G-protein Activation Research Articles

Preclinical Evaluation of the Effects of Trazpiroben (TAK-906), a Novel, Potent Dopamine D2/D3 Receptor Antagonist for the Management of Gastroparesis.

Current therapies for gastroparesis metoclopramide and domperidone carry risks of extrapyramidal symptoms and life-threatening cardiac arrhythmias. Trazpiroben, a novel, potent dopamine D2/D3 receptor antagonist, has low brain permeation and very low affinity for human ether-à-go-go-related gene (hERG) channel inhibition, potentially improving on safety profiles of existing therapies. Trazpiroben demonstrated the following receptor affinities: high for D2 and D3, moderate for D4, and minimal for D1 and D5 It demonstrated moderate affinity for adrenergic α 1B (α 1B) and 5-hydroxytryptamine (5HT) 2A receptors and low potential for off-target adverse events (AEs). Trazpiroben potently inhibited dopamine-activated D2L receptor activation of cognate G-proteins in human embryonic kidney 293 cell membranes and was a neutral D2L receptor antagonist. In vivo, trazpiroben dose-dependently increased prolactin release in orally dosed rat (0.1-1 mg/kg). Additionally, multiple oral doses in the rat (100 mg/kg) and dog (50 mg/kg) for 3 days produced robust plasma exposures and prolactin increases in both species. Trazpiroben inhibited retching/vomiting in the dog with apomorphine-induced emesis with a potency (0.1-1 mg/kg) like that of trazpiroben-mediated prolactin increases in rat. Oral trazpiroben (1, 10, and 30 mg/kg) did not affect rat rotarod performance, suggesting low brain penetration. Trazpiroben concentrations were low in cerebrospinal fluid versus plasma after multiple oral doses for 4 days in rat and dog. Trazpiroben weakly inhibited the hERG channel current (concentration causing half-maximal inhibition of control-specific binding of 15.6 µM), indicating little potential for disrupting cardiac rhythm. Overall, trazpiroben is a potent D2/D3 receptor antagonist designed to avoid the serious potential AEs associated with current gastroparesis therapies. SIGNIFICANCE STATEMENT: Trazpiroben is a novel, potent dopamine D2/D3 selective receptor antagonist designed to avoid adverse effects associated with the current pharmacological therapies metoclopramide and domperidone. Preclinical studies have demonstrated low brain penetration and weak affinity for the hERG channel, indicating that trazpiroben is not expected to be associated with central nervous system or cardiovascular safety issues. With these pharmacological properties, trazpiroben may represent a viable new treatment option for gastroparesis because of a potentially improved safety profile relative to existing therapies.

Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom.

Dry leaves of kratom (mitragyna speciosa) are anecdotally consumed as pain relievers and antidotes against opioid withdrawal and alcohol use disorders. There are at least 54 alkaloids in kratom; however, investigations to date have focused around mitragynine, 7-hydroxy mitragynine (7OH), and mitragynine pseudoindoxyl (MP). Herein, we probe a few minor indole and oxindole based alkaloids, reporting the receptor affinity, G-protein activity, and βarrestin-2 signaling of corynantheidine, corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at mouse and human opioid receptors. We identify corynantheidine as a mu opioid receptor (MOR) partial agonist, whereas its oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial agonist, while isopaynantheine was a KOR agonist which showed reduced βarrestin-2 recruitment. Corynantheidine, corynoxine, and mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally. Isopaynantheine on the other hand was identified as the first kratom derived KOR agonist in vivo. While these minor alkaloids are unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversification as well as distinct efficacy and signaling profiles with which to probe opioid actions in vivo.

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia

BackgroundAlterations of dopamine D1 (D1R) and D2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects.MethodsG-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [35S]GTPγS-binding stimulation induced by increasing concentrations (10–10–10–3 M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration–response curves to NPA stimulation of [35S]GTPγS binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17).ResultsIn caudate, [35S]GTPγS-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [35S]GTPγS-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 μM) and control (EC50H = 7.24 nM; EC50L = 15.14 μM) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters.ConclusionsFeasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [35S]GTPγS-binding assays appears to be restricted to signalling through inhibitory Gi/o proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.

