Cort108297

Steroid receptor competition binding assays are run in a buffer containing 20 mM HEPES buffer (pH=7.6), 0.2 mM EDTA, 75 mM NaCl, 1.5 mM MgCl₂, 20% glycerol, 20 mM sodium molybdate, 0.2 mM DTT, 20 μg/mL Aprotinin, and 20 μg/mL Leupeptin (assay buffer). Radiolabeled ligands are used to detect binding to cells expressing receptors including 0.25 nM [³H]Aldosterone for mineralocorticoid receptor (MR) binding, 0.3 nM [³H]Dexamethasone for GR binding, 0.36 nM [³H]Methyltrienolone for aldosterone receptor (AR) binding, and 0.29 nM [3H]methyltrienolone for PR binding. Receptors are recombinantly expressed in human embryonic kidney 293 (HEK-293) cells, and 20 μg of 293-MR lysate, 20 μg of 293-GR lysate, 22 μg of 293-AR lysate, or 40 μg of 293-PR lysate are added per well. Competing test compounds (e.g., Cort108297) are added at various concentrations from 0.01 nM to 10 μM. Nonspecific binding is determined in the presence of 500 nM Aldosterone for MR binding, 500 nM Dexamethasone for GR binding, or 500 nM methyltrienolone for AR and PR binding. The binding reactions (140 µL) are incubated overnight at 4°C, then 70 µl of cold charcoal-dextran buffer (containing per 50 mL of assay buffer, 0.75 g of Charcoal, and 0.25 g of Dextran) is added to each reaction. Plates are mixed for 8 minutes on an orbital shaker at 4°C. The plates are then centrifuged at 3000 rpm at 4°C for 10 minutes. A 120 µL aliquot of the binding reaction mixture is then transferred to another 96-well plate, and 175 µL of Wallac Optiphase Hisafe 3 scintillation fluid is added to each well. The plates are sealed and shaken vigorously using an orbital shaker. After 2 hour incubation, the plates are counted using a Wallac MicroBeta counter^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

LAPC4 and CWR-22Rv1 cells are plated in standard media and incubated overnight. Cells are washed with PBS and placed into media containing charcoal stripped FBS, 10% for LAPC4 or 1%/10% for CWR-22Rv1. Cells are treated for indicated times with media changes every other day with either vehicle control or specified treatment: 1 nM R1881, 100 nM Dexamethasone, 10 µM Enzalutamide, 100 nM Mifepristone, 1 µM CORT118335, 1 µM Cort108297. For all experiments, equimolar vehicle (ethanol±DMSO) is added to every sample for equal treatment periods. Cells are plated and treated. At indicated days cells are washed, trypsinized, pelleted, and resuspended in media. Cells are then mixed 1:1 with trypan blue and viable cells are counted in a blinded fashion. Three biological replicates are assayed per condition per time point and the mean of the biological replicates is reported^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Forty ten-week-old, male, C57BL/6J mice are fed ad libitum a diet containing 60% fat calories and water supplemented with 11% sucrose for 4 weeks. In addition, they receive one of the following five treatments: Cort108297 (80 mg/kg QD), Cort108297 (40 mg/kg BID), Mifepristone (30 mg/kg BID), Rosiglitazone, an oral glycemic medication (10 mg/kg QD), or vehicle (10% DMSO in 0.5% CMC). An additional control group (n=8) is fed a standard chow diet and tap water and does not receive any treatment.
Rats^[4]
Male Sprague Dawley rats (250-275 g) are used. Forty-eight rats are matched by body weight and are administered, Cort108297 dissolved in DMSO (30mg/kg s.c.(n=10) or 60 mg/kg s.c. (n=10), Mifepristone dissolved in DMSO 10mg/kg s.c. (n=10), Imipramine dissolved in saline 10mg/kg i.p. (n=10) or vehicle DMSO s.c. (n=4) or saline i.p. (n=4). Control groups consist of both subcutaneous (s.c.) and intraperitoneal (i.p.) groups to control for the route of administration and both DMSO and saline to control for any potential differences between the compounds on neuroendocrine and behavioral stress responsiveness.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.