Fig. 2. Responses of controls in the 384-well formatted hV2R[L⁸³Q] Pharmacoperone Assay.

(a)Comparison of vasopressin titrations in the absence of doxycycline using the heterogenous and no-wash protocols. Note, the shifted EC₅₀ for the no-wash protocol (in the presence of 100 nM SR121463) indicative of the competitive antagonistic properties of this pharmacoperone. (b)Raw luminescence values from a vasopressin titration performed using the no-wash protocol in the presence of 100 nM SR121463 fit to a one site competitive binding model. Literature values (0.94 nM) ¹³ for the K_i of SR121463 were used to perform the fit. The resulting K_d for vasopressin is consistent with the literature value of 1.6 nM ¹⁵. (c) Vasopressin titrations. In the absence of doxycycline (“Dox”), hV2R[L⁸³Q] is synthesized and retained in the ER; a vasopressin challenge yields no cAMP response (triangles). Following pre-treatment with 100 nM of the pharmacoperone SR121463 (“SR”), hV2R[L⁸³Q] is rescued and trafficked to the plasma membrane, and a robust cAMP response to vasopressin challenge is observed (circles). Also included are the results of challenging the cells with vasopressin after preincubation with SR and 1 μg/ml doxycycline (squares). (d) Pharmacoperone titrations. In the absence of Dox, increasing concentrations of pharmacoperone SR121463 increased the amount of hV2R[L⁸³Q] on the surface of the cells, with the result of increasing cAMP responses after challenge with an EC₁₀₀ of vasopressin (circles). An EC₁₀₀ challenge of vasopressin to cells in the absence of doxycycline (squares) or pretreated with 1 μg/ml doxycycline (triangles) results in no appreciable cAMP response. Each dataset is normalized to either the 100% response of either VP (left) or SR (right) dose-response curve.