G-protein activation by a metabotropic glutamate receptor.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

Dual Sphingosine Kinase and Bcl-2 Inhibition Exhibits Synergistic Cell Death in Acute Myeloid Leukemia

Background:Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their ability to suppress the apoptotic potential of pro-apoptotic Bax and Bak. In response to cell stress, BH3-only proteins sequester Bcl-2 family members, promoting apoptosis. Small molecule inhibitors such as the Bcl-2 inhibitor Venetoclax have demonstrated poor single agent efficacy in AML. This poor efficacy is a result of AML cells being highly dependent on the pro-survival Bcl-2 family member Mcl-1. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. SK1 promotes oncogenic signalling through the production of sphingosine-1-phopshate and subsequent activation of G-protein coupled receptors. In addition, SK1 has been reported to modulate Mcl-1 expression in a number of haematological malignancies including AML. Thus we sought to investigate the efficacy of targeting SK1 to sensitise AML cells to Venetoclax.Results:To determine how SK1 inhibition influences Mcl-1 levels we utilised a short hairpin RNA (shRNA) approach targeting members of the BH3 only protein family (Bid, Bim, Bad, Puma and Noxa). Targeting of Noxa significantly attenuated the cytotoxic effects of the SK1 inhibitor, MP-A08. Immunoprecipitation of Mcl-1 in response to MP-A08 treatment revealed increased association of BH3 only protein, Noxa and ubiquitin-ligase FBW7 suggesting this triggers Mcl-1 degradation in agreeance with previous studies. No interaction was observed between Mcl-1 and Bim further supporting the importance of Noxa in MP-A08 induced Mcl-1 degradation.Canonical pathway analysis of MP-A08 treated cells has previously shown to enrich for genes associated with the unfolded protein response (UPR) (Powell et al. Blood 2017). Western blot analysis recapitulated these findings with activation of the PERK arm, typified by eIF2a phosphorylation and ATF4 upregulation. ATF4 has previously been shown to bind within the Noxa promoter and drive transcription in response to ER stress (Wang et al PNAS 2009). Maintaining global translation with eIF2B agonist, ISRIB blocked Noxa transcription in response to MP-A08 suggesting ATF4 drives Noxa mediated Mcl-1 degradation.Given the Noxa dependent Mcl-1 degradation in response to MP-A08 treatment, we sought to assess the efficacy of combined treatment with the Bcl-2 inhibitor, Venetoclax. Combinational therapies with sub-cytotoxic doses of MP-A08 and Venetoclax induced synergistic cell death in AML cell lines, primary patient AML blasts and leukemic stem and progenitor cells. Synergy could be observed as early as two hours, in agreeance with Noxa upregulation and binding Mcl-1, implicating this as key for synergistic cell death.Conclusions:We have demonstrated SK1 inhibition invokes a pro-apoptotic UPR response via ATF4 driven Noxa upregulation and Mcl-1 degradation. Nullification of the pro-survival function of Mcl-1 by Noxa renders AML cells vulnerable to Bcl-2 targeting strategies such as Venetoclax providing a novel therapeutic angle to enhance patient response to Venetoclax. Current work is underway assessing MP-A08 and Venetoclax using patient-derived xenograft models. DisclosuresNo relevant conflicts of interest to declare.

Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish

The Regulator of G protein signaling 4 (Rgs4) is a member of the RGS proteins superfamily that modulates the activity of G-protein coupled receptors. It is mainly expressed in the nervous system and is linked to several neuronal signaling pathways; however, its role in neural development in vivo remains inconclusive. Here, we generated and characterized a rgs4 loss of function model (MZrgs4) in zebrafish. MZrgs4 embryos showed motility defects and presented reduced head and eye sizes, reflecting defective motoneurons axon outgrowth and a significant decrease in the number of neurons in the central and peripheral nervous system. Forcing the expression of Rgs4 specifically within motoneurons rescued their early defective outgrowth in MZrgs4 embryos, indicating an autonomous role for Rgs4 in motoneurons. We also analyzed the role of Akt, Erk and mechanistic target of rapamycin (mTOR) signaling cascades and showed a requirement for these pathways in motoneurons axon outgrowth and neuronal development. Drawing on pharmacological and rescue experiments in MZrgs4, we provide evidence that Rgs4 facilitates signaling mediated by Akt, Erk and mTOR in order to drive axon outgrowth in motoneurons and regulate neuronal numbers.

