Receptor Allosteric Modulators Research Articles

Molecular modelling studies on ɑ7 nicotinic receptor allosteric modulators yields novel filter-based virtual screening protocol

The ɑ7 receptor is a member in the nicotinic acetylcholine receptor (nAChR) which has been implicated in several neurological disorders. Beside normal agonists and antagonists of α7 nAChRs, several studies revealed other types of molecules that are able to activate or deactivate ɑ7 receptors via allosteric binding; those are called positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs), with the former having more pharmacological importance than the latter. Since both types of modulators are believed to bind to the same place in the intracavity of the transmembrane domain, it was important to differentiate between them in terms of structural features and their binding with the target receptor, and then use these specific characteristics as filters to discriminate PAMs from NAMS. To do that, modulators’ physicochemical properties were investigated using two databases of known PAMs or NAMs which were then used to elucidate a specific pharmacophore for each class. Interestingly, PAMs were found to be relatively larger and more polar compared to NAMs, which was observed to carry a positive charge with double the number of cases than PAMs. Furthermore. a pharmacophore for each class was developed and the best PAMs pharmacophore was successfully able to pass 94% of tested PAMs and to eliminate 71% of NAMs, while the best NAM pharmacophore was able to pass 82% of NAMs and to filter out 85% of PAMs. Docking these known modulators into the α7 nAChRs allosteric site identified several amino acids that are key for specifically binding PAMs compared to NAMs. Next, these findings were employed in virtual screening and then seeding experiments were conducted to validate the developed pharmacophores usage as filters prior to the final docking. Interestingly, the number of retrieved PAMs in the final docking list was improved by up to five-fold compared to the non-filtered protocol, which clearly indicates for the efficiency of our protocol to pick true PAMs over decoys. Hence, the pharmacophore-based filtering technique developed in this work can act as a valuable tool in the pursuit of new, potent PAM molecules as therapeutically useful modulators of the α7 nicotinic receptors.

Membrane‐Facilitated Allosteric Modulation of GABAA Receptor by Farnesol: An In Silico Modeling and Simulation Study

ObjectiveGABAA receptors (GABAARs) are ligand‐gated ion channels that can be activated by γ‐aminobutyric acid (GABA) and allosterically modulated by several antiepileptic drugs and endogenous neurosteroids. Using electrophysiology (patch‐clamp) and HEK cells expressing human α1β3γ2 GABAARs, our team demonstrated positive allosteric receptor modulation (PAM) by farnesol, an endogenous isoprenol. Furthermore, farnesol attenuated seizures in vivo using mouse models of GABAAR‐dependent seizures. Here, we present a computational study that provides a structural basis for the observed farnesol‐mediated allosteric modulation of GABAARs and illustrates the plausible role of membrane in facilitating access and binding of the ligand.MethodsThe membrane partitioning of farnesol were investigated in dimyristoyl phosphatidylcholine model membrane system by steered molecular dynamics (MD) and Umbrella Sampling simulations using NAMD2.12 software. To investigate farnesol's PAM mechanism, we initially constructed a computerized heteropentameric model of the human α1β3γ2 GABAAR in which two GABA molecules were sequentially docked to the extracellular orthosteric sites at the β+α‐interfaces. Subsequently, we used this GABA‐docked model to build two additional models: 1) farnesol docked at the transmembrane lipid‐facing neurosteroid‐binding site, and 2) 10 mol% farnesol molecules placed randomly in the bulk membrane around the receptor. The energetically favorable location and orientation of farnesol within the bilayer suggested the neurosteroid binding site as a potential allosteric site. Each model was subjected to 1 μs all‐atom MD simulations totaling 3 μs.ResultsThe mechanism of channel activation by farnesol was characterized by examining dynamic structural variables including global twisting of the channel, tilting of the extracellular domain, and configuration of the ion pore including pore radius, hydration status, and electrostatic potential. We observed the channel pore opening associated with an increased flux of water molecules and pore hydration that supports the conductance (or open state) of GABAAR with farnesol docked to the neurosteroid‐binding site. Remarkably, in the bulk simulation with multiple ligand molecules around the receptor, one of the farnesol molecules reached the neurosteroid binding site via lateral diffusion followed by binding in a configuration identical to the docking simulation. The membrane interactions of farnesol seemed to play an apparent role in its access and binding.ConclusionsThis study provides a structural basis for the positive allosteric modulation of the GABAAR by farnesol in atomistic details consistent with both in vitro and in vivo data. This structural and dynamic model for the PAM of GABAAR may benefit the design of novel therapeutics targeting transmembrane allosteric sites of the GABAAR in both health and disease.Support or Funding InformationNational Institutes of Health – R15GM13129This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alpha-7 nicotinic receptor allosteric modulator PNU120596 prevents lipopolysaccharide-induced anxiety, cognitive deficit and depression-like behaviors in mice

