Emerging GPCR targets for AUD: Insights from preclinical studies

G protein-coupled receptors (GPCRs) are the largest group of membrane receptors in the central nervous system and one of the key proteins for signal transduction between cells. Currently, many drugs available on the market act via GPCRs and these receptors remain attractive targets for the treatment of brain disorders, including alcohol use disorder (AUD). Here, we describe the most recent literature, with a primary focus on the past 5 years, on GPCR targets with the potential for reducing behaviours associated with excessive alcohol intake. Specifically, we focus on preclinical evidence of compounds with attractive pharmacological profiles and potential for future clinical investigation for the treatment of AUD.


Introduction
Alcohol-related deaths have increased in the last two decades and alcohol is the third leading cause of preventable death in the US [1].In 2016, 2.4 billion people consumed alcohol globally and 40% were reported to be chronic users [2].Chronic and excessive alcohol intake are characteristics of alcohol use disorder (AUD), that results in up to 3 million deaths per year [3,4].AUD can be defined as a chronic relapsing brain disorder characterised by excessive and compulsive alcohol drinking, inability to control alcohol use despite negative consequences, and the emergence of a negative emotional state in the absence of alcohol [5].These phases have been operationalised as "binge intoxication", "withdrawal/negative affect" and "anticipation/preoccupation" that may need different pharmacotherapeutic interventions.In this context, there are currently only four FDA and/or EMAapproved drugs specifically registered to treat AUD (disulfiram, naltrexone, acamprosate and nalmefene), with acamprosate being the last approved in 2004.Problematically, all three drugs have limited efficacy at a population level, undesirable side effects, and are not readily available, which contributes to the treatment gap observed in many countries [6].Despite the improvement of guidelines for pharmacological treatment of AUD in 2017, only 1.6% of people with AUD used an available FDA-approved medication in 2019 [7,8].Obtaining a further comprehensive understanding of the neural circuits underlying AUD and the best approaches to target the different phases of AUD are key to developing a broader spectrum of pharmacotherapeutic options.
G protein-coupled receptors (GPCRs) are seven transmembrane (7TM) domain receptors, crucial for regulating essential neurobiological processes and pivotal targets for medication development [9,10].GPCRs constitute the largest class of cell surface membrane proteins, with around 800 members [9,11] and are classed into five main categories based on phylogenetic studies e Glutamate (Class C), Rhodopsin (Class A), Adhesion, Secretin, and Frizzled/ Taste2 [12].When activated, these receptors undergo a conformational change that can modify their structure, function, and dynamics, offering enormous therapeutic potential [11].Over 30% of FDA-approved drugs target GPCRs, which are predominantly expressed within the nervous system and act on over 100 sole GPCR targets.Notably, opioids, endocannabinoids, and biogenic amine receptors are key targets for treating neuropsychiatric disorders [9,13].Indeed, a range of drugs targeting GPCRs are currently undergoing clinical trials for specific interventions in AUD (Figure 1).Discussing these compounds is outside our scope; rather we canvas GPCR targets that are in various stages of preclinical development, with focus on studies from the past 5 years, and where results support progress to proof of concept for clinical development.

Emerging GPCR targets to treat AUD
Here, we explore compounds acting at GPCR targets with promising preclinical results in reducing excessive alcohol intake, alcohol-related negative affect, seeking and cue-reactivity, and relapse.This includes new drug candidates and drug repurposing options.