Structures of human mGlu2 and mGlu7 homo- and heterodimers.

The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helixIV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish classC G-protein-coupled receptors from those in classesA and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.

Bile Acids Induce IQ Containing GTPase Activating Protein 1 to Alleviate Liver Injury

Bile acids (BA) are cholesterol metabolites synthesized in the liver. In addition to their role as detergents, bile acids can function as signaling molecules via the activation of nuclear and G-protein coupled receptors, FXR and TGR5. BA signaling is associated with inflammation, cell proliferation, as well as apoptosis in the liver. Excessive accumulation of bile acids in the liver (cholestasis) is observed in many diseased states. We found that the scaffold protein IQGAP1 was induced in mouse models of cholestasis and in patients. In vitro BA treatment of liver cell-line, HepG2 also induced IQGAP1 in a dose-dependent manner. To investigate the role of this IQGAP1 induction, we fed wild-type male mice 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 3days, 1week, and 2weeks. Serum bile acid and hepatic IQGAP1 induction increased concomitantly with time. We next treated Iqgap1-/- male mice similarly with DDC and found a 3-fold increase in serum bile acid levels compared to treated wild-type mice. Liver injury markers ALT, AST, and bilirubin were also increased. Although Iqgap1-/- mice had similar gene expression of key BA regulators, Fxr and Shp as wild-type mice, the alteration in expression of BA transporters and reduced cell-cell junction protein levels may contribute to the elevated BAs levels. We quantified gene expression of pro-inflammatory and proliferative mediators using qRT-PCR and examined the liver histology with immuno-staining. We found Iqgap1-/- livers expressed increased pro-inflammatory mediators and proliferative genes like Il-6 and cyclin D1 along with MAPK signaling targets, Fos and Egr-1. To further examine how IQGAP1 functions as a mediator of bile acid signaling, we have generated IQGAP1 domain deleted constructs and are examining their protein-protein interactions with Mass Spec. Based on our results, IQGAP1 induction subsequent to accumulation of BAs is protective against injury in the liver.

Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis

Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.

Targeted Deletion of Sphingosine‐1‐Phosphate Receptor Type 1 (S1PR1) Reveals a Predominant Functional Localization of S1PR1 in Astrocytes in the Spinal Cord

Sphingosine-1-phosphate (S1P) is a tightly regulated bioactive lysolipid that acts as the endogenous ligand for G-protein coupled S1P receptors (S1PR) types 1-5. The S1PR system has been targeted for immune modulation, but research indicates its potential as a target for pain relief. FTY720 is a S1PR immunomodulatory prodrug that is phosphorylated in vivo to its active form, FTY720-phosphate (FTY-P), which activates S1PR 1, 3, 4, and 5. FTY720 is currently FDA approved for the treatment of Multiple Sclerosis, but preclinical evidence has indicated both S1P and FTY720 produce antinociception in models of acute and neuropathic pain. Results with S1PR1 selective agonists and antagonists suggest S1PR ligands most likely act to alleviate pain specifically through S1PR1. Here we used conditional gene deletion to determine the cell type(s) mediating S1P-stimulated G-protein activation in the spinal cord, measured using agonist-stimulated [35S]GTPγS binding. Deletion of S1PR1 in S1PR1-floxed mice expressing cre recombinase under control of the nestin promoter, which deletes S1PR1 from neurons, astrocytes, and oligodendroglia, showed a profound decrease in G-protein activation stimulated by S1P (by ~85%) or the S1PR1 agonist SEW2871 (>95%) in the spinal cord, suggesting the majority of S1PR activity in spinal cord is mediated by S1PR1. We next explored the extent to which S1PR1-mediated G-protein activation in the lumbar spinal cord of male and female mice is specifically localized in astrocytes. The lumbar spinal cord is thought to be a major point of transduction of pain signals from nerve-injury models of neuropathic pain in rodents. S1PR1-floxed mice were crossed with cre recombinase under the control of a glial fibrillary acidic protein (GFAP)-promotor. In this model, there was a ~75% loss in G-protein activation by S1P in the astrocyte specific conditional knock out (cKO) compared to the control mice, whereas there was a near complete (>95%) loss of SEW2871-stimulated G-protein activation in these cKO mice. No sex differences in S1PR-stimulated G-protein activation or the effect of S1PR1 deletion was observed. This loss of S1PR1-stimulated G-protein activation in the lumbar spinal cord upon astrocyte specific deletion of S1PR1 suggests S1PR1 activity is mainly localized in astrocytes within the lumbar spinal cord. These results aid in the understanding of S1PR1 localization and supports recently reported evidence for a role for S1PR1 in astrocytes in FTY720-responsive neuropathic pain models. Studies are also ongoing to determine the functional localization of S1PR1 in astrocytes in brain regions associated with pain perception and regulation.

