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Updated: Feb 15, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Multiple Binding Sites Contribute to the Mechanism of Mixed Agonistic and Positive Allosteric Modulators of the
Noureldin Saleh1, Oliver Hucke2, Gert Kramer3
1Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Institute of Medical Physics and Biophysics, Charitéplatz 1, 10117, Berlin, Germany.
Abstract:
The cannabinoid CB1 receptor (CB1R) is an abundant metabotropic G-protein-coupled receptor that has been difficult to address therapeutically because of CNS side effects exerted by orthosteric drug candidates. Recent efforts have focused on developing allosteric modulators that target CB1R. Compounds from the recently discovered class of mixed agonistic and positive allosteric modulators (Ago-PAMs) based on 2-phenylindoles have shown promising functional and binding properties as CB1R ligands. Here, we identify binding modes of both the CP 55,940 agonist and GAT228, a 2-phenylindole allosteric modulator, by using our metadynamics simulation protocol, and quantify their affinity and cooperativity by atomistic simulations. We demonstrate the involvement of multiple adjunct binding sites in the Ago-PAM characteristics of the 2-phenylindole modulators and explain their ability to compete with orthosteric agonists at higher concentrations. We validate these results experimentally by showing the contribution of multiple sites on the allosteric binding of ZCZ011, another homologous member of the class, together with the orthosteric agonist.
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