The Synthetic Androgen Methyltrienolone (R1881) Acts as a Potent Antagonist of the Mineralocorticoid Receptor

Abstract

Aldosterone binds to the mineralocorticoid receptor (MR) and exerts fine control over Na ϩ absorption in renal collecting duct cells (CCDs). Many natural and synthetic steroids can also bind to the MR to produce agonist or antagonist effects. Here, we investigate whether androgenic hormones act as MR agonist or antagonist ligands in CCDs. Testosterone (T), dihydrotestosterone (DHT), and methyltrienolone (R1881), a synthetic androgen agonist, all bind to the MR. R1881 displayed the same affinity for MR as aldosterone. Androgens did not activate the MR transiently expressed in human embryonic kidney 293T cells but did antagonize aldosterone-induced MR trans-activation activity (R1881ϾDHTϾT). Short-circuit current (I sc ) experiments, used to measure transepithelial Na ϩ transport, revealed that 10 Ϫ5 M T and DHT or R1881 prevented the increase in the amiloride-sensitive component of I sc caused by aldosterone in mouse mpkCCD cl4 collecting duct cells partially and totally, respectively. In contrast, androgens had no effect on stimulated I sc elicited by the specific glucocorticoid agonist 11␤,17␤-dihydroxy-17␣-(1-propynyl)androst-1,4,6-trien-3-one (RU26988). Docking of steroids within the crystal structure of the ligandbinding domain of MR, together with trans-activation studies, revealed that the contacts between the 17␤-hydroxyl group of androgens and the Asn770, Cys942, and Thr945 residues of the ligand-binding cavity stabilize ligand binding complexes but are not strong enough to keep the receptor in its active state. Altogether, these findings indicate that androgen ligands, particularly R1881, act as MR antagonists in aldosterone target cells and provide new insights into the requirements for MR activation to occur and for the designing of new selective MR antagonists.

Compound Profiling Using a Panel of Steroid Hormone Receptor Cell-Based Assays

“…13 There were also differences in potencies in some of the estrogenic and antimineralocorticoid compounds, though importantly, the proper rank order potency was preserved between different assay formats. 14,18 The reasons for these discrepancies are not always clearly known. We speculate that various cellular backgrounds for different types of assays could lead to some differences in the activities of certain compounds.…”

“…[13][14][15] All 6 assays demonstrated good assay windows and good Z′ values ( Table 1), indicating that these assays are suitable for high-throughput profiling.…”

A High-Content Glucocorticoid Receptor Translocation Assay for Compound Mechanism-of-Action Evaluation

“…Recently, R1881 was observed to have potent antagonist activity for MR (0.75 nM) by antagonizing the aldosterone-induced MR transactivation activity. 41 Aldosterone has high affinity for GR translocation (36.3 nM) and no activity on AR translocation. 42 Nuclear translocation in response to Dex for GR was consistent with previous results, but Dex was ineffective at promoting AR nuclear translocation.…”

Cell models for studying renal physiology

“…Fibroblastoid African green monkey kidney COS-7 and Vero cells, and human embryonic HEK293 cells, the origin of which remains uncertain, are also powerful cell systems for use in transient transfection studies. For example, COS-7 cells lacking steroid receptors and HEK 293T cells have been transfected with the human glucocorticoid receptor, or wild-type and mutants of the human mineralocorticoid receptor, to analyze mineralocorticoid or glucocorticoid agonist and antagonist properties of progesterone and its derivatives, dehydroepiandrosterone and its 7-hydroxylated metabolites, or natural and synthetic androgenic hormones [82,105,131].…”