SERCA pump as a novel therapeutic target for treating neurodegenerative disorders.

The neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Spinocerebellar ataxias (SCAs), present an enormous medical, social, financial and scientific problem. Despite intense research into the causes of these disorders, only marginal progress has been made in the clinic and no cures exist for any of them. Most of the scientific effort has been focused on identification of the major causes of these diseases and on developing ways to target them, such as targeting amyloid accumulation for AD or targeting expression of mutant Huntingtin for HD. Calcium (Ca2+) signaling has long been proposed to play an important role in the pathogenesis of neurodegenerative disorders, but blockers of Ca2+ channels and Ca2+ signaling proteins have not been translated to clinic primarily due to side effects related to the important roles of target molecules for these compounds at the peripheral tissues. In this review article, we would like to discuss an idea that recently identified positive allosteric modulators (PAMs) of the sarco-endoplasmic reticulum calcium (SERCA) pump may provide a promising approach to develop therapeutic compounds for treatment of these disorders. This hypothesis is supported by the preclinical data obtained with animal models of AD and PD. The first critical test of this idea will be an imminent phase I study that will offer an opportunity to evaluate potential side effects of this class of compounds in humans.

Synthesis and biological studies of 2-aminothiophene derivatives as positive allosteric modulators of glucagon-like peptide 1 receptor.

As a step toward the development of novel small-molecule positive allosteric modulators (PAMs) of glucagon-like peptide 1 receptor (GLP-1R) for the treatment of type 2 diabetes, obesity, and heart diseases, we discovered a novel 2-amino-thiophene (2-AT) based lead compound bearing an ethyl 3-carboxylate appendage. In this work, we report the syntheses and biological studies of more than forty 2-AT analogs, that have revealed a 2-aminothiophene-3-arylketone analogue 7 (MW 299) showing approximately a 2-fold increase in insulin secretion at 5 µM when combined with the GLP-1 peptide at 10 nM. In vivo studies using CD1 mice at a dose of 10 mg/kg, clearly demonstrated that the blood plasma glucose level was lowered by 50% after 60 min. Co-treatment of 7 with sitagliptin, an inhibitor of GLP-1 degrading enzyme Dipeptidyl Peptidase IV, further confirmed 7 to be an effective PAM of GLP-1R. The small molecular weight and demonstrated allosteric modulating properties of these compound series, show the potential of these scaffolds for future drug development.

Positive allosteric modulators of the GABAB receptor: a new class of ligands with therapeutic potential for alcohol use disorder.

BACKGROUND: Positive allosteric modulators (PAMs) of the GABAB receptor constitute a new class of GABAB-receptor ligands. GABAB PAMs reproduce several pharmacological effects of the orthosteric GABAB receptor agonist, baclofen, although displaying a better safety profile. AIMS: This paper reviews the reducing or, frequently, even suppressing effects of all GABAB PAMs tested to date on multiple alcohol-related behaviours in laboratory rodents exposed to validated experimental models of human alcohol use disorder. RESULTS: Acute or repeated treatment with CGP7930, GS39783, BHF177, rac-BHFF, ADX71441, CMPPE, COR659, ASP8062, KK-92A, and ORM-27669 reduced excessive alcohol drinking, relapse- and binge-like drinking, operant alcohol self-administration, reinstatement of alcohol seeking, and alcohol-induced conditioned place preference in rats and mice. CONCLUSIONS: These effects closely mirrored those of baclofen; notably, they were associated to remarkably lower levels of tolerance and toxicity. The recent transition of ASP8062 to clinical testing will soon prove whether these highly consistent preclinical data translate to AUD patients.

M1/M4 receptors as potential therapeutic treatments for schizophrenia: A comprehensive study.

Muscarinic acetylcholine receptors (mAChRs) play a significant role in the pathophysiology of schizophrenia. Although activating mAChRs holds potential in addressing the full range of schizophrenia symptoms, clinical application of many non-selective mAChR agonists in cognitive deficits, positive and negative symptoms is hindered by peripheral side effects (gastrointestinal disturbances and cardiovascular effects) and dosage restrictions. Ligands binding to the allosteric sites of mAChRs, particularly the M1 and M4 subtypes, demonstrate activity in improving cognitive function and amelioration of positive and negative symptoms associated with schizophrenia, enhancing our understanding of schizophrenia. The article aims to critically examine current design concepts and clinical advancements in synthesizing and designing small molecules targeting M1/M4, providing theoretical insights and empirical support for future research in this field.

Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%-25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones. Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

AMPA receptor neurotransmission and therapeutic applications: A comprehensive review of their multifaceted modulation.

The neuropharmacological community has shown a strong interest in AMPA receptors as critical components of excitatory synaptic transmission during the last fifteen years. AMPA receptors, members of the ionotropic glutamate receptor family, allow rapid excitatory neurotransmission in the brain. AMPA receptors, which are permeable to sodium and potassium ions, manage the bulk of the brain's rapid synaptic communications. This study thoroughly examines the recent developments in AMPA receptor regulation, focusing on a shift from single chemical illustrations to a more extensive investigation of underlying processes. The complex interplay of these modulators in modifying the function and structure of AMPA receptors is the main focus, providing insight into their influence on the speed of excitatory neurotransmission. This research emphasizes the potential of AMPA receptor modulation as a therapy for various neurological disorders such as epilepsy and Alzheimer's disease. Analyzing these regulators' sophisticated molecular details enhances our comprehension of neuropharmacology, representing a significant advancement in using AMPA receptors for treating intricate neurological conditions.

Exploring thienothiadiazine dioxides as isosteric analogues of benzo- and pyridothiadiazine dioxides in the search of new AMPA and kainate receptor positive allosteric modulators.

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2H-1,2,4-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 µM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 (11) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators.

An integrated approach to evaluate the functional effects of disease-associated NMDA receptor variants.

The NMDA receptor (NMDAR) is a ubiquitously expressed glutamate-gated ion channel that plays key roles in brain development and function. Not surprisingly, a variety of disease-associated variants have been identified in genes encoding NMDAR subunits. A critical first step to assess whether these variants contribute to their associated disorder is to characterize their effect on receptor function. However, the complexity of NMDAR function makes this challenging, with many variants typically altering multiple functional properties. At synapses, NMDARs encode pre- and postsynaptic activity to carry a charge transfer that alters membrane excitability and a Ca2+ influx that has both short- and long-term signaling actions. Here, we characterized epilepsy-associated variants in GluN1 and GluN2A subunits with various phenotypic severity in HEK293 cells. To capture the complexity of NMDAR gating, we applied 10 glutamate pulses at 10 Hz to derive a charge integral. This assay is advantageous since it incorporates multiple gating parameters - activation, deactivation, and desensitization - into a single value. We then integrated this gating parameter with Mg2+ block and Ca2+ influx using fractional Ca2+ currents to generate indices of charge transfer and Ca2+ transfer over wide voltage ranges. This approach yields consolidated parameters that can be used as a reference to normalize channel block and allosteric modulation to better define potential patient treatment. This is especially true for variants in the transmembrane domain that affect not only receptor gating but also often Mg2+ block and Ca2+ permeation.

Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

Design, synthesis and evaluation of novel 2-phenyl-3-(1H-pyrazol-4-yl)pyridine positive allosteric modulators for the M4 mAChR.

Translation of muscarinic acetylcholine receptor (mAChR) agonists into clinically used therapeutic agents has been difficult due to their poor subtype selectivity. M4 mAChR subtype-selective positive allosteric modulators (PAMs) may provide better therapeutic outcomes, hence investigating their detailed pharmacological properties is crucial to advancing them into the clinic. Herein, we report the synthesis and comprehensive pharmacological evaluation of M4 mAChR PAMs structurally related to 1e, Me-C-c, [11C]MK-6884 and [18F]12. Our results show that small structural changes to the PAMs can result in pronounced differences to baseline, potency (pEC50) and maximum effect (Emax) measures in cAMP assays when compared to the endogenous ligand acetylcholine (ACh) without the addition of the PAMs. Eight selected PAMs were further assessed to determine their binding affinity and potential signalling bias profile between cAMP and ß-arrestin 2 recruitment. These rigorous analyses resulted in the discovery of the novel PAMs, 6k and 6l, which exhibit improved allosteric properties compared to the lead compound, and probative in vivo exposure studies in mice confirmed that they maintain the ability to cross the blood-brain barrier, making them more suitable for future preclinical assessment.

