Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gαq/11 and Gαs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gαq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.   ### Competing Interest Statement The authors have declared no competing interest. National Health and Medical Research Council, APP2021675, 1125877, 1139591, 2002947, APP2021163 Australian Research Council, FT170100392, FT220100617

Trace amine-associated receptor 1 (TAAR1) is an emerging pharmaceutical target for treating a variety of neuropsychiatric conditions, with several drug candidates in clinical and preclinical development. Multiple single-nucleotide variants have been associated with neuropsychiatric disorders, and genetic variants may influence the therapeutic outcomes of TAAR1-based therapies. Here, we utilize mutagenesis, functional assays, and computational models to profile schizophrenia-associated TAAR1 variant C182 45.50 F. In cyclic adenosine monophosphate (cAMP) assays, TAAR1 C182 45.50 F demonstrated a complete loss of activity in the homozygous state, and approximately 50% loss in the heterozygous state compared to TAAR1 WT (wild type). Furthermore, the surface expression of homozygous TAAR1 C182 45.50 F was altered, with approximately 40% reduction in surface expression compared to TAAR1 WT. Expression marginally improved in the heterozygous state. Molecular dynamics simulations of the TAAR1 C182 45.50 F model demonstrated increased extracellular loop 2 (ECL2) flexibility, with F182 45.50 forming a stable aromatic cluster (aromatic–aromatic interactions) involving F165 ECL2 and Y172 ECL2 that occludes the orthosteric binding site. The aromatic cluster is further stabilized by a transient salt bridge between E93 3.22 and K97 3.26 . Overall, our study shows the TAAR1 C182 45.50 F variant may disrupt endogenous trace amine signaling via a reduction of cell surface expression and occlusion of endogenous ligand binding; in addition, newly developed TAAR1 therapeutics may be subefficacious in carriers of this variant.

Allosteric modulation of G protein-coupled receptors (GPCRs) is an exciting strategy for developing new therapeutic agents, and it has several advantages over more commonly used orthosteric drugs. Recently determined GPCR structures have revealed allosteric pockets facing the lipid bilayer, enabling rational drug design. Here, we develop a virtual screening strategy to discover ligands of extrahelical binding pockets and apply this approach to the adenosine A1 receptor (A1R). The A1R is a high-value therapeutic target for ischemia-reperfusion injury and chronic neuropathic pain. Developing effective A1R therapeutics remains challenging due to high structural conservation across orthosteric binding sites and on-target unwanted effects stimulated by prototypical A1R agonists, such as bradycardia and atrioventricular block. However, A1R positive allosteric modulators (PAMs) acting through spatially distinct allosteric sites can fine-tune A1R activity with high subtype selectivity and spatiotemporal specificity, thereby overcoming current limitations. A chemical library of 160 million compounds was computationally docked to the allosteric pocket identified in a cryo-EM structure of the A1R, and a set of 26 top-ranked compounds were selected for experimental evaluation. Pharmacological evaluation of these, and structure-guided hit optimization, led to the discovery of subtype-selective A1R PAMs. These compounds demonstrated minimal allosteric agonism and negligible impact on A1R-mediated beat rate of an orthosteric agonist. The discovered PAMs pave the way for potential treatments for neuropathic pain and ischemia-reperfusion injury without accompanying side effects. Our results demonstrate the utility of a synergistic computational and experimental approach in GPCR drug discovery.

The metabotropic glutamate receptor 5 (mGlu5) is a Class C G protein-coupled receptor, ubiquitously expressed throughout the central nervous system (CNS). With major roles in cognition, learning and memory, mGlu5 dysfunction is linked with numerous neurodegenerative and neuropsychiatric disorders, representing a viable therapeutic target. Allosteric modulators bind topographically distinct sites from glutamate and other orthosteric agonists and enhance (positive allosteric modulators, PAMs), inhibit (negative allosteric modulators, NAMs) or do not effect (neutral allosteric ligands, NALs) mGlu5 function. While mGlu5 modulators have efficacy in in vivo rodent models of CNS disorders, none have been approved for human use. We hypothesise preclinical optimisation using non-human pharmacological data may contribute to translational failures, as functional studies are predominantly performed using rat mGlu5 and non-human brain neuronal cultures. Here we are the first to systematically assess and quantify the impact of eleven chemically and pharmacologically diverse mGlu5 PAMs, NAMs and NALs on human mGlu5 activity using radioligand binding, intracellular calcium (iCa2+) mobilisation and inositol monophosphate (IP1) accumulation assays. By comparing to published and newly generated data for rat mGlu5 we show that while modulator pharmacology is relatively consistent across species, ligand dependent species differences in allosteric modulator affinity, cooperativity and probe dependence are evident. Additionally, we report PAM-dependent effects on orthosteric agonist kinetic profiles at human mGlu5. Together, these data highlight the importance of systematic evaluation of mGlu5 allosteric ligand activity at human mGlu5 to improve drug design and overcome potential barriers to translatability to clinical settings.

