Β-arrestin Recruitment Research Articles

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers.Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation.Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment.Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.

Staphylococcus aureus-Derived PSMα Peptides Activate Neutrophil FPR2 but Lack the Ability to Mediate β-Arrestin Recruitment and Chemotaxis.

Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit β-arrestin and induce neutrophil chemotaxis, supporting the previous notion that β-arrestin translocation is of importance for cell migration. Despite the lack of β-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit β-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca2+, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of β-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.

Discovery of endogenous inverse agonists for G protein-coupled receptor 6

The orphan G protein-coupled receptor 6 (GPR6) is highly expressed in the striatum and has been linked to multiple striatal pathologies. The identification of endogenous ligands and their mechanisms of action at GPR6 will help to elucidate the physiological and pathological roles of the receptor. In the current study, we tested the concentration-dependent effects of a variety of endocannabinoid-like N-acylamides on GPR6 signaling. Here, we demonstrate for the first time that N-arachidonoyl dopamine, N-docosahexaenoyl dopamine, N-oleoyl dopamine and N-palmitoyl dopamine exert inverse agonism at GPR6. This effect was concentration-dependent, with potencies in the micromolar range, and functionally selective for β-arrestin2 recruitment. Structure-activity relationship studies demonstrate that both the N-acyl side chain and the dopamine head group are important for these ligands to act on GPR6. Our discovery of these N-acyl dopamines as endogenous inverse agonists for GPR6 moves us one step further in understanding the roles GPR6 play in neurodegenerative and neuropsychiatric disorders related to striatal dysfunction.

Residues and residue pairs of evolutionary importance differentially direct signaling bias of D2 dopamine receptors

The D2 dopamine receptor and the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A) are closely-related G-protein-coupled receptors (GPCRs) from the class A bioamine subfamily. Despite structural similarity, they respond to distinct ligands through distinct downstream pathways, whose dysregulation is linked to depression, bipolar disorder, addiction, and psychosis. They are important drug targets, and it is important to understand how their bias toward G-protein versus β-arrestin signaling pathways is regulated. Previously, evolution-based computational approaches, difference Evolutionary Trace and Evolutionary Trace-Mutual information (ET-Mip), revealed residues and residue pairs that, when switched in the D2 receptor to the corresponding residues from 5-HT2A, altered ligand potency and G-protein activation efficiency. We have tested these residue swaps for their ability to trigger recruitment of β-arrestin2 in response to dopamine or serotonin. The results reveal that the selected residues modulate agonist potency, maximal efficacy, and constitutive activity of β-arrestin2 recruitment. Whereas dopamine potency for most variants was similar to that for WT and lower than for G-protein activation, potency in β-arrestin2 recruitment for N124H3.42 was more than 5-fold higher. T205M5.54 displayed high constitutive activity, enhanced dopamine potency, and enhanced efficacy in β-arrestin2 recruitment relative to WT, and L379F6.41 was virtually inactive. These striking differences from WT activity were largely reversed by a compensating mutation (T205M5.54/L379F6.41) at residues previously identified by ET-Mip as functionally coupled. The observation that the signs and relative magnitudes of the effects of mutations in several cases are at odds with their effects on G-protein activation suggests that they also modulate signaling bias.

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC50 = 21.5 µM, Ki = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC50 = 0.800 µM, Ki = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC50 values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.

Setup of a Serotonin 2A Receptor (5-HT2AR) Bioassay: Demonstration of Its Applicability To Functionally Characterize Hallucinogenic New Psychoactive Substances and an Explanation Why 5-HT2AR Bioassays Are Not Suited for Universal Activity-Based Screening of Biofluids for New Psychoactive Substances.

Classic or serotonergic hallucinogens comprise the third largest number of reported new psychoactive substances (NPS), according to the United Nations Office on Drugs and Crime. While being structurally very divergent, they share activation of the serotonin 2A receptor (5-HT2AR), a G protein-coupled receptor, as their main pharmacological mechanism. Here, we report on the development of a 5-HT2AR bioassay, which monitors β-arrestin2 recruitment to the receptor via a split-luciferase system, as a measure of receptor activation. Possible applications of the bioassay would lie in the characterization of serotonergic hallucinogens and the screening of these compounds in biofluids based on their serotonergic activity, rather than on their specific structures. The developed bioassay allowed the determination of the potency and efficacy of representatives of different classes of hallucinogens (LSD, 5-MeO-DALT, mescaline) and of a selected group of 2C hallucinogens and their corresponding NBOMes, with EC50 values from the subnanomolar (NBOMes) to micromolar (mescaline) range. When implementing the bioassay for the screening of plasma, a pronounced receptor activation was already observed in blank samples, which could be ascribed to endogenous serotonin, as suggested by annihilation of this activity by a 5-HT2AR antagonist or after incubation with MAO-A (monoamine oxidase-A). The presence and degradability by MAO-A of serotonin in plasma extracts were confirmed by LC-HRMS. Due to the possible metabolism of certain hallucinogens by MAO-A, which would cause a bias in the detectability of compounds in biofluids, the main application potential of this bioassay lies in the characterization of these scarcely characterized serotonergic hallucinogens.