The impact of advanced age and sex on Mu Opioid Receptor signaling in the midbrain periaqueductal gray: implications on analgesia

Chronic pain is under-managed in individuals over 65 years of age due to a dearth of knowledge regarding the impact of age on opioid efficacy in the elderly. We have previously shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar administration of Complete Freund's adjuvant (CFA)), such that morphine potency is highest in adult male rats (2mos), with EC50 values 2-fold higher in aged males (18mos) and females regardless of age. Age-induced reductions in morphine potency were accompanied by reduced mu opioid receptor (MOR) expression in the ventrolateral periaqueductal gray (vlPAG), a CNS region critical in pain modulation. The present studies further explore the impact of age on opioid signaling within the PAG. MOR affinity, availability, and G-protein activation were assessed using radioligand binding assays and GTPγS assays in vlPAG tissue from adult and aged, male and female rats collected 72h following CFA administration. Regulation of opioid induced G-protein signaling was assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2. We find that aged males and females (adult and aged) exhibit reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males, suggesting age- and sex- differences in MOR machinery drive reduced opioid potency. RNAscope revealed increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subject to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR agonist binding and opioid induced G-protein activation, along with the observed increase in vlPAG RGS expression have significant implications in pain management in the aged population. Our novel findings elucidate several mechanisms mediating reduced morphine potency in aged animals, and identify potential targets to improve pain management in the elderly. R01DA041529-04.

NanoLuc (NLuc) complementation assay elucidates role of specific G‐proteins in GPR88 signaling

GPR88 is an orphan G protein-coupled receptor (GPCR) expressed at high levels in the striatum and is a target for the development of therapeutics to treat multiple neuropsychiatric disorders. While a few small-molecule synthetic agonists have been reported, no synthetic antagonists or endogenous ligands have been identified. Typical 2nd messenger, cell-based assays for agonist and antagonist identification and signaling pathway elucidation rely on complex downstream events, with interpretation of results complicated from effects of transfected receptor overexpression and interference from endogenous receptors and proteins. The NLuc complementation assay, based on an engineered luciferase from the deep-sea shrimp, Oplophorus gracilirostris, can monitor receptor-G protein interaction in live cells and profile compounds according to the G proteins they activate. The synthetic agonists, 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid], and 2-OMPP [(2R)-N-(2-hydroxy-1-(4-((2-methylpentyl)oxy)phenyl)ethyl)-2-phenylpropanamide] are suspected to activate GPR88 via a Gαi-coupled signaling pathway. In this study we show the activation of GPR88 by 2-PCCA and 2-OMPP inhibited isoproterenol induced cAMP production in transiently transfected GPR88 HEK-293T cells with relative EC50 values of 250 nM and 1.0 μM, respectively, consistent with prior reports. The EC50 value for PCCA in HEK-293T/GPR88 transients is five times that in RH-7777 cells with COS-7 cells showing no cAMP response. These results highlight the importance of the cellular background in engineered systems. Additionally, the NLuc complementation assay shows PCCA receptor binding triggers activation of several G-proteins including Gαi1(EC50= 760 nM), Gαi0 (EC50=3.3μM) and GαS (EC50=4.5 nM) indicating the effects of varying G-protein expression in different cell lines. In combination with our computational endeavors, 2nd messenger and the NLuc complementation assays are valuable tools in our research as we develop high-throughput screening processes and probe signaling pathways in search of GPCR based therapeutics.

