A New Mode of Mineralocorticoid Receptor Antagonism by a Potent and Selective Nonsteroidal Molecule

Jérôme Fagart,Alexander Hillisch,Jessica Huyet,Lars Bärfacker,Michel Fay,Ulrich Pleiss,Elisabeth Pook,Stefan Schäfer,Marie-Edith Rafestin-Oblin,Peter Kolkhof

doi:10.1074/jbc.m110.131342

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.

Highlights

Inactivating mineralocorticoid receptor (MR) mutations provoke salt wasting (4 –7), whereas an activating mutation (S810L) has been shown to induce a severe form of early onset hypertension [8]
Transactivation assays using the MR LBD fused to the GAL4 DNA-binding domain revealed that BR-4628 is as potent as spironolactone but significantly more potent than eplerenone (Table 1)
A similar inhibitory hierarchy is observed by using the entire MR, because BR-4628 is characterized by an IC50 of 33.5 nM (Table 2) compared with 50.0 nM for spironolactone and 2000 nM for eplerenone [27]

Summary

Introduction

Inactivating MR mutations provoke salt wasting (4 –7), whereas an activating mutation (S810L) has been shown to induce a severe form of early onset hypertension [8]. Transactivation assays performed with CHO-K1 cells stably expressing the MR LBD revealed that BR-4628 acts as a full MR antagonist.

Results

Conclusion