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Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A 1 R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A 1 R of an A 1 R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A 1 R antagonists and showed evidence of covalent binding and simultaneous binding across an A 1 R homodimer. Greater selectivity for A 1 R over the adenosine A 3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Epilepsy is one of the most serious and common chronic neurological conditions, characterised by recurrent hypersynchronous electrical activity in the brain that lead to seizures. Despite over 50 million people being affected worldwide, only ~70% of people with epilepsy have their seizures successfully controlled with current pharmacotherapy, and many experience significant psychiatric and physical comorbidities. Adenosine, a ubiquitous purine metabolite, is a potent endogenous anti-epileptic substance that can abolish seizure activity via the adenosine A 1 G protein-coupled receptor. Activation of A 1 receptors decreases seizure activity in animal models, including models of drug-resistant epilepsy. Recent advances have increased our understanding of epilepsy comorbidities, highlighting the potential for adenosine receptors to modulate epilepsy-associated comorbidities, including cardiovascular dysfunction, sleep and cognition. This review provides an accessible resource of the current advances in understanding the adenosine system as a therapeutic target for epilepsy and epilepsy-associated comorbidities. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Development of subtype-selective leads is essential in drug discovery campaigns targeting G protein-coupled receptors (GPCRs). Herein, a structure-based virtual screening approach to rationally design subtype-selective ligands was applied to the A 1 and A 2A adenosine receptors (A 1 R and A 2A R). Crystal structures of these closely related subtypes revealed a non-conserved subpocket in the binding sites that could be exploited to identify A 1 R selective ligands. A library of 4.6 million compounds was screened computationally against both receptors using molecular docking and 20 A 1 R selective ligands were predicted. Of these, seven antagonized the A 1 R with micromolar activities and several compounds displayed slight selectivity for this subtype. Twenty-seven analogs of two discovered scaffolds were designed, resulting in antagonists with nanomolar potency and up to 76-fold A 1 R-selectivity. Our results show the potential of structure-based virtual screening to guide discovery and optimization of subtype-selective ligands, which could facilitate the development of safer drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A 1 receptor (A 1 R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A 1 R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A 1 R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A 1 R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A 1 R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A 1 R as a potential therapeutic target and highlight recent advances in the structural understanding of A 1 R allosteric modulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nguyen, Tran, Baltos, McNeill, Nguyen and May.)

Alzheimer's disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer's disease. The adenosine A 1 and A 2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic β-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer's disease and the therapeutic potential of A 1 and A 2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response., (© 2022. The Author(s).)

Adenosine modulates many aspects of human physiology and pathophysiology through binding to the adenosine family of G protein-coupled receptors, which are comprised of four subtypes, the A 1 R, A 2A R, A 2B R and A 3 R. Modulation of adenosine receptor function by exogenous agonists, antagonists and allosteric modulators can be beneficial for a number of conditions including cardiovascular disease, Parkinson's disease, and cancer. Unfortunately, many preclinical drug candidates targeting adenosine receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning adenosine receptor ligands into the clinic, research efforts have focussed on exploiting the phenomenon of biased agonism. Biased agonism provides the opportunity to develop ligands that favour therapeutic signalling pathways, whilst avoiding signalling associated with on-target undesired effects. Recent studies have begun to define the structure-function relationships that underpin adenosine receptor biased agonism and establish how this phenomenon can be harnessed therapeutically. In this review we describe the recent advancements made towards achieving therapeutically relevant biased agonism at adenosine receptors., (Copyright © 2021. Published by Elsevier Inc.)

Adenosine receptors are a family of GPCRs containing four subtypes (A 1 , A 2A , A 2B and A 3 receptors), all of which bind the ubiquitous nucleoside adenosine. These receptors play an important role in physiology and pathophysiology and therefore represent attractive drug targets for a range of conditions. The theoretical framework surrounding drug action at adenosine receptors now extends beyond the notion of prototypical agonism and antagonism to encompass more complex pharmacological concepts. New paradigms include allostery, in which ligands bind a topographically distinct receptor site from that of the endogenous agonist, homomeric or heteromeric interactions across receptor oligomers and biased agonism, that is, ligand-dependent differential intracellular signalling. This review provides a concise overview of allostery, oligomerization and biased agonism at adenosine receptors and outlines how these paradigms may enhance future drug discovery endeavours focussed on the development of novel therapeutic agents acting at adenosine receptors., Linked Articles: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc., (© 2018 The British Pharmacological Society.)

Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 Å resolution, respectively. The dominant negative Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery., Competing Interests: The authors declare no competing financial interest., (Copyright © 2018 American Chemical Society.)

The class A adenosine A 1 receptor (A 1 R) is a G-protein-coupled receptor that preferentially couples to inhibitory G i/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A 1 R in complex with adenosine and heterotrimeric G i2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A 1 R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A 1 R primarily via amino acids in the C terminus of the Gα i α5-helix, concomitant with a 10.5 Å outward movement of the A 1 R transmembrane domain 6. Comparison with the agonist-bound β 2 adrenergic receptor-G s -protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A 1 R structure provides molecular insights into receptor and G-protein selectivity.