CARD9 Mediates Glucose‐Stimulated Insulin Secretion in Pancreatic‐β Cells

Background- Glucose stimulated insulin secretion (GSIS) from the pancreatic beta cell involves a variety of metabolic events including generation of second messenger molecules, activation of small G-proteins culminating in vesicular transport to the plasma membrane for their fusion and secretion of insulin. Caspase recruitment domain containing protein 9 (CARD9) is a major signal transducer in innate and adaptive immune responses. CARD9 has also been implicated in the onset of metabolic diseases including insulin resistance. However, potential roles of this scaffolding protein in GSIS remains unknown. Therefore, we undertook the current investigations to determine its expression and assess the roles of CARD9 in glucose- induced Rac1 activation and insulin secretion in pancreatic beta cells. Methods and Materials- Western blotting was used to demonstrate protein expression. CARD9 expression was suppressed using Horizon/ Dharmacon siRNA and transfection reagents. Membrane and cytosolic fractions were isolated using Membrane Extraction Kit from ThermoFisher Scientific. Insulin secretion was quantified by using ALPCO Rat insulin ELISA kit. The degree of activation of Rac1 was determined by pull-down assay from Cytoskeleton Inc. Results - CARD9 was expressed in a variety of insulin-secreting cells, including human islets, rat islets and clonal INS-1 832/13 cells. Subcellular fractionation studies revealed that CARD9 is predominantly cytosolic (~75%) in distribution in INS1- 832/13 cells. Transfection of INS-1 832/13 cells with CARD9 siRNA (CARD9-si), resulted in a 70% reduction of CARD9 expression. Under these conditions, GSIS was markedly suppressed (~ 50%) in CARD9-depeleted INS-1 832/13 cells. Interestingly, glucose-induced activation of Rac1 (~3- fold), a small G-protein, which is a requisite for GSIS to occur, was unaffected in siRNA-CARD9 transfected sells, suggesting that CARD9-mediates GSIS in a Rac1-independent fashion. Furthermore, insulin secretion elicited by a membrane-depolarizing concentration of KCl (60 mM) remained resistant to CARD9 depletion in INS-1 832/13 cells. These findings imply that CARD9 might regulate insulin secretion in a calcium-independent signaling module. Conclusions- We provide the first evidence to indicate that CARD9 plays regulatory roles in GSIS via Rac1- and calcium-independent signaling mechanisms. Additional studies are underway to further assess the effector proteins that might be involved in CARD9-mediated GSIS.

SARS‐CoV‐2 May Hijack GPCR Signaling Pathways to Compromise Lung Ion and Fluid Transport

The tropism of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus responsible for the ongoing coronavirus disease 2019 (COVID‐19) pandemic, towards the host cells is determined, at least in part, by the expression and distribution of its cell surface receptor, angiotensin‐converting enzyme 2 (ACE2). The virus further exploits the host cellular machinery to gain access into the cells; its spike protein is cleaved by a host cell surface transmembrane serine protease 2 (TMPRSS2) shortly after binding ACE2 followed by its proteolytic activation at a furin cleavage site. The virus primarily targets the epithelium of the respiratory tract which is covered by a tightly regulated airway surface liquid (ASL) layer that serves as a primary defense mechanism against respiratory pathogens. The volume and viscosity of this fluid layer is regulated and maintained by a coordinated function of different transport pathways in the respiratory epithelium. We argue that SARS‐CoV‐2 may potentially alter evolutionary conserved second‐messenger signaling cascades via activation of G‐protein coupled receptors (GPCRs) or by directly modulating G protein signaling. Such signaling may in turn adversely modulate transepithelial transport processes, especially those involving cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC), thereby shifting the delicate balance between anion secretion and sodium absorption that controls homeostasis of this fluid layer. As a result, activation of the secretory pathways including CFTR‐mediated Cl‐ transport may overwhelm the absorptive pathways such as ENaC‐dependent Na+ uptake and initiate a pathophysiological cascade leading to lung edema, one of the most serious and potentially deadly clinical manifestations of COVID‐19.

