A new mode of mineralocorticoid receptor antagonism by a potent and selective nonsteroidal molecule

Abstract

Limitations of current steroidal mineralocorticoid receptor (MR) antagonists have stimulated the search for a new generation of molecules. We screened for novel nonsteroidal compounds and identified MR antagonists derived from the chemical class of dihydropyridines. Chemical optimization resulted in BR-4628, which displays high in vitro and in vivo MR potency as well as selectivity with respect to the other steroid hormone receptors and the L-type calcium channel. Biochemical studies demonstrated that BR-4628 forms complexes with MR that do not promote the recruitment of transcriptional co-regulators. Docking experiments, using the crystal structure of the MR ligand-binding domain in an agonist conformation, revealed that BR-4628 accommodates in the MR ligand-binding cavity differently in comparison with the classical steroidal MR antagonists. An alanine scanning mutagenesis approach, based on BR-4628 docking, allowed identifying its anchoring mode within the ligand-binding cavity. Altogether, we propose that BR-4628 is a bulky antagonist that inactivates MR through a passive mechanism. It represents the prototype of a new class of MR antagonists.

FIGURE 1.

Representative inhibition curves of aldosterone-induced GAL4-MR-LBD and GAL4-MR_S810L-LBD activities in response to antagonists. A, CHO-K1 cells stably expressing the GAL4-MR_WT LBD fusion protein and the luciferase reporter gene under the control of a GAL4-responsive element-containing promoter (pFA-luc; Stratagene) were incubated for 6 h with increasing concentrations of BR-4628 (▽), spironolactone (△), or eplerenone ●) in the presence of 10⁻⁹ m aldosterone. B, CHO-K1 cells stably expressing the GAL4-MR_S810L LBD fusion protein were incubated for 6 h with increasing concentrations of BR-4628 alone (▲) or in the presence of 10⁻⁹ m aldosterone (●). The MR_WT and MR_S810L transactivation activities were determined in duplicate from the respective luciferase activities.

FIGURE 2.

Natriuretic in vivo activity of BR-4628 and spironolactone in conscious rats. The increase of the urinary sodium to potassium ratio after oral application of vehicle (V), BR-4628 (1 and 10 mg/kg), and spironolactone (1 and 10 mg/kg) was determined by flame spectroscopy of urine samples after a collection period of 8 h. Each bar represents the mean value of n = 8 animals ± S.E. *, p < 0.05; **, p < 0.01; and ***, p < 0.005 versus vehicle after calculation using Student's t test.

FIGURE 3.

Accommodation mode of BR-4628 within the MR LBD. A, picture showing the minimized complex between BR-4628 (gold) and the MR LBD devoid of the H12 helix (gray) superimposed to the structure of the full-length MR LBD (blue). The ligand cavity volume, as calculated with Voidoo (45), is depicted as a green wire surface. B, overall view of the BR-4628 accommodation within the MR LBD devoid of the H12 helix. Only residues that form critical contacts with the ligand are shown. Hydrogen bound between BR-4628 and the polar residues is depicted as dashed red lines. This figure was produced using DINO.

FIGURE 4.

Transactivation activity of the wild type and mutant MRs. HEK-293T cells transiently expressing the MR, MR_N770A, MR_A773G, MR_Q776A, MR_S810A, MR_S810M, MR_R817A, MR_M852A, MR_C942A, MR_T945A, or MR_A773G/S810M were incubated for 16 h with aldosterone (Aldo), 18-vinyl-4-pregnen-3,18,20-trione (18OVP), or spironolactone (Spiro). The cell extracts were assayed for luciferase and β-galactosidase activities as reported previously (30). The GraphPad Prism software was used for curve fitting and calculation of the EC₅₀ values.

FIGURE 5.

Binding properties of BR-4628 to MR. A, Scatchard plot of the binding of [³H]BR-4628 to MR. The in vitro expressed MR was incubated with [³H]BR-4628 (3 × 10⁻¹⁰ to 3 × 10⁻⁷ m) for 4 h at 4 °C. Bound and unbound ligands were separated by the dextran-charcoal method, the evolution of bound/unbound as a function of the amount bound was plotted, and the K_d value was calculated using the ScatMac program (32). A parallel experiment was performed with a rabbit reticulocyte lysate in which no receptor was expressed. B, dissociation kinetics of [³H]BR-4628 from MR. The in vitro expressed MR was incubated with 10⁻⁸ m [³H]BR-4628 for 4 h at 4 °C and then incubated for various time with 10⁻⁶ m BR-4628. The bound and free ligands were separated by the dextran-charcoal method, and the residual binding was calculated. C, limited proteolysis assays. In vitro expressed [³⁵S]MR was incubated for 10 min at 20 °C with or without 10⁻⁸ m aldosterone or BR-4628 and then for 10 min at 20 °C with trypsin (0–300 μg/ml). The digestion products were analyzed by SDS-PAGE and autoradiographed.

FIGURE 6.

Recruitment of transcriptional co-regulators by MR in mammalian two-hybrid assays. HEK293T cells transiently expressing the fusion proteins VP16-MR and the GAL4 DNA-binding domain fused to the receptor interacting domain of TIF2 (A) or NcoR (B) were incubated in triplicate with ethanol, aldosterone (Aldo, 10⁻¹⁰ to 10⁻⁸ m), spironolactone (Spiro, 10⁻⁸ to 10⁻⁶ m), or BR-4628 (10⁻⁸ to 10⁻⁶ m) alone or with 10⁻⁹ m aldosterone in the presence of 10⁻⁸ to 10⁻⁶ m spironolactone or BR-4628. After harvesting the cells, the luciferase activities were measured and normalized by the values obtained with ethanol. The results are the means ± S.E. of five to six independent experiments. *, p ≤ 0.05; **, p ≤ 0.01.