Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist

Abstract

α_1A-, α_1B-, and α_1D-adrenoceptors (α₁-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α₁-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α₁-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α₁-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α_1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α_1B-AR antagonist that has 10-15-fold selectivity over α_1A-AR and α_1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α_1B-AR and propose the molecular basis of α_1B-AR selectivity, where the nonconserved V197^45.52 residue plays a major role, with contributions from L314^6.55 within the α_1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α_1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α_1B-AR. Cpd1 and Cpd24 represent potential leads for α_1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α₁-ARs.

Figure 1

Validating the α_1B-AR selectivity of Cpd1 in binding and functional assays. (A) Chemical structure of Cpd1. (B) Equilibrium binding of the antagonist QAPB was inhibited by Cpd1 at WT α_1B-AR (white circles) to a 30-fold greater extent than at WT α_1A-AR (black circles) and Δ1–79 α_1D-AR (white squares) in COS-7 cells transiently expressing human receptors at 21 °C. (C–F) Phenylephrine (PhE) concentration response curves measuring intracellular Ca²⁺ mobilization in the absence (red circles) or presence (blue circles) of 500 μM Cpd1 in COS-7 cells stably expressing human WT α_1A-AR (C), WT α_1B-AR (D), WT α_1D-AR (E) or transiently expressing Δ1–79 α_1D-AR (F) at 37 °C. Cpd1 was preincubated with the cells for 30 min before addition of PhE. Testing the antagonistic effects of Cpd1 on PhE-induced contraction of rat isolated (G) mesenteric arteries or (H) abdominal aorta. Points represent the mean ± SE of at least three independent experiments performed in duplicate. Refer to Table S1 for values.

Figure 2

Docking and MD simulation studies of Cpd1 bound to α_1A-AR and α_1B-AR. Alternative views of docked Cpd1 (purple) in homology models of WT α_1A-AR (A & B) and α_1B-AR (C & D). Stability of docked Cpd1 in the MD simulations homology model of WT α_1A-AR (E) and α_1B-AR (F). Cpd1 remained stably bound to α_1B-AR over 400 ns simulations but is not stable in the α_1A-AR. Each simulation was replicated three times.

Figure 3

Validation of the Cpd1 binding pocket in α_1A-AR and α_1B-AR by site-directed mutagenesis. (A) Equilibrium binding of QAPB in the presence of increasing concentrations of Cpd1 at WT α_1A-AR or mutated α_1A-AR (I178V, M292L, and I178V, M292L). (B) Equilibrium binding of QAPB in the presence of increasing concentrations of Cpd1 at WT α_1B-AR or mutated α_1B-AR (V197I, L314M, and V197I, L314M). QAPB binding was inhibited by Cpd1 at WT α_1B-AR, with Cpd1 potency weakened by the presence of the mutations. (C & D) Intracellular Ca²⁺ mobilization assays were used to test the effects of receptor mutations on Cpd1 inhibition of PhE (phenylephrine) binding. Cells were preincubated with Cpd1 for 30 min before addition of an EC₅₀ concentration of PhE to cells expressing α_1A-AR variants (C) or α_1B-AR variants (D). Points represent the mean ± SE of at least three independent experiments performed in duplicate. Refer to Table S1 for values.

Figure 4

SAR screen of Cpd1 analogues and Cpd24 binding profile. QAPB equilibrium binding inhibition profile of Cpd1 and structural analogues tested at (A) WT α_1A-AR or (B) α_1B-AR. QAPB equilibrium binding inhibition profile of Cpd24 was tested at (C) WT α_1A-AR and the α_1A-AR mutants (I178V, M292L, and I178V, M292L) or (D) WT α_1B-AR and the α_1B-AR mutants (V197I, L314M, and V197I, L314M). Points represent the mean ± SE of at least three independent experiments performed in duplicate. Refer to Tables S1 and S2 for values.

Figure 5

Cpd24 docking and MD simulation studies on α_1A-AR and α_1B-AR. Docked structure of Cpd24 (green) in the homology models of (A) WT α_1A-AR and (B) WT α_1B-AR. (C) MD simulations were run on the Cpd24-bound α_1B-AR homology model, revealing that Cpd24 remained stably bound in the α_1B-AR in two out of three replicate 400 ns simulations.