Role of mGlu5 in Persistent Forms of Hippocampal Synaptic Plasticity and the Encoding of Spatial Experience.

The metabotropic glutamate (mGlu) receptor family consists of group I receptors (mGlu1 and mGlu5) that are positively coupled to phospholipase-C and group II (mGlu2 and mGlu3) and III receptors (mGlu4-8) that are negatively coupled to adenylyl cyclase. Of these, mGlu5 has emerged as a key factor in the induction and maintenance of persistent (>24 h) forms of hippocampal synaptic plasticity. Studies in freely behaving rodents have revealed that mGlu5 plays a pivotal role in the stabilisation of hippocampal long-term potentiation (LTP) and long-term depression (LTD) that are tightly associated with the acquisition and retention of knowledge about spatial experience. In this review article we shall address the state of the art in terms of the role of mGlu5 in forms of hippocampal synaptic plasticity related to experience-dependent information storage and present evidence that normal mGlu5 function is central to these processes.

mGlu2 mechanism-based interventions to treat alcohol relapse.

Recently we identified a deficiency in metabotropic glutamate receptor 2 (mGlu2) function in the corticoaccumbal pathway, as a common pathological mechanism underlying alcohol-seeking and relapse behavior. Based on this mechanism, we hypothesized that mGlu2/3 agonists and mGlu2 positive allosteric modulators (PAMs) may be effective in reducing relapse-like behavior. Two mGlu2/3 agonists, LY379268 and LY354740 (a structural analog of LY379268 six-fold more potent in activating mGlu2 over mGluR3), were tested in a well-established rat model of relapse, the alcohol deprivation effect (ADE) with repeated deprivation phases. Since these agonists do not readily discriminate between contributions of mGlu2 and mGluR3, we also tested LY487379, a highly specific PAM that potentiates the effect of glutamate on the mGlu2 with less specificity on other mGlu receptor subtypes. Both LY379268 and LY354740 significantly and dose-dependently reduced the expression of the ADE. No significant changes in water intake, body weight and locomotor activity were observed. Importantly, repeated administration of mGlu2/3 agonist did not lead to tolerance development. mGlu2 PAM LY487379 treatment significantly reduced expression of the ADE in both male and female rats. Combination treatment of mGlu2/3 agonist and PAM had similar effect on relapse-like drinking to that seen in mGlu2/3 agonist treatment alone. Together with other preclinical data showing that PAMs can reduce alcohol-seeking behavior we conclude that mGlu2 PAMs should be considered for clinical trials in alcohol-dependent patients.

Binding of a positive allosteric modulator CDPPB to metabotropic glutamate receptor type 5 (mGluR5) probed by all-atom molecular dynamics simulations.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor type 5 (mGluR5) potentiate positive receptor response and may be effective for the treatment of schizophrenia and cognitive disorders. Although crystal structures of mGluR5 complexed with the negative allosteric modulators (NAMs) are available, no crystal structure of mGluR5 complexed with PAM has been reported to date. Thus, conformational changes associated with the binding of PAMs to mGluR5 remain elusive. Here, a PAM CDPPB, and two NAMs MTEP and MFZ10-7 used as a negative control, were docked to the crystal structure. The docked complexes were submitted to molecular dynamics simulations to examine the activation of the PAM system. An MM/GBSA binding energy calculation was performed to estimate binding strength. Furthermore, molecular switch analysis was done to get insights into conformational changes of the receptor. The PAM CDPPB displays a stronger binding affinity for mGluR5 and induces conformational changes. Also, a salt bridge between TM3 and TM7, corresponding to the ionic lock switch in class A GPCRs is found to be broken. The PAM-induced receptor conformation is more like the agonist-induced conformation than the antagonist-induced conformation, suggesting that PAM works by inducing conformation change and stabilizing the active receptor conformation.

Allosteric modulators of the δ GABAA receptor subtype demonstrate a therapeutic effect in morphine-antinociceptive tolerance and withdrawal in mice.

