Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular “canonical” site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/β− sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.

Background: Human pentameric ligand-gated ion channels (pLGICs) comprise nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type 3 receptors (5-HT3Rs), zinc-activated channels (ZAC), γ-aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs). They are recognized therapeutic targets of some of the most prescribed drugs like general anesthetics, anxiolytics, smoking cessation aids, antiemetics and many more. Currently, approximately 100 experimental structures of pLGICs with ligands bound exist in the protein data bank (PDB). These atomic-level 3D structures enable the generation of a comprehensive binding site inventory for the superfamily and the in silico prediction of binding site properties.Methods: A panel of high throughput in silico methods including pharmacophore screening, conformation analysis and descriptor calculation was applied to a selection of allosteric binding sites for which in vitro screens are lacking. Variant abundance near binding site forming regions and computational docking complement the approach.Results: The structural data reflects known and novel binding sites, some of which may be unique to individual receptors, while others are broadly conserved. The membrane spanning domain, comprising four highly conserved segments, contains ligand interaction sites for which in vitro assays suitable for high throughput screenings are critically lacking. This is also the case for structurally more variable novel sites in the extracellular domain. Our computational results suggest that the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) can utilize multiple pockets which are likely to exist on most superfamily members.Conclusion: With this study, we explore the potential for polypharmacology among pLGICs. Our data suggest that ligands can display two forms of promiscuity to an extent greater than what has been realized: 1) Ligands can interact with homologous sites in many members of the superfamily, which bears toxicological relevance. 2) Multiple pockets in distinct localizations of individual receptor subtypes share common ligands, which counteracts efforts to develop selective agents. Moreover, conformational states need to be considered for in silico drug screening, as certain binding sites display considerable flexibility. In total, this work contributes to a better understanding of polypharmacology across pLGICs and provides a basis for improved structure guided in silico drug development and drug derisking.

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABA A receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal α5 subunitcontaining GABA A receptors. The purpose of this study is to investigate the effects of tricyclic compounds on α5 subunit-containing receptor subtypes.Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABA A receptor subtypes by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the α5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings.

Background and Purpose: Many psychotherapeutic drugs including clozapine have a polypharmacological profile and act on GABA receptors, where subtype-specific information is often lacking. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal α5 subunit containing GABA receptors. Experimental Approach: Functional studies of GABA modulatory effects by antipsychotic and antidepressant medications were performed in several GABA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the α5 subunit, probing a novel binding site. Computational ligand analysis complemented the functional and mutational data. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine have negative modulatory effects on multiple GABA receptor subtypes, including α5-containing. On the latter we show negative modulatory effects for five additional antipsychotic and antidepressant drugs. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in α5 GABA receptor subunits. Conclusion and Implications: Our findings support previous studies suggesting a link between some of the therapeutic effects of clozapine and its negative modulatory action on certain GABA receptor subtypes. The novel site we describe in this study is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology.

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