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

A New Mode of Mineralocorticoid Receptor Antagonism by a Potent and Selective Nonsteroidal... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 39, pp. 29932–29940, September 24, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A New Mode of Mineralocorticoid Receptor Antagonism by a □ S Potent and Selective Nonsteroidal Molecule Received for publication, April 7, 2010, and in revised form, July 22, 2010 Published, JBC Papers in Press, July 22, 2010, DOI 10.1074/jbc.M110.131342 ‡§1 ¶ ‡§ ¶ ‡§ Je´roˆ me Fagart , Alexander Hillisch , Jessica Huyet , Lars Ba¨rfacker , Michel Fay , Ulrich Pleiss , ‡‡2 ‡§ ‡‡3 Elisabeth Pook**, Stefan Scha¨fer , Marie-Edith Rafestin-Oblin , and Peter Kolkhof ¶  ‡‡ From the Departments of Medicinal Chemistry, Isotope Chemistry, **Lead Discovery, and Cardiology Research, Bayer Schering Pharma AG, Global Drug Discovery, 42096 Wuppertal, Germany, INSERM U773, Centre de Recherche Biome´dicale Bichat-Beaujon, CRB3, 75870 Paris, France, and the Universite´ Paris 7-Denis Diderot, Site Bichat, 75018 Paris, France Limitations of current steroidal mineralocorticoid receptor inactivating MR mutations provoke salt wasting (4–7), whereas (MR) antagonists have stimulated the search for a new genera- an activating mutation (S810L) has been shown to induce a tion of molecules. We screened for novel nonsteroidal com- severe form of early onset hypertension (8). In addition to its pounds and identified MR antagonists derived from the chemi- renal effects, aldosterone acts in nonepithelial tissues such as cal class of dihydropyridines. Chemical optimization resulted in brain, vasculature, and heart, in which it has deleterious effects. BR-4628, which displays high in vitro and in vivo MR potency as In the cardiovascular system, aldosterone provokes inflamma- well as selectivity with respect to the other steroid hormone tion and fibrosis, combined with ventricular hypertrophy (9). receptors and the L-type calcium channel. Biochemical studies Whether these adverse effects are mediated by aldosterone demonstrated that BR-4628 forms complexes with MR that do and/or cortisol at conditions of inappropriate salt or redox sta- not promote the recruitment of transcriptional co-regulators. tus has not yet been established (10). Docking experiments, using the crystal structure of the MR Spironolactone and eplerenone, both of which display struc- ligand-binding domain in an agonist conformation, revealed tural elements of progesterone, have been developed as MR that BR-4628 accommodates in the MR ligand-binding cavity antagonists (11–14). These molecules inhibit aldosterone bind- differently in comparison with the classical steroidal MR antag- ing to MR and render it transcriptionally inactive. As a conse- onists. An alanine scanning mutagenesis approach, based on quence, they prevent the aldosterone-induced sodium reab- BR-4628 docking, allowed identifying its anchoring mode sorption and are effective in decreasing blood pressure (15, 16). within the ligand-binding cavity. Altogether, we propose that Remarkably, these molecules have been shown to reduce the BR-4628 is a bulky antagonist that inactivates MR through a deleterious aldosterone effects in the cardiovascular system. In passive mechanism. It represents the prototype of a new class of the randomized aldactone evaluation study (RALES) and MR antagonists. eplerenone post-acute myocardial infarction heart failure effi- cacy and survival study (EPHESUS), spironolactone and eplerenone, respectively, significantly reduced mortality and The first and best documented effects of aldosterone are morbidity in patients with heart failure (17, 18). those observed on the kidney distal tubule. In this epithelial Despite their renal and cardiac benefits, these two steroidal spirolactones suffer from substantial drawbacks that limit their tissue, aldosterone promotes sodium reabsorption and potas- sium secretion, increasing the blood pressure by expanding the clinical use. Although spironolactone is highly potent, it lacks extracellular volume (1, 2). These aldosterone effects are medi- selectivity toward other members of the oxo-steroid receptor ated by the mineralocorticoid receptor (MR), a ligand-acti- family. Indeed, its prolonged use is associated with sexual vated transcription factor belonging to the nuclear receptor side effects that are related to its progestogenic and antian- superfamily (3). According to the aldosterone renal effects, drogenic activities (19). Eplerenone is characterized by an improved selectivity as compared with spironolactone. However, eplerenone has a low affinity for MR (20) and is * Portions of these results have been presented at the Congress of the Euro- less efficient than spironolactone with respect to blood pean Society of Cardiology in Vienna in 2007 and at the German Society of pressure lowering in patients with mild-to-moderate hyper- Cardiology Congress in 2007 in Mannheim. □ S tension (21). In addition, both steroidal molecules are not The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Video S1, and Figs. S1–S3. only unable to block the constitutive activity of the mutant Present address: INSERM U693, 53 rue Gabriel Pe´ri, 94270 Le Kremlin-Biceˆ- MR , a gain-of function mutation that is linked clini- S810L tre, France. cally to early onset hypertension in men and gestational Present address: Synthon, Microweg 22, 6545 CM Nijmegen, The Netherlands. hypertension in women, but paradoxically activate this To whom correspondence should be addressed: Cardiology Research, Bayer mutant receptor (8, 22). Therefore, the use of spirolactones Schering Pharma AG, Global Drug Discovery, Aprather Weg 18a, 42096, to treat hypertensive patients carrying the S810L mutation is Wuppertal, Germany. Fax: 49-202-36-8009; E-mail: Peter.Kolkhof@ bayerhealthcare.com. inappropriate. The abbreviations used are: MR, mineralocorticoid receptor; AR, androgen The known disadvantages of spirolactones stimulated receptor; GR, glucocorticoid receptor; PR, progesterone receptor; TIF2, research for new, selective MR antagonists (23). An ultrahigh transcriptional intermediary factor 2; NCoR, nuclear receptor co-repressor 1; LBD, ligand-binding domain; DHP, dihydropyridine. throughput screening revealed substituted dihydropyridines 29932 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 This is an Open Access article under the CC BY license. A Potent and Selective Nonsteroidal MR Antagonist (DHP) as novel nonsteroidal MR antagonists. Chemical optimi- compounds were given in eight dilutions to the cells followed by zation of these DHP lead compounds resulted in BR-4628. Here the relevant EC concentration of each agonist. After an incu- we present the in vitro and in vivo pharmacological character- bation time of 5–6 h, luciferase activity was determined using a ization of BR-4628, together with studies that highlight the luminescence detecting video camera system. The GraphPad binding mode on MR and the molecular reasons for the distinct Prism software (version 3.02; GraphPad Software Inc., San antagonism. Diego, CA) was used for curve fitting and calculation of the IC and EC values. The IC and EC values from the luciferase 50 50 50 EXPERIMENTAL PROCEDURES assay were determined in at least three independent experi- Compounds—BR-4628 and [ H]BR-4628 were synthesized as ments performed in duplicate. The IC and EC values are 50 50 described in the supplemental data. Aldosterone and spirono- given as the means  S.E. lactone were purchased from Sigma. Eplerenone was purified Transactivation Assays in Transiently Transfected Cells— from commercially available tablets (Inspra). Nitrendipine HEK-293T cells were cultured and transfected with the ex- was obtained by Hantzsch dihydropyridine synthesis using pression vectors pchMR, pchMRN770A, pchMRA773G, classical published methods (24). 18-Oxo-18-vinylprogester- pchMRQ776A, pchMRS810A, pchMRS810M, pchMRR817A, one (18-vinyl-4-pregnen-3,18,20-trione) was a gift from A. pchMRM852A, pchMRC942A, pchMRT945A, or pchMRA773G/ Marquet (Paris, France). S810M, the reporter vector pFC31Luc, and the pcgal vector Expression Vectors—The expression vectors pchMR, according to the method described previously (30). 24 h after pchMRN770A, pchMRA773G, pchMRQ776A, pchMRR817A, transfection, ligands were added, and after a 16-h incubation, pchMRM852A, pchMRC942A, and pchMRT945A code for the cell extracts were assayed for luciferase and -galactosidase MR, MR ,MR ,MR ,MR MR , N770A A773G Q776A R817A M852A activities as reported previously (30). The GraphPad Prism soft- MR , and MR , respectively (25–28). The pchMRS810A, C942A T945A ware (version 3.02; GraphPad Software Inc., San Diego, CA) pchMRS810M, and pchMRA773G/S810M coding for was used for curve fitting and calculation of the IC and EC 50 50 MR ,MR , and MR , respectively, were S810A S810M A773G/S810M values. The IC values were determined from at least three obtained from pchMR using the site-directed mutagenesis pro- independent experiments performed in triplicate. The IC val- cedure (QuikChange; Agilent, Massy, France). The plasmid ues are given as the means  S.E. The EC values were calcu- pFC31Luc contains the murine mammary tumor virus pro- lated from one experiment performed in triplicate. moter that drives the luciferase gene (29). The coding Mammalian Two-hybrid Assays—HEK 293T cells were sequences of the respective LBDs of MR, MR , AR, GR, and S810L transfected with 2 g of pVPMR, pGALTIF2, or pGALNcoR; PR have been PCR-amplified and fused to the coding sequence 5 g of pg5luc; and 1 gofpcgal. 