Calculation of Bias Factor.

Bias factors were calculated using the method previously described (Kenakin, 2017). Briefly, Log(E_max/EC₅₀) values for both GloSensor and Tango assays were separately generated for each ligand using potency (EC₅₀) and maximal efficacy (E_max) values obtained from these assays. ΔLog(E_max/EC₅₀) was calculated in each assay as the difference in Log(E_max/EC₅₀) for each ligand from the reference ligand (the unbiased KOR agonist U50,488). ΔΔLog(E_max/EC₅₀) was then calculated as the difference in ΔLog(E_max/EC₅₀) between GloSensor and Tango assays for each ligand. The 95% confidence interval (CI) for each bias factor was calculated using the E_max and EC₅₀ values generated in individual experiments for each ligand in each assay.