For over seven decades, dopamine receptor 2 (D2 receptor) antagonists remained the mainstay treatment for neuropsychiatric disorders. Although it is effective for treating hyperdopaminergic symptoms, it is often ineffective for treating negative and cognitive deficits. Trace amine-associated receptor 1 (TAAR1) is a novel, pharmacological target in the treatment of schizophrenia and other neuropsychiatric conditions. Several TAAR1 agonists are currently being developed and are in various stages of clinical and preclinical development. Previous efforts to identify TAAR1 agonists have been hampered by challenges in pharmacological characterisation, the absence of experimentally determined structures, and species-specific preferences in ligand binding and recognition. Further, poor insights into the functional selectivity of the receptor led to the characterisation of ligands with analogous signalling mechanisms. Such approaches limited the understanding of divergent receptor signalling and their potential clinical utility. Recent cryogenic electron microscopic (cryo-EM) structures of human and mouse TAAR1 (hTAAR1 and mTAAR1, respectively) in complex with agonists and G proteins have revealed detailed atomic insights into the binding pockets, binding interactions and binding modes of several agonists including endogenous trace amines (β-phenylethylamine, 3-Iodothyronamine), psychostimulants (amphetamine, methamphetamine), clinical compounds (ulotaront, ralmitaront) and repurposed drugs (fenoldopam). The in vitro screening of drug libraries has also led to the discovery of novel TAAR1 agonists (asenapine, guanabenz, guanfacine) which can be used in clinical trials or further developed to treat different neuropsychiatric conditions. Furthermore, an understanding of unappreciated signalling mechanisms (Gq, Gs/Gq) by TAAR1 agonists has come to light with the discovery of selective compounds to treat schizophrenia-like phenotypes. In this review, we discuss the emergence of structure-based approaches in the discovery of novel TAAR1 agonists through drug repurposing strategies and structure-guided designs. Additionally, we discuss the functional selectivity of TAAR1 signalling, which provides important clues for developing disorder-specific compounds.

Metabotropic glutamate receptors are a family of eight class C G protein-coupled receptors regulating higher order brain functions including cognition and motion. Metabotropic glutamate receptors have thus been heavily investigated as potential drug targets for treating neurological disorders. Drug discovery efforts directed toward metabotropic glutamate receptor subtype 5 (mGlu5) have been particularly fruitful, with a wealth of drug candidates and pharmacological tools identified. mGlu5 negative allosteric modulators (NAMs) are promising novel therapeutics for developmental, neuropsychiatric and neurodegenerative disorders (e.g., Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, autism spectrum disorders, substance use disorders, stroke, anxiety and depression) and show promise in ameliorating adverse effects induced by other medications (e.g., L-dopa induced dyskinesia in Parkinson's Disease). However, despite preclinical success, mGlu5 NAMs are yet to reach the market due to poor safety and efficacy profiles in clinical trials. Herein, we review the physiology and signal transduction of mGlu5. We provide a comprehensive critique of therapeutic options with respect to mGlu5 inhibitors, spanning from orthosteric antagonists to NAMs. Finally, we address the challenges associated with drug development and highlight future directions to guide rational drug discovery of safe and effective novel therapeutics.

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.

Background and Purpose: Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and linked symptoms to mGlu1 mutations. However, how mutations alter mGlu1 function remains unknown, as does amenability of receptor function to pharmacological rescue. Here, we explored SCA-associated mutation effects on mGlu1 cell surface expression, canonical signal transduction and allosteric ligand pharmacology. Experimental Approach: Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery. Key Results: mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1. Conclusion and Implications: Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.

