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

Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.

A structural explanation of the effects of dissociated glucocorticoids on glucocorticoid receptor transactivation.

There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11ß,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.

Mineralocorticoid receptor mutations and a severe recessive pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease of mineralocorticoid resistance characterized by salt wasting and failure to thrive in infancy. Here we describe the first case of a newborn with severe recessive PHA1 caused by two heterozygous mutations in NR3C2, gene coding for the mineralocorticoid receptor (MR). Independent segregation of the mutations occurred in the family, with p.Ser166X being transmitted from the affected father and p.Trp806X from the asymptomatic mother Whereas the truncated MR(166X) protein was degraded, MR(806X) was expressed both at the mRNA and protein level. Functional studies demonstrated that despite its inability to bind aldosterone, MR(806X) had partial ligand-independent transcriptional activity. Partial nuclear localization of MR(806X) in the absence of hormone was identified as a prerequisite to initiate transcription. This exceptional case broadens the spectrum of clinical phenotypes of PHA1 and demonstrates that minimal residual activity of MR is compatible with life. It also suggests that rare hypomorphic NR3C2 alleles may be more common than expected from the prevalence of detected PHA1 cases. This might prove relevant for patient's care in neonatal salt losing disorders and may affect renal salt handling and blood pressure in the general population.

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

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.

Steroid profiling in preeclamptic women: evidence for aromatase deficiency.

OBJECTIVE: Experimental data have revealed the critical role played by 2-methoxy-estradiol, a metabolite of 17ß-estradiol, in the pathophysiology of preeclampsia. We used gas chromatography/mass spectrometry to measure a whole panel of hormonal steroids in the plasma from women during the third trimester of their pregnancy. STUDY DESIGN: The population study consists of 24 pregnant patients with different outcomes: normal, or complicated by isolated preeclampsia or by severe preeclampsia with Hemolysis Enzyme Liver Low Platelets (HELLP) syndrome. RESULTS: 17ß-estradiol was reduced by 50% in isolated preeclampsia, and by 70% in severe preeclampsia with HELLP syndrome (normal: 8.54 ± 0.9 ng/mL; isolated preeclampsia: 4.65 ± 1.0 ng/mL; severe preeclampsia with HELLP syndrome: 2.64 ± 0.4 ng/mL), as is estrone. Downstream, 2-methoxy-estradiol was decreased only in severe preeclampsia with HELLP syndrome. The concentrations of estrone and 17ß-estradiol precursors were comparable between groups, suggesting that placental aromatase is deficient in preeclampsia. CONCLUSION: The gradual decrease of estrogen levels with increasing severity of preeclampsia suggests an impairment of placental steroidogenesis.

Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension.

The S810L mutation within the human mineralocorticoid receptor (MR S810L) induces severe hypertension and switches progesterone from antagonist to agonist. Here we report the crystal structures of the ligand-binding domain of MR S810L in complex with progesterone and deoxycorticosterone, an agonist of both wild-type and mutant MRs. These structures, the first for MR, identify the specific contacts created by Leu810 and clarify the mechanism of activation of MR S810L.

Aldosterone activates NF-kappaB in the collecting duct.

Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-kappaB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-kappaB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-kappaB signaling pathway, leading to increased expression of several NF-kappaB-targeted genes (IkappaBalpha, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1beta, and IL-6). Small interfering RNA-mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal-regulated kinase and p38 kinase, attenuated aldosterone-induced NF-kappaB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-kappaB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-kappaB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-kappaB-induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-kappaB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1.

Met909 plays a key role in the activation of the progesterone receptor and also in the high potency of 13-ethyl progestins.

Many progestins have been developed for use in contraception, menopausal hormone therapy, and treatment of gynecological diseases. They are derived from either progesterone or testosterone, and they act by binding to the progesterone receptor (PR), a hormone-inducible transcription factor belonging to the nuclear receptor superfamily. Unlike mineralocorticoid, glucocorticoid, and androgen receptors, the steroid-receptor contacts that trigger the switch of the ligand-binding domain from an inactive to an active conformation have not yet been identified for the PR. With this aim, we solved the crystal structure of the ligand-binding domain of the human PR complexed with levonorgestrel, a potent testosterone-derived progestin characterized by a 13-ethyl substituent. Via mutagenesis analysis and functional studies, we identified Met909 of the helix 12 as the key residue for PR activation by both testosterone- and progesterone-derived progestins with a 13-methyl or a 13-ethyl substituent. We also showed that Asn719 contributes to PR activation by testosterone-derived progestins only, and that Met759 and Met909 are responsible for the high potency of 19-norprogestins and of 13-ethyl progestins, respectively. Our findings provide a structural guideline for the rational synthesis of potent PR agonist and antagonist ligands that could have therapeutic uses in women's health.