Serotonergic
Dysfunctional serotonin (5-hydroxytryptamine, 5-HT) signalling has been implicated in the pathophysiology of AUD and agonists of serotonin GPCRs have been studied as potential treatments [14].Lorcaserin, a 5-HT2C receptor agonist was effective in reducing alcohol binge-like drinking and relapse-like behaviour in mice and rats [15,16].In humans, an open-label study reported that lorcaserin was effective in reducing selfreported alcohol use, craving, and psychological distress in patients with AUD [17].Although lorcaserin was withdrawn due to safety issues, selective positive allosteric modulators (PAMs) are now available [18].For example, CYD-1-79, a brain-penetrant PAM, was effective in reducing relapse-like behaviour in a rat model of cocaine self-administration [19].
Psilocybin, a naturally occurring psychedelic that targets 5HT2AR is currently being tested as a novel therapeutic approach to treat AUD (see Figure 1).Interest in targeting these receptors has contributed to the development of non-hallucinogenic, psychedelic analogues [20], although their clinical remains to be determined.Tabernanthalog (TBG), a non-hallucinogenic ibogaine analogue and 5-HT2AR agonist, promotes neural plasticity in cortical rat neurons, has antidepressant-like effects and reduced alcohol-seeking and bingeing in mice [20].TBG also effectively reduced motivation to acquire alcohol and heroin in a polysubstance use rat model [21].Historically, ibogaine demonstrated potential in reducing excessive intake of alcohol and other psychoactive substances in preclinical [22] and clinical [23] studies, but given its adverse, sometimes fatal, side effects, ibogaine's promise as a candidate drug to treat substance use disorder has been stalled.Nonetheless, TBG has a safer profile and long-lasting therapeutic effect, and might be a worthy candidate for further assessment.

Cholinergic
It is well established that alcohol leads to changes in the cholinergic system, including dysregulation of Compounds with GPCR action currently under clinical trial for AUD.Dashed line under pregnenolone is used to highlight that this compound and its metabolite also binds to other non-GPCR receptors.Note that complete, withdrawn, or terminated trials are not listed.nicotinic (nAChR) and muscarinic acetylcholine receptor (mAChR) expression in rodent and human brains [24e26].Targeting nAChRs with varenicline, a drug prescribed for smoking cessation, can reduce alcohol drinking in mice [27]; as does the a4b2 PAM (NS9283), demonstrating that selective engagement of a4b2 is sufficient to reduce drinking in rodents [28] and may avoid undesired side effects of varenicline.While these compounds might not have the desired pharmacological profile for human use, they are useful for target validation.
M4 muscarinic receptors are also emerging as a novel therapeutic approach for AUD in preclinical models [25,26,29,30].Historically, muscarinic receptors have been targeted for the treatment of mental health disorders and neurodegenerative conditions [31].For example, xanomeline, a M1/M4 mAChR-preferring ago-PAM that was assessed in patients with Alzheimer's disease and schizophrenia [32,33], also reduced cocaineseeking in mice [34] but due to gastrointestinal side effects, clinical development ceased.Nonetheless, KarXT, a combination drug of xanomeline and trospium (peripherally acting M2/3 antagonist) has shown clinical potential in patients with schizophrenia [35] and is expected to receive FDA approval in the near future.Similarly, emraclidine (an M4 receptor PAM) was also efficient in reducing the Positive and Negative Syndrome (PANSS) scale in patients with schizophrenia [36] and is currently under phase 2 trials (NCT05227703, NCT05227690).Hence, repurposing these drugs could be a novel and effective treatment to reduce alcohol intake and the negative affect associated with chronic alcohol consumption.Human studies investigating the effectiveness of KarXT and other M4 agonists/PAMs to treat AUD are warranted.

