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Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

ABSTRACT: BACKGROUND AND PURPOSE:Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-?-thiotriphosphate (GTP?S) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M? muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH:Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³?S]GTP?S and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M? muscarinic acetylcholine receptor. KEY RESULTS:Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTP?S (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTP?S binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS:These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTP?S binding.

SUBMITTER: Jakubik J

PROVIDER: S-EPMC3051377 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

Jakubík J J Janíčková H H El-Fakahany E E EE Doležal V V

British journal of pharmacology 20110301 5

<h4>Background and purpose</h4>Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy.<h4>Experimental approach</h4>Filtration and scintillation p ...[more]

PMID: 20958290

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Project description:BackgroundThe muscarinic receptor agonist xanomeline has antipsychotic properties and is devoid of dopamine receptor-blocking activity but causes cholinergic adverse events. Trospium is a peripherally restricted muscarinic receptor antagonist that reduces peripheral cholinergic effects of xanomeline. The efficacy and safety of combined xanomeline and trospium in patients with schizophrenia are unknown.MethodsIn this double-blind, phase 2 trial, we randomly assigned patients with schizophrenia in a 1:1 ratio to receive twice-daily xanomeline-trospium (increased to a maximum of 125 mg of xanomeline and 30 mg of trospium per dose) or placebo for 5 weeks. The primary end point was the change from baseline to week 5 in the total score on the Positive and Negative Syndrome Scale (PANSS; range, 30 to 210, with higher scores indicating more severe symptoms of schizophrenia). Secondary end points were the change in the PANSS positive symptom subscore, the score on the Clinical Global Impression-Severity (CGI-S) scale (range, 1 to 7, with higher scores indicating greater severity of illness), the change in the PANSS negative symptom subscore, the change in the PANSS Marder negative symptom subscore, and the percentage of patients with a response according to a CGI-S score of 1 or 2.ResultsA total of 182 patients were enrolled, with 90 assigned to receive xanomeline-trospium and 92 to receive placebo. The PANSS total score at baseline was 97.7 in the xanomeline-trospium group and 96.6 in the placebo group. The change from baseline to week 5 was -17.4 points with xanomeline-trospium and -5.9 points with placebo (least-squares mean difference, -11.6 points; 95% confidence interval, -16.1 to -7.1; P<0.001). The results for the secondary end points were significantly better in the xanomeline-trospium group than in the placebo group, with the exception of the percentage of patients with a CGI-S response. The most common adverse events in the xanomeline-trospium group were constipation, nausea, dry mouth, dyspepsia, and vomiting. The incidences of somnolence, weight gain, restlessness, and extrapyramidal symptoms were similar in the two groups.ConclusionsIn a 5-week trial, xanomeline-trospium resulted in a greater decrease in the PANSS total score than placebo but was associated with cholinergic and anticholinergic adverse events. Larger and longer trials are required to determine the efficacy and safety of xanomeline-trospium in patients with schizophrenia. (Funded by Karuna Therapeutics and the Wellcome Trust; ClinicalTrials.gov number, NCT03697252.).

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Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

Publications

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

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