Nestorone® as a Novel Progestin for Nonoral Contraception: Structure-Activity Relationships and Brain Metabolism Studies.

Nestorone® (NES) is a potent nonandrogenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (PR). This was confirmed by docking experiments where NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD, supporting its higher potency than progesterone. The analog 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor LBD, leading to negligible androgen receptor transactivation. Because progesterone acts in the brain by both PR binding and indirectly via binding of the metabolite allopregnanolone to γ-aminobutyric acid type A receptor (GABAAR), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABAAR. In female mice, low concentrations of reduced NES metabolites were identified by gas chromatography/mass spectrometry in both plasma and brain. Electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABAARs of mouse cortical neurons. Thus, the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABAAR suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABAAR.

The severe form of hypertension caused by the activating S810L mutation in the mineralocorticoid receptor is cortisone related.

A gain of function mutation resulting in the substitution of leucine for serine at codon 810 (S810L) in the human mineralocorticoid receptor (MR) is responsible for early-onset hypertension that is exacerbated in pregnancy. All steroids, including progesterone, that display antagonist properties when bound to the wild-type MR are able to activate the mutant receptor (MR(L810)). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MR(L810) carriers during pregnancy. However, the steroid(s) responsible for hypertension in MR(L810) carriers (men and nonpregnant women) has not yet been identified. Here we show that cortisone and 11-dehydrocorticosterone, the main cortisol and corticosterone metabolites produced in the distal nephron, where sodium reabsorption stimulated by aldosterone takes place, bind with high affinity to MR(L810). The potency with which cortisone and 11-dehydrocorticosterone bind to the mutant MR contrasts sharply with their low wild-type MR-binding capacity. In addition, cotransfection assays demonstrate that cortisone and 11-dehydrocorticosterone are potent activators of the MR(L810) trans-activation function. Because the plasma concentration of cortisol in humans is about 30-fold higher than that of corticosterone, these findings strongly suggest that cortisone is one of the endogenous steroids responsible for early-onset hypertension in men and nonpregnant women carrying the MR(L810) mutation.

Identification of steroid ligands able to inactivate the mineralocorticoid receptor harboring the S810L mutation responsible for a severe form of hypertension.

The ability of steroid ligands to inactivate the human mineralocorticoid receptor (MR(WT)) has been shown to be due to their inability to contact Asn770, a residue of the H3 helix involved in stabilizing contacts with the H11-H12 loop region. However, all steroid ligands that display antagonist properties when bound to MR(WT), have been shown to activate a mutant receptor (MR(L810)) associated with a severe form of hypertension. Biochemical studies revealed that S810L mutation induces a change in the receptor conformation and increases the steroid-receptor complexes stability. From a three-dimensional model of the MR ligand-binding domain, it is likely that the S810L mutation causes a steric hindrance between the side chains of Leu810 (H5) and Gln776 (H3) that provokes a bending of the H3 helix. As a consequence, the positioning of MR(WT) antagonists within the ligand-binding cavity is modified in such a way that they can activate the mutant MR(L810). The results from biochemical studies also revealed that 5alpha-pregnan-20-one, 4,9-androstadiene-3,17-dione and RU486, unable to bind MR(WT), acted as potent MR(L810) antagonists.

Glucocorticoid metabolism by 11-beta hydroxysteroid dehydrogenase type 2 modulates human mineralocorticoid receptor transactivation activity.

The mineralocorticoid receptor (MR) binds aldosterone, but also glucocorticoid hormones (corticosterone in rodents, cortisol in humans), which largely prevail in the plasma. To prevent permanent and maximal occupancy of MR by glucocorticoid hormones in aldosterone-target cells, specific effects of aldosterone require metabolism of glucocorticoid hormones into 11-dehydroderivatives by 11-beta hydroxysteroid dehydrogenase (11-HSD2). We analyzed the effect of corticosterone or 11-dehydrocorticosterone (11-DHC) on the transactivation activity of the MR, transiently expressed in a new renal cell line expressing 11-HSD2. We show that, because of its metabolism by 11-HSD2, corticosterone is a poor activator of MR transactivation, except at micromolar concentrations, where the enzyme is saturated. We also show that high micromolar concentrations of 11 DHC are required to activate the MR. The weak antagonist property of 11-DHC on aldosterone-induced hMR transactivations is also documented. Such partial agonist activity of 11-DHC is discussed in the light of its positioning in a three-dimensional model of the MR ligand-binding domain.

Dehydroepiandrosterone and its 7-hydroxylated metabolites do not interfere with the transactivation and cellular trafficking of the glucocorticoid receptor.