Major Metabolites of the Synthetic Cannabinoid 5F‐ADB Retain High Affinity and Full Efficacy at CB1 Receptors; Potential Mechanism Contributing to Enhanced Toxicity?

Synthetic cannabinoids (SCs) are a large group of abused psychoactive compounds that elicit numerous toxic effects not observed with marijuana, including death. SCs are abused worldwide and metabolites of these drugs are often detected as a means of drug identification. SC metabolites resulting from Phase I metabolism have been shown to retain affinity and activity at cannabinoid receptors. However, whether these active compounds contribute to toxicity of SCs is unknown. 5-Fluoro-ADB (5F-ADB), also known as 5-Fluoro-MDMB-PINACA, is a commonly abused third-generation synthetic cannabinoid agonist based on an indazole-structure. Significantly, over 40 recent fatalities have been attributed to 5F-ADB overdose. A potential mechanism underlying 5F-ADB-induced mortality is drug-related dysrhythmia resulting in sudden death. Analysis of postmortem central and peripheral blood samples have revealed abnormally high concentrations of an ester-hydrolysis Phase I metabolite of 5F-ADB, identified as metabolite 7 (5F-ADB-M7). We propose that high concentrations of potent and efficacious active metabolites, such as 5F-ADB-M7 and others, accumulate with the parent drug 5F-ADB and produce additive and/or synergistic effects that possibly contribute to fatal intoxication following overdose. As a first step to test this hypothesis, in vitro studies were conducted to compare the affinity and activity of tetrahydrocannabinol (THC; the primary psychoactive component in marijuana), 5F-ADB, 5F-ADB-M7 and a second major metabolite, 5F-ADB-M2 at cannabinoid type-1 (CB1) receptors. Competition receptor binding studies, using mouse brain membranes, showed that all four cannabinoids bound with high affinity to CB1 receptors with a rank order of affinity (Ki values) of 5F-ADB > 5F-ADB-M2 > THC > 5F-ADB-M7. Furthermore, functional experiments examining modulation of G-protein activity demonstrated that 5F-ADB and both Phase I metabolites acted as full agonists at CB1 receptors, relative to partial agonist effects produced by THC. Subsequently, in vivo studies were conducted utilizing the cannabinoid tetrad in mice to compare the effects of THC with those of 5F-ADB-M2. Both 5F-ADB-M2 and THC produced CB1-mediated dose-dependent hypothermia, analgesia, and locomotor suppression, within 30 minutes of injection; however, catalepsy was not elicited by the metabolite. Consistent with higher CB1 affinity determined from in vitro studies, 5F-ADB-M2 produced tetrad effects with higher potency compared to THC. Future studies are planned to examine in vivo effects of 5F-ADB-M7 compared to THC. If 5F-ADB metabolites exhibit in vivo activity similar to that predicted from the in vitro studies presented here, this suggests that the enhanced, often fatal, effects of 5F-ADB may be due to an accumulation of the parent and active, high affinity metabolites acting at CB1 receptors.

Natural and Synthetic Cannabinoids Reduce Cell Viability of Ewing Sarcoma TC‐71 Cells Potentially via Non‐canonical CB receptors