Selective Allosteric Modulation of N-Terminally Cleaved, but Not Full Length CCL3 in CCR1

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.

DDIS-20. IMIPRIDONE STRUCTURE ACTIVITY RELATIONSHIP UNCOVERS ONC206 AS THE NEXT BITOPIC DRD2 ANTAGONIST FOR ONCOLOGY WITH DIFFERENTIATED RECEPTOR PHARMACOLOGY

Abstract ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) in oncology clinical trials, which have shown that the small molecule is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC201 possesses a unique tri-heterocyclic core structure that prompted a medicinal chemistry exploration following its successful clinical translation. Chemical derivatization of the ONC201 pharmacophore from an angular to a linear isomer ablated DRD2 antagonist activity and anti-cancer activity, indicating the core structure integrity is critical. A series of analogs that share the same core structure, called imipridones, were profiled for modulation of β-arrestin recruitment to GPCRs and anti-cancer efficacy. The profiled imipridones exhibited a heterogeneous spectrum of GPCR agonist/antagonist activity that was exclusive to Class A GPCRs. The addition of electron withdrawing groups to one of two peripheral benzyl rings enhanced the potency of GPCR engagement and anti-cancer effects, while derivatization of the other benzyl ring was inactivating. Among the GPCR hits identified, maximal variance in imipridone GPCR antagonism was identified for DRD2/DRD3. ONC206 emerged as the most selective and potent antagonist for DRD2/DRD3 with a Ki of ~320nM for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. Six residues were critical for ONC201 and ONC206, however the impact varied between the two compounds and one allosteric residue was exclusive to ONC206. Structural mapping revealed that some ONC206-critical allosteric residue interactions are located at the interface of TM-IV and –V that mediates the DRD2 homodimer interface. Thus, ONC206 is a bitopic DRD2 antagonist that may be poised to address oncogenic DRD2 monomers or dimers.

Assessment of Biased Agonism among Distinct Synthetic Cannabinoid Receptor Agonist Scaffolds

Cannabinoid receptor 1 (CB1) is a key drug target for a number of diseases, including metabolic syndromes and neuropathic pain. Most of the typical cannabinoid ligands provoke psychotropic side effects that impair their therapeutic utility. As of today, it is not yet clearly known which structural features of cannabinoid ligands determine a preference toward specific signaling pathways. Distinct bioassays are typically used to elucidate signaling preferences. However, these are often based on different cell lines and use different principles and/or read-outs, which makes straightforward assessment of "ligand bias" difficult. Within this context, this study is the first to investigate ligand bias among synthetic cannabinoid receptor agonists (SCRAs) in as closely analogous conditions as possible, by applying a new functional complementation-based assay panel to assess the recruitment of Gαi protein or β-arrestin2 to CB1. In a panel of 21 SCRAs, chosen to cover a broad diversity in chemical structures, distinct, although often subtle, preferences toward specific signaling pathways were observed. Relative to CP55940, here considered as a "balanced" reference agonist, most of the selected SCRAs (e.g., 5F-APINACA, CUMYL-PEGACLONE, among others) displayed preferred signaling through the β-arrestin2 pathway, whereas MMB-CHMICA could serve as a potential "balanced" agonist. Interestingly, EG-018 was the only SCRA showing a significant (10-fold) preference toward G protein over β-arrestin2 recruitment. While it is currently unclear what this exactly means in terms of abuse potential and/or toxicity, the approach proposed here may allow construction of a knowledge base that in the end may allow better insight into the structure-"functional" activity relationship of these compounds. This may aid the development of new therapeutics with less unwanted psychoactive effects.

ATHENA: A Phase 3, Open-Label Study Of The Safety And Effectiveness Of Oliceridine (TRV130), A G-Protein Selective Agonist At The µ-Opioid Receptor, In Patients With Moderate To Severe Acute Pain Requiring Parenteral Opioid Therapy.