24 h after transfection, of the DNA-binding domain of GAL4 (amino acids 1–147) 10 8 aldosterone (10 to 10 M) or spironolactone or BR-4628 under the control of a CMV promoter, leading to the expression 8 6 (10 to 10 M) were added. Parallel experiments were per- plasmids pGAL4-MR, pGAL4-MR , pGAL4-GR, pGAL4- S810L 9 8 formed with 10 M aldosterone in the presence of 10 to AR, and pGAL4-PR. The pVPMR (kindly provided by Dr. G. 10 M spironolactone or BR-4628. After 16 h, the cell Pinon) codes for the fusion protein between the VP16 activat- extracts were assayed for luciferase and -galactosidase as ing domain and the full-length MR. The pGALTIF2 and reported previously (30). pGALNcoR vectors, which code for fusion protein between the Animals—Male Wistar rats (Charles River Germany, 300 GAL4 DNA-binding domain and the nuclear activating domain g) were used in the experiment. They were housed with free of TIF2 and NCoR, respectively, were kindly provided by Dr. P. access to food and water and maintained on a light-dark cycle at Balaguer. The pG5luc (kindly provided by Prof. P. Fuller) 22–24 °C. All of the animal experiments were conducted in contains the luciferase gene driven by a GAL4-responsive accordance with the National Institutes of Health Guide for the promoter. Care and Use of Laboratory Animals and German legislation on Generation of Stable Steroid Hormone Receptor Cell Lines— animal welfare. The pGAL4-MR, pGAL4-MR , pGAL4-GR, pGAL4-AR, S810L Model of Acute Natriuresis—A similar protocol as published and pGAL4-PR vectors were transfected into CHO-K1 cells previously (31) was used. Briefly, after 7 days of acclimatization, stably expressing a thymidine kinase promoter construct con- male Wistar rats were placed on a low salt diet containing taining five GAL4-binding elements in front of the firefly lucif- 0.02% (w/v) sodium chloride (S0602-E081; ssniff Spezial- erase gene. For each receptor a stable cell line has been gener- dia¨ten GmbH, Soest, Germany) for 72 h. BR-4628 and spi- ated by several rounds of limiting dilutions. ronolactone were administered in 1 ml/kg of vehicle Transactivation Assays in Stable Cell Lines—The stable MR, (PEG400, 85.8%; glycerine, 5.3%, water, 8.9%, v/v/v) by oral MR , GR, AR, and PR cell lines were cultured at 37 °C and S810L gavage, and the animals (n  8/group) were placed in meta- 5% CO in DMEM/Ham’s F-12 medium with GlutaMAX sup- bolic cages (Tecniplast Deutschland GmbH, Hohenpeissen- plemented with 10% (v/v) inactivated fetal calf serum, 20 mM berg, Germany) for8hon water ad libitum. The urine sam- HEPES, 1.4 mM sodium pyruvate, 1.8 mM sodium bicarbonate, ples were analyzed for volume as well as sodium and and 1 mg/ml Geneticin. Subconfluent cultures were passaged using Accutase. All of the cell culture reagents were obtained potassium concentrations by flame spectroscopy. from Invitrogen. The cells were seeded 24 h before testing in Coupled Cell-free Transcription and Translation—The Optimem medium containing 2.5% FCS (v/v), 2 mM glutamine, human MR was expressed in vitro in the rabbit reticulocyte and 10 mM HEPES in 96- or 384-well plates. On the test day lysate system as described previously (27). SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29933 A Potent and Selective Nonsteroidal MR Antagonist TABLE 1 Half-maximally inhibitory/effective concentrations of spironolactone, eplerenone, nitrendipine, and BR-4628 determined for the oxo-steroid receptor and for the L-type calcium channel The IC or EC values (nM) are the means  S.E. of at least four independent experiments, performed in duplicate. The IC values at the L-type calcium channel were 50 50 50 determined in two independent experiments, performed in duplicate using homogenates of rat cerebral cortex as described (46). BR-4628 Binding Characteristics at Equilibrium—The ly- using the program Desmond was run (for details, see the sates containing the in vitro expressed MR were diluted 4-fold supplemental text). with TEGWM buffer (20 mM Tris-HCl, 1 mM EDTA, 20 mM sodium tungstate, 1 mM -mercaptoethanol, and 10% glycerol RESULTS (v/v), pH 7.4) and incubated with 0.3–300 nM [ H]BR-4628 for BR-4628 Is a Potent and Selective MR Antagonist—An ultra- 4 h at 4 °C. Bound and unbound ligands were separated by the high throughput screening was performed with a cellular trans- dextran-charcoal method (27). The change in bound/unbound activation assay based on a recombinant CHO cell line stably as a function of the amount bound was analyzed, and the K expressing the MR LBD. Screening of almost 1,000,000 com- value was calculated as described previously (32). A parallel pounds revealed 677 confirmed primary hits. Approximately experiment was performed with an untranslated rabbit reticu- 300 screening hits were selected for further evaluation after locyte lysate. elimination of toxic, unspecific, and noncompetitive com- Kinetic Experiments—The lysates containing the in vitro pounds. Among them, a single cluster of 100 compounds expressed MR were 4-fold diluted with TEGWM buffer and comprised dihydropyridines (WO2007/025604) (33). This incubated with 10 nM [ H]BR-4628 for4hat4 °C.One half of finding was surprising because dihydropyridines constitute the the labeled lysate was kept at 4 °C and was used to determine well known class of L-type calcium channel antagonists. Chem- the stability of the [ H]BR-4628MR complexes, and the other ical optimization with respect to potency, selectivity, and met- half was incubated with 1 M BR-4628 for various periods. abolic stability lead to the synthesis of BR-4628 as a drug can- Bound and free ligands were separated using charcoal-dextran. didate (see formula in Table 1). The findings were corrected for receptor stability and were Transactivation assays performed with CHO-K1 cells stably expressed as percentages of the binding measured at time 0. expressing the MR LBD revealed that BR-4628 acts as a full MR Dissociation was represented in a semi-logarithmic scale giving antagonist. It inactivates the aldosterone-induced MR-LBD a linear manner representation. 35 transactivation activity with an IC of 27.9 nM, a value compa- Limited Proteolysis Assays—In vitro expressed [ S]MR was rable with that of spironolactone (24.2 nM) but much lower than incubated for 10 min at 20 °C with or without aldosterone or that of eplerenone (990 nM) (Fig. 1A and Table 1). Furthermore, BR-4628 (10 M) and then for 10 min at 20 °C with increasing its antagonist potency is 2 orders of magnitude higher than that concentrations of trypsin (0–300 g/ml). The digestion prod- of nitrendipine, the well known dihydropyridine-based calcium ucts were analyzed by SDS-PAGE and autoradiographed. channel blocker (Table 1). Spironolactone and eplerenone lose Protein Modeling, Docking, and Molecular Dynamics Sim- their antagonist feature upon the S810L mutation (8, 22, 30). ulations—The x-ray structure of the wild type MR LBD com- Thus, we wondered whether BR-4628 still acts as an antagonist plexed with deoxycorticosterone (Protein Data Bank code when bound to the MR . CHO-K1 cells stably expressing 2ABI) (30) served as a model for docking of BR-4628. The size of S810L the MR LBD are characterized by a weak constitutive the binding site was enlarged by modifying several side chain S810L activity (5-fold increase in the base-line luciferase activity; Fig. conformations. The program Glide SP (version 3.5) was used to 1B). Remarkably, BR-4628 inhibits the constitutive MR dock BR-4628 into the resulting binding cleft. The complex S810L LBD activity (IC , 3630 nM) and the aldosterone-induced with the most probable binding pose was energy-minimized MR LBD activity (IC , 813 nM; Fig. 1B) in a dose-depen- using the OPLS-AA/L force field. To check for the stability of S810L 50 dent manner. Thus, BR-4628 is a potent MR antagonist that hydrogen bonds or alternative contacts, a 4-ns molecular dynamics simulation with 11,522 explicit water molecules retains its antagonist character at the MR mutant. S810L 29934 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist 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. potassium concentrations were measured in the collected urine. BR-4628 administration increased the urinary sodium/ potassium ratio in a dose-dependent manner with a significant effect for a dose as low as 1 mg/kg (Fig. 2). For comparison, spironolactone was found to increase the Na /K at a dose of 10 mg/kg (Fig. 2). FIGURE 1. Representative inhibition curves of aldosterone-induced Docking of BR-4628 within the MR LBD Reveals an Unusual GAL4-MR-LBD and GAL4-MR -LBD activities in response to antago- S810L nists. A, CHO-K1 cells stably expressing the GAL4-MR LBD fusion protein WT Anchoring Mode—The crystal structures of the LBD of MR and and the luciferase reporter gene under the control of a GAL4-responsive ele- MR complexed with steroidal agonists have been recently S810L ment-containing promoter (pFA-luc; Stratagene) were incubated for 6 h with increasing concentrations of BR-4628 (ƒ), spironolactone (‚), or eplerenone solved, allowing their anchoring mode to be studied in detail F) in the presence of 10 M aldosterone. B, CHO-K1 cells stably expressing (28, 30, 35, 36). The nonsteroidal nature of BR-4628 raised the the GAL4-MR LBD fusion protein were incubated for 6 h with increasing S810L question of its binding mode within the MR binding pocket. To concentrations of BR-4628 alone (Œ) or in the presence of 10 M aldosterone (F). The MR and MR transactivation activities were determined in answer this question, a large amount of purified MRBR-4628 WT S810L duplicate from the respective