Ewing Sarcoma (EWS) is the second most common bone tumor in children, characterized by rapid tumor growth and extensive bone destruction. Treatment for EWS is multimodal: surgery, radiation, and chemotherapy. However, these approaches are effective in only 70% of the patients with localized disease and thus new drugs are needed. WIN-55,212-2 (WIN-2), a synthetic cannabinoid in the aminoalkyindole (AI) class, has been shown to slow tumor growth in several types of cancer and human Kaposi sarcoma via cannabinoid (CB) and non-CB mechanisms. Therefore, we hypothesize that WIN-2 and other synthetic CBs will exhibit anti-proliferative effects in EWS TC-71 cells via CB receptors. Competitive binding screens using TC-71 membranes showed that binding of the nonselective CB1/CB2 radiolabeled agonist [3H]CP-55,940 could not be displaced by selective CB1 or CB2 ligands, or even non-radioactive CP-55,940 itself. However, binding of a second nonselective CB1/CB2 radiolabeled agonist [3H]WIN-2 could be displaced with a CB1 but not CB2 antagonist, and non-radioactive WIN-2 itself. This suggests that TC-71 cells express a novel non-canonical CB binding site (WIN-2 site). Subsequent homologous receptor binding studies with [3H]WIN-2 showed that TC-71 cells do indeed express a WIN-2 site at a density (Bmax) of 3.7 +/- 1.6 pmole/mg, to which WIN-2 binds with an affinity (Ki) of 4.5 +/- 0.6 nM. Next, thirty-eight different synthetic CBs were screened for potential binding to WIN-2 receptors in TC-71 cells. Only four compounds exhibited a high affinity for the WIN-2 receptor in the low to the mid-nanomolar range. Interestingly, four other compounds increased [3H]WIN-2 binding, indicating that these ligands might act as positive allosteric modulators of the WIN-2 site. When compounds with the highest affinity were examined in G-protein activation assays for functional activity, two of them reduced [35S]GTPgS binding below basal levels indicating that they may act as inverse agonists at the novel WIN-2 site. Most importantly, WIN-2 and all four compounds with high affinity for the WIN-2 site produced a significant time-dependent reduction in viability of EWS TC-71 cells as measured by trypan blue exclusion and lactate dehydrogenase (LDH) release. In conclusion, WIN-2 and other synthetic CBs reduce EWS cell viability potentially via a non-canonical CB binding site. These results suggest that the WIN-2 site might be a novel therapeutic target for drug development to treat EWS and perhaps other types of cancer as well.

Structural basis of GABAB receptor\u2013Gi protein coupling

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Disulfiram Abrogates Morphine Tolerance-A Possible Role of µ-Opioid Receptor-Related G-Protein Activation in the Striatum.

One of the key strategies for effective pain management involves delaying analgesic tolerance. Early clinical reports indicate an extraordinary effectiveness of off-label disulfiram—an agent designed for alcohol use disorder—in potentiating opioid analgesia and abrogation of tolerance. Our study aimed to determine whether sustained µ-opioid signaling upon disulfiram exposure contributes to these phenomena. Wistar rats were exposed to acute and chronic disulfiram and morphine cotreatment. Nociceptive thresholds were assessed with the mechanical Randal-Selitto and thermal tail-flick tests. µ-opioid receptor activation in brain structures important for pain processing was carried out with the [35S]GTPγS assay. The results suggest that disulfiram (12.5–50 mg/kg i.g.) augmented morphine antinociception and diminished morphine (25 mg/kg, i.g.) tolerance in a supraspinal, opioid-dependent manner. Disulfiram (25 mg/kg, i.g.) induced a transient enhancement of µ-opioid receptor activation in the periaqueductal gray matter (PAG), rostral ventromedial medulla (RVM), hypothalamus, prefrontal cortex and the dorsal striatum at day 1 of morphine treatment. Disulfiram rescued µ-opioid receptor signaling in the nucleus accumbens and caudate-putamen 14 days following morphine and disulfiram cotreatment. The results of this study suggest that striatal µ-opioid receptors may contribute to the abolition of morphine tolerance following concomitant treatment with disulfiram.

Functional Selectivity of a Biased Cannabinoid-1 Receptor (CB1R) Antagonist.

Seven-transmembrane receptors signal via G-protein- and β-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced β-arrestin-2 (βArr2) recruitment over G-protein activation. In obese wild-type and βArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy. By contrast, the unbiased global CB1R antagonist rimonabant elicits anxiety in both strains, indicating no βArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in βArr2-KO mice. In C2C12 myoblasts, CB1R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, βArr2 knockdown or overexpression of CB1R-interacting protein. MRI-1891, but not rimonabant, interacts with nonpolar residues on the N-terminal loop, including F108, and on transmembrane helix-1, including S123, a combination that facilitates βArr2 bias. Thus, CB1R promotes muscle insulin resistance via βArr2 signaling, selectively mitigated by a biased CB1R antagonist at reduced risk of central nervous system (CNS) side effects.