The Met852 Residue Is a Key Organizer of the Ligand-Binding Cavity of the Human Mineralocorticoid Receptor

Abstract

Spirolactones harboring various C7 substituents are aldosterone antagonists, and some of them are used in the treatment of essential hypertension. They bind to the human mineralocorticoid receptor and render it transcriptionally inactive. Structural analysis using a three-dimensional homology model of the ligand-binding domain of the receptor has revealed that the Met852 residue of the ligand-binding cavity faces the C7 substituent of spirolactones. We therefore tested the binding capacities of C7-substituted spirolactones in an in vitro system expressing either the mutant receptor, in which Met852 was replaced by alanine, or the wild-type receptor. The M852A mutation had almost no effect on the binding of C7-substituted spirolactones to mineralocorticoid receptor but dramatically reduced the capacity of the receptor to bind steroids with no C7 substituent (aldosterone, cortisol, deoxycorticosterone, and canrenone). cis-trans Cotransfection assays revealed that two spirolactones characterized by having a propyl group [7␣-propyl-17␣-hydroxy-3-oxo-preg-4-ene-21-carboxylic acid ␥-lactone (RU26752)] or a thioacetyl group (spironolactone) at the C7 position acquired agonist properties when bound to the mutant receptor. In contrast, mexrenone and eplerenone, both of which harbor an acetyl group at the C7 position, retained antagonist properties when bound to the mutant receptor. Overall, these findings indicate that Met852 acts as an organizer residue that plays two major roles: 1) it allows steroids with no substituent at the C7 position to be accommodated within the ligand-binding cavity; and 2) it is involved in the steric hindrance that prevents C7-substituted spirolactones from folding the receptor in its active state.

Structural Basis of Spirolactone Recognition by the Mineralocorticoid Receptor

“…Thus, all the spirolactones tested activate MR S810L , and their potencies depend on the C7 substituents. It is interesting that the MR S810L -activating and MR WT -inactivating (Fagart et al, 2005a) potencies of the C7-substituted spirolactones follow the same order, suggesting that the C7 substituent is accommodated in the same way in MR S810L and MR WT .…”

“…The expression vector pchMR S810L/N770A was created by cutting out the Bpu1102I-AflII fragment from pchMR S810L and inserting it into pchMR N770A . The plasmid pc␤gal contains the coding sequence for the ␤-galactosidase (Fagart et al, 2005a). The plasmid pFC31Luc contains the MMTV promoter that drives the luciferase gene (Gouilleux et al, 1991).…”

“…Spirolactones are synthetic molecules with a C17 ␥-lactone that is responsible for their antagonist activity and various other substituents on the steroid skeleton that modulate their potency (Corvol et al, 1977;Nickisch et al, 1985;de Gasparo et al, 1987;Elger et al, 2003;Fagart et al, 2005a); this is particularly evident in the case of the C7 substituents. SC9420, a member of the spirolactone family, characterized by the presence of a C7 thioacetyl group, and RU26752, which has a C7 propyl group, more potently inactivate MR WT than mexrenone, which harbors a C7 carboxymethyl ester group, or canrenone, which has no C7 substituent (Fagart et al, 2005a). In this study, we set out to elucidate how the C7 substituent regulates the antagonist potency of spirolactones.…”

“…They improve survival in heart failure, and they have beneficial effects in preventing the development of cardiac fibrosis and renal damage in patients with essential hypertension (Pitt et al, 1999(Pitt et al, , 2003Garthwaite and McMahon, 2004). Spirolactones are synthetic molecules with a C17 ␥-lactone that is responsible for their antagonist activity and various other substituents on the steroid skeleton that modulate their potency (Corvol et al, 1977;Nickisch et al, 1985;de Gasparo et al, 1987;Elger et al, 2003;Fagart et al, 2005a); this is particularly evident in the case of the C7 substituents. SC9420, a member of the spirolactone family, characterized by the presence of a C7 thioacetyl group, and RU26752, which has a C7 propyl group, more potently inactivate MR WT than mexrenone, which harbors a C7 carboxymethyl ester group, or canrenone, which has no C7 substituent (Fagart et al, 2005a).…”

Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1

“…Lysates were diluted 4-fold with TEGWM buffer (20 mM Tris-HCl, 1 mM EDTA, 20 mM sodium tungstate, 1 mM ␤-mercaptoethanol, and 10% glycerol (v/v), pH 7.4) and incubated with 0.3 to 300 nM [ 3 H]finerenone for 4 h at 4°C. Bound and Unbound ligands were separated by the dextran-charcoal method (23). The change in bound/unbound as a function of bound was analyzed, and the K d value was calculated as previously described (30).…”