Trace amine associated receptor 1: predicted effects of single nucleotide variants on structure-function in geographically diverse populations.

Trace Amine Associated Receptor 1 (TAAR1) is a novel pharmaceutical target under investigation for the treatment of several neuropsychiatric conditions. TAAR1 single nucleotide variants (SNV) have been found in patients with schizophrenia and metabolic disorders. However, the frequency of variants in geographically diverse populations and the functional effects of such variants are unknown. In this study, we aimed to characterise the distribution of TAAR1 SNVs in five different WHO regions using the Database of Genotypes and Phenotypes (dbGaP) and conducted a critical computational analysis using available TAAR1 structural data to identify SNVs affecting ligand binding and/or functional regions. Our analysis shows 19 orthosteric, 9 signalling and 16 micro-switch SNVs hypothesised to critically influence the agonist induced TAAR1 activation. These SNVs may non-proportionally influence populations from discrete regions and differentially influence the activity of TAAR1-targeting therapeutics in genetically and geographically diverse populations. Notably, our dataset presented with orthosteric SNVs D1033.32N (found only in the South-East Asian Region and Western Pacific Region) and T1945.42A (found only in South-East Asian Region), and 2 signalling SNVs (V1253.54A/T2526.36A, found in African Region and commonly, respectively), all of which have previously demonstrated to influence ligand induced functions of TAAR1. Furthermore, bioinformatics analysis using SIFT4G, MutationTaster 2, PROVEAN and MutationAssessor predicted all 16 micro-switch SNVs are damaging and may further influence the agonist activation of TAAR1, thereby possibly impacting upon clinical outcomes. Understanding the genetic basis of TAAR1 function and the impact of common mutations within clinical populations is important for the safe and effective utilisation of novel and existing pharmacotherapies.

Development of Putative Bivalent Dicovalent Ligands for the Adenosine A1 Receptor.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) 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 A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 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.

International Union of Basic and Clinical Pharmacology. CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors.

Metabotropic glutamate (mGlu) receptors respond to glutamate, the major excitatory neurotransmitter in the mammalian brain, mediating a modulatory role that is critical for higher-order brain functions such as learning and memory. Since the first mGlu receptor was cloned in 1992, eight subtypes have been identified along with many isoforms and splice variants. The mGlu receptors are transmembrane-spanning proteins belonging to the class C G protein-coupled receptor family and represent attractive targets for a multitude of central nervous system disorders. Concerted drug discovery efforts over the past three decades have yielded a wealth of pharmacological tools including subtype-selective agents that competitively block or mimic the actions of glutamate or act allosterically via distinct sites to enhance or inhibit receptor activity. Herein, we review the physiologic and pathophysiological roles for individual mGlu receptor subtypes including the pleiotropic nature of intracellular signal transduction arising from each. We provide a comprehensive analysis of the in vitro and in vivo pharmacological properties of prototypical and commercially available orthosteric agonists and antagonists as well as allosteric modulators, including ligands that have entered clinical trials. Finally, we highlight emerging areas of research that hold promise to facilitate rational design of highly selective mGlu receptor-targeting therapeutics in the future. SIGNIFICANCE STATEMENT: The metabotropic glutamate receptors are attractive therapeutic targets for a range of psychiatric and neurological disorders. Over the past three decades, intense discovery efforts have yielded diverse pharmacological tools acting either competitively or allosterically, which have enabled dissection of fundamental biological process modulated by metabotropic glutamate receptors and established proof of concept for many therapeutic indications. We review metabotropic glutamate receptor molecular pharmacology and highlight emerging areas that are offering new avenues to selectively modulate neurotransmission.

Calcium-Sensing Receptor Negative Allosteric Modulators Oppose Contraction of Mouse Airways.

Asthma is a heterogeneous chronic airway disease with an unmet need for improved therapeutics in uncontrolled severe disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor upregulated in asthma. The CaSR agonist, spermine, is also increased in asthmatic airways and contributes to bronchoconstriction. CaSR negative allosteric modulators (NAMs) oppose chronic airway inflammation, remodeling, and hyperresponsiveness in murine and guinea pig asthma models, but whether CaSR NAMs are effective acute bronchodilators compared with standard of care has not yet been established. Furthermore, the ability of different classes of NAMs to inhibit spermine-induced CaSR signaling or methacholine (MCh)-induced airway contraction has not been quantified. Here, we show CaSR NAMs differentially inhibit spermine-induced intracellular calcium mobilization and inositol monophosphate accumulation in HEK293 cells stably expressing the CaSR. NAMs reverse MCh-mediated airway contraction in mouse precision-cut lung slices with similar maximal relaxation compared with the standard treatment, salbutamol. Of note, the bronchodilator effects of CaSR NAMs are maintained under conditions of ß2-adrenergic receptor desensitization when salbutamol efficacy is abolished. Furthermore, overnight treatment with some, but not all, CaSR NAMs prevents MCh-mediated bronchoconstriction. These findings further support the CaSR as a putative drug target and NAMs as alternative or adjunct bronchodilators in asthma.

Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy, and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilization revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilization and IP1 accumulation. The PAMs Ro67-4853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilization assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu1) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu1. Such complexities in allosteric ligand pharmacology should be considered in future mGlu1 allosteric drug discovery programs.

Regulation and functional consequences of mGlu4 RNA editing.

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.

Binding of SEP-363856 within TAAR1 and the 5HT1A receptor: implications for the design of novel antipsychotic drugs.

Current medications for schizophrenia typically modulate dopaminergic neurotransmission. While affecting positive symptoms, antipsychotic drugs have little clinical effect on negative symptoms and cognitive impairment. Moreover, newer 'atypical' antipsychotic drugs also have significant metabolic adverse-effects. The recent positive clinical trial of the novel drug candidate SEP-363856, which targets non-dopamine receptors (trace amine-associated receptor and the 5HT1A receptor), is a potentially promising development for the management of schizophrenia. In this perspective, we briefly overview the role of TAAR1 and the 5HT1A receptor in schizophrenia and explore the specific binding characteristics of SEP-363856 at these receptors. Molecular dynamics simulations (MDS) indicate that SEP-363856 interacts with a small, common set of conserved residues within the TAAR1 and 5HT1A ligand-binding domain. The primary interaction of SEP-363856 involves binding to the negatively charged aspartate residue (Asp1033.32, TAAR1; Asp1163.32, 5HT1A). In general, the binding of SEP-363856 within TAAR1 involves a greater number of aromatic contacts compared to 5HT1A. MDS provides important insights into the molecular basis of binding site interactions of SEP-363856 with TAAR1 and the 5HT1A receptor, which will be beneficial for understanding the pharmacological uniqueness of SEP-363856 and for the design of novel drug candidates for these newly targeted receptors in the treatment of schizophrenia and related disorders.

Adenosine receptor signalling in Alzheimer's disease.

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 A1 and A2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic ß-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 A1 and A2A 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.

Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias.

Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu5) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu5 PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu5 PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu5 using Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca2+ mobilization over IP1 accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca2+ mobilization over IP1 accumulation. Our characterization of three structurally diverse mGlu5 PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu5 internalization. SIGNIFICANCE STATEMENT: Enhancing metabotropic glutamate receptor subtype 5 (mGlu5) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu5 are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu5 PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu5 PAMs and is the first study of the effects of mGlu5 PAMs on receptor internalization.

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites.

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M4 muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.

Detailed In Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5.

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.

Metabotropic glutamate receptor 5 (mGlu5 )-positive allosteric modulators differentially induce or potentiate desensitization of mGlu5 signaling in recombinant cells and neurons.

Allosteric modulators of metabotropic glutamate receptor 5 (mGlu5 ) are a promising therapeutic strategy for a number of neurological disorders. Multiple mGlu5 -positive allosteric modulator (PAM) chemotypes have been discovered that act as either pure PAMs or as PAM-agonists in recombinant and native cells. While these compounds have been tested in paradigms of receptor activation, their effects on receptor regulatory processes are largely unknown. In this study, acute desensitization of mGlu5 mediated intracellular calcium mobilization by structurally diverse mGlu5 orthosteric and allosteric ligands was assessed in human embryonic kidney 293 cells and primary murine neuronal cultures from both striatum and cortex. We aimed to determine the intrinsic efficacy and modulatory capacity of diverse mGlu5 PAMs [(R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide (VU0424465), N-cyclobutyl-6-((3-fluorophenyl)ethynyl)picolinamide (VU0360172), 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE), ((4-fluorophenyl) (2-(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)methanone) (VU0409551), 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB)] on receptor desensitization and whether cellular context influences receptor regulatory processes. Only VU0424465 and VU0409551 induced desensitization alone in human embryonic kidney 293-mGlu5 cells, while all PAMs enhanced (S)-3,5-dihydroxyphenylglycine (DHPG)-induced desensitization. All mGlu5 PAMs induced receptor desensitization alone and enhanced DHPG-induced desensitization in striatal neurons. VU0424465 and VU0360172 were the only PAMs that induced desensitization alone in cortical neurons. With the exception of (CDPPB), PAMs enhanced DHPG-induced desensitization in cortical neurons. Moreover, differential apparent affinities, efficacies, and cooperativities with DHPG were observed for VU0360172, VU0409551, and VU0424465 when comparing receptor activation and desensitization in a cell type-dependent manner. These data indicate that biased mGlu5 allosteric modulator pharmacology extends to receptor regulatory processes in a tissue dependent manner, adding yet another layer of complexity to rational mGlu5 drug discovery.

"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu5 Allosteric Ligands.

Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu5 as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu5 but exhibited neutral cooperativity with mGlu5 agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa2+) mobilization, and inositol monophosphate (IP1) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu5 (HEK293A-mGlu5-low) for diverse allosteric chemotypes. Numerous "non-mGlu5" class C GPCR allosteric modulators incompletely displaced allosteric mGlu5 radioligand [3H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu1 ligand), PHCCC (mGlu4 ligand), GS39783 (GABAB ligand), AZ12216052 (mGlu8 ligand), and CGP7930 (GABAB ligand) at mGlu5 were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu5 agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu5 agonists for IP1 accumulation, but not iCa2+ mobilization. By using mGlu5 as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.

