A cell-based,high-throughput homogeneous time- resolved fluorescence assay for the screening of potential K-opioid receptor agonists

Aim:

The aim of this study was to identify κ-opioid receptor (KOR) agonists from a library of 80 000 small-molecule compounds and provide the experimental basis for the development of new analgesic candidates.

Methods:

The cell-based, high-throughput screen for human KOR agonists was based on the LANCE^™ cAMP assay. Preliminary structure-activity relationship (SAR) analysis was applied according to the compounds'' structures. An acetic acid twisting experiment was used to verify the pharmacodynamics.

Results:

In total, 31 compounds were identified as KOR agonists after preliminary and secondary screening. Of these compounds, five demonstrated significant KOR-stimulating activity that was comparable to U-50,488, a selective KOR agonist. The EC₅₀ values for I-7, I-8, I-10, II-5, and II-8 were 13.34±1.65, 14.01±1.84, 9.57±0.19, 14.94±0.64, and 8.74±0.72 nmol/L, respectively. Based on SAR studies, the stimulating activity of compounds with 5-phenyl-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo [1, 5-a] pyrimidine (group I) and 3,4-dimethoxy-N-(2-oxoethyl)-N-p-tolylbenzenesulfonamide (group II) parent structures were higher than the compound with a 5-hydroxy-2-methylbenzofuran-3-carboxylic acid (group III) parent structure. Pharmacodynamic experiments indicated that 20–40 μg/kg ip of compounds I-10 and II-8 significantly decreased the number of writhes induced by acetic acid; this finding is consistent with the SAR studies. Furthermore, the analgesic effects of compounds I-10 and II-8 were significantly antagonized in the presence of the selective KOR antagonist nor-BNI.

Conclusion:

These findings collectively indicate that compounds I-10 and II-8 exhibit significant analgesic activities, providing evidence, at least in part, for their clinical application as new analgesic drugs.