luciferase activities. complex was produced. However, despite numerous trials, crystallization of a complex was unsuccessful. Thus, a mutagen- The high sequence similarity among the LBDs of oxo-steroid esis approach guided by BR-4628 docking within the x-ray receptors results in the cross-binding of ligands to the various structure of the wild type MR LBD (Protein Data Bank code receptors. To evaluate whether BR-4628 was MR-selective, 2ABI) (30) was followed. Initial docking into the agonist con- transactivation assays were performed in CHO-K1 cells stably formation of the MR LBD was unsuccessful because the binding expressing the LBD of AR, GR, and PR. Spironolactone is a pocket is simply too small to accommodate the highly branched strong AR antagonist (IC 77 nM), a weak GR antagonist BR-4628 (supplemental Fig. S1). Indeed, the 5-acetyl moiety is (IC 2.4 M), and a weak PR agonist (EC 740 nM). In in close contact with Trp and might displace it for accom- 50 50 contrast, BR-4628, like nitrendipine and eplerenone, is a weak modation, resulting in a clash with the H12 helix (Fig. 3A). antagonist of AR, GR, and PR, as revealed by IC values higher Moreover, the neighboring 6-methyl group of the DHP core than 4 M (Table 1). Thus, BR-4628 is at least 160-fold more clashes with Leu located in helix H12 (Fig. 3A). Both of these selective for MR than for AR, whereas spironolactone exhibits structural elements suggest that this molecule might have bulky only a 3-fold selectivity. antagonist features by impairing the H12 helix to adopt its ago- The dihydropyridine-derived nitrendipine is a potent cal- nist position. Therefore, the H12 helix was omitted from fur- cium channel blocker used as a antihypertensive agent (34). ther docking experiments and from a constrained 4-ns molec- Binding assays show that BR-4628 has a low calcium channel ular dynamics simulation (supplemental Video S1 and Fig. S2). blocker activity, as revealed by an IC value 3 orders of magni- The chromenone carbonyl group is anchored to Gln and 817 770 tude higher than that of nitrendipine (1.99 M versus 0.26 nM; Arg , the DHP NH group is hydrogen-bonded to the Asn Table 1). In summary, we could demonstrate that minor chem- carbonyl oxygen, and the carbonyl group of the 5-acetyl moiety ical modifications from a classical dihydropyridine calcium forms a hydrogen bond with Ser (Fig. 3B). During the course antagonist lead to a potent and highly specific MR antagonist. of the 4-ns molecular dynamics simulation, Asn is hydrogen- BR-4628 Is a Potent MR Antagonist in Vivo—The acute in bonded in 92.8% of the conformations, Ser is hydrogen- vivo activity of a MR antagonist can be monitored by measuring bonded in 65.3%, Arg is hydrogen-bonded in 34.5%, and its effects on the urinary Na /K ratio. BR-4628 was orally Gln is hydrogen-bonded in 0.6%. No hydrogen bond is administered to conscious rats by gavage, and the sodium and observed for Cys ; however, the thiol group packs against the SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29935 A Potent and Selective Nonsteroidal MR Antagonist core of the DHP ring, forming van der Waal’s contacts (average dis- tance from the BR-4628 NH to the sulfur of Cys is 3.9 Å). The methyl side chain of Ala fits tightly into a notch on the BR-4628 surface, formed by the 6-methyl carbon (3.6 Å), the C5 in the DHP core (3.9 Å), the 5-acetyl carbon (4.0 Å), and the C7 in the chromenone ring (3.7 Å). Interestingly, in such a position, BR-4628 does not interact with Thr (average distance from the BR-4628 NH to Thr C is 7.4 Å). The 3-ethyl ester fills a lipophilic 814 829 pocket formed by Leu , Phe , FIGURE 3. Accommodation mode of BR-4628 within the MR LBD. A, picture showing the minimized 845 852 938 Met , Met , and Leu (Fig. 3B). 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 The methyl substituent on the depicted as a green wire surface. B, overall view of the BR-4628 accommodation within the MR LBD devoid 4-chromenone ring interacts with of the H12 helix. Only residues that form critical contacts with the ligand are shown. Hydrogen bound close van der Waal’s contacts with between BR-4628 and the polar residues is depicted as dashed red lines. This figure was produced using 811 807 DINO. the side chain of Ser , Met , 852 938 Met , and Leu . Based on this binding mode, 10 amino acids in the binding site were selected for mutation studies. We 770 776 817 focused on Asn , Gln , Arg , and Thr , known to anchor the steroids (25), and on the Ala , 810 852 942 Ser , Met , and Cys residues that play a critical role in delimiting the volume of the cavity (25, 26, 28). Each residue was replaced by an ala- nine, except Ala , which was replaced by a glycine. All of the mutations were introduced within the full-length MR gene, and the corresponding mutant receptors were transiently expressed in HEK293T cells and tested for their transcriptional activity. Aldoster- one activates MR and A773G MR with ED values identical S810A 50 to that of MR (0.025 and 0.019 nM, respectively, versus 0.014 nM; Fig. 4). Aldosterone is less efficient in activating MR and MR C942A T945A (ED , 9.5 and 4.9 nM, respectively). The activity of MR and Q776A MR in response to 10 M R817A aldosterone is 20 and 70% that of MR, respectively (Fig. 4). Aldoster- one is unable to activate MR N770A and MR (25, 27). Neverthe- M852A less, 18-vinyl-4-pregnen-3,18,20- trione and spironolactone activate FIGURE 4. Transactivation activity of the wild type and mutant MRs. HEK-293T cells transiently expressing the MR, MR ,MR ,MR ,MR ,MR ,MR ,MR ,MR ,MR ,orMR N770A A773G Q776A S810A S810M R817A M852A C942A T945A A773G/S810M MR and MR , respec- N770A M852A were incubated for 16 h with aldosterone (Aldo), 18-vinyl-4-pregnen-3,18,20-trione (18OVP), or spironolactone tively (ED , 8.4 and 0.41 nM; Fig. 4). (Spiro). The cell extracts were assayed for luciferase and -galactosidase activities as reported previously (30). The GraphPad Prism software was used for curve fitting and calculation of the EC values. The effect of the mutations on the 29936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist TABLE 2 Half-maximally inhibitory concentrations of BR-4628 and spironolactone for the full-length wild-type and mutant MRs The IC values were calculated using the GraphPad Prism software and are the means  S.E. of three independent experiments performed in triplicate. The EC values for spironolactone were calculated using the Prism software for a represent- ative experiment performed in triplicate. BR-4628 SPIRO IC Fold IC Fold EC 50 50 50 nM nM nM MR 34  4 1 74.0  15 1 WT MR 1334  384 39.2 1313  167 17.7 N770A MR 958  321 28.2 84  15 1.1 A773G MR 317  87 9.3 301  77 4.1 Q776A MR 345  21 10.1 109  24 1.5 S810A MR 179  30 5.3 6 S810M MR 323  22 9.5 331  20 4.5 R817A MR 270  23 7.9 0.4 M852A MR 219  21 6.4 506  55 6.8 C942A MR 30  5 0.9 444  43 6.0 T945A MR 12100  3000 356 8.4 A773G/S810M BR-4628 and spironolactone antagonist potency was analyzed in transfection assays (supplemental Fig. S3 and Table 2). The T945A mutation had no effect on the BR-4628 potency, as FIGURE 5. Binding properties of BR-4628 to MR. A, Scatchard plot of the revealed by an IC identical to that of wild type MR, and con- binding of [ H]BR-4628 to MR. The in vitro expressed MR was incubated with firmed that BR-4628 does not interact with this residue. In 3 10 7 [ H]BR-4628 (3  10 to 3  10 M)for4hat4 °C. Bound and unbound sharp contrast, the N770A and A773G mutations have a strong 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 effect, increasing BR-4628 IC values by 39- and 25-fold, value was calculated using the ScatMac program (32). A parallel experiment respectively. From these results it can be proposed that Asn was performed with a rabbit reticulocyte lysate in which no receptor was and Ala are critical for BR-4628 binding. The Q776A, S810A, expressed. B, dissociation kinetics of [ H]BR-4628 from MR. The in vitro 8 3 expressed MR was incubated with 10 M [ H]BR-4628 for4hat4 °Cand then R817A, M852A, and C942A mutations have intermediate incubated for various time with 10 M BR-4628. The bound and free ligands effects, increasing the IC of BR-4628 by 6–10-fold. This 50 were separated by the dextran-charcoal method, and the residual binding 776 810 817 852 was calculated. C, limited proteolysis assays. In vitro expressed [ S]MR was suggests that the amino acids Gln , Ser , Arg , Met , 942 incubated for 10 min at 20 °C with or without 10 M aldosterone or BR-4628 and Cys contribute to the BR-4628 binding. Interestingly, and then for 10 min at 20 °C with trypsin (0 –300 g/ml). The digestion prod- the A773G and S810A mutations have no effect on the spirono- ucts were analyzed by SDS-PAGE and autoradiographed. lactone antagonist potency, whereas these two mutations increase the BR-4628 IC value. Moreover, the T945A muta- properties to the in vitro expressed MR. A dissociation constant tion induces a 6-fold increase in the spironolactone IC value, at equilibrium (K )of2nM was measured (Fig. 5A), indicating 50 d whereas it does not modify BR-4628. Altogether, these results an affinity for MR in the same order of magnitude as that of fully agree with the docking experiments showing striking dif- aldosterone (25). Interestingly, we observed that, at 4 °C, ferences between BR-4628 and spironolactone accommodation [ H]BR-4628 dissociates quickly from MR with a half-life time mode within the ligand-binding pocket of MR. of 90 min (Fig. 5B), a value identical to that of the tritiated Previous reports have highlighted remarkable differences spirolactone RU26752 (t1 90 min) (27). We further