The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA. The antiglucocorticoid effects of both 7-hydroxylated metabolites have been investigated with evidence in mice that neither form of DHEA interfered with the binding of GC to its glucocorticoid receptor (GR), but contributed to a decreased nuclear uptake of the activated GR. Our objective was to use COS-7 cell culture to research DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7alpha-hydroxylation of DHEA and the oxidation of cortisol into cortisone. The cDNA of the human GR was inserted into pcDNA3 for a transient transfection of COS-7 cells. Human GR transactivation activity was measured from a luciferase-MMTV reporter gene. The transfected COS-7 cells were cultured using 10(-12) to 10(-5) M dexamethasone (DEX) or cortisol, which triggered the reporter expression. Treatment with 10(-12) to 10(-5) M DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA caused no change in the GC-induced GR transactivation. A reconstruction of the process associated EGFP to the human GR cDNA. Confocal microscopic examination of COS-7 cells transiently expressing the fusion protein EGFP-GR showed nuclear fluorescence 60 min after incubation with 10(-8) M DEX or cortisol. The addition of 10(-5) M DHEA, 7alpha-hydroxy-DHEA or 7beta-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA, in relation to antiglucocorticoid activity. We conclude that direct interference with GR trafficking can be discounted in the case of these hormones, therefore proposing new possibilities of investigation.

A new strategy for selective targeting of progesterone receptor with passive antagonists.

Currently available progesterone (P4) receptor (PR) antagonists, such as mifepristone (RU486), lack specificity and display partial agonist properties, leading to potential drawbacks in their clinical use. Recent x-ray crystallographic studies have identified key contacts involved in the binding of agonists and antagonists with PR opening the way for a new rational strategy for inactivating PR. We report here the synthesis and characterization of a novel class of PR antagonists (APRn) designed from such studies. The lead molecule, the homosteroid APR19, displays in vivo endometrial anti-P4 activity. APR19 inhibits P4-induced PR recruitment and transactivation from synthetic and endogenous gene promoters. Importantly, it exhibits high PR selectivity with respect to other steroid hormone receptors and is devoid of any partial agonist activity on PR target gene transcription. Two-hybrid and immunostaining experiments reveal that APR19-bound PR is unable to interact with either steroid receptor coactivators 1 and 2 (SRC1 and SCR2) or nuclear receptor corepressor (NcoR) and silencing mediator of retinoid acid and thyroid hormone receptor (SMRT), in contrast to RU486-PR complexes. APR19 also inhibits agonist-induced phosphorylation of serine 294 regulating PR transcriptional activity and turnover kinetics. In silico docking studies based on the crystal structure of the PR ligand-binding domain show that, in contrast to P4, APR19 does not establish stabilizing hydrogen bonds with the ligand-binding cavity, resulting in an unstable ligand-receptor complex. Altogether, these properties highly distinguish APR19 from RU486 and likely its derivatives, suggesting that it belongs to a new class of pure antiprogestins that inactivate PR by a passive mechanism. These specific PR antagonists open new perspectives for long-term hormonal therapy.

Structural determinants of ligand binding to the mineralocorticoid receptor.

The first and critical step in the mechanism of aldosterone action is its binding to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Over the last 40 years, numerous studies have attempted to determine the structural determinants of ligand-binding to MR. An initial set of data showed that hsp90 is bound to the receptor via specific regions and maintains it in a ligand-binding competent state. Site-directed mutagenesis and functional studies guided by a 3D model of the MR ligand-binding domain (LBD) made it possible to identify the residues responsible for the high affinity and selectivity for aldosterone, and to characterize the mechanisms of MR activation and inactivation. The recent determination of the X-ray crystal structures of the LBD of the wild-type MR and MR(S810L), which is responsible for a familial form of hypertension, has made it possible to elucidate the peculiar mechanism of activation of MR(S810L) and established a clear structure/activity relationship for steroidal and non-steroidal MR antagonists.

Mineralocorticoid receptor mutations differentially affect individual gene expression profiles in pseudohypoaldosteronism type 1.

CONTEXT: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, is due to inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). OBJECTIVE: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. DESIGN AND METHODS: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotransactivation assays and by measuring the induction of endogenous gene transcription. RESULTS: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wild-type MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression of endogenous sgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. CONCLUSIONS: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease.

Structural basis of spirolactone recognition by the mineralocorticoid receptor.

