Receptor Allosteric Modulators Research Articles

Design, synthesis, and pharmacological profiling of cannabinoid 1 receptor allosteric modulators: Preclinical efficacy of C2-group GAT211 congeners for reducing intraocular pressure

Allosteric modulators of cannabinoid 1 receptor (CB1R) show translational promise over orthosteric ligands due to their potential to elicit therapeutic benefit without cannabimimetic side effects. The prototypic 2-phenylindole CB1R allosteric modulator, GAT211 (1), demonstrates preclinical efficacy in various disease models. The limited systematic structure–activity relationship (SAR) data at the C2 position of the indole ring within GAT211 invites the opportunity for further modifications to improve GAT211′s pharmacological profile while serving to amplify and variegate this library of therapeutically attractive agents. These considerations prompted this focused SAR study in which we substituted the GAT211 C2-phenyl ring with heteroaromatic substituents. The synthesized GAT211 analogs were then evaluated in vitro as CB1R allosteric modulators in cAMP and β-arrestin2 assays with CP55,940 as the orthosteric ligand. Furan and thiophene rings (15c-f and 15m) were the best-tolerated substituents at the C2 position of GAT211 for engagement with human CB1R (hCB1R). The SAR around the novel ligands reported allowed direct experimental characterization of the interaction profile of that pharmacophore with its binding domain in functional, human CB1R, thus offering guidance for accessing subsequent-generation hCB1R allosteric modulators as potential therapeutics. The most potent analog, 15d, markedly promoted orthosteric ligand binding to hCB1R. Pharmacological profiling in the GTPγS and mouse vas deferens assays demonstrated that 15d behaves as a CB1R agonist-positive allosteric modulator (ago-PAM), as confirmed electrophysiologically in autoptic neurons. In vivo, 15d was efficacious as a topical agent that significantly reduced intraocular pressure (IOP) in the ocular normotensive murine model of glaucoma. Since elevated IOP is a decisive risk factor for glaucoma and attendant vision loss, our data support the proposition that the 2-phenylindole class of CB1R ago-PAMs has therapeutic potential for glaucoma and other diseases where potentiation of CB1R signaling may be therapeutic.

Multiple ligand recognition sites in free fatty acid receptor 2 (FFA2R) direct distinct neutrophil activation patterns

The allosteric modulating free fatty acid receptor 2 ligands Cmp58 and AZ1729, increased the activity induced by orthosteric receptor agonists mediating a rise in intracellular calcium ions and activation of the neutrophil NADPH-oxidase. Together, the two modulators triggered an orthosteric-agonist-independent activation of the oxidase without any rise in the concentration of intracellular calcium ions. In this study, structurally diverse compounds presumed to be ligands for free fatty acid receptor 2 were used to gain additional insights into receptor-modulation/signaling. We identified two molecules that activate neutrophils on their own and we classified one as allosteric agonist and the other as orthosteric agonist. Ten compounds were classified as allosteric FFA2R modulators. Of these, one activated neutrophils when combined with AZ1729; the nine remaining compounds activated neutrophils solely when combined with Cmp58. The activation signals were primarily biased when stimulated by two allosteric modulators interacting with different binding sites, such that two complementary modulators together triggered an activation of the NADPH-oxidase but no increase in the intracellular concentration of calcium ions. No neutrophil activation was induced when allosteric receptor modulators suggested to be recognized by the same binding site were combined, results in agreement with our proposed model for activation, in which the receptor has two different sites that selectively bind allosteric modulators. The down-stream signaling mediated by cross-sensitizing allosteric receptor modulators, occurring independent of any orthosteric agonist, represent a new mechanism for activation of the neutrophil NADPH oxidase.

Muscarinic M4 and M5 receptors in the ventral subiculum differentially modulate alcohol seeking versus consumption in male alcohol-preferring rats.