Opioid
Nalfurafine (TRK-820), a KOR agonist currently approved in Japan to treat pruritus, successfully reduced excessive alcohol intake and alcohol preference in a dose-dependent manner, and combination of nalfurafine with low-dose naltrexone had an even more profound effect in mice [37].In addition, this combination reduced relapse-like behaviour in female and male mice [38].Nalfurafine is thought to reduce alcohol consumption by inhibiting GPCR kinase 2/3, which in turn activates kappa opioid receptors [39].Although classic KOR agonists increased alcohol intake [39] and triggered relapse-like alcohol drinking [40] in mice, nalfurafine's potential in reducing alcohol intake could be through competition with dynorphins for KOR binding [41].This is interesting given that the dynorphin/KOR system has historically been associated with the dysphoric state observed following alcohol consumption [42] and traditionally KOR antagonists were considered more suitable candidates for reducing stress-related substance use and relapse (For comprehensive reviews see [43e45]).
Aticaprant (JNJ-67953964), a new generation shortacting KOR antagonist, reduced anxiety-like behaviour in rats during acute withdrawal, prevented escalation of alcohol self-administration and attenuated stressinduced, but not cue-induced relapse, demonstrating its potential in reducing negative affective states associated with alcohol-seeking and consumption [46].Similarly, aticaprant in combination with naltrexone reduced relapse-like drinking in both male and female mice [47].Hence, given naltrexone's role in reducing alcohol reward and cue-induced relapse, the combination of naltrexone with aticaprant poses an attractive potential combinatorial pharmacotherapy strategy for AUD.Importantly, this antagonist is safe and well-tolerated in humans and has shown promising potential to reduce symptoms of depression in patients with major depressive disorder (NCT03559192, 2023).Butorphanol, a MOR/KOR partial agonist, could potentially circumvent issues seen with pure MOR antagonists and be given to patient populations with liver insufficiency, a major complication frequently observed in patients with AUD; and renal dysfunction, which is often seen following liver damage [48e50].

Amino acids
Chronic alcohol exposure causes adaptations in the expression and function of multiple metabotropic glutamate (mGlu) receptors.mGlu5 negative allosteric modulators (NAM) such as MTEP [51,52] and auglurant have shown promising results in reducing motivation to self-administer alcohol in baboons [53] and rats [54].Yet, lack of specificity, poor oral availability and toxicity impeded the progress of these compounds.More recently, TMP-301 (HTL0014242), a mGlu5 NAM with a safer profile and better oral availability has progressed to clinical trial (NCT03785054) and received investigational new drug (IND) clearance by the FDA to advance its development for the treatment of AUD (https://www.temperobio.com/news2023-jan04).Although this drug has not been tested preclinically in models of AUD, rapid progress is a result of extensive prior knowledge of this NAM class safety and mGlu5 involvement in substance use disorders.Targeting mGlu2 receptors with PAMs is also potentially a viable alternative.LY487379 administration reduced relapselike behaviour in both male and female rats [55] and this compound also promotes cognitive flexibility and reduces impulsive-like responses in rats [56].
GABAergic dysfunctional signalling following alcohol consumption has been associated with the emergence and maintenance of AUD [57,58].Baclofen (GABA-B receptor agonist) is available as a treatment for AUD but given intermittent serious adverse effects, contrasting results depending on severity of AUD, and development of tolerance following continued use [59,60], the focus has shifted to the development of GABA-BR PAMs.For example, ADX71441 reduced motivation to alcohol acquisition and intake in rats and has a better therapeutic profile when compared to baclofen [61], yet the development of this compound has been halted.Nonetheless, there is continuing interest on the development of novel GABA-B PAMs (https://www.addextherapeutics.com/en/investors/press-releases/addex-and-indivior-extend-ga bab-positive-allosteric-modulator-research-collaborationsubstance-use-disorders/).
Similarly, ASP8062 reduced alcohol self-administration in rats [62] and a phase 1 study confirmed a favourable pharmacokinetic profile and potential to be given to patients during abstinence [63].A clinical study investigating ASP8062 treatment for reduction of alcohol intake and craving has been recently completed but results are not yet available (NCT05096117), while a phase 2 study to assess ASP8062 as an add-on therapy to buprenorphine/naloxone in people with opioid use disorder was initiated but withdrawn due to corporate strategic considerations (NCT05062577).

