A cell-based, high-throughput homogeneous time-resolved fluorescence assay for the screening of potential κ-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.