Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor

Abstract

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

Figure 1

Two partial β2AR agonists with new activating chemotypes in their docked poses. (A) The docked pose of isoproterenol, with a salt-bridge between the amine group and key residue Asp113^3.32 and hydrogen bonds to Ser203^5.42, Ser204^5.43, and Ser207^5.46 in TM5. The co-crystal ligand BI-167107 is shown in black sticks. Previously unreported (B) imidazole, compound 10, and (C) amino-purine, compound 14, polar head groups make activating hydrogen bonds with TM5.

Figure 2

Functional assays for β2AR agonists. Six compounds considerably increased cAMP formation and β-arrestin recruitment, consistent with agonism (compounds 1, 4, 10, 12, 14, and 22 as indicated colors). (A) Dose–respone curves measuring G-protein activation through cAMP formation using the GloSensor assay. (B) Known β2AR agonists used as controls in the GloSensor assay: isoproterenol (ISO, black), epinephrine (Epi, green), hydroxybenzylisoproterenol (HBI, blue), and BI-167107 (BI, red). (C) Dose–response curves measuring β-arrestin recruitment using the β2 V2R Tango assay. For compound 4, a connected line of each data point is presented instead of its dose–response curve since its fitting was not converged. (D) The control β2AR agonists in the Tango assay as described for panel B. Each data point represents mean ± SE obtained from three independent experiments done in duplicates. Dose–response curves for each compound were obtained using the nonlinear iterative curve-fitting computer program Prism.

Figure 3

An additional DiscoveRx PathHunter β-arrestin recruitment assay and bias factors calculated using the operational model. (A) Dose–response curves for the six β2AR agonists discovered in the virtual screen. (B) Control compounds as described for Figure 2B. Each data point represents mean ± SE, and dose–response curves for each compound were obtained from three independent data sets. (C) The bias factors of indicated compounds were calculated from the Tau value analysis by the Operational Model using the data sets in Figure 2 and panels A and B of this figure, as well as the binding affinity values obtained in Supplementary Figure S2. Each bar represents mean ± SE. The statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the reference value of isoproterenol (ISO).

Figure 4

Functional assays for DRD2 agonists and inverse-agonists. (A) Two compounds (29 and 33, green and blue squares, respectively) activated Gi in GloSensor assays, consistent with partial agonism. QUI is the known agonist quinpirole (black circle). (B) The GloSensor assay was run in inverse-agonist mode with addition of 100 nM quinpirole. One compound (38, red square) inhibited Gi activation. HAL is the known inhibitor haloperidol (black diamond).