Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands

Abstract

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and β-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-β-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3–10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

In vitro pharmacological characterization of standard and new lysophosphatidic acid receptor antagonists using dynamic mass redistribution assay

“…DMR assay allows the system to reach equilibrium and offers a more precise instrument than calcium mobilization assay for qualitative definition of the type of antagonism exerted. Of note, similar differences between calcium mobilization and DMR have previously been reported for other antagonists and GPCRs, namely, nor-binaltorphimine at the kappa receptor ( Sturaro et al, 2022 ) and [ t Bu-D-Gly 5 ]NPS at the neuropeptide S receptor ( Ruzza et al, 2012 ; Ruzza et al, 2018) . Importantly, the use in the present study of a single antagonist concentration strongly limits our ability to firmly define the nature of the antagonism exerted.…”

Functional selectivity of EM-2 analogs at the mu-opioid receptor

“…With the BRET assay for mu receptor-transducer interaction, an already well-established readout for distinguishing G protein vs. βarrestin coupling at several GPCRs, important pharmacological aspects of the developed analogs could be assessed (Malfacini et al, 2015;Piekielna-Ciesielska et al, 2018;Sturaro et al, 2022). Importantly, calcium mobilization and BRET mu-G protein interaction assays nicely correlated, in terms of both potency and efficacy (Supplementary Figure S6), thus confirming their value for investigating the pharmacological profile of novel ligands at the mu opioid receptor.…”

Decoding the κ Opioid Receptor (KOR): Advancements in Structural Understanding and Implications for Opioid Analgesic Development

“…In particular, MOR agonists induce euphoria and enhance stress management, KOR agonists evoke dysphoria, stress-related reactions, and negative emotional states, whereas DOR agonists alleviate anxiety and foster positive emotional states [34,35]. Extensive studies have been conducted to explore the biology, biochemistry, pharmacology, and mechanism of action of opioid receptors, aiming to deepen our understanding of these receptors [36][37][38][39][40][41][42][43][44][45][46][47]. This knowledge is crucial not only for addressing the opioid crisis but also for the development of novel therapeutic agents geared towards more effective pain management.…”

“…This knowledge is crucial not only for addressing the opioid crisis but also for the development of novel therapeutic agents geared towards more effective pain management. While progress has been made in the development of Extensive studies have been conducted to explore the biology, biochemistry, pharmacology, and mechanism of action of opioid receptors, aiming to deepen our understanding of these receptors [36][37][38][39][40][41][42][43][44][45][46][47]. This knowledge is crucial not only for addressing the opioid crisis but also for the development of novel therapeutic agents geared towards more effective pain management.…”

“…Extensive studies have been conducted to explore the biology, biochemistry, pharmacology, and mechanism of action of opioid receptors, aiming to deepen our understanding of these receptors [ 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. This knowledge is crucial not only for addressing the opioid crisis but also for the development of novel therapeutic agents geared towards more effective pain management.…”