Inhibition Of cAMP Accumulation Research Articles

In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled β2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein‐coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25–81) and a C‐terminal peptide fragment GPR15L(71–81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3′,5′‐monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs, Gq/11 and G12/13 is unclear. Here, GPR15L(25–81) and GPR15L(71–81) are used as pharmacological tool compounds to delineate the GPR15 receptor‐mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1, Gi2, Gi3, GoA, GoB and Gz). The Emax and activation rates of Gi1, Gi2, Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

MiR-139/PDE2A-Notch1 feedback circuit represses stemness of gliomas by inhibiting Wnt/β-catenin signaling.

Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation.Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically.Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and β-catenin, which induced Wnt/β-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/β-catenin signaling by inhibiting cAMP accumulation and GSK-3β phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression.Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa

The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent effects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by ∆9-THC is known to bring about the ‘high’ associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating effects that are potentially therapeutic. CB2R activation by certain cannabinoids has been associated with anti-inflammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifically, ∆9-THC, ∆9-tetrahydrocannabinolic acid (∆9-THCa), ∆9-tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their affinity to receptors, ability to inhibit cAMP accumulation, βarrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic effects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis-derived molecules.

The CB1 cannabinoid receptor‐mediated inhibition of cAMP accumulation in neuroblastoma cells: role of different Gi/o protein subtypes

The CB1 cannabinoid receptor (CB1R) is a G protein coupled receptor with robust expression in the nervous system. CB1R signaling is mediated by pertussis toxin (PTX)‐sensitive Gi /o proteins, and leads to inhibition of adenylyl cyclase. Because of the importance of the cyclic AMP pathway in differentiated neuronal functions, the investigation of G protein subtypes involved in the inhibition of adenylyl cyclase activity is of particular interest. The aim of the present study is to investigate the effect of individual Gi/o proteins on CB1R‐promoted inhibition of forskolin‐ stimulated cAMP accumulation in N18TG2 neuroblastoma cells. We utilized [3H]cAMP binding competition assay and cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) as previously described ( Blume et al 2015 and Eldeeb et al 2017).The CB1R agonist, CP55940 (100 nM) inhibited forskolin‐stimulated cAMP accumulation to 34±8% of forskolin alone. Pretreatment with 100 ng/ml of PTX for 20 hrs attenuated CP55940 induced inhibition to 85±24% of forskolin control. In N18TG2 cells that were stably expressing either PTX‐resistant Gαo, Gαi1, Gαi2 or Gαi3 proteins, treatment of PTX‐pretreated with CP55940 (100 nM) inhibited cAMP accumulation to 38±3% Gαo, 53±4 Gαi1, 76±24 Gαi2 and 28±15% Gαi3 of forsklin alone. Using SPA, CP55940 (100 nM) stimulated [35S]GTPγS binding mediated by Gi/o protein subtypes, Gαo, Gαi1, Gαi2 and Gαi3 in N18TG2 Gαi3 PTX‐resistant cell membranes. In contrast, in PTX‐pretreated membranes CP55940 was able to stimulate [35S] GTPγS binding mediated by Gαi3 only. In conclusion, these data show that Gi/o proteins contribute differently to CB1R‐promoted inhibition of cAMP in N18TG2 cells, with greater contribution by Gαi3. This is consistent with our earlier findings of the high level of Gαi3 expression and its contribution toward total G protein activation in N18TG2 membranes ( Eldeeb et al 2017). The contribution of different G protein subtypes to CB1R signaling is necessary to consider as we evaluate CB1R functions in the nervous system.Support or Funding InformationNIH grants R01‐DA042157, P50‐DA006634

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

BackgroundIncreasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PurposeThe purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. MethodsA gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. ResultsCelastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. ConclusionTaken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Prolonged Sumatriptan Treatment Differentially Alters 5HT1 Receptor‐mediated Signaling In Rat Trigeminal Ganglion