Neuroinflammation is often associated with the development of major depressive disorder (MDD)-related symptoms. Previous studies have indicated that activation of glial cells, upregulation of proinflammatory cytokines and dysregulation of adrenergic system in the central nervous system (CNS) could be the key mediators to modulate depression-related behaviors after peripheral immune activation. Central α7 nicotinic acetylcholine receptor (α7 nAChR) has a role in the regulation of the cholinergic anti-inflammatory pathway. The present study determined the effects of PNU120596, α7 nAChR positive allosteric modulator (PAM), on lipopolysaccharide (LPS)-induced anxiety, cognitive deficit, and depression-like behaviors in mice. These behaviors were evaluated 24 h after LPS (1 mg/kg) administration using elevated plus-maze, Y-maze, and forced swim test, respectively. The effects of PNU120596 on mRNA of neuroinflammatory markers and norepinephrine (NE) level in behaviorally-relevant brain regions such as the hippocampus and prefrontal cortex were examined. PNU120596 administration (1 or 4 mg/kg) showed anxiolytic, pro-cognitive, and antidepressant-like effects by preventing LPS-induced behavioral abnormalities. Following LPS treatment, PNU120596 hindered activation markers of the microglia and astrocytes (cluster of differentiation molecule 11b and glial fibrillary acidic protein) and upregulation of proinflammatory cytokines such as interleukin 1 beta and tumor necrosis factor-alpha in the hippocampus and prefrontal cortex. NE level that was reduced by peripheral LPS challenge was normalized by PNU120596 effects in both brain regions. Overall, the results in this study indicate that activation of α7 nAChR by PAM effectively prevents LPS-induced anxiety, cognitive deficit, and depression-like behaviors and regulates relevant neuroinflammatory markers in the hippocampus and prefrontal cortex.

Flumazenil but not bicuculline counteract the impairing effects of anesthetic ketamine on recognition memory in rats. Evidence for a functional interaction between the GABAA-benzodiazepine receptor and ketamine?

Experimental evidence indicates that anesthetic doses of the non-competitive NMDA receptor antagonist ketamine impair memory abilities in rodents. The mechanism by which anesthetic ketamine produces its adverse behavioural effects is not yet clarified. In this context, it has been proposed that the effects of anesthetic ketamine on memory might be attributed to its agonistic properties on the GABA type A receptor. The present study was designed to address this issue. Thus, we investigated the ability of the benzodiazepine receptor antagonist flumazenil (1, 3, 6 mg/kg, i.p.) and the GABAA receptor antagonist bicuculline (0.5, 1.5, 3 mg/kg, i.p.) to counteract recognition memory deficits produced by anesthetic ketamine (100 mg/kg, i.p.) in rats. For this purpose, the novel object recognition task, a behavioural paradigm assessing recognition memory abilities in rodents was used. Compounds were coadministered 24 h before testing or retention. Pre (24 h before testing) or post-training (24 h before retention) administration of flumazenil (6 mg/kg, i.p.) counteracted anesthetic ketamine-induced performance deficits in the novel object recognition memory task. Conversely, bicuculline failed to attenuate the recognition memory deficits caused by anesthetic ketamine. Our findings propose a functional interaction between anesthetic ketamine and the GABAA receptor allosteric modulator flumazenil on recognition memory.