Neuropeptides
In AUD an increase in negative affect and stress sensitivity is observed in the absence of alcohol intake [64].This state has been termed hyperkatifeia where peptide systems including corticotrophin-releasing factor (CRF), glucocorticoids, and orexin/hypocretin are implicated [5].CRF1 receptor antagonists were developed but largely failed to progress into clinical products [65].CRF signalling is only one component of stress neurobiology and targeting other systems could be an effective alternative for treating the maladaptive stress regulation observed in AUD.Substance P (SP) and neurokinin A and B are tachykinin neuropeptides that modulate alcohol-related behaviours and stress responses [66,67].Chronic alcohol exposure leads to signalling adaptations within the central amygdala (CeA), where administration of SP-induced GABA release is accompanied by a decreased expression of the Neurokinin-1 receptor (NK1R) in alcohol-dependent rats, an effect that persisted during protracted alcohol withdrawal.In contrast, the selective NK1R antagonist L822429 blocked alcohol-induced GABA release in the CeA of alcohol-dependent rats undergoing withdrawal, but had no effect in naı ¨ve rats, suggesting that alcohol dependence engages the SP/NK1R system [68].In mouse models of stress-induced alcohol intake, another NK1R antagonist, L-733060, prevented escalation of alcohol consumption following inflammatory stress in male and female mice [69] and vicarious defeat stress in female mice [70].Indeed, the NK1R receptor antagonist LY686017 (tradipitant) has shown preliminary clinical potential for reducing alcohol craving in patients with high anxiety and alcohol dependence in a Phase 2a trial (NCT00805441).Unfortunately, a phase 2b study was unable to replicate this primary outcome, possibly due to recruitment of an inadequate patient population that did not represent the participants from phase 2a, as well as the use of a sub-optimal dose [44].Nonetheless, this drug might still be useful for treating a subpopulation of people with AUD and co-morbid anxiety.A recent preclinical study showed that rolapitant, an NK1R antagonist marketed for delaying chemotherapy-induced nausea, reduced anxiety-like behaviour and relapse-like alcohol drinking in male Wistar rats, without affecting voluntary alcohol intake in the two-bottle choice paradigm [71].Therefore, preclinical studies continue to support the ongoing development and repurposing of NK1R antagonists as a potential treatment to maintain abstinence in patients with AUD.
Relaxin-3, is a neuropeptide important for essential homeostatic functions and within the past decade, a role for relaxin-3 and its cognate GPCR RXFP3 in alcohol drinking-related behaviours has been established [72,73]; however, a lack of brain-penetrant small molecules hindered further progress.Recently, RLX-33 has been developed as the first-in-class small molecule RXFP3-selective NAM [74].Systemic injections of RLX-33 reduced alcohol-self administration in both female and male Wistar rats, and sucrose selfadministration in male rats only.In contrast, in alcohol-preferring P rats, reduction of alcohol selfadministration was only seen in males and no effect on sucrose self-administration was reported in either sex [75].Further studies are warranted to investigate the potential role of sex and individual differences in the relaxin-3/RXFP3 system to support the progress of RXFP3 inhibition as a treatment strategy for AUD.

