GRK5 regulates endocytosis of FPR2 independent of β-Arrestins.

The formyl-peptide receptor 2 (FPR2) is a G-protein-coupled receptor (GPCR) that responds to pathogen-derived peptides and regulates both pro-inflammatory and pro-resolution cellular processes. While ligand selectivity and G-protein-signalling of FPR2 have been well characterized, molecular mechanisms controlling subsequent events such as endocytosis and recycling to the plasma membrane are less understood. Here we show the key role of the GPCR kinase 5 (GRK5) in facilitating FPR2 endocytosis and post-endocytic trafficking.

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited.

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for β-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail TREHPSTANT motif harbours Ser/Thr residues important for these regulatory functions.

Enantioselective synthesis of 14-hydroxy-6-oxomorphinans.

The enantioselective synthesis of pharmacologically important 14-hydroxy-6-oxomorphinans is described. 4,5-Desoxynaltrexone and 4,5-desoxynaloxone were prepared using this route and their biological activities against the opioid receptors were measured.

Arrestin-centred interactions at the membrane and their conformational determinants.

More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions.

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as μ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach.

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized.

The functional profiles of fentanyl and nitazene analogs at the μ-opioid receptor - high efficacy is dangerous regardless of signaling bias.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the μ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the functional profiles of various NSOs at MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach.

Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity.

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties of receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and the resulting effects is important.

Assessment of the potential of novel and classical opioids to induce respiratory depression in mice.

Background And Purpose: Opioid-induced respiratory depression limits the use of μ-opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti-nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy.

Pharmacological and Physicochemical Properties Optimization for Dual-Target Dopamine D (DR) and μ-Opioid (MOR) Receptor Ligands as Potentially Safer Analgesics.

A new generation of dual-target μ opioid receptor (MOR) agonist/dopamine D receptor (DR) antagonist/partial agonists with optimized physicochemical properties was designed and synthesized. Combining in vitro cell-based on-target/off-target affinity screening, in silico computer-aided drug design, and BRET functional assays, we identified new structural scaffolds that achieved high affinity and agonist/antagonist potencies for MOR and DR, respectively, improving the dopamine receptor subtype selectivity (e.g.

OZITX, a pertussis toxin-like protein for occluding inhibitory G protein signalling including Gα.

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB toxin, inhibits Gα G proteins and has proven useful for interrogating inhibitory G protein signalling.

The respiratory depressant effects of mitragynine are limited by its conversion to 7-OH mitragynine.

Background And Purpose: Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), is a partial agonist at the μ opioid receptor. CYP3A-dependent oxidation of mitragynine yields the metabolite 7-OH mitragynine, a more efficacious μ receptor agonist. While both mitragynine and 7-OH mitragynine can induce anti-nociception in mice, recent evidence suggests that 7-OH mitragynine formed as a metabolite is sufficient to explain the anti-nociceptive effects of mitragynine.

Opioid-Induced Pronociceptive Signaling in the Gastrointestinal Tract Is Mediated by Delta-Opioid Receptor Signaling.

Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration.

Anxiety enhances pain in a model of osteoarthritis and is associated with altered endogenous opioid function and reduced opioid analgesia.

Introduction: Negative affect, including anxiety and depression, is prevalent in chronic pain states such as osteoarthritis (OA) and associated with greater use of opioid analgesics, potentially contributing to present and future opioid crises.

Objectives: We tested the hypothesis that the interaction between anxiety, chronic pain, and opioid use results from altered endogenous opioid function.

Methods: A genetic model of negative affect, the Wistar-Kyoto (WKY) rat, was combined with intra-articular injection of monosodium iodoacetate (MIA; 1 mg) to mimic clinical presentation.

Positive allosteric modulation of endogenous delta opioid receptor signaling in the enteric nervous system is a potential treatment for gastrointestinal motility disorders.

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models.

Atypical opioid receptors: unconventional biology and therapeutic opportunities.

Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating four opioid receptors, namely μ (mu, MOP), δ (delta, DOP), κ (kappa, KOP) and the nociceptin/orphanin FQ receptor (NOP). Interestingly, several other receptors are also activated by endogenous opioid peptides and influence opioid-driven signaling and biology. However, they do not meet the criteria to be recognized as classical opioid receptors, as they are phylogenetically distant from them and are insensitive to classical non-selective opioid receptor antagonists (e.

Experimental considerations for the assessment of in vivo and in vitro opioid pharmacology.

Morphine and other mu-opioid receptor (MOR) agonists remain the mainstay treatment of acute and prolonged pain states worldwide. The major limiting factor for continued use of these current opioids is the high incidence of side effects that result in loss of life and loss of quality of life. The development of novel opioids bereft, or much less potent, at inducing these side effects remains an intensive area of research, with multiple pharmacological strategies being explored.

Novel Dual-Target μ-Opioid Receptor and Dopamine D Receptor Ligands as Potential Nonaddictive Pharmacotherapeutics for Pain Management.

The need for safer pain-management therapies with decreased abuse liability inspired a novel drug design that retains μ-opioid receptor (MOR)-mediated analgesia, while minimizing addictive liability. We recently demonstrated that targeting the dopamine D receptor (DR) with highly selective antagonists/partial agonists can reduce opioid self-administration and reinstatement to drug seeking in rodent models without diminishing antinociceptive effects. The identification of the DR as a target for the treatment of opioid use disorders prompted the idea of generating a class of ligands presenting bitopic or bivalent structures, allowing the dual-target binding of the MOR and DR.

Glycosylation Regulates N-Terminal Proteolysis and Activity of the Chemokine CCL14.

Chemokines are secreted proteins that regulate leukocyte migration during inflammatory responses by signaling through chemokine receptors. Full length CC chemokine ligand 14, CCL14(1-74), is a weak agonist for the chemokine receptor CCR1, but its activity is substantially enhanced upon proteolytic cleavage to CCL14(9-74). CCL14 is O-glycosylated at Ser7, adjacent to the site of proteolytic activation.

Systematic Assessment of Chemokine Signaling at Chemokine Receptors CCR4, CCR7 and CCR10.

Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism.

New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D receptor regulation and signaling.

The dopamine D receptor (DR) is the target of drugs used to treat the symptoms of Parkinson's disease and schizophrenia. The DR is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, DR arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NKR) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NKR antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NKR signaling, and causes prolonged antinociception.