Muscarinic acetylcholine receptors mediate alcohol consumption and seeking in rats. While M4 and M5 receptors have recently been implicated to mediate these behaviours in the striatum, their role in other brain regions remain unknown. The ventral tegmental area (VTA) and ventral subiculum (vSub) both densely express M4 and M5 receptors and modulate alcohol-seeking, via their projections to the nucleus accumbens shell (AcbSh). In Indiana alcohol-preferring (iP) male rats, we examined Chrm4 (M4 ) and Chrm5 (M5 ) expression in the VTA and vSub following long-term alcohol consumption and abstinence using RT-qPCR. Using a combination of retrograde tracing and RNAscope, we examined the localisation of Chrm4 and Chrm5 on vSub cells that project to the AcbSh. Using selective allosteric modulators, we examined the functional role of M4 and M5 receptors within the vSub in alcohol consumption, context-induced alcohol-seeking, locomotor activity, and food/water consumption. Long-term alcohol and abstinence dysregulated the expression of genes for muscarinic receptors in the vSub, not in the VTA. Chrm4 was down-regulated following long-term alcohol and abstinence, while Chrm5 was up-regulated following long-term alcohol consumption. Consistent with these data, a positive allosteric modulator (VU0467154) of intra-vSub M4 receptors reduced context-induced alcohol-seeking, but not motivation for alcohol self-administration, while M5 receptor negative allosteric modulator (ML375) reduced initial motivation for alcohol self-administration, but not context-induced alcohol-seeking. Collectively, our data highlight alcohol-induced cholinergic dysregulation in the vSub and distinct roles for M4 and M5 receptor allosteric modulators to reduce alcohol consumption or seeking.

Selective actions of insecticides on desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach (Periplaneta americana) neurons.

Insect desensitizing nicotinic acetylcholine (nAChD) receptors are desensitized by low concentrations of agonists, including neonicotinoid insecticides, but are essentially insensitive to spinosyns, while non-desensitizing nicotinic acetylcholine (nAChN) receptors are selectively activated by spinosyns and relatively insensitive to neonicotinoids. The single-electrode voltage-clamp technique was used to measure the actions of newer nicotinic insecticides dinotefuran, sulfoxaflor, triflumezopyrim, spinetoram and GS-ω/k-hexatoxin-Hv1a on cockroach neuronal nAChD and nAChN currents. Like imidacloprid and clothianidin, newer orthosteric nicotinic agonist insecticides dinotefuran and sulfoxaflor act by desensitizing nAChD receptors. The mesoionic insecticide triflumezopyrim selectively inhibited nAChD current with an half maximal inhibitory concentration (IC50 ) of 1.2nmol L-1 , with no activation. Unlike other Group 4 insecticides, it did not activate nAChN current, but inhibited it with an IC50 of 3.8μmol L-1 , indicating that the compound is a true antagonist. Spinosad and the spinosyn-derived insecticide spinetoram potently and selectively activated nAChN receptors. GS-ω/k-hexatoxin-Hv1a had no effect on nAChN currents and it had a complex action on nAChD currents, inhibiting at sub-nanomolar concentrations and causing some activation and enhancement of ACh-evoked currents at 30 nmol L-1 and above. Some cells express GS-ω/k-hexatoxin-Hv1a-resistant nAChD receptors. Nicotinic acetylcholine receptor competitive modulators (IRAC Group 4) and nicotinic acetylcholine receptor allosteric modulators, site II (hexatoxins, IRAC Group 32) are selective for nAChD receptors, while nicotinic acetylcholine receptor allosteric modulators, site I (spinosyns, IRAC Group 5) are selective for nAChN receptors. It is proposed that IRAC Groups 5 and 32 be re-named non-desensitizing nicotinic acetylcholine receptor allosteric modulators and desensitizing nicotinic acetylcholine receptor allosteric modulators, respectively. © 2021 Society of Chemical Industry.