Conclusions
This review highlights the most recent preclinical literature of compounds with promising pharmacological properties and/or effects in reducing excessive alcohol intake-associated behaviours.The health and economic burden of AUD continue to grow and the development of novel pharmacotherapies that are safe, effective, and well-tolerated is overdue.The studies presented here demonstrate the complexity of AUD and cover some of the dynamic neurobiological mechanisms underlying this condition.Preclinical models of excessive alcohol intake are key to the development of new pharmacological agents to treat AUD, as they allow for investigation of the potential of compounds in reducing behaviours that are specific to each of the distinct phases of the addiction process.This targeted approach could result in better compliance and treatment outcome [76].Importantly, a better understanding of the neurobiology associated with different stages of the addiction cycle could facilitate the development of combination drugs that target more than one stage.Further, given that GPCRs are expressed throughout the body and regulate a variety of biological processes, preclinical testing allows for the necessary safety screening of GPCR targeting in a living organism.These, when combined with reverse translation strategies form a powerful toolbox for the development and/or repurposing of drug candidates [25,57].
Nalfurafine is an example of a compound with potential for combinatorial treatment and repurposing.This commercially available drug is a highly selective KOR agonist that unlike other drugs of this class, does not activate p38 MAPK (p38) signalling, a potentially beneficial fact given that p38 activation can contribute to some of the adverse side effects of other KOR agonists [77].Importantly, nalfurafine in combination with a sub-threshold dose of naltrexone appears to potentiate naltrexone's effect in reducing alcohol-seeking and drinking in rodents, demonstrating its clinical potential to reduce motivation and binge drinking, possibly with less side-effects [37].Similarly, rolapitant, an NK1R antagonist prescribed to delay chemotherapy-induced nausea, has demonstrated potential in reducing negative affect associated with chronic alcohol intake in rodents [71].As recently suggested, treatments that focus on reducing stress, anhedonia, and negative affective states, which are major symptoms of AUD, could present better potential as chronic treatments and be more appealing to patients [44].See Figure 2 for other examples of drug candidates and their potential to target maladaptive behaviours and processes associated with distinct stages of the addiction cycle.
These approaches together with emerging technologies that allow faster and more precise screening of GPCR targets and their neurophysiological roles should support development of safer and more effective pharmacotherapeutics.
have appeared to influence the work reported in this paper.

Figure 1
Figure 1 Nature 2021, 589:474-479.This work reported the development of tabernanthalog (TBG)a water soluble, non-hallucinogenic, non-toxic analog of ibogaine.TBG promoted structural neural plasticity, reduced alcohol-and heroin-seeking behaviour, and produced antidepressant-like effects in rodents.This work demonstrates that through careful chemical design, it is possible to modify a psychedelic compound to produce a safer, nonhallucinogenic variant with therapeutic potential.VU0467154, authors were able to reduce alcohol seeking and relapse-like behaviour in rats, demonstrating the role of this receptor in alcohol intake and potential of the M4 mAChR as a therapeutic target.26.Walker LC, Huckstep KL, Chen NA, Hand LJ, Lindsley CW, Langmead CJ, Lawrence AJ: Muscarinic M 4 and M 5 receptors in the ventral subiculum differentially modulate alcohol seeking versus consumption in male alcohol-preferring rats.Cue-induced reinstatement of seeking behavior in male rats is independent from the rewarding value of the primary reinforcer: effect of mGluR5 blockade.Neuropharmacology 2023, 240, 109694.55.Vengeliene V, Spanagel R: mGlu2 mechanism-based interventions to treat alcohol relapse.Front Pharmacol 2022, 13, 985954.56.Nikiforuk A, Popik P, Drescher KU, van Gaalen M, Relo AL, Mezler M, Marek G, Schoemaker H, Gross G, Bespalov A: Effects of a positive allosteric modulator of group II metabotropic glutamate receptors, LY487379, on cognitive flexibility and impulsive-like responding in rats.J Pharmacol Exp Ther 2010, 335:665-673.Barbier E, Dulman RS, Licheri V, Augier G, Domi E, Barchiesi R, Farris S, Nätt D, Mayfield RD, Adermark L, Heilig M: A molecular mechanism for choosing alcohol over an alternative reward.Science 2018, 360:1321-1326.This work used back-translation to identify, for the first time, that downregulation of GABA transporter GAT-3 in the amygdala of rats and humans was associated with the development of alcohol dependence.58.Heinz A, Schäfer M, Higley JD, Krystal JH, Goldman D: Neurobiological correlates of the disposition and maintenance of alcoholism.Pharmacopsychiatry 2003, 36(Suppl.3): S255-S258.GPCRs and alcohol use disorder Anversa et al. 7 Br J Pharmacol 2021, 178:3730-3746.27.Kamens HM, Andersen J, Picciotto MR: The nicotinic acetylcholine receptor partial agonist varenicline increases the 54.Kallupi M, Ciccocioppo R: www.sciencedirect.comCurrent Opinion in Neurobiology 2024, 87:102896