Molecular Basis of Action of a Small-Molecule Positive Allosteric Modulator Agonist at the Type 1 Cholecystokinin Holoreceptor.

Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.

An investigation of the molecular and biochemical basis underlying chlorantraniliprole-resistant Drosophila strains and their cross-resistance to other insecticides.

Chlorantraniliprole is an anthranilic diamide insecticide that binds to the insect ryanodine receptor (RyR) and induces an uncontrolled release of Ca2+ , resulting in paralysis and ultimately death of the target insects. Recently, it was reported that chlorantraniliprole-resistant diamondback moths, Plutella xylostella Linnaeus, have mutations in their RyR. In this study, we developed two different chlorantraniliprole-resistant Drosophila melanogaster strain. The resistance ratio (RR) of the low-concentration chlorantraniliprole-treated resistant (Low-Res) strain was 2.3, while that of the high-concentration chlorantraniliprole-treated resistant (High-Res) strain was 21.3. The LC 50 of the untreated control (Con) strain was 23.8~25.9 ppm, which was significantly higher than that reported for the susceptible diamondback moth (0.03~0.51 ppm). The high LC 50 of the Con may be because the helix S2 amino acid sequence of D. melanogaster RyR ( DmRyR) is identical to the I4790M mutation of the chlorantraniliprole-resistant diamondback moths, resulting in a lower binding affinity of DmRyR for chlorantraniliprole. Among the tested detoxification enzymes, the activity of esterase was significantly increased in the two Res strains, but glutathione S-transferases and acetylcholinesterase were significantly decreased in the two Res strains. The cross-resistance of the High-Res strain to other insecticides with different modes of actions (MoAs) revealed that the RRs of the neuronal acetylcholine receptor allosteric and competitive modulators were significantly increased, while those of the Na 2+ channel modulators were significantly reduced. Our studies showed that RRs against the same insecticide vary with the treatment concentration, and that RRs against other insecticides with different MoAs can be altered.

Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation.

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and [35S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940 binding assay, indicating greater CB1 receptor affinity and/or cooperativity.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the “druggable genome” that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Allosteric and orthosteric pharmacology of cannabidiol and cannabidiol-dimethylheptyl at the type 1 and type 2 cannabinoid receptors.

We sought to understand why (-)-cannabidiol (CBD) and (-)-cannabidiol-dimethylheptyl (CBD-DMH) exhibit distinct pharmacology, despite near identical structures. HEK293A cells expressing either human type 1 cannabinoid (CB1 ) receptors or CB2 receptors were treated with CBD or CBD-DMH with or without the CB1 and CB2 receptor agonist CP55,940, CB1 receptor allosteric modulator Org27569 or CB2 receptor inverse agonist SR144528. Ligand binding, cAMP levels and βarrestin1 recruitment were measured. CBD and CBD-DMH binding was simulated with models of human CB1 or CB2 receptors, based on the recently published crystal structures of agonist-bound (5XRA) or antagonist-bound (5TGZ) human CB1 receptors. At CB1 receptors, CBD was a negative allosteric modulator (NAM), and CBD-DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist-bound model of CB1 receptors (5TGZ) but shared a binding site with CP55,940 in the agonist-bound model of CB1 receptors (5XRA). The binding site for CBD-DMH in the CB1 receptor models overlapped with CP55,940 and Org27569. At CB2 receptors, CBD was a partial agonist, and CBD-DMH was a positive allosteric modulator of cAMP modulation but a NAM of βarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB2 receptor models that was separate from CBD-DMH. The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB1 and CB2 receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB1 and CB2 receptors. This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.

Differential Pharmacology and Binding of mGlu2 Receptor Allosteric Modulators.

Allosteric modulation of metabotropic glutamate receptor 2 (mGlu2) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu2 allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu2 allosteric modulators, we developed and characterized a novel mGlu2 positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu2 PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu2, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu2 PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu2 allosteric site labeled with the novel mGlu2 PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu2 allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).