exam- ⁄2 between the oxo-steroid receptors ligand-binding cavity resi- ined the stability of the MRBR-4628 complex by measuring the 773 810 dues. It concerns the MR Ala and Ser , which correspond ability of BR-4628 to protect MR against proteolysis. Upon to glycine and methionine residues at the corresponding posi- treatment of the unbound [ S]MR for 10 min at 20 °C by 15 tions of the human AR, GR, and PR (26, 37). Because BR-4628 g/ml trypsin, a major fragment of 41 kDa and a minor frag- possesses remarkable selectivity toward MR, we wondered ment of 30 kDa both encompassing the LBD (38) were recov- whether these residues are involved in the high MR selectivity. ered (Fig. 5C). They were completely digested when the trypsin The A773G and S810M single mutations and the A773G/ concentration was increased to 120 g/ml. In contrast, the S810M double mutation have no effect on the aldosterone aldosteroneMR complex was highly resistant to the trypsin potency to activate MR as illustrated by identical ED values action. Although the proteolysis pattern is similar for the (Fig. 4). In sharp contrast, these mutations lead to an increase of MRBR-4628 complex, the intensity of the 30-kDa fragment is BR-4628 IC values by 28-, 5-, and 356-fold, respectively much lower upon BR-4628 treatment (Fig. 5C). This result indi- (supplemental Fig. S3 and Table 2). These results suggest that cates that BR-4628 is less efficient than aldosterone in protect- the alanine/serine pair at the 773 and 810 positions favor ing the MR against proteolysis, confirming the high instability BR-4628 binding to MR. of the BR-4628MR complex. The MRBR-4628 Complex Is Unstable and Unable to Recruit To further characterize the mechanism by which BR-4628 Transcriptional Co-regulators—To further characterize the inactivates MR, we evaluated the capacity of MR to recruit tran- MRBR-4628 interaction, we synthesized the H-labeled scriptional co-regulators upon BR-4628 binding. Mammalian BR-4628 (see supplemental data) and characterized its binding two-hybrid assays revealed that aldosterone, but not BR-4628 SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29937 A Potent and Selective Nonsteroidal MR Antagonist contacts with Gln , explaining the importance of this amino acid in our mutational studies. The second noteworthy feature of BR-4628 is its MR selec- tivity. It inhibits the transcriptional activity of the other oxo- steroid receptors (AR, GR, and PR) with IC values higher than 4 M, pointing out only residual affinity for these receptors. MR is the unique oxo-steroid receptor, having an alanine residue 773 810 (Ala ) in the H3 helix and a serine residue (Ser ) in the H5 helix. AR, PR, and GR each harbor a glycine and a methionine at the corresponding positions, respectively. Interestingly, BR-4628 docking within the MR binding pocket underscores that Ser forms a hydrogen bond with the C5-acetyl group and that the Ala residue is surrounded by the aromatic phe- nyl moiety of the chromenone, the C5-acetyl, and the C6-methyl group. The dramatic decrease in the BR-4628 inhib- FIGURE 6. Recruitment of transcriptional co-regulators by MR in mamma- lian two-hybrid assays. HEK293T cells transiently expressing the fusion pro- itory potency upon the A773G, S810M, and A773G/S810M teins VP16-MR and the GAL4 DNA-binding domain fused to the receptor mutations demonstrates the importance of the interactions interacting domain of TIF2 (A) or NcoR (B) were incubated in triplicate with 10 8 8 between BR-4628 and these residues for its potency and ethanol, aldosterone (Aldo,10 to 10 M), spironolactone (Spiro,10 to 6 8 6 9 10 M), or BR-4628 (10 to 10 M) alone or with 10 M aldosterone in the selectivity. 8 6 presence of 10 to 10 M spironolactone or BR-4628. After harvesting the BR-4628 is a dihydropyridine derivative. Classic dihydropy- 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 inde- ridine-derived molecules, such as nifedipine and nitrendipine, pendent experiments. *, p  0.05; **, p  0.01. are known to act as potent L-type calcium channel blockers (34). These molecules have been reported to also have a weak MR antagonist activity (39). However, we show here that a clas- and spironolactone, promoted a dose-dependent binding of the TIF2 to MR (Fig. 6A). Moreover, the two antagonists are able to sical L-type channel blocker like nitrendipine has no selectivity inhibit the aldosterone-induced MR/TIF2 interaction in a dose- toward MR and rather nonselectively antagonizes all steroid hormone receptors in the micromolar range (Table 1). In sharp dependent manner. Interestingly, the NCoR is not recruited by contrast, BR-4628 is a highly potent MR antagonist, with a low MR upon the binding of BR-4628 and spironolactone, whereas a weak interaction is observed after aldosterone binding (Fig. L-type calcium channel binding activity in the micromolar 6B). Furthermore, the two MR antagonists inhibit the binding range. BR-4628 differs from classical dihydropyrine derivatives mainly by its 2-methylchromenonyl substituent. It is likely that of NCoR to the MR-aldosterone complex. Thus, despite a high this substituent drastically reduces calcium channel inhibitory affinity of BR-4628 for MR, the BR-4628MR complex is highly unstable and unable to recruit transcriptional co-modulators. activity, and it is responsible for high MR binding affinity, because it rigidifies the active conformation with respect to the DISCUSSION MR. The methyl group is involved in close van der Waal’s con- In this study, we performed thorough mutagenesis, struc- tact with four lipophilic side chains in MR, explaining the pro- tural, biochemical, and pharmacological investigations of a nounced differences in activity between nitrendipine and novel dihydropyridine-based MR antagonist, which allowed BR-4628 on MR. Thus, the presence of the 2-methylchrom- elucidation of its mechanism of MR inactivation. Transactiva- enonyl substituents on the dihydropyridine ring ensures the tion assays using the MR LBD fused to the GAL4 DNA-binding high MR selectivity versus calcium channel. domain revealed that BR-4628 is as potent as spironolactone Functional and structural data have clearly shown that the but significantly more potent than eplerenone (Table 1). A sim- inability of progesterone and spironolactone to establish a ilar inhibitory hierarchy is also observed by using the entire MR, strong contact with Asn is responsible for their antagonistic because BR-4628 is characterized by an IC of 33.5 nM (Table features (25). BR-4628 strongly interacts with Asn , raising 2) compared with 50.0 nM for spironolactone and 2000 nM for the question of the mechanism by which it inactivates MR. Two eplerenone (27). Molecular dynamics and point mutation data classes of antagonists have been described for steroid receptors. suggest that binding of BR-4628 is mediated by hydrogen bonds The first one, called “passive,” refers to molecules that are 770 776 810 817 to Asn , Gln , Ser , and Arg and lipophilic contacts to unable to stabilize the receptor in a conformation able to recruit 773 852 942 Ala , Met , and Cys . The calculated hydrogen bond the transcriptional co-regulators. These antagonists are usually probabilities in the molecular dynamics simulation agree well small molecules that dissociate quickly from the receptor. This for all point mutation data; the higher the observed H-bond is namely the case for the MR antagonists progesterone and frequency, the more important the amino acid is in the trans- spironolactone, the estrogen receptor  antagonist (R,R)-5,11- activation experiment. Only Gln is underestimated in the cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (40), and the molecular dynamics simulation. However, even in the pub- AR antagonist hydroxyflutamide (41). The second class of lished x-ray structures, the distance between the steroidal antagonists, called “active,” comprises molecules that permit 3-oxo function and the Gln side chain NH is relatively large the LBD to recruit transcriptional co-repressors. This type of (3.2–3.3 Å), and an additional H-bond acceptor in a ligand (like antagonism is observed namely with RU486 for PR and GR and the 5-acetyl group in BR-4628) might form water-mediated fulvestrant for estrogen receptor . These molecules are char- 29938 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist REFERENCES acterized by bulky side chains that impair H12 helix from adopting its active conformation. The docking experiments we 1. Horisberger, J. 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Pharmacol. 58, 684–691 as well as in rare forms of hypertension (including the hyperten- 27. Fagart, J., Seguin, C., Pinon, G. M., and Rafestin-Oblin, M. E. (2005) Mol. sion associated with the S810L mutation), where available steroi- Pharmacol. 67, 1714–1722 dal antagonists are even contraindicated. 28. Fagart, J., Huyet, J., Pinon, G. M., Rochel, M., Mayer, C., and Rafestin- Altogether these findings suggest that BR-4628 represents a Oblin, M. E. (2005) Nat. Struct. Mol. Biol. 12, 554–555 prototype of an at least third generation MR antagonist (44) 29. Gouilleux, F., Sola, B., Couette, B., and Richard-Foy, H. (1991) Nucleic that acts through a new bulky-passive mechanism. Several in Acids Res. 19, 1563–1569 30. Huyet, J., Pinon, G. M., Fay, M. R., Fagart, J., and Rafestin-Oblin, M. E. vivo investigations are underway to evaluate its end organ pro- (2007) Mol. Pharmacol. 72, 563–571 tective effects in preclinical animal models. 31. Brandish, P. 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(1994) Acta Crystallogr. D Biol. Crystal- 39. Dietz, J. D., Du, S., Bolten, C. W., Payne, M. A., Xia, C., Blinn, J. R., Funder, logr. 50, 178–185 J. W., and Hu, X. (2008) Hypertension 51, 742–748 46. Ehlert, F. J., Roeske, W. R., Itoga, E., and Yamamura, H. I. (1982) Life Sci. 40. Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Kat- 30, 2191–2202 29940 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