Medication Overuse Headache (MOH) is caused by repeated use of analgesics, such as triptans, which are used to treat migraines. Triptans are agonists at serotonin (5‐HT) type 1 receptors (5‐HT1B, 5‐HT1D, and 5‐HT1F), which are G protein‐coupled receptors known to couple to both Gi protein‐dependent (e.g., inhibition of cAMP production) and Gi protein‐independent (e.g., extracellular signal regulated kinase, ERK) signaling. Activation of 5HT1 receptors by triptans produce relief of migraine pain largely by inhibiting peripheral pain sensing neurons (nociceptors) of the trigeminal ganglia. However, the mechanism by which repeated/prolonged use of triptans causes MOH is unknown. We hypothesize that repeated or prolonged triptan exposure alters 5‐HT1 receptor‐mediated signaling from inhibitory to excitatory as a result of differential desensitization and/or signal switching in nociceptors. In this study, we examined the effects of prolonged treatment with the prototypical triptan, sumatriptan, on 5‐HT1 receptor‐mediated inhibition of PGE2‐stimulated cAMP accumulation and activation of ERK in rat trigeminal ganglia (TG) primary cultures.Incubation of TG cultures with the 5‐HT1 receptor agonists, 5‐carboxamidotryptamine (5‐CT) or sumatriptan (SUMA) produced a dose dependent inhibition of PGE2‐stimulated cAMP accumulation with an EC50 of 7.7 nM and 6.8 nM and a maximal inhibition of 59% and 45% for 5‐CT and SUMA, respectively. Pretreatment of TG cultures for 24h with SUMA (1 μM) followed by a rigorous washout step had no effect on the ability of 5‐CT to inhibit PGE2‐stimulation of cAMP accumulation. By contrast, rather than an inhibition of cAMP accumulation, SUMA increased PGE2‐mediated cAMP accumulation in TG cultures pretreated for 24h with SUMA suggesting that a switch from 5‐HT1 receptor‐mediated activation of Gi‐ to Gs‐proteins may have occurred. Although both 5‐CT and SUMA increased ERK activity by 40% above basal within 5 min of incubation, by contrast to effects on cAMP signaling, ERK activation in response to 5‐CT was lost whereas responses to SUMA were unaltered in TG cultures pretreated 24h with SUMA. Interestingly, similar to effects on SUMA‐mediated signaling, following 24h pretreatment with SUMA, 5‐HT‐mediated ERK activation was unaltered whereas 5‐HT‐mediated inhibition of cAMP accumulation was no longer observedThese results support our hypothesis that prolonged exposure to a triptan may produce MOH by inducing differential desensitization of 5‐HT1 receptor signaling pathways and signal switching in nociceptors. Future experiments will determine if repeated in vivo administration of sumatriptan will similarly alter 5‐HT1 receptor signaling in rat trigeminal ganglia nociceptors.Support or Funding InformationSupported by NIH/NINDS R21 NS094488 and NIH/T32 NS082145This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CB1 cannabinoid receptor-phosphorylated fourth intracellular loop structure-function relationships.

A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

Antinociceptive effects of novel histamine H3 and H4 receptor antagonists and their influence on morphine analgesia of neuropathic pain in the mouse.

The histaminergic system is a promising target for the development of new analgesics, as histamine H3 and H4 receptors are expressed in regions concerned with nociceptive transmission. Here we have determined the analgesic effects of new H3 and H4 receptor antagonists in naive and neuropathic mice. We used chronic constriction injury (CCI) to the sciatic nerve in mice to model neuropathy. Effects of a new H3 receptor antagonist, E-162(1-(5-(naphthalen-1-yloxy)pentyl)piperidine) and H4 receptor antagonist, TR-7(4-(4-chlorophenyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine) were assessed on mechanical (von Frey) and thermal (cold plate, tail flick) stimuli in mice with and without CCI (7days after injury). Effects of these antagonists on morphine analgesia were also evaluated, along with the possible participation of H1 receptors in their effects. We analysed the compounds in binding and functional cAMP assays at the H3 and H4 receptors and determined metabolic stability. E-162 and TR-7 attenuated nociceptive responses and profound morphine analgesia in males with CCI. These antagonists showed analgesia in naive mice (tail flick test) and produced prolonged analgesia in neuropathic females. E-162-induced analgesia was reversed by pyrilamine, an H1 receptor antagonist. E-162 bound potently to H3 receptors (Ki =55nM) and inhibited cAMP accumulation (IC50 =165nM). TR-7 showed lower affinity for H4 receptors (Ki =203nM) and IC50 of 512nM. We describe a therapeutic use for new H3 (E-162) and H4 receptor (TR-7) antagonists in neuropathy. Targeting H3 and H4 receptors enhanced morphine analgesia, consistent with multimodal pain therapy.

