From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.
Cannabinoid Receptor Agonists , Signal Transduction , Humans , Rats , Animals , Receptors, Cannabinoid , beta-Arrestins , Corpus Striatum/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptor, Cannabinoid, CB2 , Receptor, Cannabinoid, CB1
We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.
Pyrimidines/pharmacology , Receptors, G-Protein-Coupled/agonists , Allosteric Regulation/drug effects , Dose-Response Relationship, Drug , Humans , Molecular Structure , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Receptors, G-Protein-Coupled/metabolism , Structure-Activity Relationship
AIMS: Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. METHODS AND RESULTS: Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. CONCLUSION: cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.
Cyclic AMP/pharmacology , Early Growth Response Protein 1/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Myocytes, Smooth Muscle/metabolism , Adenosine/pharmacology , Aminopyridines/pharmacology , Animals , Cell Proliferation/drug effects , Colforsin/pharmacology , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Early Growth Response Protein 1/antagonists & inhibitors , Early Growth Response Protein 1/metabolism , Epoprostenol/analogs & derivatives , Epoprostenol/pharmacology , Gene Expression Regulation , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Organ Specificity , Primary Cell Culture , Protein Binding , Rats , Rats, Sprague-Dawley , Serum Response Factor/genetics , Serum Response Factor/metabolism , Signal Transduction , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism
Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2'-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2'-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.
Acetylcysteine/analogs & derivatives , Cell Proliferation/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Erythromycin/analogs & derivatives , Myocytes, Smooth Muscle/drug effects , Acetylcysteine/metabolism , Animals , Blotting, Western , Cells, Cultured , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Erythromycin/metabolism , Male , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Paxillin/metabolism , Phalloidine/metabolism , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism