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

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m110.131342
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 39, pp. 29932–29940, September 24, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A New Mode of Mineralocorticoid Receptor Antagonism by a □ S Potent and Selective Nonsteroidal Molecule Received for publication, April 7, 2010, and in revised form, July 22, 2010 Published, JBC Papers in Press, July 22, 2010, DOI 10.1074/jbc.M110.131342 ‡§1 ¶ ‡§ ¶ ‡§ Je´roˆ me Fagart , Alexander Hillisch , Jessica Huyet , Lars Ba¨rfacker , Michel Fay , Ulrich Pleiss , ‡‡2 ‡§ ‡‡3 Elisabeth Pook**, Stefan Scha¨fer , Marie-Edith Rafestin-Oblin , and Peter Kolkhof ¶  ‡‡ From the Departments of Medicinal Chemistry, Isotope Chemistry, **Lead Discovery, and Cardiology Research, Bayer Schering Pharma AG, Global Drug Discovery, 42096 Wuppertal, Germany, INSERM U773, Centre de Recherche Biome´dicale Bichat-Beaujon, CRB3, 75870 Paris, France, and the Universite´ Paris 7-Denis Diderot, Site Bichat, 75018 Paris, France Limitations of current steroidal mineralocorticoid receptor inactivating MR mutations provoke salt wasting (4–7), whereas (MR) antagonists have stimulated the search for a new genera- an activating mutation (S810L) has been shown to induce a tion of molecules. We screened for novel nonsteroidal com- severe form of early onset hypertension (8). In addition to its pounds and identified MR antagonists derived from the chemi- renal effects, aldosterone acts in nonepithelial tissues such as cal class of dihydropyridines. Chemical optimization resulted in brain, vasculature, and heart, in which it has deleterious effects. BR-4628, which displays high in vitro and in vivo MR potency as In the cardiovascular system, aldosterone provokes inflamma- well as selectivity with respect to the other steroid hormone tion and fibrosis, combined with ventricular hypertrophy (9). receptors and the L-type calcium channel. Biochemical studies Whether these adverse effects are mediated by aldosterone demonstrated that BR-4628 forms complexes with MR that do and/or cortisol at conditions of inappropriate salt or redox sta- not promote the recruitment of transcriptional co-regulators. tus has not yet been established (10). Docking experiments, using the crystal structure of the MR Spironolactone and eplerenone, both of which display struc- ligand-binding domain in an agonist conformation, revealed tural elements of progesterone, have been developed as MR that BR-4628 accommodates in the MR ligand-binding cavity antagonists (11–14). These molecules inhibit aldosterone bind- differently in comparison with the classical steroidal MR antag- ing to MR and render it transcriptionally inactive. As a conse- onists. An alanine scanning mutagenesis approach, based on quence, they prevent the aldosterone-induced sodium reab- BR-4628 docking, allowed identifying its anchoring mode sorption and are effective in decreasing blood pressure (15, 16). within the ligand-binding cavity. Altogether, we propose that Remarkably, these molecules have been shown to reduce the BR-4628 is a bulky antagonist that inactivates MR through a deleterious aldosterone effects in the cardiovascular system. In passive mechanism. It represents the prototype of a new class of the randomized aldactone evaluation study (RALES) and MR antagonists. eplerenone post-acute myocardial infarction heart failure effi- cacy and survival study (EPHESUS), spironolactone and eplerenone, respectively, significantly reduced mortality and The first and best documented effects of aldosterone are morbidity in patients with heart failure (17, 18). those observed on the kidney distal tubule. In this epithelial Despite their renal and cardiac benefits, these two steroidal spirolactones suffer from substantial drawbacks that limit their tissue, aldosterone promotes sodium reabsorption and potas- sium secretion, increasing the blood pressure by expanding the clinical use. Although spironolactone is highly potent, it lacks extracellular volume (1, 2). These aldosterone effects are medi- selectivity toward other members of the oxo-steroid receptor ated by the mineralocorticoid receptor (MR), a ligand-acti- family. Indeed, its prolonged use is associated with sexual vated transcription factor belonging to the nuclear receptor side effects that are related to its progestogenic and antian- superfamily (3). According to the aldosterone renal effects, drogenic activities (19). Eplerenone is characterized by an improved selectivity as compared with spironolactone. However, eplerenone has a low affinity for MR (20) and is * Portions of these results have been presented at the Congress of the Euro- less efficient than spironolactone with respect to blood pean Society of Cardiology in Vienna in 2007 and at the German Society of pressure lowering in patients with mild-to-moderate hyper- Cardiology Congress in 2007 in Mannheim. □ S tension (21). In addition, both steroidal molecules are not The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Video S1, and Figs. S1–S3. only unable to block the constitutive activity of the mutant Present address: INSERM U693, 53 rue Gabriel Pe´ri, 94270 Le Kremlin-Biceˆ- MR , a gain-of function mutation that is linked clini- S810L tre, France. cally to early onset hypertension in men and gestational Present address: Synthon, Microweg 22, 6545 CM Nijmegen, The Netherlands. hypertension in women, but paradoxically activate this To whom correspondence should be addressed: Cardiology Research, Bayer mutant receptor (8, 22). Therefore, the use of spirolactones Schering Pharma AG, Global Drug Discovery, Aprather Weg 18a, 42096, to treat hypertensive patients carrying the S810L mutation is Wuppertal, Germany. Fax: 49-202-36-8009; E-mail: Peter.Kolkhof@ bayerhealthcare.com. inappropriate. The abbreviations used are: MR, mineralocorticoid receptor; AR, androgen The known disadvantages of spirolactones stimulated receptor; GR, glucocorticoid receptor; PR, progesterone receptor; TIF2, research for new, selective MR antagonists (23). An ultrahigh transcriptional intermediary factor 2; NCoR, nuclear receptor co-repressor 1; LBD, ligand-binding domain; DHP, dihydropyridine. throughput screening revealed substituted dihydropyridines 29932 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 This is an Open Access article under the CC BY license. A Potent and Selective Nonsteroidal MR Antagonist (DHP) as novel nonsteroidal MR antagonists. Chemical optimi- compounds were given in eight dilutions to the cells followed by zation of these DHP lead compounds resulted in BR-4628. Here the relevant EC concentration of each agonist. After an incu- we present the in vitro and in vivo pharmacological character- bation time of 5–6 h, luciferase activity was determined using a ization of BR-4628, together with studies that highlight the luminescence detecting video camera system. The GraphPad binding mode on MR and the molecular reasons for the distinct Prism software (version 3.02; GraphPad Software Inc., San antagonism. Diego, CA) was used for curve fitting and calculation of the IC and EC values. The IC and EC values from the luciferase 50 50 50 EXPERIMENTAL PROCEDURES assay were determined in at least three independent experi- Compounds—BR-4628 and [ H]BR-4628 were synthesized as ments performed in duplicate. The IC and EC values are 50 50 described in the supplemental data. Aldosterone and spirono- given as the means  S.E. lactone were purchased from Sigma. Eplerenone was purified Transactivation Assays in Transiently Transfected Cells— from commercially available tablets (Inspra). Nitrendipine HEK-293T cells were cultured and transfected with the ex- was obtained by Hantzsch dihydropyridine synthesis using pression vectors pchMR, pchMRN770A, pchMRA773G, classical published methods (24). 18-Oxo-18-vinylprogester- pchMRQ776A, pchMRS810A, pchMRS810M, pchMRR817A, one (18-vinyl-4-pregnen-3,18,20-trione) was a gift from A. pchMRM852A, pchMRC942A, pchMRT945A, or pchMRA773G/ Marquet (Paris, France). S810M, the reporter vector pFC31Luc, and the pcgal vector Expression Vectors—The expression vectors pchMR, according to the method described previously (30). 24 h after pchMRN770A, pchMRA773G, pchMRQ776A, pchMRR817A, transfection, ligands were added, and after a 16-h incubation, pchMRM852A, pchMRC942A, and pchMRT945A code for the cell extracts were assayed for luciferase and -galactosidase MR, MR ,MR ,MR ,MR MR , N770A A773G Q776A R817A M852A activities as reported previously (30). The GraphPad Prism soft- MR , and MR , respectively (25–28). The pchMRS810A, C942A T945A ware (version 3.02; GraphPad Software Inc., San Diego, CA) pchMRS810M, and pchMRA773G/S810M coding for was used for curve fitting and calculation of the IC and EC 50 50 MR ,MR , and MR , respectively, were S810A S810M A773G/S810M values. The IC values were determined from at least three obtained from pchMR using the site-directed mutagenesis pro- independent experiments performed in triplicate. The IC val- cedure (QuikChange; Agilent, Massy, France). The plasmid ues are given as the means  S.E. The EC values were calcu- pFC31Luc contains the murine mammary tumor virus pro- lated from one experiment performed in triplicate. moter that drives the luciferase gene (29). The coding Mammalian Two-hybrid Assays—HEK 293T cells were sequences of the respective LBDs of MR, MR , AR, GR, and S810L transfected with 2 g of pVPMR, pGALTIF2, or pGALNcoR; PR have been PCR-amplified and fused to the coding sequence 5 g of pg5luc; and 1 gofpcgal. 