Assessing the real-time activation of the cannabinoid CB1 receptor and the associated structural changes using a FRET biosensor.

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.

GNAS‐PKA Oncosignaling Network in Colorectal Cancer

G protein coupled receptors (GPCRs) represent the largest class of drug target among currently approved small molecules. However, there is a dearth of oncology drugs targeting GPCRs. Analysis of cancer genomes reveals that G proteins often harbor activating mutations, inherently making their cognate receptor resistant to modulation. In particular, activated GNAS mutants, encoding a constitutively active, GTPase defective Gαs, can be identified in approximately 5% of all sequenced tumors, including 4% of colorectal cancers (CRCs). By stimulating adenylyl cyclases, GNAS activation leads to the intracellular accumulation of cAMP, a central second messenger, which in turn, activates protein kinase A (PKA). Similarly, 10% of CRC patients harbor gene amplification of GNAS, and another 45% have genetic alterations in PKA subunits, adenylyl cyclases, or phosphodiesterases (which inhibit cAMP accumulation). These findings highlight the importance of understanding oncogenic signaling downstream of GPCRs and the potential role of GNAS‐PKA signaling in CRC. The aim of our study is to define the contribution of the GNAS‐PKA pathway in CRCs and explore the cancer‐associated GNAS‐PKA signaling network. We hypothesize that increased PKA activity downstream of GNAS mutations, or in response to persistent GPCR activation, plays a central role in the growth and progression of CRCs. Preliminary results demonstrate that PKA inhibition significantly reduces tumor burden in xenograft mouse models. Interestingly, activation of GNAS alone in both transgenic and chemical carcinogen mouse models is insufficient to induce tumorigenesis. However, upon the co‐activation of GNAS and RAS, mice show rapid development of squamous cell carcinoma. This finding is strongly supported by bioinformatic analysis revealing a co‐occurrence of GNAS and KRAS mutations clinically. We are currently profiling the GNAS‐PKA pathway by large scale transcriptomic and proteomic experiments to understand signaling circuitries and mechanisms of tumorigenesis. Emerging results from our studies will be presented.Support or Funding InformationDana Steffen was supported by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752). This study is supported in part by the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ca2+ ]i modulation of cAMP-stimulated ciliary beat frequency via PDE1 in airway ciliary cells of mice.

What is the central question of this study? The ciliary beat frequency (CBF) of the airway is controlled by [Ca2+ ]i . However, the effects of a reduction in [Ca2+ ]i on CBF are still controversial (an increase, a decrease or no change). What is the main finding and its importance? This study demonstrated that [Ca2+ ]i directly regulates CBF (direct action) and also indirectly regulates CBF via cAMP accumulation controlled by Ca2+ -dependent PDE1 activity (indirect action). The final CBF is determined by the balance of direct and indirect actions. PDE1 plays crucial roles in the regulation of airway CBF. ABSTRACT: [Ca2+ ]i plays crucial roles in the regulation of ciliary beat frequency (CBF) and ciliary bend angle (CBA) of airway cilia. Moreover, Ca2+ -dependent PDE1A existing in the CBF-regulating metabolon of cilia modifies the CBF by regulating the cAMP accumulation. This study demonstrated that the CBF is regulated by a direct and an indirect action of [Ca2+ ]i ; the direct action changes CBF mediated via [Ca2+ ]i , and the indirect action changes CBF mediated via cAMP, the accumulation of which is controlled by PDE1 activity. Upon reducing [Ca2+ ]i to various levels, the direct action decreases CBF and the indirect action increases CBF. The final CBF is determined by the extent of cAMP accumulation, which is determined by the amount of inhibition of PDE1 activity, dependent on a reduction in [Ca2+ ]i ; a slight decrease induced by a nominally Ca2+ -free solution (no cAMP accumulation via PDE1) decreases CBF, and an extreme decrease induced by 50μm BAPTA-AM increases CBF via cAMP accumulation by inhibiting PDE1 in a similar manner to a PDE1 inhibitor (8MmIBMX). The increase in CBA in response to a reduction in [Ca2+ ]i is smaller than the increase in CBF, because no PDE1A exists in the CBA-regulating metabolon. On the contrary, an increase in [Ca2+ ]i induced by ionomycin, which decreases cAMP accumulation by PDE1A activation, caused a slower procaterol-stimulated increase in CBF than that decreased by a Ca2+ -free solution. A decrease in [Ca2+ ]i stimulates cAMP accumulation, whereas an increase in [Ca2+ ]i inhibits cAMP accumulation in airway ciliary cells. Thus, changes in [Ca2+ ]i modulate CBF and CBA via cAMP accumulation by controlling the activity of PDE1.