The present study evaluated the effects of compounds targeting extrasynaptic δ subunit-containing γ-aminobutyric acid type A receptors (δ*-GABAARs) to interrogate the role of tonic inhibition in the development of antinociceptive tolerance caused by repeated morphine administration. We investigated the effect of subchronic or acute treatment with non-steroidal positive allosteric modulators (PAMs) of δ*-GABAARs, such as 2-261, on the morphine-antinociceptive tolerance. Mice were treated twice daily with morphine for 9 days and antinociception was measured using the hot water tail immersion test. Co-treatment with 2-261 and morphine prevented morphine-antinociceptive tolerance and acute administration of 2-261 on day 9 was sufficient to reverse the tolerance. Other compounds with activity at δ*-GABAARs also reversed morphine tolerance, whereas an enaminone that lacked activity at δ*-GABAARs did not. Acute administration of 2-261 did not cause an additive or synergistic antinociceptive effect when combined with an acute submaximal dose of morphine. We then used Cre/LoxP recombination to generate GABAA δ-subunit knockout mice to corroborate the pharmacological results. Observations of male δ-knockout mice demonstrated that the δ*-GABAARs was necessary for 2-261 modulation of both analgesic tolerance and somatic withdrawal symptoms produced by subchronic morphine. While female mice still benefited from the positive effects of 2-261, the δ-subunit was not necessary for these effects, highlighting a distinction of the different pathways that could have implications for some of the sex-related differences seen in human opioid-induced outcomes. Consequently, subtype-specific allosteric modulators of GABAARs may warrant further investigation as pharmacological targets to manage tolerance and withdrawal from opioids.

Positive Allosteric Modulation of α5-GABAA Receptors Reverses Stress-Induced Alterations in Dopamine System Function and Prepulse Inhibition of Startle.

BACKGROUND: Up to 64% of patients diagnosed with posttraumatic stress disorder (PTSD) experience psychosis, likely attributable to aberrant dopamine neuron activity. We have previously demonstrated that positive allosteric modulators of α5-GABAARs can selectively decrease hippocampal activity and reverse psychosis-like physiological and behavioral alterations in a rodent model used to study schizophrenia; however, whether this approach translates to a PTSD model remains to be elucidated. METHODS: We utilized a 2-day inescapable foot shock (IS) procedure to induce stress-related pathophysiology in male Sprague-Dawley rats. We evaluated the effects of intra-ventral hippocampus (vHipp) administration GL-II-73, an α5-GABAAR, or viral overexpression of the α5 subunit, using in vivo electrophysiology and behavioral measures in control and IS-treated rats. RESULTS: IS significantly increased ventral tegmental area dopamine neuron population activity, or the number of dopamine neurons firing spontaneously (n = 6; P = .016), consistent with observation in multiple rodent models used to study psychosis. IS also induced deficits in sensorimotor gating, as measured by reduced prepulse inhibition of startle (n = 12; P = .039). Interestingly, intra-vHipp administration of GL-II-73 completely reversed IS-induced increases in dopamine neuron population activity (n = 6; P = .024) and deficits in prepulse inhibition (n = 8; P = .025), whereas viral overexpression of the α5 subunit in the vHipp was not effective. CONCLUSIONS: Our results demonstrate that pharmacological intervention augmenting α5-GABAAR function, but not α5 overexpression in itself, can reverse stress-induced deficits related to PTSD in a rodent model, providing a potential site of therapeutic intervention to treat comorbid psychosis in PTSD.

Synthesis and evaluation of avermectin-imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators.

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin-imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin-imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

6-Aryl-quinazolines as novel GABAB receptor positive allosteric modulators.

The systemic use of GABAB orthosteric agonist baclofen might be limited due to its detrimental properties: sedation and motor impairment. In contrast, GABAB positive allosteric modulators produce less adverse effects. Using BHF-177 as a starting point, we found a new active scaffold: the 6-aryl-quinazoline scaffold. Further elaborating the scaffold, we identified several in vitro and in vivo active compounds.

2-Aminothiophene derivatives as a new class of positive allosteric modulators of glucagon-like peptide 1 receptor.