24 h after transfection, of the DNA-binding domain of GAL4 (amino acids 1–147) 10 8 aldosterone (10 to 10 M) or spironolactone or BR-4628 under the control of a CMV promoter, leading to the expression 8 6 (10 to 10 M) were added. Parallel experiments were per- plasmids pGAL4-MR, pGAL4-MR , pGAL4-GR, pGAL4- S810L 9 8 formed with 10 M aldosterone in the presence of 10 to AR, and pGAL4-PR. The pVPMR (kindly provided by Dr. G. 10 M spironolactone or BR-4628. After 16 h, the cell Pinon) codes for the fusion protein between the VP16 activat- extracts were assayed for luciferase and -galactosidase as ing domain and the full-length MR. The pGALTIF2 and reported previously (30). pGALNcoR vectors, which code for fusion protein between the Animals—Male Wistar rats (Charles River Germany, 300 GAL4 DNA-binding domain and the nuclear activating domain g) were used in the experiment. They were housed with free of TIF2 and NCoR, respectively, were kindly provided by Dr. P. access to food and water and maintained on a light-dark cycle at Balaguer. The pG5luc (kindly provided by Prof. P. Fuller) 22–24 °C. All of the animal experiments were conducted in contains the luciferase gene driven by a GAL4-responsive accordance with the National Institutes of Health Guide for the promoter. Care and Use of Laboratory Animals and German legislation on Generation of Stable Steroid Hormone Receptor Cell Lines— animal welfare. The pGAL4-MR, pGAL4-MR , pGAL4-GR, pGAL4-AR, S810L Model of Acute Natriuresis—A similar protocol as published and pGAL4-PR vectors were transfected into CHO-K1 cells previously (31) was used. Briefly, after 7 days of acclimatization, stably expressing a thymidine kinase promoter construct con- male Wistar rats were placed on a low salt diet containing taining five GAL4-binding elements in front of the firefly lucif- 0.02% (w/v) sodium chloride (S0602-E081; ssniff Spezial- erase gene. For each receptor a stable cell line has been gener- dia¨ten GmbH, Soest, Germany) for 72 h. BR-4628 and spi- ated by several rounds of limiting dilutions. ronolactone were administered in 1 ml/kg of vehicle Transactivation Assays in Stable Cell Lines—The stable MR, (PEG400, 85.8%; glycerine, 5.3%, water, 8.9%, v/v/v) by oral MR , GR, AR, and PR cell lines were cultured at 37 °C and S810L gavage, and the animals (n  8/group) were placed in meta- 5% CO in DMEM/Ham’s F-12 medium with GlutaMAX sup- bolic cages (Tecniplast Deutschland GmbH, Hohenpeissen- plemented with 10% (v/v) inactivated fetal calf serum, 20 mM berg, Germany) for8hon water ad libitum. The urine sam- HEPES, 1.4 mM sodium pyruvate, 1.8 mM sodium bicarbonate, ples were analyzed for volume as well as sodium and and 1 mg/ml Geneticin. Subconfluent cultures were passaged using Accutase. All of the cell culture reagents were obtained potassium concentrations by flame spectroscopy. from Invitrogen. The cells were seeded 24 h before testing in Coupled Cell-free Transcription and Translation—The Optimem medium containing 2.5% FCS (v/v), 2 mM glutamine, human MR was expressed in vitro in the rabbit reticulocyte and 10 mM HEPES in 96- or 384-well plates. On the test day lysate system as described previously (27). SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29933 A Potent and Selective Nonsteroidal MR Antagonist TABLE 1 Half-maximally inhibitory/effective concentrations of spironolactone, eplerenone, nitrendipine, and BR-4628 determined for the oxo-steroid receptor and for the L-type calcium channel The IC or EC values (nM) are the means  S.E. of at least four independent experiments, performed in duplicate. The IC values at the L-type calcium channel were 50 50 50 determined in two independent experiments, performed in duplicate using homogenates of rat cerebral cortex as described (46). BR-4628 Binding Characteristics at Equilibrium—The ly- using the program Desmond was run (for details, see the sates containing the in vitro expressed MR were diluted 4-fold supplemental text). with TEGWM buffer (20 mM Tris-HCl, 1 mM EDTA, 20 mM sodium tungstate, 1 mM -mercaptoethanol, and 10% glycerol RESULTS (v/v), pH 7.4) and incubated with 0.3–300 nM [ H]BR-4628 for BR-4628 Is a Potent and Selective MR Antagonist—An ultra- 4 h at 4 °C. Bound and unbound ligands were separated by the high throughput screening was performed with a cellular trans- dextran-charcoal method (27). The change in bound/unbound activation assay based on a recombinant CHO cell line stably as a function of the amount bound was analyzed, and the K expressing the MR LBD. Screening of almost 1,000,000 com- value was calculated as described previously (32). A parallel pounds revealed 677 confirmed primary hits. Approximately experiment was performed with an untranslated rabbit reticu- 300 screening hits were selected for further evaluation after locyte lysate. elimination of toxic, unspecific, and noncompetitive com- Kinetic Experiments—The lysates containing the in vitro pounds. Among them, a single cluster of 100 compounds expressed MR were 4-fold diluted with TEGWM buffer and comprised dihydropyridines (WO2007/025604) (33). This incubated with 10 nM [ H]BR-4628 for4hat4 °C.One half of finding was surprising because dihydropyridines constitute the the labeled lysate was kept at 4 °C and was used to determine well known class of L-type calcium channel antagonists. Chem- the stability of the [ H]BR-4628MR complexes, and the other ical optimization with respect to potency, selectivity, and met- half was incubated with 1 M BR-4628 for various periods. abolic stability lead to the synthesis of BR-4628 as a drug can- Bound and free ligands were separated using charcoal-dextran. didate (see formula in Table 1). The findings were corrected for receptor stability and were Transactivation assays performed with CHO-K1 cells stably expressed as percentages of the binding measured at time 0. expressing the MR LBD revealed that BR-4628 acts as a full MR Dissociation was represented in a semi-logarithmic scale giving antagonist. It inactivates the aldosterone-induced MR-LBD a linear manner representation. 35 transactivation activity with an IC of 27.9 nM, a value compa- Limited Proteolysis Assays—In vitro expressed [ S]MR was rable with that of spironolactone (24.2 nM) but much lower than incubated for 10 min at 20 °C with or without aldosterone or that of eplerenone (990 nM) (Fig. 1A and Table 1). Furthermore, BR-4628 (10 M) and then for 10 min at 20 °C with increasing its antagonist potency is 2 orders of magnitude higher than that concentrations of trypsin (0–300 g/ml). The digestion prod- of nitrendipine, the well known dihydropyridine-based calcium ucts were analyzed by SDS-PAGE and autoradiographed. channel blocker (Table 1). Spironolactone and eplerenone lose Protein Modeling, Docking, and Molecular Dynamics Sim- their antagonist feature upon the S810L mutation (8, 22, 30). ulations—The x-ray structure of the wild type MR LBD com- Thus, we wondered whether BR-4628 still acts as an antagonist plexed with deoxycorticosterone (Protein Data Bank code when bound to the MR . CHO-K1 cells stably expressing 2ABI) (30) served as a model for docking of BR-4628. The size of S810L the MR LBD are characterized by a weak constitutive the binding site was enlarged by modifying several side chain S810L activity (5-fold increase in the base-line luciferase activity; Fig. conformations. The program Glide SP (version 3.5) was used to 1B). Remarkably, BR-4628 inhibits the constitutive MR dock BR-4628 into the resulting binding cleft. The complex S810L LBD activity (IC , 3630 nM) and the aldosterone-induced with the most probable binding pose was energy-minimized MR LBD activity (IC , 813 nM; Fig. 1B) in a dose-depen- using the OPLS-AA/L force field. To check for the stability of S810L 50 dent manner. Thus, BR-4628 is a potent MR antagonist that hydrogen bonds or alternative contacts, a 4-ns molecular dynamics simulation with 11,522 explicit water molecules retains its antagonist character at the MR mutant. S810L 29934 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist 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. potassium concentrations were measured in the collected urine. BR-4628 administration increased the urinary sodium/ potassium ratio in a dose-dependent manner with a significant effect for a dose as low as 1 mg/kg (Fig. 2). For comparison, spironolactone was found to increase the Na /K at a dose of 10 mg/kg (Fig. 2). FIGURE 1. Representative inhibition curves of aldosterone-induced Docking of BR-4628 within the MR LBD Reveals an Unusual GAL4-MR-LBD and GAL4-MR -LBD activities in response to antago- S810L nists. A, CHO-K1 cells stably expressing the GAL4-MR LBD fusion protein WT Anchoring Mode—The crystal structures of the LBD of MR and and the luciferase reporter gene under the control of a GAL4-responsive ele- MR complexed with steroidal agonists have been recently S810L ment-containing promoter (pFA-luc; Stratagene) were incubated for 6 h with increasing concentrations of BR-4628 (ƒ), spironolactone (‚), or eplerenone solved, allowing their anchoring mode to be studied in detail F) in the presence of 10 M aldosterone. B, CHO-K1 cells stably expressing (28, 30, 35, 36). The nonsteroidal nature of BR-4628 raised the the GAL4-MR LBD fusion protein were incubated for 6 h with