Although the gene DUSP6 has been implicated as a risk gene for ADHD in recent GWAS studies, its functional role in the aetiology of the condition remains poorly understood. DUSP6 is reported to regulate dopaminergic neurotransmission by decreasing available synaptic dopamine, suggesting a potential mechanism by which DUSP6 may confer risk to ADHD. In this study, we employed CRISPR-Cas9 to knockout DUSP6 in induced pluripotent stem cells (iPSCs) derived from an individual with ADHD. These isogenic engineered cells were then differentiated into highly homogeneous dopaminergic neurons, including both heterozygous (Het) and homozygous (Hom) knockout lines, along with parental control lines, to assess changes in dopaminergic neurotransmission at both cellular and transcriptomic levels. Enzyme Linked Immunosorbent Assay (ELISA) analyses showed a dose-dependent trend of increasing dopamine levels, at extracellular level, in the knockout lines. RNA sequencing further supported this finding, showing that downregulation of DUSP6 lead to the upregulation of differentially expressed genes (DEGs) associated with the regulation of dopamine secretion and synaptic functions critical for dopamine signalling. These DEGs appear to encode components of the synaptic machinery essential for effective dopamine transmission or act as regulators of dopaminergic neurotransmission. Additionally, RNA sequencing uncovered other potential biological mechanisms through which DUSP6 may contribute to increase the risk for ADHD, including effects on neurogenesis, extracellular matrix-associated processes, lipid metabolism, and sex-specific gene expression. Furthermore, we identified overlaps between DUSP6 knockout DEGs and those associated with other neuropsychiatric disorders, including major depression (MDD), bipolar disorder (BD), and schizophrenia (SCZ), suggesting shared genetic pathways potentially influenced by DUSP6. Together, this study provides deeper insights into the molecular underpinnings of the role played by DUSP6 in the genetic aetiology of ADHD and its broader implications across related neuropsychiatric conditions. ### Competing Interest Statement The authors have declared no competing interest.

Background Attention deficit hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental condition globally. It is characterized by inattention, hyperactivity/impulsivity, or the combination of both. The heritability of ADHD estimated at 75% – 91%. Recent genome-wide association studies (GWAS) have identified 76 genes associated with ADHD, particularly those expressed in early brain development and midbrain dopaminergic neurons. While GWAS identifies genetic loci associated with certain traits or diseases, it often doesn't provide detailed insights into the underlying biological mechanisms. Transcriptomic analysis, on the other hand, focuses on the study of gene expression patterns. It offers a direct approach to understand gene expression and regulatory mechanisms, providing a means to explore the molecular mechanisms of ADHD. Methods This study employs a cutting-edge combination of pluripotent stem cell programming and RNA sequence (RNA-Seq) analysis to examine the molecular mechanisms underlying ADHD. Based on the dopaminergic hypothesis of ADHD, induced pluripotent stem cells (iPSCs) derived from donors with and without a diagnosis of ADHD were differentiated in parallel into dopaminergic neurons. The neurons were subsequently subjected to RNA-Seq to investigate the gene expression profiles of ADHD in the cell type specific context. Results Comparative gene expression analysis between the donor lines revealed several important findings. The top 10 differentially expressed genes (DEG) (false discovery rate [FDR] ≤ 1.03  ×  10−9 and log2 fold change of ⩾1.73) were involved in various nervous system functions including dopaminergic system regulation (INPP5F, CBLN1, EPHA5), neuronal excitability and synaptic function (DPP10, DPP6, GIRK3, PTPRT, RBFOX1), neurodevelopment (LARGE1, EPHA5, RBFOX1), and behavioural modulation (RTL1, PTPRT). Interestingly, 14 of the DEGs (FDR ≤ 0.05) including six of top reported GWAS hits (DCC, FOXP1, FOXP2, PTPRF, TMEM200C, and VGLL3) were found to overlap with genes associated with genome-wide significant loci of the latest ADHD-GWAS. Further, gene set enrichment analysis of the top 100 GO categories revealed significant enrichment in processes related to neuronal development (e.g. GO:0021675) and differentiation (e.g. GO:0021953), synaptic assembly (e.g. GO:0007416) and function (e.g. GO:0060076), ion channel activity (e.g. GO:0005245), and various signalling pathways (e.g. GO:0019933) essential for neural communication and regulation.Association of DEGs in processes related to neuronal development and differentiation highlight the brain maturational delay hypothesis of ADHD. To explore this, we conducted neurite outgrowth analysis during the iPSc differentiation at days in vitro 7, 14, and 42. We observed significantly fewer neuronal projections in the ADHD donor-derived line compared to the non-ADHD donor line on day 14 (p = 0.012). This aligned with the transcriptomic findings, and further supports the maturational delay hypothesis in ADHD. Discussion These findings provide significant insights into the molecular mechanisms of ADHD associated with dopaminergic dysregulation and further support the hypothesis of maturational delay in neuronal development. Our study underscores the importance of integrating iPSC-derived cell type specific models and transcriptomics to uncover the genetic and molecular mechanisms driving ADHD. However, replication of these finding in a bigger sample size is warranted.