Spirolactones are potent antagonists of the mineralocorticoid receptor (MR), a ligand-induced transcription factor belonging to the nuclear receptor superfamily. Spirolactones are synthetic molecules characterized by the presence of a C17 gamma-lactone, which is responsible for their antagonist character. They harbor various substituents at several positions of the steroid skeleton that modulate their potency in ways that remain to be determined. This is particularly obvious for C7 substituents. The instability of antagonist-MR complexes makes them difficult to crystallize. We took advantage of the S810L activating mutation in MR (MR(S810L)), which increases the stability of ligand-MR complexes to crystallize the ligand-binding domain (LBD) of MR(S810L) associated with 7alpha-acetylthio-17beta-hydroxy-3-oxopregn-4-en-21-carboxylic acid gamma-lactone (SC9420), a spirolactone with a C7 thioacetyl group. The crystal structure makes it possible to identify the contacts between SC9420 and MR and to elucidate the role of Met852 in the mode of accommodation of the C7 substituent of SC9420. The transactivation activities of MR(S810L/Q776A), MR(S810L/R817A), and MR(S810L/N770A) reveal that the contacts between SC9420 and the Gln776 and Arg817 residues are crucial to maintaining MR(S810L) in its active state, whereas the contact between SC9420 and the Asn770 residue contributes only to the high affinity of SC9420 for MR. Moreover, docking experiments with other C7-substituted spirolactones revealed that the MR(S810L)-activating potency of spirolactones is linked to the ability of their C7 substituent to be accommodated in LBD. It is remarkable that the MR(S810L)-activating and MR(WT)-inactivating 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). Thus, the MR(S810L) structure may provide a powerful tool for designing new, more effective, MR antagonists.

The synthetic androgen methyltrienolone (r1881) acts as a potent antagonist of the mineralocorticoid receptor.

Aldosterone binds to the mineralocorticoid receptor (MR) and exerts fine control over Na+ 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 (R1881>DHT>T). Short-circuit current (Isc) experiments, used to measure transepithelial Na+ transport, revealed that 10(-5) M T and DHT or R1881 prevented the increase in the amiloride-sensitive component of Isc caused by aldosterone in mouse mpkCCDcl4 collecting duct cells partially and totally, respectively. In contrast, androgens had no effect on stimulated Isc elicited by the specific glucocorticoid agonist 11beta,17beta-dihydroxy-17alpha-(1-propynyl) and rost-1,4,6-trien-3-one (RU26988). Docking of steroids within the crystal structure of the ligand-binding domain of MR, together with trans-activation studies, revealed that the contacts between the 17beta-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.

The Met852 residue is a key organizer of the ligand-binding cavity of the human mineralocorticoid receptor.

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 alpha-propyl-17 alpha-hydroxy-3-oxo-preg-4-ene-21-carboxylic acid gamma-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.

11beta-hydroxyprogesterone acts as a mineralocorticoid agonist in stimulating Na+ absorption in mammalian principal cortical collecting duct cells.

The binding of mineralocorticoid hormones to the mineralocorticoid receptor is the first step in a cascade of events leading to the stimulation of Na(+) reabsorption by renal cortical collecting duct (CCD) principal cells. The agonist properties of mineralocorticoid hormones are linked to contacts between their 21-hydroxyl group and Asn770, a residue of the ligand-binding domain of the human mineralocorticoid receptor (hMR). Here, we investigate whether the presence of a hydroxyl group at position 11, 17, or 20 could also alter the activity of progesterone (P), a mineralocorticoid antagonist without the 21-hydroxyl group. Both 17 alpha-hydroxyprogesterone (17OHP) and 20 alpha-hydroxyprogesterone (20OHP) antagonized the aldosterone-induced trans-activation activity (IC(50): 17OHP, 10(-7) M; 20OHP, 10(-8) M) of the hMR transiently expressed in COS-7 cells lacking steroid receptors. In cultured mouse mpkCCD(cl4) principal cells, 17OHP and 20OHP also prevented the aldosterone-stimulated amiloride-sensitive component of the short-circuit current (Ams I(sc)), reflecting Na(+) absorption mediated by the epithelial Na(+) channel (ENaC). In contrast, 11 beta-hydroxyprogesterone (11OHP) activated the transiently expressed hMR in COS-7 cells in a dose-dependent manner (ED(50): 10(-8) M) and, like aldosterone, stimulated Ams I(sc) in mpkCCD(cl4) cells. Docking 11OHP within the hMR-ligand-binding domain homology model revealed that the agonist activity of 11OHP is caused by contacts between its 11 beta-hydroxyl group and Asn770. Furthermore, 11OHP was unable to activate the mutant hMR/N770A, in which Ala is substituted for Asn at position 770. These findings demonstrate that in the absence of the 21-hydroxyl group, the 11 beta-hydroxyl group can produce the contact with the hMR-Asn770 required for the hMR activation leading to stimulated Na(+) absorption.