increasing S810L question of its binding mode within the MR binding pocket. To concentrations of BR-4628 alone (Œ) or in the presence of 10 M aldosterone (F). The MR and MR transactivation activities were determined in answer this question, a large amount of purified MRBR-4628 WT S810L duplicate from the respective luciferase activities. complex was produced. However, despite numerous trials, crystallization of a complex was unsuccessful. Thus, a mutagen- The high sequence similarity among the LBDs of oxo-steroid esis approach guided by BR-4628 docking within the x-ray receptors results in the cross-binding of ligands to the various structure of the wild type MR LBD (Protein Data Bank code receptors. To evaluate whether BR-4628 was MR-selective, 2ABI) (30) was followed. Initial docking into the agonist con- transactivation assays were performed in CHO-K1 cells stably formation of the MR LBD was unsuccessful because the binding expressing the LBD of AR, GR, and PR. Spironolactone is a pocket is simply too small to accommodate the highly branched strong AR antagonist (IC 77 nM), a weak GR antagonist BR-4628 (supplemental Fig. S1). Indeed, the 5-acetyl moiety is (IC 2.4 M), and a weak PR agonist (EC 740 nM). In in close contact with Trp and might displace it for accom- 50 50 contrast, BR-4628, like nitrendipine and eplerenone, is a weak modation, resulting in a clash with the H12 helix (Fig. 3A). antagonist of AR, GR, and PR, as revealed by IC values higher Moreover, the neighboring 6-methyl group of the DHP core than 4 M (Table 1). Thus, BR-4628 is at least 160-fold more clashes with Leu located in helix H12 (Fig. 3A). Both of these selective for MR than for AR, whereas spironolactone exhibits structural elements suggest that this molecule might have bulky only a 3-fold selectivity. antagonist features by impairing the H12 helix to adopt its ago- The dihydropyridine-derived nitrendipine is a potent cal- nist position. Therefore, the H12 helix was omitted from fur- cium channel blocker used as a antihypertensive agent (34). ther docking experiments and from a constrained 4-ns molec- Binding assays show that BR-4628 has a low calcium channel ular dynamics simulation (supplemental Video S1 and Fig. S2). blocker activity, as revealed by an IC value 3 orders of magni- The chromenone carbonyl group is anchored to Gln and 817 770 tude higher than that of nitrendipine (1.99 M versus 0.26 nM; Arg , the DHP NH group is hydrogen-bonded to the Asn Table 1). In summary, we could demonstrate that minor chem- carbonyl oxygen, and the carbonyl group of the 5-acetyl moiety ical modifications from a classical dihydropyridine calcium forms a hydrogen bond with Ser (Fig. 3B). During the course antagonist lead to a potent and highly specific MR antagonist. of the 4-ns molecular dynamics simulation, Asn is hydrogen- BR-4628 Is a Potent MR Antagonist in Vivo—The acute in bonded in 92.8% of the conformations, Ser is hydrogen- vivo activity of a MR antagonist can be monitored by measuring bonded in 65.3%, Arg is hydrogen-bonded in 34.5%, and its effects on the urinary Na /K ratio. BR-4628 was orally Gln is hydrogen-bonded in 0.6%. No hydrogen bond is administered to conscious rats by gavage, and the sodium and observed for Cys ; however, the thiol group packs against the SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29935 A Potent and Selective Nonsteroidal MR Antagonist core of the DHP ring, forming van der Waal’s contacts (average dis- tance from the BR-4628 NH to the sulfur of Cys is 3.9 Å). The methyl side chain of Ala fits tightly into a notch on the BR-4628 surface, formed by the 6-methyl carbon (3.6 Å), the C5 in the DHP core (3.9 Å), the 5-acetyl carbon (4.0 Å), and the C7 in the chromenone ring (3.7 Å). Interestingly, in such a position, BR-4628 does not interact with Thr (average distance from the BR-4628 NH to Thr C is 7.4 Å). The 3-ethyl ester fills a lipophilic 814 829 pocket formed by Leu , Phe , FIGURE 3. Accommodation mode of BR-4628 within the MR LBD. A, picture showing the minimized 845 852 938 Met , Met , and Leu (Fig. 3B). 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 The methyl substituent on the depicted as a green wire surface. B, overall view of the BR-4628 accommodation within the MR LBD devoid 4-chromenone ring interacts with of the H12 helix. Only residues that form critical contacts with the ligand are shown. Hydrogen bound close van der Waal’s contacts with between BR-4628 and the polar residues is depicted as dashed red lines. This figure was produced using 811 807 DINO. the side chain of Ser , Met , 852 938 Met , and Leu . Based on this binding mode, 10 amino acids in the binding site were selected for mutation studies. We 770 776 817 focused on Asn , Gln , Arg , and Thr , known to anchor the steroids (25), and on the Ala , 810 852 942 Ser , Met , and Cys residues that play a critical role in delimiting the volume of the cavity (25, 26, 28). Each residue was replaced by an ala- nine, except Ala , which was replaced by a glycine. All of the mutations were introduced within the full-length MR gene, and the corresponding mutant receptors were transiently expressed in HEK293T cells and tested for their transcriptional activity. Aldoster- one activates MR and A773G MR with ED values identical S810A 50 to that of MR (0.025 and 0.019 nM, respectively, versus 0.014 nM; Fig. 4). Aldosterone is less efficient in activating MR and MR C942A T945A (ED , 9.5 and 4.9 nM, respectively). The activity of MR and Q776A MR in response to 10 M R817A aldosterone is 20 and 70% that of MR, respectively (Fig. 4). Aldoster- one is unable to activate MR N770A and MR (25, 27). Neverthe- M852A less, 18-vinyl-4-pregnen-3,18,20- trione and spironolactone activate FIGURE 4. Transactivation activity of the wild type and mutant MRs. HEK-293T cells transiently expressing the MR, MR ,MR ,MR ,MR ,MR ,MR ,MR ,MR ,MR ,orMR N770A A773G Q776A S810A S810M R817A M852A C942A T945A A773G/S810M MR and MR , respec- N770A M852A were incubated for 16 h with aldosterone (Aldo), 18-vinyl-4-pregnen-3,18,20-trione (18OVP), or spironolactone tively (ED , 8.4 and 0.41 nM; Fig. 4). (Spiro). The cell extracts were assayed for luciferase and -galactosidase activities as reported previously (30). The GraphPad Prism software was used for curve fitting and calculation of the EC values. The effect of the mutations on the 29936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist TABLE 2 Half-maximally inhibitory concentrations of BR-4628 and spironolactone for the full-length wild-type and mutant MRs The IC values were calculated using the GraphPad Prism software and are the means  S.E. of three independent experiments performed in triplicate. The EC values for spironolactone were calculated using the Prism software for a represent- ative experiment performed in triplicate. BR-4628 SPIRO IC Fold IC Fold EC 50 50 50 nM nM nM MR 34  4 1 74.0  15 1 WT MR 1334  384 39.2 1313  167 17.7 N770A MR 958  321 28.2 84  15 1.1 A773G MR 317  87 9.3 301  77 4.1 Q776A MR 345  21 10.1 109  24 1.5 S810A MR 179  30 5.3 6 S810M MR 323  22 9.5 331  20 4.5 R817A MR 270  23 7.9 0.4 M852A MR 219  21 6.4 506  55 6.8 C942A MR 30  5 0.9 444  43 6.0 T945A MR 12100  3000 356 8.4 A773G/S810M BR-4628 and spironolactone antagonist potency was analyzed in transfection assays (supplemental Fig. S3 and Table 2). The T945A mutation had no effect on the BR-4628 potency, as FIGURE 5. Binding properties of BR-4628 to MR. A, Scatchard plot of the revealed by an IC identical to that of wild type MR, and con- binding of [ H]BR-4628 to MR. The in vitro expressed MR was incubated with firmed that BR-4628 does not interact with this residue. In 3 10 7 [ H]BR-4628 (3  10 to 3  10 M)for4hat4 °C. Bound and unbound sharp contrast, the N770A and A773G mutations have a strong 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 effect, increasing BR-4628 IC values by 39- and 25-fold, value was calculated using the ScatMac program (32). A parallel experiment respectively. From these results it can be proposed that Asn was performed with a rabbit reticulocyte lysate in which no receptor was and Ala are critical for BR-4628 binding. The Q776A, S810A, expressed. B, dissociation kinetics of [ H]BR-4628 from MR. The in vitro 8 3 expressed MR was incubated with 10 M [ H]BR-4628 for4hat4 °Cand then R817A, M852A, and C942A mutations have intermediate incubated for various time with 10 M BR-4628. The bound and free ligands effects, increasing the IC of BR-4628 by 6–10-fold. This 50 were separated by the dextran-charcoal method, and the residual binding 776 810 817 852 was calculated. C, limited proteolysis assays. In vitro expressed [ S]MR was suggests that the amino acids Gln , Ser , Arg , Met , 942 incubated for 10 min at 20 °C with or without 10 M aldosterone or BR-4628 and Cys contribute to the BR-4628 binding. Interestingly, and then for 10 min at 20 °C with trypsin (0 –300 g/ml). The digestion prod- the A773G and S810A mutations have no effect on the spirono- ucts were analyzed by SDS-PAGE and autoradiographed. lactone antagonist potency, whereas these two mutations increase the BR-4628 IC value. Moreover, the T945A muta- properties to the in vitro expressed MR. A dissociation constant tion induces a 6-fold increase in the spironolactone IC value, at equilibrium (K )of2nM was measured (Fig. 5A), indicating 50 d whereas it does not modify BR-4628. Altogether, these results an affinity for MR in the same order of magnitude as that of fully agree with the docking experiments showing striking dif- aldosterone (25). Interestingly, we observed that, at 4 °C, ferences between BR-4628 and spironolactone accommodation [ H]BR-4628 dissociates quickly from MR with a half-life time mode within the ligand-binding pocket of MR. of 90 min (Fig. 5B), a value identical to that of the tritiated Previous reports have highlighted remarkable differences spirolactone RU26752 (t1 90 min) (27). We further exam- ⁄2 between the oxo-steroid receptors ligand-binding cavity resi- ined the stability of the MRBR-4628 complex by measuring the 773 810 dues. It concerns the MR Ala and Ser , which correspond ability of BR-4628 to protect MR against proteolysis. Upon to glycine and methionine residues at the corresponding posi- treatment of the unbound [ S]MR for 10 min at 20 °C by 15 tions of the human AR, GR, and PR (26, 37). Because BR-4628 g/ml trypsin, a major fragment of 41 kDa and a minor frag- possesses remarkable selectivity toward MR, we wondered ment of 30 kDa both encompassing the LBD (38) were recov- whether these residues are involved in the high MR selectivity. ered (Fig. 5C). They were completely digested when the trypsin The A773G and S810M single mutations and the A773G/ concentration was increased to 120 g/ml. In contrast, the S810M double mutation have no effect on the aldosterone aldosteroneMR complex was highly resistant to the trypsin potency to activate MR as illustrated by identical ED values action. Although the proteolysis pattern is similar for the (Fig. 4). In sharp contrast, these mutations lead to an increase of MRBR-4628 complex, the intensity of the 30-kDa fragment is BR-4628 IC values by 28-, 5-, and 356-fold, respectively much lower upon BR-4628 treatment (Fig. 5C). This result indi- (supplemental Fig. S3 and Table 2). These results suggest that cates that BR-4628 is less efficient than aldosterone in protect- the alanine/serine pair at the 773 and 810 positions favor ing the MR against proteolysis, confirming the high instability BR-4628 binding to MR. of the BR-4628MR complex. The MRBR-4628 Complex Is Unstable and Unable to Recruit To further characterize the mechanism by which BR-4628 Transcriptional Co-regulators—To further characterize the inactivates MR, we evaluated the capacity of MR to recruit tran- MRBR-4628 interaction, we synthesized the H-labeled scriptional co-regulators upon BR-4628 binding. Mammalian BR-4628 (see supplemental data) and characterized its binding two-hybrid assays revealed that aldosterone, but not BR-4628 SEPTEMBER 24, 2010• VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 29937 A Potent and Selective Nonsteroidal MR Antagonist contacts with Gln , explaining the importance of this amino acid in our mutational studies. The second noteworthy feature of BR-4628 is its MR selec- tivity. It inhibits the transcriptional activity of the other oxo- steroid receptors (AR, GR, and PR) with IC values higher than 4 M, pointing out only residual affinity for these receptors. MR is the unique oxo-steroid receptor, having an alanine residue 773 810 (Ala ) in the H3 helix and a serine residue (Ser ) in the H5 helix. AR, PR, and GR each harbor a glycine and a methionine at the corresponding positions, respectively. Interestingly, BR-4628 docking within the MR binding pocket underscores that Ser forms a hydrogen bond with the C5-acetyl group and that the Ala residue is surrounded by the aromatic phe- nyl moiety of the chromenone, the C5-acetyl, and the C6-methyl group. The dramatic decrease in the BR-4628 inhib- FIGURE 6. Recruitment of transcriptional co-regulators by MR in mamma- lian two-hybrid assays. HEK293T cells transiently expressing the fusion pro- itory potency upon the A773G, S810M, and A773G/S810M teins VP16-MR and the GAL4 DNA-binding domain fused to the receptor mutations demonstrates the importance of the interactions interacting domain of TIF2 (A) or NcoR (B) were incubated in triplicate with 10 8 8 between BR-4628 and these residues for its potency and ethanol, aldosterone (Aldo,10 to 10 M), spironolactone (Spiro,10 to 6 8 6 9 10 M), or BR-4628 (10 to 10 M) alone or with 10 M aldosterone in the selectivity. 8 6 presence of 10 to 10 M spironolactone or BR-4628. After harvesting the BR-4628 is a dihydropyridine derivative. Classic dihydropy- 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 inde- ridine-derived molecules, such as nifedipine and nitrendipine, pendent experiments. *, p  0.05; **, p  0.01. are known to act as potent L-type calcium channel blockers (34). These molecules have been reported to also have a weak MR antagonist activity (39). However, we show here that a clas- and spironolactone, promoted a dose-dependent binding of the TIF2 to MR (Fig. 6A). Moreover, the two antagonists are able to sical L-type channel blocker like nitrendipine has no selectivity inhibit the aldosterone-induced MR/TIF2 interaction in a dose- toward MR and rather nonselectively antagonizes all steroid hormone receptors in the micromolar range (Table 1). In sharp dependent manner. Interestingly, the NCoR is not recruited by contrast, BR-4628 is a highly potent MR antagonist, with a low MR upon the binding of BR-4628 and spironolactone, whereas a weak interaction is observed after aldosterone binding (Fig. L-type calcium channel binding activity in the micromolar 6B). Furthermore, the two MR antagonists inhibit the binding range. BR-4628 differs from classical dihydropyrine derivatives mainly by its 2-methylchromenonyl substituent. It is likely that of NCoR to the MR-aldosterone complex. Thus, despite a high this substituent drastically reduces calcium channel inhibitory affinity of BR-4628 for MR, the BR-4628MR complex is highly unstable and unable to recruit transcriptional co-modulators. activity, and it is responsible for high MR binding affinity, because it rigidifies the active conformation with respect to the DISCUSSION MR. The methyl group is involved in close van der Waal’s con- In this study, we performed thorough mutagenesis, struc- tact with four lipophilic side chains in MR, explaining the pro- tural, biochemical, and pharmacological investigations of a nounced differences in activity between nitrendipine and novel dihydropyridine-based MR antagonist, which allowed BR-4628 on MR. Thus, the presence of the 2-methylchrom- elucidation of its mechanism of MR inactivation. Transactiva- enonyl substituents on the dihydropyridine ring ensures the tion assays using the MR LBD fused to the GAL4 DNA-binding high MR selectivity versus calcium channel. domain revealed that BR-4628 is as potent as spironolactone Functional and structural data have clearly shown that the but significantly more potent than eplerenone (Table 1). A sim- inability of progesterone and spironolactone to establish a ilar inhibitory hierarchy is also observed by using the entire MR, strong contact with Asn is responsible for their antagonistic because BR-4628 is characterized by an IC of 33.5 nM (Table features (25). BR-4628 strongly interacts with Asn , raising 2) compared with 50.0 nM for spironolactone and 2000 nM for the question of the mechanism by which it inactivates MR. Two eplerenone (27). Molecular dynamics and point mutation data classes of antagonists have been described for steroid receptors. suggest that binding of BR-4628 is mediated by hydrogen bonds The first one, called “passive,” refers to molecules that are 770 776 810 817 to Asn , Gln , Ser , and Arg and lipophilic contacts to unable to stabilize the receptor in a conformation able to recruit 773 852 942 Ala , Met , and Cys . The calculated hydrogen bond the transcriptional co-regulators. These antagonists are usually probabilities in the molecular dynamics simulation agree well small molecules that dissociate quickly from the receptor. This for all point mutation data; the higher the observed H-bond is namely the case for the MR antagonists progesterone and frequency, the more important the amino acid is in the trans- spironolactone, the estrogen receptor  antagonist (R,R)-5,11- activation experiment. Only Gln is underestimated in the cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (40), and the molecular dynamics simulation. However, even in the pub- AR antagonist hydroxyflutamide (41). The second class of lished x-ray structures, the distance between the steroidal antagonists, called “active,” comprises molecules that permit 3-oxo function and the Gln side chain NH is relatively large the LBD to recruit transcriptional co-repressors. This type of (3.2–3.3 Å), and an additional H-bond acceptor in a ligand (like antagonism is observed namely with RU486 for PR and GR and the 5-acetyl group in BR-4628) might form water-mediated fulvestrant for estrogen receptor . These molecules are char- 29938 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010 A Potent and Selective Nonsteroidal MR Antagonist REFERENCES acterized by bulky side chains that impair H12 helix from adopting its active conformation. The docking experiments we 1. Horisberger, J. 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(1994) Acta Crystallogr. D Biol. Crystal- 39. Dietz, J. D., Du, S., Bolten, C. W., Payne, M. A., Xia, C., Blinn, J. R., Funder, logr. 50, 178–185 J. W., and Hu, X. (2008) Hypertension 51, 742–748 46. Ehlert, F. J., Roeske, W. R., Itoga, E., and Yamamura, H. I. (1982) Life Sci. 40. Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Kat- 30, 2191–2202 29940 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 39 •SEPTEMBER 24, 2010

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Sep 24, 2010

Keywords: Drug Action; Drug Design; Heart; Kidney; Steroid Hormone Receptor; Cardiovascular Diseases; Drug Discovery; Mechanism of Action; Steroid Receptors; Antagonist

References