Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmaco-dynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and β-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF > FENT=OCF > ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the β-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote β-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT > FUF > OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.

OBJECTIVE: β-Arrestin2 signaling has emerged as a promising therapeutic target for the management of insulin resistance and related complications. Moreover, recent studies have shown that certain G protein-coupled receptor (GPCR) ligands can modulate β-arrestin2 signaling. The current study examined the effects of the β-blocker propranolol and a low dose of the agonist isoproterenol (L-D-ISOPROT) on β-arrestin2 signaling, insulin resistance, and cardiac remodeling in high-fructose, high-fat diet (HFrHFD)-fed mice. In addition, the effects of these agents were compared to those of the clinical antidiabetic agent, metformin.
METHODS: Insulin resistance was induced by HFrHFD feeding for 16 weeks. Mice were then randomly allocated to groups receiving propranolol, L-D-ISOPROT, metformin, or vehicle (control) for 4 weeks starting on week 13 of HFrHFD feeding. Survival rate, body weight, visceral fat weight, blood glucose, serum insulin, insulin resistance index, hepatic β-arrestin2 signaling, heart weight, left and right ventricular thicknesses, cardiac fibrosis severity, serum endothelin-1, cardiac cardiotrophin-1, and cardiac β-arrestin2 signaling were then compared among groups.
RESULTS: HFrHFD for 16 weeks significantly increased insulin resistance index, cardiac fibrosis area, and serum endothelin-1, and reduced hepatic β-arrestin2 signaling, cardiac cardiotrophin-1, and cardiac β-arrestin2 signaling without significant changes in survival rate, body weight, visceral fat weight, heart weight, or left and right ventricular thicknesses. All three drugs reduced insulin resistance and cardiac remodeling parameters and enhanced β-arrestin2 signaling with variable efficacies.
CONCLUSIONS: Propranolol and L-D-ISOPROT, like metformin, can reduce insulin-resistance and cardiac remodeling in HFrHFD-fed mice, possibly by upregulating β-arrestin2 signaling activity. Therefore, β-arrestin2-signaling modulation might be a promising strategy for insulin-resistance treatment.

The C-X-C motif ligand 8 and C-X-C chemokine receptor 2 (CXCL8-CXCR2) axis is involved in pathogenesis of various diseases including inflammation and cancers. Various CXCR2 antagonists are under development for several diseases. Our previous high-throughput cell-based assay specific for CXCR2 has identified a pyrimidine-based compound CX797 acting on CXCR2 down-stream signaling. A lead optimization campaign through scaffold-hopping strategy led to a series of 2-thioureidothiophene-3-carboxylates (TUTP) as novel CXCR2 antagonists. Structure-activity relationship study of TUTPs led to the identification of compound 52 that significantly inhibited CXCR2-mediated β-arrestin recruitment signaling (IC50 = 1.1±0.01 μM) with negligible effect on CXCL8-mediated cAMP signaling and calcium flux. Similar to the known CXCR2 antagonist SB265610, compound 52 inhibited CXCL8-CXCR2 induced phosphorylation of ERK1/2. TUTP compounds also inhibited CXCL8-mediated cell migration and showed synergy with doxorubicin in ovarian cancer cells, thereby supporting TUTPs as promising compounds for cancer treatment.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and β-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

The rewarding properties of drugs of abuse are mediated by the mu-opioid receptor (MOR). Genetic variations in MOR and MOR interacting proteins (MORIPs) involved in MOR signaling may increase the risk for drug dependence. The MORIP β-arrestin plays an important role in the regulation of MOR trafficking, thereby highlighting it as a candidate gene for addiction phenotypes. In this case-control association study, DNA samples from cocaine-dependent (n=336) and opioid-dependent (n=335) patients and controls (n=656) were genotyped for seven single nucleotide polymorphisms (rs11868227, rs3786047, rs4522461, rs1045280, rs2271167, rs2036657, and rs4790694) across ARRB2, the gene encoding the β-arrestin 2 protein. No significant differences were observed in genotype or allele frequency between drug-dependent and control individuals for any of the single nucleotide polymorphisms analyzed. Haplotype analysis was similarly negative. Further studies are needed to determine whether variations in ARRB2 (or other MORIPs) are relevant to cocaine or opioid dependence in different ethnic populations or whether they confer a risk that is specific to dependence on other drugs of abuse.

β-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of β-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged β-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of β-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of β-arrestin2-YFP for B2R within endosomes in HEK293 cells. β-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached β-arrestin2-YFP is monitored over time as a measure of the rate exchange of β-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/β-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of β-arrestins in the intracellular trafficking and signaling of GPCRs.

The neuropeptide Y (NPY) family peptides NPY, peptide YY (PYY), and pancreatic polypeptide (PP) bind to four G protein-coupled receptors (GPCRs): Y1, Y2, Y4, and Y5. A key step in the desensitization and internalization of GPCRs is the association of the receptor with beta-arrestins. In the present study, these receptors were analyzed with respect to their ability to interact with GFP2-tagged beta-arrestin 2 using the new bioluminescence resonance energy transfer 2 method. Agonists induced a concentration-dependent association of beta-arrestin 2 with all four receptors. Whereas the Y1 receptor exhibited the highest maximum response and rapid association (t(1/2) = 3.4 min), the maximal signals for the association of Y2 and Y4 receptors were less than half of that of Y1, and the association rates were much slower. Interestingly, when evaluated at the Y4 receptor, the Y4 agonist 1229U91 [(Ile,Glu,Pro,Dpr,Tyr,Arg, Leu,Arg,Try-NH2)-2-cyclic(2,4\'),(2\',4)-diamide] was unable to provoke the same maximal response as human PP, suggesting that 1229U91 is a partial agonist. When stimulated by PYY, the Y5 receptor responded with a t(1/2) of 4.6 min and a maximal response approximately 60% of what was observed with Y1. Because beta-arrestins are key components in GPCR internalization, it is interesting to note that the receptor that is known to internalize rapidly (Y1) exhibits the most rapid association with beta-arrestin 2, whereas the receptor that is known to internalize slowly, or not at all (Y2) associates slowly with beta-arrestin 2.