Slowly Signaling G Protein–Biased CB2 Cannabinoid Receptor Agonist LY2828360 Suppresses Neuropathic Pain with Sustained Efficacy and Attenuates Morphine Tolerance and Dependence

The CB2 cannabinoid agonist LY2828360 lacked both toxicity and efficacy in a clinical trial for osteoarthritis. Whether LY2828360 suppresses neuropathic pain has not been reported, and its signaling profile is unknown. In vitro, LY2828360 was a slowly acting but efficacious G protein-biased CB2 agonist, inhibiting cAMP accumulation and activating extracellular signal-regulated kinase 1/2 signaling while failing to recruit arrestin, activate inositol phosphate signaling, or internalize CB2 receptors. In wild-type (WT) mice, LY2828360 (3 mg/kg per day i.p. × 12 days) suppressed chemotherapy-induced neuropathic pain produced by paclitaxel without producing tolerance. Antiallodynic efficacy of LY2828360 was absent in CB2 knockout (KO) mice. Morphine (10 mg/kg per day i.p. × 12 days) tolerance developed in CB2KO mice but not in WT mice with a history of LY2828360 treatment (3 mg/kg per day i.p. × 12 days). LY2828360-induced antiallodynic efficacy was preserved in WT mice previously rendered tolerant to morphine (10 mg/kg per day i.p. × 12 days), but it was absent in morphine-tolerant CB2KO mice. Coadministration of LY2828360 (0.1 mg/kg per day i.p. × 12 days) with morphine (10 mg/kg per day × 12 days) blocked morphine tolerance in WT but not in CB2KO mice. WT mice that received LY2828360 coadministered with morphine exhibited a trend (P = 0.055) toward fewer naloxone-precipitated jumps compared with CB2KO mice. In conclusion, LY2828360 is a slowly signaling, G protein-biased CB2 agonist that attenuates chemotherapy-induced neuropathic pain without producing tolerance and may prolong effective opioid analgesia while reducing opioid dependence. LY2828360 may be useful as a first-line treatment in chemotherapy-induced neuropathic pain and may be highly efficacious in neuropathic pain states that are refractive to opioid analgesics.

Cannabidiol, a novel inverse agonist for GPR12

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

GNAS‐PKA Oncosignaling Network in Colorectal Cancer

Despite the successes of early detection and polyp removal, colorectal cancer (CRC) remains the second leading cause of cancer death in the United States. Substantial evidence supports the efficacy of nonsteroidal anti‐inflammatory drugs (NSAIDs), like aspirin and ibuprofen, for the prevention of CRC. NSAIDs inhibit cyclooxygenase‐2 (COX2), an enzyme that converts arachidonic acid to prostaglandins, including prostaglandin E2 (PGE2). Increased synthesis of PGE2 due to COX2 overexpression is consistently observed in patients with colorectal neoplasia and has been shown to promote CRC. Activation of Gαs‐linked, G protein‐coupled receptors by PGE2 has also been linked to CRC progression. Furthermore, analysis of cancer genomes reveals that activated GNAS mutants, encoding a constitutively active, GTPase defective Gαs, can be identified in approximately 5% of all sequenced tumors, including 4% of CRC. By stimulating adenylyl cyclases, GNAS activation leads to the intracellular accumulation of cyclic adenosine monophosphate (cAMP), a central second messenger, which in turn, activates protein kinase A (PKA). Similarly, 10% of CRC patients harbor gene amplification of GNAS, and another 45% have mutations in PKA subunits, adenylyl cyclase genes, or homozygous deletions of phosphodiesterases (which inhibit cAMP accumulation). These findings highlight the potential oncogenic role of GNAS‐PKA signaling. The aim of our study is to define the contribution of the GNAS‐PKA pathway in CRCs and explore the cancer‐associated GNAS‐PKA signaling network. We hypothesize that increased PKA activity downstream of GNAS mutations plays a central role in the growth and progression of CRCs. We utilized human colorectal cancer cell lines, genetic mouse models of colorectal cancer, and organoids derived from mouse and human tumor samples. We are able to precisely modulate GNAS‐PKA pathway activity in our in vitro and in vivo models with genetically encoded, inducible expression systems. We performed RNA sequencing, phospho‐proteomics, and computational analysis to identify candidate signaling modalities downstream of GNAS‐PKA. We have demonstrated the ability of the activating GNASR201C mutation to drive a greater than 3‐fold increase in cell proliferation and the ability of PKA inhibition to significantly reduce tumor burden in xenograft mouse models. Preliminary analysis of genomic, transcriptomic, and proteomic data suggest a signaling interaction between GNAS‐PKA and RAS. Emerging results from our studies will be presented. We have demonstrated the ability of GNAS‐PKA signaling to promote growth and identified a potential signaling connection between GNAS‐PKA and RAS.Support or Funding InformationDana Steffen was supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752) and the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot Grant.

Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization.

The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.

Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D.

Identification of a novel dopaminergic agonist that displays locational bias and functional selectively at the D 2 dopamine receptor

To identify novel ligands for the D2 dopamine receptor (D2R), we screened small molecule chemical libraries using high throughput screening. This identified a compound that selectively activates the D2R in a functionally biased manner. The chemically optimized lead compound (VU207) exhibits full agonist activity in three different D2R signaling assays: Ca2+ mobilization (Gqi5), inhibition of cAMP accumulation (Gi/o), and β-arrestin recruitment. However, VU207 fails to activate D2R-Gβγ mediated responses including GIRK channel activation and adenylyl cyclase potentiation. VU207 was also found to exhibit potent D3R antagonism with little activity at other DA receptors. Further, behavioral paradigms support that VU207 acts as a D2R agonist and a D3R antagonist in vivo. Using ex vivo brain slices, we investigated the activity of VU207 on D2Rs located on dopamine neurons. In these cells, D2Rs on the cell bodies activate GIRK channels whereas presynaptic D2Rs on the nerve terminals inhibit DA release. We found that VU207 failed to activate GIRK currents through somatodendritic D2Rs whereas it inhibited DA release via the D2Rs at the terminals. These findings suggest that VU207 exhibits a form of “location bias” acting as either an agonist or an antagonist depending on where in the cell the D2R is located.

Identification of Small Molecule Inhibitors Selective for Type 1 Adenylyl Cyclase

Adenylyl cyclases (ACs) are important integrators of signaling through G protein‐coupled receptors. There are nine isoforms of membrane‐bound ACs that have unique regulatory properties and expression patterns. Adenylyl cyclase 1 (AC1) belongs to a group of ACs that are stimulated by calcium in a calmodulin‐dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating neuropathic/inflammatory pain, opioid dependence, and preventing neuronal excitotoxicity. However, previous reports in mice lacking both AC1 and AC8 suggest that non‐selective inhibition of AC1 and AC8 would result in significant memory impairments and possibly depression. These observations suggest that AC1‐selective inhibitors have potential in a variety of neurological diseases. Accordingly, inhibition of AC1 with the small molecule NB001 had analgesic properties in animal models of chronic pain in the absence of alterations of hippocampal LTP and fear memory. Unfortunately, the mechanism of action of NB001 remains unknown as it failed to directly inhibit AC1. The present study identified several novel classes of AC1 inhibitors through both chemical library screening and structure‐activity relationship studies. Compounds were initially tested against forskolin and Ca2+ (i.e. A23187) stimulated cAMP accumulation in HEK‐AC1 cells. Robust inhibitors were also evaluated against members from other AC families (i.e. AC2 and AC5) revealing selective AC1 inhibitors. Further comparisons between AC1 and AC8 revealed ligands that showed selectivity for AC1. Subsequent studies determined the effect of these AC1 modulators on Gαi/o‐coupled receptor‐mediated inhibition of cAMP accumulation as well as heterologous sensitization of AC.