolar to micromolar concentration range and correlate well with the concentrations reported to elicit specific responses in behavioral and electrophysiologic studies. Modulation of [35S]TBPS binding in vitro is affected by micromolar quantities of gamma-aminobutyric acid in a (+)-bicuculline-sensitive fashion and is unaffected by the central BZ receptor "antagonist" Ro15 1788. Collectively, the evidence presented suggests the existence of a novel drug binding site that is functionally coupled to a gamma-aminobutyric acid-A receptor and a [35S]TBPS-labeled chloride ionophore. Moreover, this site is distinct from the central BZ receptor recognized by clonazepam and the high-affinity peripheral BZ binding site labeled by [3H] Ro5 4864. The hypothesis is proposed that the novel "Ro5 4864 site" identified in the present study is a functionally relevant binding site that mediates some of the pharmacologic effects of Ro5 4864, PK 8165, PK 9084 and PK 11195 in the mammalian central nervous system.

Animals↗
PubMed2006

Structure-activity relationships of substituted N-benzyl piperidines in the GBR series: Synthesis of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine, an allosteric modulator of the serotonin transporter.

A series of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-(substituted benzyl) piperidines with substituents at the ortho and meta positions in the aromatic ring of the N-benzyl side chain were synthesized and their affinities and selectivities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined. One analogue, 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine (the C(2)-trifluoromethyl substituted compound), has been found to act as an allosteric modulator of hSERT binding and function. It had little affinity for any of the transporters. Several compounds showed affinity for the DAT in the low nanomolar range and displayed a broad range of SERT/DAT selectivity ratios and very little affinity for the NET. The pharmacological tools provided by the availability of compounds with varying transporter affinity and selectivity could be used to obtain additional information about the properties a compound should have to act as a useful pharmacotherapeutic agent for cocaine addiction and help unravel the pharmacological mechanisms relevant to stimulant abuse.

Allosteric Regulation↗
PubMed1984

Capped mRNAs may stimulate the influenza virion polymerase by allosteric modulation.

Analogues of the mRNA 5'-terminal methyl cap structure were found to stimulate the influenza virion RNA-dependent RNA polymerase. The single nucleotide analogue m7GMP was incorporated into RNA during transcription in vitro, and the stimulatory effect was not additive with the primer ApG, suggesting that m7GMP stimulates the virion polymerase by priming virus-specific mRNA synthesis, as has been shown for ApG. By contrast, stimulation by m7G(5')ppp(5')m6AM2-O was additive with that by ApG, and we could not demonstrate incorporation of the similar analogue m7G(5')ppp(5')Am2-O into RNA during transcription. We propose that these dinucleotide cap analogues stimulate the virion polymerase by allosteric modulation, independent of priming. This stimulation can be abolished by mutation, without loss of other activities associated with the cap-dependent endonuclease.

Allosteric Regulation↗
PubMed2001

Positive allosteric modulation of native and recombinant gamma-aminobutyric acid(B) receptors by 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501.

The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of gamma-aminobutyric acid(B) (GABA(B)) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate) (GTP gamma[(35)S]) binding to membranes from a GABA(B(1b/2)) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTP gamma[(35)S] binding assay using a native GABA(B) receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTP gamma[(35)S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B(1b/2)) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABA(B(1b)) receptors. In addition to GTP gamma[(35)S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca(2+) signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect of L-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABA(B) receptors may represent a novel means of therapeutic interference with the GABA-ergic system.

Allosteric Regulation↗