Physiological role for GABAA receptor desensitization in the induction of long-term potentiation at inhibitory synapses

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

Allosterism vs. Orthosterism: Recent Findings and Future Perspectives on A2B AR Physio-Pathological Implications.

The development of GPCR (G-coupled protein receptor) allosteric modulators has attracted increasing interest in the last decades. The use of allosteric modulators in therapy offers several advantages with respect to orthosteric ones, as they can fine-tune the tissue responses to the endogenous agonist. Since the discovery of the first A1 adenosine receptor (AR) allosteric modulator in 1990, several efforts have been made to develop more potent molecules as well as allosteric modulators for all adenosine receptor subtypes. There are four subtypes of AR: A1, A2A, A2B, and A3. Positive allosteric modulators of the A1 AR have been proposed for the cure of pain. A3 positive allosteric modulators are thought to be beneficial during inflammatory processes. More recently, A2A and A2B AR allosteric modulators have also been disclosed. The A2B AR displays the lowest affinity for its endogenous ligand adenosine and is mainly activated as a consequence of tissue damage. The A2B AR activation has been found to play a crucial role in chronic obstructive pulmonary disease, in the protection of the heart from ischemic injury, and in the process of bone formation. In this context, allosteric modulators of the A2B AR may represent pharmacological tools useful to develop new therapeutic agents. Herein, we provide an up-to-date highlight of the recent findings and future perspectives in the field of orthosteric and allosteric A2B AR ligands. Furthermore, we compare the use of orthosteric ligands with positive and negative allosteric modulators for the management of different pathological conditions.

The endocannabinoid system - current implications for drug development.

In this review, the state of the art for compounds affecting the endocannabinoid (eCB) system is described with a focus on the treatment of pain. Amongst directly acting CB receptor ligands, clinical experience with ∆9‐tetrahydracannabinol and medical cannabis in chronic non‐cancer pain indicates that there are differences between the benefits perceived by patients and the at best modest effect seen in meta‐analyses of randomized controlled trials. The reason for this difference is not known but may involve differences in the type of patients that are recruited, the study conditions that are chosen and the degree to which biases such as reporting bias are operative. Other directly acting CB receptor ligands such as biased agonists and allosteric receptor modulators have not yet reached the clinic. Amongst indirectly acting compounds targeting the enzymes responsible for the synthesis and catabolism of the eCBs anandamide and 2‐arachidonoylglycerol, fatty acid amide hydrolase (FAAH) inhibitors have been investigated clinically but were per se not useful for the treatment of pain, although they may be useful for the treatment of post‐traumatic stress disorder and cannabis use disorder. Dual‐acting compounds targeting this enzyme and other targets such as cyclooxygenase‐2 or transient potential vanilloid receptor 1 may be a way forward for the treatment of pain.

The GABAA-Benzodiazepine Receptor Antagonist Flumazenil Abolishes the Anxiolytic Effects of the Active Constituents of Crocus sativus L. Crocins in Rats.

Anxiety is a chronic severe psychiatric disorder. Crocins are among the various bioactive components of the plant Crocus sativus L. (Iridaceae) and their implication in anxiety is well-documented. However, which is the mechanism of action underlying the anti-anxiety effects of crocins remains unknown. In this context, it has been suggested that these beneficial effects might be ascribed to the agonistic properties of these bioactive ingredients of saffron on the GABA type A receptor. The current experimentation was undertaken to clarify this issue in the rat. For this research project, the light/dark and the open field tests were used. A single injection of crocins (50 mg/kg, i.p., 60 min before testing) induces an anti-anxiety-like effect revealed either in the light-dark or open field tests. Acute administration of the GABAA-benzodiazepine receptor antagonist flumazenil (10 mg/kg, i.p., 30 min before testing) abolished the above mentioned anxiolytic effects of crocins. The current findings suggest a functional interaction between crocins and the GABAA receptor allosteric modulator flumazenil on anxiety.