BackgroundPain management with conventional opioids can be challenging due to dose-limiting adverse events (AEs), some of which may be related to the simultaneous activation of β-arrestin (a signaling pathway associated with opioid-related AEs) and G-protein pathways. The investigational analgesic oliceridine is a G-protein-selective agonist at the µ-opioid receptor with less recruitment of β-arrestin. The objective of this phase 3, open-label, multi-center study was to evaluate the safety and tolerability, of IV oliceridine for moderate to severe acute pain in a broad, real-world patient population, including postoperative surgical patients and non-surgical patients with painful medical conditions.MethodsAdult patients with a score ≥4 on 11-point NRS for pain intensity received IV oliceridine either by bolus or PCA; multimodal analgesia was permitted. Safety was assessed using AE reports, study discontinuations, clinical laboratory and vital sign measures.ResultsA total of 768 patients received oliceridine. The mean age (SD) was 54.1 (16.1) years, with 32% ≥65 years of age. Most patients were female (65%) and Caucasian (78%). Surgical patients comprised the majority of the study population (94%), most common being orthopedic (30%), colorectal (15%) or gynecologic (15%) procedures. Multimodal analgesia was administered to 84% of patients. Oliceridine provided a rapid reduction in NRS pain score by 2.2 ± 2.3 at 30 mins from a score of 6.3 ± 2.1 (at baseline) which was maintained to the end of treatment. No deaths or significant cardiorespiratory events were reported. The incidence of AEs leading to early discontinuation and serious AEs were 2% and 3%, respectively. Nausea (31%), constipation (11%), and vomiting (10%) were the most common AEs. AEs were mostly of mild (37%) or moderate (25%) severity and considered possibly or probably related to oliceridine in 33% of patients.ConclusionOliceridine IV for the management of moderate to severe acute pain was generally safe and well tolerated in the patients studied.ClinicalTrials.gov identifierNCT02656875.

Arrestin‐independent constitutive endocytosis of GPR125/ADGRA3

The orphan receptor GPR125 (ADGRA3) belongs to subgroup III of the adhesion G protein−coupled receptor (aGPCR) family. aGPCRs, also known as class B2 GPCRs, share basic structural and functional properties with other GPCRs. Many of them couple to G proteins and activate G protein−dependent and −independent signaling pathways, but little is known about aGPCR internalization and β‐arrestin recruitment. GPR125 was originally described as a spermatogonial stem cell marker and studied for its role in Wnt signaling and cell polarity. Here, using cell‐based assays and confocal microscopy, we show that GPR125 is expressed on the cell surface and undergoes constitutive endocytosis in a β‐arrestin−independent, but clathrin‐dependent manner, as indicated by colocalization with transferrin receptor 1, an early endosome marker. These data support that the constitutive internalization of GPR125 contributes to its biological functions by controlling receptor surface expression and accessibility for ligands. Our study sheds light on a new property of aGPCRs, namely internalization; a property described to be important for signal propagation, signal termination, and desensitization of class A (rhodopsin‐like) and B1 (VIP/secretin) GPCRs.

Indomethacin Enhances Type 1 Cannabinoid Receptor Signaling.

In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [3H]CP55940 to hCB1R and enhanced AEA-dependent [35S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 μM) also enhanced CP55940-dependent βarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCβ3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems.

Benzyl Phenylsemicarbazides: A Chemistry-Driven Approach Leading to G Protein-Biased Dopamine D4 Receptor Agonists with High Subtype Selectivity.

Many subtype-selective dopamine receptor ligands developed for the D2-D4 family incorporate a 1-arylpiperazine-derived primary recognition motif, which is connected to a lipophilic moiety occupying an extended binding pocket (EBP) of the receptor via an aliphatic linker of variable lengths. The evaluation of a novel group of dopamine receptor ligands now showed that highly subtype-selective ligands [up to Ki(D4.4) = 0.25 nM, D2L/D4.4 = 320, D3/D4.4 = 710 for APH199 (17)] can be obtained by choosing a relatively large and conformationally flexible 1-benzyl-1-phenylsemicarbazide substructure to fill the EBP. The novel chemotype APH199 (17) was found to act as a full agonist at the D4 receptor showing significant bias toward G protein activation over β-arrestin recruitment in comparison to quinpirole.

Obesity-associated mutant melanocortin-4 receptors with normal Gαs coupling frequently exhibit other discoverable pharmacological and biochemical defects.

