Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches.

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor.

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.

Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor.

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M1 muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M1 mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85(2.64) in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397(7.32) and Trp-400(7.35) in TMVII as residues that contribute to the BQCA binding pocket at the M1 mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described "common" allosteric site in the extracellular vestibule of the M1 mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102(3.29) and Asp-105(3.32)) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.

The 8-hydroxyquinoline derivative, clioquinol, is an alpha-1 adrenoceptor antagonist.

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.

Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

Allosteric modulation of a chemogenetically modified G protein-coupled receptor.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetically modified muscarinic acetylcholine receptors (mAChRs) that have minimal responsiveness to acetylcholine (ACh) but are potently and efficaciously activated by an otherwise inert synthetic ligand, clozapine-N-oxide (CNO). DREADDs have been used as tools for selectively modulating signal transduction pathways in vitro and in vivo. Recent comprehensive studies have validated how the pharmacology of a CNO-bound DREADD mirrors that of an ACh-bound wild-type (WT) mAChR. However, nothing is known about whether this equivalence extends to the allosteric modulation of DREADDs by small molecules. To address this, we investigated the actions at an M(1) DREADD of benzyl quinolone carboxylic acid (BQCA), a positive allosteric modulator of ACh binding and function that is known to behave according to a simple two-state mechanism at the WT receptor. We found that allosteric modulation of the CNO-bound DREADD receptor is not equivalent to the corresponding modulation of the ACh-bound WT receptor. We also found that BQCA engenders stimulus bias at the M(1) DREADD, having differential types of cooperativity depending on the signaling pathway. Furthermore, the modulation of ACh itself by BQCA at the DREADD is not compatible with the two-state model that we previously applied to the M(1) WT receptor.

Diazepam is not a direct allosteric modulator of α1-adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.

α1A- and α1B-adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A- and α1B-AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1-ARs. Here, using thermostabilized, purified, α1A- and α1B-ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A- and α1B-ARs. Similarly, in cell-based assays expressing unmodified α1A- and α1B-ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A- and α1B-ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

Gram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs.

Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing in vivo functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine N-oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: •A simple extraction method of clozapine from commercial tablets;•A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and•That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq in vivo. This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.

Novel Irreversible Agonists Acting at the A1 Adenosine Receptor.

The A1 adenosine receptor (A1AR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the A1AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesized, and four compounds, 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the A1AR. Of these compounds, 15b and 15d were more directly validated as irreversible agonists of the A1AR using membrane-based [3H]DPCPX and [35S]GTPγS binding experiments.

Regulation of G protein-coupled receptors by allosteric ligands.

Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs.