Identification and In Vivo Evaluation of Myelination Agent PIPE-3297, a Selective Kappa Opioid Receptor Agonist Devoid of ß-Arrestin-2 Recruitment Efficacy.

Structure-activity relationship studies led to the discovery of PIPE-3297, a fully efficacious and selective kappa opioid receptor (KOR) agonist. PIPE-3297, a potent activator of G-protein signaling (GTPγS EC50 = 1.1 nM, 91% Emax), did not elicit a ß-arrestin-2 recruitment functional response (Emax < 10%). Receptor occupancy experiments performed with the novel KOR radiotracer [3H]-PIPE-3113 revealed that subcutaneous (s.c.) administration of PIPE-3297 at 30 mg/kg in mice achieved 90% occupancy of the KOR in the CNS 1 h post dose. A single subcutaneous dose of PIPE-3297 in healthy mice produced a statistically significant increase of mature oligodendrocytes (P < 0.0001) in the KOR-enriched striatum, an effect that was not observed in animals predosed with the selective KOR antagonist norbinaltorphimine. An equivalent dose given to mice in an open-field activity-monitoring system revealed a small KOR-independent decrease in total locomotor activity versus vehicle measured between 60 and 75 min post dose. Daily doses of PIPE-3297 at both 3 and 30 mg/kg s.c. reduced the disease score in the mouse experimental autoimmune encephalomyelitis (EAE) model. Visually evoked potential (VEP) N1 latencies were also significantly improved versus vehicle in both dose groups, and latencies matched those of untreated animals. Taken together, these findings highlight the potential therapeutic value of functionally selective G-protein KOR agonists in demyelinating disease, which may avoid the sedating side effects typically associated with classical nonbiased KOR agonists.

Understanding the Molecular Regulation of Serotonin Receptor 5-HTR1B-ß-Arrestin1 Complex in Stress and Anxiety Disorders.

The serotonin receptor subtype 5-HTR1B is widely distributed in the brain with an important role in various behavioral implications including neurological conditions and psychiatric disorders. The neuromodulatory action of 5-HTR1B largely depends upon its arrestin mediated signaling pathway. In this study, we tried to investigate the role of unusually long intracellular loop 3 (ICL3) region of the serotonin receptor 5-HTR1B in interaction with ß-arrestin1 (Arr2) to compensate for the absence of the long cytoplasmic tail. Molecular modeling and docking tools were employed to obtain a suitable molecular conformation of the ICL3 region in complex with Arr2 which dictates the specific complex formation of 5-HTR1B with Arr2. This reveals the novel molecular mechanism of phosphorylated ICL3 mediated GPCR-arrestin interaction in the absence of the long cytoplasmic tail. The in-cell disulfide cross-linking experiments and molecular dynamics simulations of the complex further validate the model of 5-HTR1B-ICL3-Arr2 complex. Two serine residues (Ser281 and Ser295) within the 5-HTR1B-ICL3 region were found to be occupying the electropositive pocket of Arr2 in our model and might be crucial for phosphorylation and specific Arr2 binding. The alignment studies of these residues showed them to be conserved only across 5-HTR1B mammalian species. Thus, our studies were able to predict a molecular conformation of 5-HTR1B-Arr2 and identify the role of long ICL3 in the signaling process which might be crucial in designing targeted drugs (biased agonists) that promote GPCR-Arr2 signaling to deter the effects of stress and anxiety-like disorders.

Astrocytic AT1R deficiency ameliorates Aß-induced cognitive deficits and synaptotoxicity through ß-arrestin2 signaling.

Alzheimer's disease (AD) seriously influences human health, and there is no effective treatment to prevent or cure AD. Recent studies have shown that angiotensin II type 1 receptor (AT1R) blockers significantly reduce the prevalence of AD, while the precise role and mechanism of AT1R in AD remain obscure. In this study, for the first time, we identified that astrocytic but not neuronal AT1R levels were significantly increased in AD model rats and found that astrocyte-specific knockout of AT1R significantly ameliorated amyloid ß (Aß)-induced cognitive deficits and synaptotoxicity. Pretreating astrocytes with an AT1R blocker also alleviated Aß-induced synaptotoxicity in the coculture system of hippocampal neurons and astrocytes. Moreover, AT1R could directly bind to Aß1-42 and activate the astrocytic ß-arrestin2 pathway in a biased manner, and biased inhibition of the astrocytic AT1R/ß-arrestin2 pathway relieved Aß-induced neurotoxicity. Furthermore, we demonstrated that astrocytic AT1R/ß-arrestin2 pathway-mediated synaptotoxicity was associated with the aggregation of autophagosomes, which triggered the disordered degradation of Aß. Our findings reveal a novel molecular mechanism of astrocytic AT1R in Aß-induced neurodegeneration and might contribute to establishing new targets for AD prevention and therapy.

ß-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice.

BACKGROUND: ß-Arrestin 2 (ß-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking ß-arr 2, suggesting a role for ß-arr 2 in the anabolic effects of PTH. The role of ß-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. OBJECTIVE: To assess the effects of cPTH administration on bone in mice lacking ß-arr 2 compared to wild-type (WT). METHODS: Groups of male and female WT or ß-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. RESULTS: ß-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. ß-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female ß-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. CONCLUSIONS: These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. ß-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Paeoniflorin-6'-O-benzene sulfonate protected the intestinal epithelial barrier by restoring the inhibitory effect of GRK2 and ß-arrestin 2 on ERK1/2-NF-κB.