The most recent ADHD GWAS meta analysis highlighted the potential role of 76 genes enriched among genes expressed in early brain development and associated with midbrain dopaminergic neurons. However, the precise functional importance of the GWAS-identified single nucleotide polymorphisms (SNPs) remain unknown. In contrast to GWAS, transcriptome analysis directly investigates gene products by assessing the transcribed RNA. This allows one to gain functional insights into gene expression paving the way for a better understanding of the molecular risk mechanisms of conditions, such as ADHD. In this study, we performed transcriptome profiling of highly homogeneous dopamine neurons developed from induced pluripotent cells (iPSCs) that were derived from an individual with ADHD and a neurotypical comparison individual. Comparative gene expression analysis between the examined lines revealed that the top differentially expressed genes (DEGs) were predominantly associated with nervous system functions related to neuronal development and dopaminergic regulation. Notably, 29 of the DEGs overlapped with those identified by ADHD GWAS meta analysis. These genes are overrepresented in biological processes including developmental growth regulation, axonogenesis, and nervous system development. In addition, gene set analysis revealed significant enrichment for meta categories such as ion channel activity, synaptic function and assembly, neuronal development and cell differentiation. Further, we observed significantly reduced projections in the ADHD dopamine neurons at the mid differentiation stage (day 14 in vitro), providing preliminary support for the delayed neuronal maturation hypotheses of ADHD. This study underscores the potential of using iPSC derived cell type specific models that integrate genome and transcriptome analyses for biological discovery in ADHD. ### Competing Interest Statement The authors have declared no competing interest.

Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and clinical symptoms of mGlu1 mutations have been described. However, how mutations alter mGlu1 function remains unknown. We explored SCA-associated mutation effects on mGlu1 cell surface expression and canonical signal transduction. Orthosteric agonists and positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and IP1 accumulation). mGlu1 mutants exhibited differential impacts on receptor expression, with a truncating C-terminus mutation significantly reducing mGlu1 expression. Mutations differentially influenced orthosteric ligand affinity, efficacy, and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. Gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations, and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1. Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a means to restore aberrant mGlu1 function in rare SCA subtypes.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically dis-tinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modula-tors have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess un-appreciated probe-dependent or biased pharmacology. Using hu-man embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemi-cal scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allos-terism 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 accumula-tion 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 orthos-teric 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.

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.

Nicotinic acetylcholine receptors are evolutionarily conserved ligand gate ion channels that play pivotal roles in neuromuscular and neuronal transmission in a wide range of species. Unsurprisingly, an equally wide range of toxins targeting nicotinic receptors have evolved in numerous terrestrial and marine species. Pinnatoxins and α-conotoxins represent two such groups of marine toxins. While pinnatoxins are macrocyclic small molecules and α-conotoxins are peptidic toxins, both act as potent and selective nicotinic receptor antagonist toxins. As such, both toxin groups can be utilized to study different aspects of nicotinic receptor structure and function. The pharmacological and toxicological insights gained from studying these toxins at an ecological, biological and molecular level have enhanced our knowledge of the importance of nicotinic receptors in both health and disease. Here, the threat of these toxins to animal and human health, their pharmacological and toxicological characteristics, their use as tool compounds and the therapeutic potential of nicotinic toxins are discussed.

Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu(5)) 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 mGlu(5) PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu(5) 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 mGlu(5) PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu(5) internalization. SIGNIFICANCE STATEMENT Enhancing metabotropic glutamate receptor subtype 5 (mGlu(5)) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu(5) are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu(5) PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu(5) PAMs and is the first study of the effects of mGlu(5) PAMs on receptor internalization.