Identification of Global and Ligand-Specific Calcium Sensing Receptor Activation Mechanisms.

Calcium sensing receptor (CaSR) positive allosteric modulators (PAMs) are therapeutically important. However, few are approved for clinical use, in part due to complexities in assessing allostery at a receptor where the endogenous agonist (extracellular calcium) is present in all biologic fluids. Such complexity impedes efforts to quantify and optimize allosteric drug parameters (affinity, cooperativity, and efficacy) that dictate PAM structure-activity relationships (SARs). Furthermore, an underappreciation of the structural mechanisms underlying CaSR activation hinders predictions of how PAM SAR relates to in vitro and in vivo activity. Herein, we combined site-directed mutagenesis and calcium mobilization assays with analytical pharmacology to compare modes of PAM binding, positive modulation, and agonism. We demonstrate that 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (NPS R568) binds to a 7 transmembrane domain (7TM) cavity common to class C G protein-coupled receptors and used by (αR)-(-)-α-methyl-N-[3-[3-[trifluoromethylphenyl]propyl]-1-napthalenemethanamine (cinacalcet) and 1-benzothiazol-2-yl-1-(2,4-dimethylphenyl)-ethanol (AC265347); however, there are subtle distinctions in the contribution of select residues to the binding and transmission of cooperativity by PAMs. Furthermore, we reveal some common activation mechanisms used by different CaSR activators, but also demonstrate some differential contributions of residues within the 7TM bundle and extracellular loops to the efficacy of the PAM-agonist, AC265347, versus cooperativity. Finally, we show that PAMS potentiate the affinity of divalent cations. Our results support the existence of both global and ligand-specific CaSR activation mechanisms and reveal that allosteric agonism is mediated in part via distinct mechanisms to positive modulation.

Biased agonism and allosteric modulation of metabotropic glutamate receptor 5.

Metabotropic glutamate receptors belong to class C G-protein-coupled receptors and consist of eight subtypes that are ubiquitously expressed throughout the central nervous system. In recent years, the metabotropic glutamate receptor subtype 5 (mGlu5) has emerged as a promising target for a broad range of psychiatric and neurological disorders. Drug discovery programs targetting mGlu5 are primarily focused on development of allosteric modulators that interact with sites distinct from the endogenous agonist glutamate. Significant efforts have seen mGlu5 allosteric modulators progress into clinical trials; however, recent failures due to lack of efficacy or adverse effects indicate a need for a better understanding of the functional consequences of mGlu5 allosteric modulation. Biased agonism is an interrelated phenomenon to allosterism, describing how different ligands acting through the same receptor can differentially influence signaling to distinct transducers and pathways. Emerging evidence demonstrates that allosteric modulators can induce biased pharmacology at the level of intrinsic agonism as well as through differential modulation of orthosteric agonist-signaling pathways. Here, we present key considerations in the discovery and development of mGlu5 allosteric modulators and the opportunities and pitfalls offered by biased agonism and modulation.

Practical Strategies and Concepts in GPCR Allosteric Modulator Discovery: Recent Advances with Metabotropic Glutamate Receptors.

Allosteric modulation of GPCRs has initiated a new era of basic and translational discovery, filled with therapeutic promise yet fraught with caveats. Allosteric ligands stabilize unique conformations of the GPCR that afford fundamentally new receptors, capable of novel pharmacology, unprecedented subtype selectivity, and unique signal bias. This review provides a comprehensive overview of the basics of GPCR allosteric pharmacology, medicinal chemistry, drug metabolism, and validated approaches to address each of the major challenges and caveats. Then, the review narrows focus to highlight recent advances in the discovery of allosteric ligands for metabotropic glutamate receptor subtypes 1-5 and 7 (mGlu1-5,7) highlighting key concepts ("molecular switches", signal bias, heterodimers) and practical solutions to enable the development of tool compounds and clinical candidates. The review closes with a section on late-breaking new advances with allosteric ligands for other GPCRs and emerging data for endogenous allosteric modulators.

Molecular insights into allosteric modulation of Class C G protein-coupled receptors.

Class C G protein-coupled receptors (GPCRs) recognise diverse extracellular stimuli and are highly tractable drug targets for a host of different psychiatric, neurological and metabolic disorders. Discovery efforts focussed on allosteric modulators for Class C GPCRs have been highly fruitful, with diverse chemotypes identified for multiple Class C members. Indeed, a positive allosteric modulator of the calcium-sensing receptor, cinacalcet, was one of the first GPCR allosteric ligands to enter the clinic. Despite this success, allosteric modulator discovery and development remains challenging. In particular, the prevalence of probe dependence and biased pharmacology (both agonism and modulation) adds considerable complexity. Recent studies have yielded new insights into the structural basis for allosteric interactions at Class C GPCRs. This information coupled with rigorous analytical approaches has increased our understanding of the rich molecular pharmacology and biology for Class C GPCRs.