[35S]GTPɣS binding assay

Binding of [³⁵S]GTPγS to membranes from CHO cells stably expressing the human MOR (CHO-hMOR) was conducted according to a previously published procedure²⁶. Cell membranes (5–10 µg in 20 mM HEPES, 10 mM MgCl₂, and 100 mM NaCl, pH 7.4) were incubated with 0.05 nM [³⁵S]GTPγS, 10 µM GDP, and test compounds in a final volume of 1 ml for 60 min at 25 °C. Non-specific binding was determined using 10 µM GTPγS, and the basal binding was determined in the absence of test ligand. Samples were filtered over glass Whatman glass GF/B fiber filters and counted as described for binding assays. The increase in [³⁵S]GTPγS binding above the basal activity was used to determine potency (EC₅₀, in nM) and efficacy (as % stimulation of maximum stimulation with respect to the reference MOR full agonist, DAMGO, which was set as 100%), from concentration–response curves by nonlinear regression analysis. All experiments were performed in duplicate and repeated at least three times with independently prepared samples.