Mechanism of Allosteric Modulator Binding to the Adenosine A1 Receptor

The adenosine A1 receptor (A1AR) is a key G-protein-coupled receptor (GPCR) that has served as a therapeutic target for treating cardiovascular and neurological diseases. Activation of the A1AR is able to protect the myocardium against ischemia-reperfusion injury and reduce neuropathic pain. In contrast to traditional agonists that often cause off-target side effects, positive allosteric modulators (PAMs) that increase the receptor responsiveness to endogenous adenosine in local regions are promising selective drug candidates of the A1AR. However, the binding modes of PAMs in the A1AR and mechanism of the receptor allosteric modulation remain unclear. Here, all-atom computer simulations using the robust Gaussian accelerated molecular dynamics (GaMD) method are performed to capture spontaneous binding of prototypical PAMs to the A1AR. The GaMD simulations allow free energy calculations to identify low-energy binding modes of the PAMs in the receptor. The predicted binding modes are consistent with previous mutation and binding assay experiments. These results provide important insights into the mechanism of A1AR allostery at an atomistic level. The work will be also useful for future computer-aided drug design efforts targeting the A1AR and other GPCRs.

Species differences and mechanism of action of A3 adenosine receptor allosteric modulators.

Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5'-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide as well as adenosine >2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

Selective Serotonin 5-HT2A Receptor Antagonists and Inverse Agonists Specifically Promote Slow Wave Sleep (Stage N3) in Man

Several drug classes are widely prescribed when difficulties with sleep induction or sleep maintenance occur, as is the case in patients with an insomnia disorder. These include the benzodiazepine and non-benzodiazepine receptor allosteric modulators, the melanin receptor agonist ramelteon, low-dose doxepin, and the orexin receptor antagonist suvorexant. Benzodiazepines are often less than satisfactory, since they are known to produce reductions in both N3 sleep and rapid-eye movement (REM) sleep. Similarly, low-dose doxepin has been associated with a reduction in REM sleep. Initially, it was shown that the non-selective serotonin 5-HT2A/2C receptor antagonists’ ritanserin, ketanserin, seganserin, and ICI-169,369 increase N3 sleep in subjects with normal sleep. Ritanserin produced also an increase of N3 sleep in poor sleepers, patients with a chronic insomnia disorder, and psychiatric patients with a generalized anxiety disorder or a mood disorder. More recent evidence indicates that the selective 5-HT2A receptor antagonist volinanserin and the 5-HT2A receptor inverse agonists’ nelotanserin and pimavanserin significantly increase N3 sleep in subjects with normal sleep. Nelotanserin was also shown to augment N3 sleep in patients with a chronic insomnia disorder. N2 sleep tended to decrease in most of these studies, while REM sleep showed no significant changes. The present review which summarizes these findings is the basis for a proposal for a new therapeutic strategy. It is proposed that the co-administration of a selective 5-HT2A receptor antagonist or inverse agonist along with a hypnotic drug could be a valid clinical strategy for normalizing sleep induction and maintenance and for promoting N3 sleep in patients with an insomnia disorder.

Neuroactive Steroids and Affective Symptoms in Women Across the Weight Spectrum

3α-5α-Tetrahydroprogesterone, a progesterone metabolite also known as allopregnanolone, and 5α-androstane-3α,17β-diol, a testosterone metabolite also known as 3α-androstanediol, are neuroactive steroids and positive GABAA receptor allosteric modulators. Both anorexia nervosa (AN) and obesity are complicated by affective comorbidities and hypothalamic-pituitary-gonadal dysregulation. However, it is not known whether neuroactive steroid levels are abnormal at the extremes of the weight spectrum. We hypothesized that serum allopregnanolone and 3α-androstanediol levels would be decreased in AN compared with healthy controls (HC) and negatively associated with affective symptoms throughout the weight spectrum, independent of body mass index (BMI). Thirty-six women were 1 : 1 age-matched across three groups: AN, HC, and overweight/obese (OW/OB). AN were amenorrheic; HC and OW/OB were studied in the follicular phase. Fasting serum neuroactive steroids were measured by gas chromatography/mass spectrometry. Mean Hamilton depression and anxiety scores were highest in AN (p<0.0001). Mean serum allopregnanolone was lower in AN and OW/OB than HC (AN 95.3±56.4 vs OW/OB 73.8±31.3 vs HC 199.5±167.8 pg/ml, p=0.01), despite comparable mean serum progesterone. Allopregnanolone levels, but not progesterone levels, were negatively associated with depression and anxiety symptom severity, independent of BMI. Serum 3α-androstanediol levels did not differ among groups and were not associated with depression or anxiety scores, despite a significant negative association between free testosterone levels and both anxiety and depression severity. In conclusion, women at both extremes of the weight spectrum have low mean serum allopregnanolone, which is associated with increased depression and anxiety severity, independent of BMI. Neuroactive steroids such as allopregnanolone may be potential therapeutic targets for depression and anxiety in traditionally treatment-resistant groups, including AN.