The adenosine A 1 receptor (A 1 AR) is a potential novel therapeutic target for myocardial ischemia-reperfusion injury. However, to date, clinical translation of prototypical A 1 AR agonists has been hindered due to dose limiting adverse effects. Recently, we demonstrated that the biased bitopic agonist 1, consisting of an adenosine pharmacophore linked to an allosteric moiety, could stimulate cardioprotective A 1 AR signaling in the absence of unwanted bradycardia. Therefore, this study aimed to investigate the structure-activity relationship of compound 1 biased agonism. A series of novel derivatives of 1 were synthesized and pharmacologically profiled. Modifications were made to the orthosteric adenosine pharmacophore, linker, and allosteric 2-amino-3-benzoylthiophene pharmacophore to probe the structure-activity relationships, particularly in terms of biased signaling, as well as A 1 AR activity and subtype selectivity. Collectively, our findings demonstrate that the allosteric moiety, particularly the 4-(trifluoromethyl)phenyl substituent of the thiophene scaffold, is important in conferring bitopic ligand bias at the A 1 AR.

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M 2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N -methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a K B of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M 2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [ 35 S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α 1A -adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α 1A -AR signalling in CHO-K1 cells co-expressing the human α 1A -AR and GLUT4 (CHOα 1A GLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca 2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α 1A -AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α 1 -AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOα 1A GLUT4myc cells, siRNA directed against rictor but not raptor suppressed α 1A -AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α 1A -AR agonists. Our findings identify a novel link between the α 1A -AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α 1A -AR selective agonists as tools in the treatment of cardiac dysfunction., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The adenosine A 2B receptor (A 2B AR) has been identified as an important therapeutic target in cardiovascular disease, however in vitro and in vivo targeting has been limited by the paucity of pharmacological tools, particularly potent agonists. Interestingly, 2-((6-amino-3,5-dicyano-4-(4-(cyclopropylmethoxy)phenyl)-2-pyridinyl)thio)acetamide (BAY60-6583), a potent and subtype-selective A 2B AR agonist, has the same core structure as 2-amino-6-[[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methylsulfanyl]-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitril (capadenoson). Capadenoson, currently classified as an adenosine A 1 receptor (A 1 AR) partial agonist, has undergone two Phase IIa clinical trials, initially in patients with atrial fibrillation and subsequently in patients with stable angina. Capadenoson has also been shown to decrease cardiac remodeling in an animal model of advanced heart failure and a capadenoson derivative, neladenoson bialanate, recently entered clinical development for the treatment of chronic heart failure. The therapeutic effects of capadenoson are currently thought to be mediated through the A 1 AR. However, the ability of capadenoson to stimulate additional adenosine receptor subtypes, in particular the A 2B AR, has not been rigorously assessed. In this study, we demonstrate that capadenoson does indeed have significant A 2B AR activity in physiologically relevant cells, cardiac fibroblasts and cardiomyocytes, which endogenously express the A 2B AR. Relative to the non-selective adenosine receptor agonist NECA, capadenoson was a biased A 2B AR agonist with a preference for cAMP signal transduction over other downstream mediators in cells with recombinant and endogenous A 2B AR expression. These findings suggest the reclassification of capadenoson as a dual A 1 AR/A 2B AR agonist. Furthermore, a potential A 2B AR contribution should be an important consideration for the future clinical development of capadenoson-like therapeutics, as the A 2B AR can promote cardioprotection and modulate cardiac fibrosis in heart disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The adenosine A 1 G protein-coupled receptor (A 1 AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A 1 AR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A 1 AR-ECL2 to explore the role of this domain on A 1 AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A 1 AR orthosteric ligand binding and receptor activation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with significant N(6) or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5'-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding., (U.S. Government work not protected by U.S. copyright.)

Adenosine A1 receptor (A1AR) stimulation is a powerful protective mechanism in cerebral and cardiac ischemia-reperfusion injury. Despite this, therapeutic targeting of the A1AR for the treatment of ischemia-reperfusion injury has been largely unsuccessful, as high concentrations of prototypical A1AR agonists impart significant hemodynamic effects, particularly pronounced bradycardia, atrioventricular block and hypotension. Exploiting the phenomenon of biased agonism to develop ligands that promote A1AR cytoprotection in the absence of adverse hemodynamic effects remains a relatively unexplored, but exciting, approach to overcome current limitations. In native systems, the atypical A1AR agonists VCP746 and capadenoson retain cytoprotective signaling in the absence of bradycardia, a phenomenon suggestive of biased agonism. The current study used pharmacological inhibitors to investigate A1AR mediated cytoprotective signal transduction in a CHO FlpIn cell background, thus identifying candidate pathways for quantitative bias profiling, including cAMP, extracellular signal-regulated kinases 1 and 2 and Akt1/2/3. Subsequently, effects on cell survival and the bias profile of VCP746 and capadenoson were determined and compared to that of the prototypical A1AR agonists, NECA, R-PIA, MeCCPA and CPA. We found that prototypical agonists do not display significant bias for any of the pathways assessed. In contrast, VCP746 and capadenoson show significant bias away from calcium mobilization relative to all pathways tested. These studies demonstrate that quantitative "fingerprinting" of biased agonism within a model system can enable ligands to be clustered by their bias profile, which in turn may be predictive of preferential physiologically relevant in vivo pharmacology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A1 GPCR (A1AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A1AR ligand (VCP746)--a hybrid molecule comprising adenosine linked to a positive allosteric modulator--specifically to engender biased signaling at the A1AR. We validate that the interaction of VCP746 with the A1AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A1AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.