Mutations in the melanocortin-4 receptor (MC4R) are the most common cause of early syndromic obesity known. Most of these mutations result in a loss of protein expression, α-melanocyte-stimulating hormone binding, receptor trafficking or coupling to the stimulatory G-protein, Gαs . However, approximately 26% of the obesity-associated mutations characterised to date exhibit none of these pharmacological defects. In the present study, we investigated seven of these apparently normal mutant MC4R in more detail and found that the majority (five of the seven) exhibit marked defects including defective binding of another endogenous melanocortin ligand, defective glycosylation, and defective recruitment of β-arrestin. These data provide support for two hypotheses: (i) that the majority of these rare, obesity-associated mutations are likely defective and causative of obesity and (ii) that β-arrestin recruitment is a valuable marker of normal MC4R function. Recent work has demonstrated a statistical correlation between the efficacy of β-arrestin recruitment to the MC4R and body mass index; however, the data reported here demonstrate both decreased and increased β-arrestin signalling in obesity-associated MC4R mutations.

Abaloparatide exhibits greater osteoanabolic response and higher cAMP stimulation and β-arrestin recruitment than teriparatide.

Teriparatide and abaloparatide are parathyroid hormone receptor 1 (PTHR1) analogs with unexplained differential efficacy for the treatment of osteoporosis. Therefore, we compared the effects of abaloparatide and teriparatide on bone structure, turnover, and levels of receptor activator of nuclear factor‐kappa B ligand (RANKL) and osteoprotegerin (OPG). Wild‐type (WT) female mice were injected daily with vehicle or 20–80 µg/kg/day of teriparatide or abaloparatide for 30 days. Femurs and spines were examined by microcomputed tomography scanning and serum levels of bone turnover markers, RANKL, and OPG, were measured by ELISA. Both analogs similarly increased the distal femoral fractional trabecular bone volume, connectivity, and number, and reduced the structure model index (SMI) at 20–80 µg/kg/day doses. However, only abaloparatide exhibited a significant increase (13%) in trabecular thickness at 20 µg/kg/day dose. Femoral cortical evaluation showed that abaloparatide caused a greater dose‐dependent increase in cortical thickness than teriparatide. Both teriparatide and abaloparatide increased lumbar 5 vertebral trabecular connectivity but had no or modest effect on other indices. Biochemical analysis demonstrated that abaloparatide promoted greater elevation of procollagen type 1 intact N‐terminal propeptide, a bone formation marker, and tartrate‐resistant acid phosphatase 5b levels, a bone resorption marker, and lowered the RANKL/OPG ratio. Furthermore, PTHR1 signaling was compared in cells treated with 0–100 nmol/L analog. Interestingly, abaloparatide had a markedly lower EC50 for cAMP formation (2.3‐fold) and β‐arrestin recruitment (1.6‐fold) than teriparatide. Therefore, abaloparatide‐improved efficacy can be attributed to enhanced bone formation and cortical structure, reduced RANKL/OPG ratio, and amplified Gs‐cAMP and β‐arrestin signaling.

Discovery of Novel Biased Opioid Receptor Ligands through Structure-Based Pharmacophore Virtual Screening and Experiment.

Gi -protein-biased agonists with minimal β-arrestin recruitment represent opportunities to overcome the serious adverse effects of human mu opioid receptor (μ-OR) agonists and developing alternative and safe treatments for pain. In order to discover novel non-morphinan opioid receptor agonists, we applied hierarchical virtual screening of our in-house database against a pharmacophore based on modeling the active conformations of opioid receptors. We discovered an initial hit compound, a novel μ-OR agonist with a pyrazoloisoquinoline scaffold. We applied computational R-group screening to this compound and synthesized 14 derivatives predicted to be the best. Of these, a new Gi -protein-biased compound, 1-{5-(3-chlorophenyl)-7,8-dimethoxy-3-[4-(methylsulfonyl)benzyl]-3H-pyrazolo[3,4-c]isoquinolin-1-yl}-N,N-dimethylmethanamine, showed an EC50 value of 179 nm against the μ-OR. This resulted in significant pain relief for mice in the phase II period of formalin response tests. This study provides a new strategy to identify diverse sets of promising compounds that might prove useful for the development of drugs that target other G-protein-coupled receptors.

Kinetics of CXCL12 binding to atypical chemokine receptor 3 reveal a role for the receptor N terminus in chemokine binding.