The great divide: Separation between in vitro and in vivo effects of PSNCBAM-based CB1 receptor allosteric modulators

While allosteric modulators of the cannabinoid type-1 receptor (CB1) continue to be developed and characterized, the gap between the in vitro and in vivo data is widening, raising questions regarding translatability of their effects and biological relevance. Among the CB1 allosteric modulators, PSNCBAM-1 has received little attention regarding its effects in vivo. Recently, pregnenolone was reported to act as an allosteric modulator of CB1, blocking THC's effects in vitro and in vivo, highlighting the potential of CB1 allosteric modulators for treatment of cannabis intoxication. We investigated the pharmacological effects of PSNCBAM-1 and two structural analogs, RTICBM-15 and -28, as well as pregnenolone, in both signaling and behavioral assays including [35S]GTPγS binding, the cannabinoid tetrad and drug discrimination. While the CB1 allosteric modulator PSNCBAM-1 attenuated THC-induced anti-nociception and its structural analog RTICBM-28 reduced THC's potency in drug discrimination, most cannabinoid effects in mice were unaffected. In contrast to the mouse studies, PSNCBAM-1 and analogs insurmountably antagonized CP55,940- and THC-stimulated [35S]GTPγS binding and exhibited negative binding cooperativity with [3H]SR141716 with similar apparent affinities. Notably, RTICBM-28, which contains a cyano substitution at the 4-chlorophenyl position of PSNCBAM-1, exhibited enhanced binding cooperativity with CP55,940. In contrast to previous findings, pregnenolone did not block THC's effects in drug discrimination or [35S]GTPγS. These data further highlight the difficulty in translating pharmacological effects of CB1 allosteric modulators in vivo but confirm the established pharmacology of PSNCBAM-1 and analogs in molecular assays of CB1 receptor function.

Elucidation of the neural circuits activated by a GABAB receptor positive modulator: Relevance to anxiety

Although there is much evidence for a role of GABAB receptors in the pathophysiology of anxiety, the underlying neuronal mechanisms are largely unclear. The GABAB receptor allosteric positive modulator, GS39783, exerts anxiolytic effects without interfering with GABAB-mediated modulation of body temperature, cognitive performance and locomotor activity thus offering advantages over GABAB receptor agonists. However, the precise neural circuits underlying the anxiolytic effects of GS39783 are unknown. The aim of the present study was to identify brain structures and associated neuronal circuits that are modulated by GS39783 under either basal or mild stress conditions. To this end, the expression pattern of c-Fos, a marker of neuronal activation, was examined in mice acutely treated with GS39783 under basal conditions or following a mild anxiogenic challenge induced by exposure to the Open Arm (OA) of an Elevated Plus Maze.OA exposure enhanced c-Fos expression in vehicle-treated animals in several brain regions, including the medial prefrontal cortex, lateral septum, amygdala, hippocampus, paraventricular nucleus of the hypothalamus and the periaqueductal gray (PAG). Under basal conditions, GS39783 increased c-Fos in a restricted panel of areas notably amygdala nuclei, cortical areas and PAG subregions, while it inhibited c-Fos expression in the dorsal raphe nucleus (DRN). Under stress conditions, GS39783 reversed OA-induced c-Fos expression in the granular cell layer of the dentate gyrus, no longer increased c-Fos expression in the amygdala nor reduced c-Fos expression in the DRN. These specific patterns of neural activation by GS39783 might explain the neurobiological correlates implicated in GABAB-mediated anti-anxiety effects.This article is part of the “Special Issue Dedicated to Norman G. Bowery”.