A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, β-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including β-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

The Effect of PAOPA, a Novel Allosteric Modulator of Dopamine D2 Receptors, on Signaling Proteins Following Sub-Chronic Administration in Rats.

Allosteric modulators of G-protein coupled receptors regulate receptor activity by binding to sites other than the active site and have emerged as a new and highly desirable class of drugs. PAOPA (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide), a peptidomimetic analog of L-prolyl-L-leucyl-glycinamide, is a potent dopamine D2 receptor allosteric modulator. PAOPA has shown therapeutic effects in pre-clinical models of schizophrenia and extrapyramidal dysfunction. In this study, we sought to examine the biomolecular underpinnings of PAOPA's therapeutic outcomes in pre-clinical models of schizophrenia. Following sub-chronic (daily for 7 days) administration of PAOPA, we assessed levels of dopamine D2 receptors, receptor kinases (GRK2 (G protein-coupled receptor kinase 2) and Arrestin- 3), and phosphorylated mitogen-activated protein kinase (MAPKs), namely, extracellular signal- regulated kinases (ERK1/2) in the hippocampus, medial pre-frontal cortex, nucleus accumbens, pre-frontal cortex, and dorsal striatum via protein quantification. Following 7 days of daily PAOPA treatment, we observed decreased GRK2 and increased dopamine D2 receptor expression in the dorsal striatum. These findings potentially underscore the therapeutic mechanism of action of PAOPA for the positive-like symptoms of schizophrenia in pre-clinical animal models. Additionally, we observed a decline in GRK2 in the hippocampus and an increase in phosphorylated ERK1 in the pre-frontal cortex, suggesting a role of PAOPA in treating cognitive and/or affective dysfunction in pre-clinical models. While further studies are required to elucidate the mechanism of action of PAOPA, this study discusses prior investigations and develops an early framework to describe the therapeutic mechanism of action of PAOPA.

The anxiolytic effect of perampanel and possible mechanisms mediating its anxiolytic effect in mice

AimsThe aim of this study is to investigate the anxiolytic activity of perampanel, a non-competitive antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors, which is approved for partial-onset seizures in patients with epilepsy, and its mechanism of action. Main methodsThe anxiolytic activity of perampanel at the doses of 0.25, 0.5, 1, 2, and 4 mg/kg intraperitoneally (i.p.) was investigated in mice using elevated plus-maze, hole-board, and open-field tests. The findings were compared to the anxiolytic activity of gamma-aminobutyric acid type A benzodiazepine (GABAA/BZ) receptor allosteric modulator diazepam (1 mg/kg, i.p.) and AMPA antagonist GYKI-53655 (5 mg/kg, i.p.). The mechanisms of action of perampanel were evaluated by pre-treatment with GABAA/BZ receptor antagonist flumazenil (3 mg/kg, i.p.), serotonin 5-hydroxytryptamine 1A (5-HT1A) antagonist WAY-100635 (1 mg/kg, i.p.), and α2-adrenoreceptor antagonist yohimbine (5 mg/kg, i.p.). Key findingsIn the elevated plus-maze and open-field tests, perampanel at the dose of 0.5 mg/kg, and in the hole-board test, at the doses of 0.25, 0.5, and 1 mg/kg demonstrated an anxiolytic effect without altering the locomotor activity. The effect of perampanel was comparable to the effect of diazepam. Stimulation of GABAA/BZ and α2-adrenergic receptors contributed to the anxiolytic effect of perampanel, since significant antagonisms were determined in various behavioral parameters by the antagonist pre-treatments. SignificanceAMPA antagonism is believed to provide the determined anxiolytic activity of perampanel. Increased GABAergic tonus induced by AMPA receptor antagonism along with other systems, especially the noradrenergic system, might be involved in the anxiolytic activity.

