作者: Kendall, David ; Palandri, Josephine ; Starke, Ingemar ; von Mentzer, Bengt ; Asghar, Junaid ; Henderson, Graeme ; Borbely, Eva ; Conibear, Alexandra E ; Hill, Rob ; Kelly, Eamonn ; Tekus, Valeria ; Helyes, Zsuzsanna ; Bailey, Chris

Agonists at the δ opioid receptor are known to be potent antihyperalgesics in chronic pain models and effective in models of anxiety and depression. However, some δ opioid agonists have proconvulsant properties while tolerance to the therapeutic effects can develop. Previous evidence indicates that different agonists acting at the δ opioid receptor differentially engage signaling and regulatory pathways with significant effects on behavioral outcomes. As such, interest is now growing in the development of biased agonists as a potential means to target specific signaling pathways and potentially improve the therapeutic profile of δ opioid agonists. Here, we report on PN6047 (3-[[4-(dimethylcarbamoyl)phenyl]-[1-(thiazol-5-ylmethyl)-4-piperidylidene]methyl]benzamide), a novel G protein-biased and selective δ opioid agonist. In cell-based assays, PN6047 fully engages G protein signaling but is a partial agonist in both the arrestin recruitment and internalization assays. PN6047 is effective in rodent models of chronic pain but shows no detectable analgesic tolerance following prolonged treatment. In addition, PN6047 exhibited antidepressant-like activity in the forced swim test, and importantly, the drug had no effect on chemically induced seizures. PN6047 did not exhibit reward-like properties in the conditioned place preference test or induce respiratory depression. Thus, δ opioid ligands with limited arrestin signaling such as PN6047 may be therapeutically beneficial in the treatment of chronic pain states. SIGNIFICANCE STATEMENT: PN6047 (3-[[4-(dimethylcarbamoyl)phenyl]-[1-(thiazol-5-ylmethyl)-4-piperidylidene]methyl]benzamide) is a selective, G protein-biased δ opioid agonist with efficacy in preclinical models of chronic pain. No analgesic tolerance was observed after prolonged treatment, and PN6047 does not display proconvulsant activity or other opioid-mediated adverse effects. Our data suggest that δ opioid ligands with limited arrestin signaling will be beneficial in the treatment of chronic pain.

Frontiers in Pharmacology

Development of a novel cell-based, In-Cell Western/ERK assay system for the high-throughput screening of agonists acting on the delta-opioid receptor

Article

作者: Alexander, Stephen P H ; Asghar, Junaid ; Latif, Liaque ; Kendall, David A

Background: Extracellular signal-regulated kinases (ERKs) are important signaling mediators in mammalian cells and, as a result, one of the major areas of research focus. The detection and quantification of ERK phosphorylation as an index of activation is normally conducted using immunoblotting, which does not allow high-throughput drug screening. Plate-based immunocytochemical assays provide a cheaper and relatively high-throughput alternative method for quantifying ERK phosphorylation. Here, we present optimization steps aimed to increase assay sensitivity and reduce variance and cost using the LI-COR In-Cell Western (I-CW) system in a recombinant CHO-K1 cell line, over-expressing the human delta-opioid receptor (hDOPr) as a model.Methods: Cells cultured in 96-well microassay plates were stimulated with three standard/selective DOPr agonists (SNC80, ADL5859, and DADLE) and a novel selective DOPr agonist (PN6047) to elicit a phospho-ERK response as an index of activation. A number of experimental conditions were investigated during the assay development.Key results: Preliminary experiments revealed a clearly visible edge-effect which significantly increased assay variance across the plate and which was reduced by pre-incubation for 30 min at room temperature. ERK phosphorylation was detectable as early as 1 min after agonist addition, with a distinct peak at 3–5 min. Optimization of the cell seeding densities showed that 25,000 cells per well have the lowest basal phospho-ERK response and an optimal agonist ERK1/2 signal. Pre-incubation with apyrase (an ATPase) did not reduce the basal or agonist responses. All agonists produced concentration-dependent increases in phospho-ERK activation, and pertussis toxin was able to attenuate these ERK responses. Naltrindole, which is a selective DOPr antagonist, was able to antagonize the DOPr-mediated ERK activation of the ligands.Conclusion: We have developed an optimization protocol and highlighted a number of considerations when performing this high-throughput fluorescence immunocytochemical (ICC) assay measuring ERK phosphorylation in the human DOPr. The optimized protocol was found to be a more conducive option for the screening of delta agonists. This provides a basis for additional assay development to investigate opioid pharmacology. This protocol should be widely applicable for measuring ERK phosphorylation in any cell line and investigating other protein targets in GPCR drug discovery.

100 项与 PN-6047 相关的药物交易

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