Chemokines bind to membrane-spanning chemokine receptors, which signal through G proteins and promote cell migration. However, atypical chemokine receptor 3 (ACKR3) does not appear to couple to G proteins, and instead of directly promoting cell migration, it regulates the extracellular concentration of chemokines that it shares with the G protein-coupled receptors (GPCRs) CXCR3 and CXCR4, thereby influencing the responses of these receptors. Understanding how these receptors bind their ligands is important for understanding these different processes. Here, we applied association and dissociation kinetic measurements coupled to β-arrestin recruitment assays to investigate ACKR3:chemokine interactions. Our results showed that CXCL12 binding is unusually slow and driven by the interplay between multiple binding epitopes. We also found that the amino terminus of the receptor played a key role in chemokine binding and activation by preventing chemokine dissociation. It was thought that chemokines initially bind receptors through interactions between the globular domain of the chemokine and the receptor amino terminus, which then guides the chemokine amino terminus into the transmembrane pocket of the receptor to initiate signaling. On the basis of our kinetic data, we propose an alternative mechanism in which the amino terminus of the chemokine initially forms interactions with the extracellular loops and transmembrane pocket of the receptor, which is followed by the receptor amino terminus wrapping around the core of the chemokine to prolong its residence time. These data provide insight into how ACKR3 competes and cooperates with canonical GPCRs in its function as a scavenger receptor.

Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. β-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although β-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on β-arrestin expression and β-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases β-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). β-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between β-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced β-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting β-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced β-arrestin2 levels and delayed agonist-induced β-arrestin2 recruitment to the membrane. We conclude that β-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate β-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.

Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

We investigated the effects of resolvin E (RvE) 1, RvE2, and RvE3 on IL-4- and IL-33-stimulated bone marrow-derived dendritic cells (BMDCs) from house dust mite (HDM)-sensitized mice. We also investigated the role of RvE3 in a murine model of HDM-induced airway inflammation. In vitro, BMDCs from HDM-sensitized mice were stimulated with IL-4 and IL-33 and then treated with RvE1, RvE2, RvE3, or vehicle. RvE1, RvE2, and RvE3 suppressed IL-23 release from BMDCs. In vivo, RvE3 administrated to HDM-sensitized and challenged mice in the resolution phase promoted a decline in total numbers of inflammatory cells and eosinophils, reduced levels of IL-23 and IL-17 in lavage fluid, and suppressed IL-23 and IL-17A mRNA expression in lung and peribronchial lymph nodes. RvE3 also reduced resistance in the lungs of HDM-sensitized mice. A NanoBiT β-arrestin recruitment assay using human embryonic kidney 293 cells revealed that pretreatment with RvE3 suppressed the leukotriene B4 (LTB4)-induced β-arrestin 2 binding to LTB4 receptor 1 (BLT1R), indicating that RvE3 antagonistically interacts with BLT1R. Collectively, these findings indicate that RvE3 facilitates the resolution of allergic airway inflammation, partly by regulating BLT1R activity and selective cytokine release by dendritic cells. Our results accordingly identify RvE3 as a potential therapeutic target for the management of asthma.-Sato, M., Aoki-Saito, H., Fukuda, H., Ikeda, H., Koga, Y., Yatomi, M., Tsurumaki, H., Maeno, T., Saito, T., Nakakura, T., Mori, T., Yanagawa, M., Abe, M., Sako, Y., Dobashi, K., Ishizuka, T., Yamada, M., Shuto, S., Hisada, T. Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

A G protein signaling-biased agonist at the μ-opioid receptor reverses morphine tolerance while preventing morphine withdrawal.

It has been demonstrated that opioid agonists that preferentially act at μ-opioid receptors to activate G protein signaling over βarrestin2 recruitment produce antinociception with less respiratory suppression. However, most of the adverse effects associated with opioid therapeutics are realized after extended dosing. Therefore, we tested the onset of tolerance and dependence, and assessed for neurochemical changes associated with prolonged treatment with the biased agonist SR-17018. When chronically administered to mice, SR-17018 does not lead to hot plateantinociceptive tolerance, receptor desensitization in periaqueductal gray, nor a super-sensitization of adenylyl cyclase in the striatum, which are hallmarks of opioid neuronal adaptations that are seen with morphine. Interestingly, substitution with SR-17018 in morphine-tolerant mice restores morphine potency and efficacy, whereas the onset of opioid withdrawal is prevented. This is in contrast to buprenorphine, which can suppress withdrawal, but produces and maintains morphine antinociceptive tolerance. Biased agonists of this nature may therefore be useful for the treatment of opioid dependence while restoring opioid antinociceptive sensitivity.