Identification and Characterization of ‘Biased’ A3 Adenosine Receptor Allosteric Modulators

IntroductionA3 adenosine receptor agonists are currently being evaluated in clinical trials for treatment of inflammation, cancer, and neuropathic pain. To circumvent complications associated with the use of direct agonists of GPCRs, we have pursued development of A3 adenosine receptor positive allosteric modulators (PAMs).MethodsIn prior studies, we synthesized a series of 1H‐imidazo[4,5]quinoline‐4‐amines that display allosteric modulatory activity versus the human A3 receptor. In prior screens, members of this class of modulators were found to increase the maximal efficacy of orthosteric agonists ~2–3‐fold in G protein‐dependent signaling assays, although they had the tendency to reduce agonist potency. In addition, lead compounds (LUF6000) were discovered to be substantially less potent at rodent A3ARs. The purpose of the present investigation was to re‐evaluate our expanded compound series based on LUF6000 in greater detail, with emphasis on examining effects on orthosteric ligand potency, G protein‐dependent versus G protein‐independent signaling, and efficacy versus mouse A3 adenosine receptors. Based on known species differences, human/mouse chimeric receptors were prepared to explore the possibility extracellular loops 1 & 2 that form an extracellular vestibule comprise the allosteric ligand binding site.ResultsOur evaluation centered on a series of 4‐(3,4‐dichlorophenylamino) compounds with 2‐substitutions consisting of 3–8 cycloalkyl rings. This was based on our observation during an initial screen that the 2‐cycloheptyl and 2‐cyclooctyl analogues increased affinity of the orthosteric radioligand ([125I]I‐AB‐MECA) in equilibrium binding assays in a manner that did not slow its (apparent) association binding rate. Among this series, compounds with larger 2‐substituents (c4–8) were found to increase the maximal efficacy of the orthosteric agonist Cl‐IB‐MECA in [35S]GTPgS binding assays ~2‐fold. Most prominently, the cyclooctyl derivative increased the efficacy of Cl‐IB‐MECA (~1.5‐fold) while also increasing its potency. A similar pattern was evident in cAMP assays, which also detected allo‐agonist activity. In contrast to these findings, analogues within the series with bulkier 2‐substituents substantially reduced (up to 95%), rather than enhanced, the efficacy of Cl‐IB‐MECA in arrestin2 recruitment assays (TANGO assay). This occurred at potencies that mirrored those observed in assays of G protein‐dependent signaling. Unfortunately, none of the derivatives investigated displayed improved activity versus mouse A3 adenosine receptors. Studies with human/mouse chimeric receptors indicated that modulators based on the imidazoquinolinamine template do not function as ‘vestibule’ modulators.ConclusionsWe have identified improved PAMs for the human A3 receptor that: 1) not only increase agonist efficacy, but also increase agonist potency (and binding affinity), 2) display allo‐agonist activity, and 3) may bias towards G protein‐dependent signaling.Support or Funding InformationNIH R01HL133589 and Therapeutic Accelerator Program

Computational insights into the molecular mechanisms of differentiated allosteric modulation at the mu opioid receptor by structurally similar bitopic modulators.

Targeting the mu opioid receptor (MOR) by applying orthosteric ligands is the most frequently employed method to treat opioid use disorder (OUD). Unfortunately, most of MOR orthosteric ligands produce severe side effects, mainly due to their low selectivity over other opioid receptors. In contrast, some G protein-coupled receptor allosteric modulators have been reported to exhibit high subtype selectivity and can effectively modulate the potency and/or efficacy of orthosteric ligands. Recently, NAQ and its analog NCQ were identified as novel MOR bitopic modulators. Interestingly, NAQ and NCQ were similar in structure but exhibited different efficacy profiles to the MOR. NAQ exhibited an antagonism activity to the MOR while NCQ showed a partial agonism activity to the MOR. In the present study, molecular modeling methods were applied to explore the putative molecular mechanisms of their different functional profiles to the MOR. When NAQ binding with the inactive MOR, the 'address' portion of NAQ interacted with the MOR allosteric site but showed no significant allosteric modulation of the efficacy of the 'message' portion of NAQ. However, when NCQ binding with the inactive and active MOR, the 'address' portion of NCQ seemed to be able to positively modulate the efficacy of the 'message' portion of NCQ at varying levels. Evidentially, the substituents at the 1'- and 4'-positions of the isoquinoline ring of NCQ seemed to play a critical role in the modulatory function of the 'address' portion of NCQ. These findings will be invaluable to develop our next generation of MOR bitopic modulators with high affinity and subtype selectivity to potentially treat OUD.

Allosteric modulation of muscarinic receptors in alcohol and substance use disorders.

Alcohol and substance use disorders are chronic relapsing disorders, characterized by a compulsion to take drugs and loss of control in limiting intake. The impact of alcohol and substance abuse on morbidity and mortality is very high worldwide, and treatment options are inadequate at a population level. There is a need for the discovery and development of novel pharmacotherapies to treat alcohol and substance use disorders. The recent development of subtype selective muscarinic acetylcholine receptor (mAChR) allosteric modulators, is rapidly increasing our understanding of this system and revealing great promise for disease treatment. In this chapter, we will present and integrate the current literature regarding the role of mAChRs in drug and alcohol abuse with emerging evidence using novel allosteric modulators targeting specific mAChR subtypes. We will focus on the potential of M1, M4 and M5 mAChRs in the treatment of alcohol, psychostimulant and opioid dependence, using evidence from preclinical studies to predict their utility in treating different stages of the addiction cycle. Overall, preclinical evidence suggests allosteric modulation of mAChRs may provide promise as potential treatment options for alcohol and substance abuse in reducing consumption, facilitating abstinence and preventing relapse.

GABAB Receptors: Anxiety and Mood Disorders.

Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, acts at the ionotropic GABAA and GABAC receptors, and the metabotropic GABAB receptor. This chapter summarizes the studies that have investigated the role of the GABAB receptor in stress-related psychiatric disorders including anxiety and mood disorders. Overall, clinical and preclinical evidences strongly suggest that the GABAB receptor is a therapeutic candidate for depression and anxiety disorders. However, the clinical development of GABAB receptor-based drugs to treat these disorders has been hampered by their potential side-effects, particularly those of agonists. Nevertheless, the discovery of novel GABAB receptor allosteric modulators, and increasing understanding of the influence of specific intracellular GABAB receptor-associated proteins on GABAB receptor activity, may now pave the way towards GABAB receptor therapeutics in the treatment of mood and anxiety disorders.

The different aspects of the GABAB receptor allosteric modulation.

The GABAB receptor is activated by the main inhibitory neurotransmitter of the central nervous system, the γ-aminobutyric acid (GABA). The receptor is expressed in almost all neuronal and glial cells and plays a central role in the modulation of many physiological and pathological processes. The GABAB receptor has been considered for years as an interesting target for the treatment of spasticity, pain, addiction, anxiety or depression. This has prompted many studies aiming at understanding the activation of the receptor and its modulation. While it belongs to the super-family of G protein-coupled receptors (GPCRs), it was rapidly evident that the GABAB receptor is peculiar in the variety of allosteric modulations governing its activation. Here, I wish to gather the different aspects of the GABAB receptor allosteric modulation. After presenting the main small molecule allosteric modulators known to date, the intramolecular transitions controlling the receptor activation will be summarized. In addition, recent findings obtained in the last decade on the existence of GABAB receptor complexes and their influence on the receptor function will be introduced, including the GABAB receptor oligomers and the auxiliary proteins associated with the receptor. These new concepts will certainly be of major interest in the future analysis of GABAB receptor allosteric modulation.

In vivo evaluation of the CB1 allosteric modulator LDK1258 reveals CB1-receptor independent behavioral effects

In the present study, we examined whether LDK1258, which produces strong CB1 receptor allosteric effects in in vitro assays, would elicit in vivo effects consistent with allosteric activity. In initial studies, LDK1258 reduced food consumption and elicited delayed antinociceptive effects in the chronic constrictive injury of the sciatic nerve (CCI) model of neuropathic pain, which unexpectedly emerged 4 h post-injection. UPLC-MS/MS analysis quantified significant levels of LDK1258 in both blood and brain tissue at 30 min post-administration that remained stable up to 4 h. The observation that LDK1258 also produced respective antinociceptive and anorectic effects in rimonabant-treated wild type mice and CB1 (−/−) mice suggests an off-target mechanism of action. Likewise, LDK1258 produced a partial array of common cannabimimetic effects in the tetrad assay, which were not CB1 receptor mediated. Additionally, LDK1258 did not substitute for the CB1 receptor orthosteric agonists CP55,940 or anandamide in the drug discrimination paradigm. In other in vivo assays sensitive to CB1 receptor allosteric modulators, LDK1258 failed to shift the dose-response curves of either CP55,940 or anandamide in producing thermal antinociception, catalepsy, or hypothermia, and did not alter the generalization curve of either drug in the drug discrimination assay. Thus, this battery of tests yielded results demonstrating that LDK1258 produces antinociceptive effects in the CCI model of neuropathic pain, anorectic effects, and other in vivo pharmacological effects in a manner inconsistent with CB1 receptor allosterism. More generally, this study offers a straightforward screening assay to determine whether newly synthesized CB1 receptor allosteric modulators translate to the whole animal.

Tricyclic derivatives of bispidine as AMPA receptor allosteric modulators

Four new tricyclic derivatives of 3,7-diazabicyclo[3.3.1]-nonane (bispidine) have been synthesized. In vitro patch clamp investigation has revealed for one of them the positive modulation of the kainate-induced AMPA receptor currents in a nanomolar concentration range, and for two other compounds the corresponding negative modulation has been demonstrated.

Synthesis and Pharmacological Evaluation of 1-Phenyl-3-Thiophenylurea Derivatives as Cannabinoid Type-1 Receptor Allosteric Modulators.

We previously reported diarylurea derivatives as cannabinoid type-1 receptor (CB1) allosteric modulators, which were effective in attenuating cocaine-seeking behavior. Herein, we extended the structure-activity relationships of PSNCBAM-1 (2) at the central phenyl ring directly connected to the urea moiety. Replacement with a thiophene ring led to 11 with improved or comparable potencies in calcium mobilization, [35S]GTPγS binding, and cAMP assays, whereas substitution with nonaromatic rings led to significant attenuation of the modulatory activity. These compounds had no inverse agonism in [35S]GTPγS binding, a characteristic that is often thought to contribute to adverse psychiatric effects. While 11 had good metabolic stability in rat liver microsomes, it showed modest solubility and blood-brain barrier permeability. Compound 11 showed an insignificant attenuation of cocaine seeking behavior in rats, most likely due to its limited CNS penetration, suggesting that pharmacokinetics and distribution play a role in translating the in vitro efficacy to in vivo behavior.

Allosteric modulators targeting cannabinoid cb1 and cb2 receptors: implications for drug discovery.

Allosteric modulators of cannabinoid receptors hold great therapeutic potential, as they do not possess intrinsic efficacy, but instead enhance or diminish the receptor's response of orthosteric ligands allowing for the tempering of cannabinoid receptor signaling without the desensitization, tolerance and dependence. Allosteric modulators of cannabinoid receptors have numerous advantages over the orthosteric ligands such as higher receptor type selectivity, probe dependence and biased signaling, so they have a great potential to separate the therapeutic benefits from side effects own of orthosteric ligands. This review aims to give an overview of the CB1 and CB2 receptor allosteric modulators highlighting the structure-activity relationship and pharmacological profile of each classes, and their future promise.