Peoniflorin-6'-O-benzene sulfonate (CP-25) inhibited the activity of GRK2 to exert anti-inflammatory and immunomodulatory effects. This study aimed to investigate the effect of CP-25 the intestinal epithelial barrier and the mechanism. CaCO-2 cell monolayer and dextran sulfate salt (DSS)-induced colitis mouse model was used to evaluate intestinal epithelial barrier function in vitro and in vivo, respectively. Results showed that CP-25 prevented dysfunction of the intestinal epithelial barrier and inhibited NF-κB p65 activation in TNF-α-induced CaCO-2 cells. The colon structure destroyed in DSS-induced colitis mice was improved by CP-25. CP-25 has a role in inhibition membrane translocation of GRK2-ß-arrestin 2 complex, stabilization of the binding of GRK2 and ß-arrestin 2 to ERK1/2 in cytoplasm. Subsequently down-regulated the nuclear transcription and transactivation of NF-κB p65 via inhibiting its phosphorylation of Ser536, and Ser276, respectively and restored the epithelial barrier function. In conclusion, CP-25 inhibited ERK1/2-NF-κB activation and thereby protected the intestinal epithelial barrier, which was associated with restoring the inhibition of GRK2 and ß-arrestin 2 on ERK1/2.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

Kisspeptin-54 attenuates oxidative stress and neuronal apoptosis in early brain injury after subarachnoid hemorrhage in rats via GPR54/ARRB2/AKT/GSK3ß signaling pathway.

Oxidative stress-induced neuron apoptosis plays a crucial role in the early brain injury (EBI) after subarachnoid hemorrhage (SAH). Kisspeptin has been reported as antioxidant to reduce oxidative stress-induced neuronal cell death through G protein-coupled receptor 54 (GPR54). The goal of this study was to determine the neuroprotection of the Kisspeptin/GRP54 signaling pathway against EBI after SAH. Two hundred and ninety-two Sprague Dawley male rats were used and SAH was induced by the endovascular perforation. Exogenous Kisspeptin 54 (KP54) was delivered intranasally. Small interfering ribonucleic acid (siRNA) for endogenous KISS1, a selective GPR54 antagonist kisspeptin 234, or ß-arrestin 2 siRNA for ARRB2 (a functional adaptor of GPR54) were administered intracerebroventricularly. Post-SAH evaluations included neurobehavioral tests, SAH grade, Western blot, immunofluorescence, Fluoro-Jade C, TUNEL, and Nissl staining. The results showed that endogenous KISS1 knockdown aggravated but exogenous KP54 (1.0 nmol/kg) treatment attenuated neurological deficits, brain oxidative stress, and neuronal apoptosis at 24 h after SAH. The benefits of KP54 persisted to 28 days after SAH, which significantly improved cognitive function in SAH rats. The GPR54 blockade or the ARRB2 knockout offset the neuroprotective effects of KP54 in SAH rats. In conclusion, our results suggested that administration of KP54 attenuated oxidative stress, neuronal apoptosis and neurobehavioral impairments through GPR54/ARRB2/AKT/GSK3ß signaling pathway after SAH in rat. Thus, KP54 may provide an effective treatment strategy for SAH patients.

ß-arrestin2 Inhibits Apoptosis and Liver Inflamation Induced by Ischemia-reperfusion in Mice via AKT and TLR4 Pathway.

BACKGROUND: Liver ischemia and reperfusion (I/R) is a common but severe clinical problem. Previous studies have revealed that the expression level of ß-arrestin2 affects serum deprivation (SD)-induced cell apoptosis and was involved in lipopolysaccharide (LPS) stimulated TLR4 signaling pathway. However, little is known about ß-arrestin2 in liver apoptosis and immune response induced by I/R. METHODS: A non-lethal model of segmental (70%) hepatic ischemia was utilized. Histology examination, cell apoptosis and cytokine levels were measured using H&E staining, TUNEL assay, and ELISA, respectively. Apoptosis-related protein and gene level of cytokines were respectively detected using Western blot and Real-time PCR. RESULTS: Our data showed that knockout (KO) of ß-arrestin2 gene significantly deteriorated the injury of liver caused by I/R according to liver histology, higher serum liver enzyme, and increased level of cell apoptosis. ß-arrestin2 KO could result in increased level of apoptosis related protein and decreased level of Akt phosphorylation. Furthermore, decreased levels of Bcl-2 and Bad phosphorylation, but increased level of Bax were found in ß-arrestin2 KO group. In addition, the levels of p-ERK1/2, p-p38MAPKs, and p-NF-κB in ß-arrestin2 KO group were significantly higher than that in WT group. CONCLUSIONS: ß-arrestin2 protected liver from I/R injury and this effect may be due to the regulating of Akt pathway, Bcl-2/Bax ratio, MAPKs and NF-κB pathway.

ß-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins ßarr (ß-arrestin)-1 and ßarr-2 are functionally distinct in the heart. ßarr1 is cardiotoxic, decreasing contractility by opposing ß1AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, ßarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether ßarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that ßarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca2+-ATPase) in vivo and in vitro in a ß1AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. ßarr1 lacks this effect. Moreover, ßarr2 does not desensitize ß1AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to ßarr1. In vivo, post-MI heart failure mice overexpressing cardiac ßarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, ßarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac ßAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac ßarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac ßarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure.