RU 26752

Corticosterone is a preferable ligand for measuring rat brain corticosteroid receptors: competition by RU 28362 and RU 26752 for dexamethasone binding in rat hippocampal cytosol.[Pubmed:8242402]

Brain Res. 1993 Oct 15;625(1):84-92.

It is unclear whether in vitro corticosteroid receptor binding assays have used inappropriately high concentrations of synthetic corticosteroid competitors, thereby potentially introducing error into estimates of type I (mineralocorticoid) and type II (glucocorticoid) receptor binding. To determine more accurately the concentration of blockers necessary to discriminate between these two sites, we have derived Ki values for the competition of dexamethasone, RU 28362 and RU 26752 for [3H]corticosterone and [3H]dexamethasone binding in rat hippocampus. Non-specific binding of both radioligands was defined with unlabeled dexamethasone to exclude transcortin. The type II agonist RU 28362 competed for only a portion of [3H]corticosterone binding, exhibiting a Ki of 0.5 nM for this binding. In contrast, RU 28362 fully competed all binding of a saturating concentration of [3H]dexamethasone, even though [3H]dexamethasone also recognized type I receptors, defined as specific [3H]corticosterone binding in the presence of 80 nM RU 28362. RU 28362 competition for [3H]dexamethasone binding exhibited characteristics of a 2-site interaction, with Kis of 0.3 and 194 nM. The type I receptor antagonist RU 26752 competed less effectively for [3H]corticosterone and [3H]dexamethasone binding, but nonetheless competed fully within a 1000-fold concentration range. Even at a level less than 125 x its Ki for type I binding, RU 26752 still inhibited virtually all type II receptor binding by [3H]corticosterone. We conclude that type I and II receptors in rat brain are best distinguished using [3H]corticosterone as the labelling ligand, with cold RU 28362 and dexamethasone to eliminate binding to type II and transcortin sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of antimineralocorticoid RU 26752 on steroid-induced hypertension in rats.[Pubmed:2333987]

Am J Physiol. 1990 May;258(5 Pt 1):E737-9.

The effect of mineralocorticoid antagonist RU 26752 on the development and maintenance of hypertension produced by long-term administration of mineralocorticoid agonist aldosterone has been investigated. Uninephrectomized, saline-drinking male Sprague-Dawley rats were subcutaneously implanted with either placebo (control) pellets or pellets containing 100 micrograms aldosterone, 50 mg RU 26752, or 100 micrograms aldosterone plus 50 mg RU 26752. Aldosterone treatment resulted in an increase in blood pressure to 165 +/- 5 mmHg over the control value of 105 +/- 2 mmHg within 3 wk of experimental period. RU 26752 given alone had no observable hypertensinogenic effect. However, RU 26752 administered with aldosterone significantly prevented the hypertension produced by aldosterone alone. RU 26752 when given with aldosterone was able to prevent the aldosterone-induced increase in saline consumption, increase urine output, and reduce urinary Na+ excretion. The results presented suggest that long-term administration of antimineralocorticoid RU 26752 in vivo to Sprague-Dawley rats prevents the aldosterone-induced hypertension.

Structural basis of spirolactone recognition by the mineralocorticoid receptor.[Pubmed:17569793]

Mol Pharmacol. 2007 Sep;72(3):563-71.

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.