Molecular Switches of Allosteric Modulation of the Metabotropic Glutamate 2 Receptor

Metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) crucial for CNS function and important drug discovery targets. Glutamate triggers receptor activation from an extracellular domain binding site while allosteric modulators bind in the seven-transmembrane domain. Little is known about how allosteric modulators produce their functional effects at the molecular level. Here we address this topic with combined experimental and computational approaches and reveal that mGlu receptor allosteric modulators interact with the homologous "trigger switch" and "transmission switch" amino acids as seen in class A GPCRs, in short, the characteristic hallmarks of class A agonist activation translate to the mGlu allosteric modulator.The proposed "trigger switch" for the mGlu2 involves the side chains of F6433.36a.40c, N7355.47a.47c, and W7736.48a.50c, whereas the "transmission switch" involves the Y6473.40a.44c, L7385.50a.50c, and T7696.44a.46c amino acids. The work has wide impact onunderstanding mGlu GPCR function and for future allosteric modulator drugs.

GABAB receptor allosteric modulators exhibit pathway-dependent and species-selective activity.

Positive modulation of the GABAB receptor (GABABR) represents a potentially useful therapeutic approach for the treatment of nicotine addiction. The positive allosteric modulators (PAMs) of GABABR GS39783 and BHF177 enhance GABA‐stimulated [35S]GTP γS‐binding, and have shown efficacy in a rodent nicotine self‐administration procedure reflecting aspects of nicotine dependence. Interestingly, the structural related analog, NVP998, had no effect on nicotine self‐administration in rats despite demonstrating similar pharmacokinetic properties. Extensive in vitro characterization of GS39783, BHF177, and NVP998 activity on GABABR‐regulated signaling events, including modulation of cAMP, intracellular calcium levels, and ERK activation, revealed that these structurally related molecules display distinct pathway‐specific signaling activities that correlate with the dissimilarities observed in rodent models and may be predictive of in vivo efficacy. Furthermore, these GABABR allosteric modulators exhibit species‐dependent activity. Collectively, these data will be useful in guiding the development of GABABR allosteric modulators that display optimal in vivo efficacy in a preclinical model of nicotine dependence, and will identify those that have the potential to lead to novel antismoking therapies.

Ligand-guided homology modelling of the GABAB2 subunit of the GABAB receptor.

γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, and disturbances in the GABAergic system have been implicated in numerous neurological and neuropsychiatric diseases. The GABAB receptor is a heterodimeric class C G protein-coupled receptor (GPCR) consisting of GABAB1a/b and GABAB2 subunits. Two GABAB receptor ligand binding sites have been described, namely the orthosteric GABA binding site located in the extracellular GABAB1 Venus fly trap domain and the allosteric binding site found in the GABAB2 transmembrane domain. To date, the only experimentally solved three-dimensional structures of the GABAB receptor are of the Venus fly trap domain. GABAB receptor allosteric modulators, however, show great therapeutic potential, and elucidating the structure of the GABAB2 transmembrane domain may lead to development of novel drugs and increased understanding of the allosteric mechanism of action. Despite the lack of x-ray crystal structures of the GABAB2 transmembrane domain, multiple crystal structures belonging to other classes of GPCRs than class A have been released within the last years. More closely related template structures are now available for homology modelling of the GABAB receptor. Here, multiple homology models of the GABAB2 subunit of the GABAB receptor have been constructed using templates from class A, B and C GPCRs, and docking of five clusters of positive allosteric modulators and decoys has been undertaken to select models that enrich the active compounds. Using this ligand-guided approach, eight GABAB2 homology models have been chosen as possible structural representatives of the transmembrane domain of the GABAB2 subunit. To the best of our knowledge, the present study is the first to describe homology modelling of the transmembrane domain of the GABAB2 subunit and the docking of positive allosteric modulators in the receptor.