We report the discovery of two new 2-aminothiophene based small molecule positive allosteric modulators (PAMs) of glucagon-like peptide 1 receptor (GLP-1R) for the treatment of type 2 diabetes. One of the chemotypes, (S-1), has a molecular weight of 239 g/mol, the smallest molecule among all reported GLP-1R PAMs. When combined with GLP-1 peptide, S-1 increased the GLP-1R activity in a dose-dependent manner in a cell-based assay. When combined with the peptide agonist of vasoactive intestinal polypeptide receptor 1 (VIPR1), S-1 showed no specific activity on VIPR1, another class B GPCR present in the same HEK293-CREB cell line. Insulin secretion studies found S-1 combined with GLP-1 increased insulin secretion by 1.5-fold at 5 µM. In a mechanistic study, evidence is provided that the synergistic effect of S-1 with GLP-1 may be partly due to the enhanced impact on CREB based phosphorylation. Given the favorable profile of these chemotypes, the work reported herein suggests that 2-aminothiophene derivatives are a new and promising class of GLP-1R PAMs.

Personalised medicines for familial hypercalcemia and hyperparathyroidism.

Loss-of-function calcium-sensing receptor (CASR) mutations cause mineral metabolism disorders, familial hypocalciuric hypercalcemia, or neonatal severe hyperparathyroidism and increase the risk of femoral fracture, chronic kidney disease, coronary heart disease, and other diseases. In severe cases, CaSR mutations are lethal. Off-label use of the CaSR-positive allosteric modulator (PAM), cinacalcet, corrects hypercalcemia in some patients with CaSR mutations. However, other patients remain unresponsive to cinacalcet, attesting to the need for novel treatments. Here, we compared the effects of cinacalcet to two other clinically approved synthetic CaSR activators, evocalcet and etelcalcetide, as well as a novel PAM, 1-(2,4-dimethylphenyl)-1-(4,5-dimethylthiazol-2-yl)ethan-1-ol (MIPS-VD-836-108) on clinically relevant CaSR mutations. We assessed the compounds in CaSR-expressing HEK293 cells for correction of mutation-induced impairments in intracellular calcium (Ca2+i) mobilization and cell surface expression. While cinacalcet, MIPS-VD-836-108 and evocalcet rescued the signaling of cell surface-expressed mutants, albeit to varying degrees, etelcalcetide was ineffective. Cinacalcet and evocalcet, but not MIPS-VD-836-108 or etelcalcetide, restored the expression of a R680H mutant. However, no compound rescued expression of I81K and C582R mutants or a receptor missing 77 amino acids in the extracellular domain mimicking deletion of CASRexon 5, which impairs CaSR function. These data suggest specific compounds may be clinically effective in some patients with CaSR mutations, but other patients will remain refractory to treatment with currently available CaSR-targeting activators, highlighting the need for new generation drugs to rescue both the signaling and expression of mutant CaSRs.

(+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the ß(+)/α(-) interface near the TM2-TM3 loop.

(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at ß2/3-containing GABAARs 2.4-3.5 times more efficaciously than at ß1-containing GABAARs. The activity of (+)-catharanthine is reduced by the ß2(N265S) mutation that decreases GABAAR potentiation by loreclezole, but not by the ß3(M286C) or α1(Q241L) mutations that reduce receptor potentiation by R(+)-etomidate or neurosteroids, respectively. Competitive functional experiments indicated that the binding site for (+)-catharanthine overlaps that for loreclezole, but not those for R(+)-etomidate or potentiating neurosteroids. Molecular docking experiments suggested that (+)-catharanthine binds at the ß(+)/α(-) intersubunit interface near the TM2-TM3 loop, where it forms H-bonds with ß2-D282 (TM3), ß2-K279 (TM2-TM3 loop), and ß2-N265 and ß2-R269 (TM2). Site-directed mutagenesis experiments supported the in silico results, demonstrating that the K279A and D282A substitutions, that lead to a loss of H-bonding ability of the mutated residue, and the N265S mutation, impair the gating efficacy of (+)-catharanthine. We infer that (+)-catharanthine potentiates the GABAAR through several H-bond interactions with a binding site located in the ß(+)/α(-) interface in the transmembrane domain, near the TM2-TM3 loop, where it overlaps with loreclezole binding site.