Glucocorticoid receptor (GR) (Ki = 210 nM)

GR is selectively bound by AL 082D06 (D06) with nanomolar affinity. When half-maximal DEX concentration was used to stimulate the MMTV:Luc reporter, transcriptional activation decreased in a dose-dependent manner upon the addition of AL 082D06. Several glucocorticoid-responsive promoter-reporter systems are used by AL 082D06, including a less complex one made up of isolated glucocorticoid-responsive element (GRE) sequences and the 3-kb tyrosine aminotransferase (TAT) promoter. promoter to sabotage the work of reporters. With nanomolar affinity, AL 082D06 faces off against 3H-Dex for baculovirus-expressed GR. Using the appropriate receptor and tritiated ligand (>2500 nM), similar structural binding assays revealed no affinity for AL 082D06 for other intracellular receptors (AR, ER, PR, and MR). Progesterone, androgens, mineralocorticoids, retinoic acid, glucocorticoids, and estrogen receptors are not activated by AL 082D06. When tested against other steroid receptors, AL 082D06 is much less effective than the reference antagonist used as a control, despite having a very strong ability to oppose GR activity [1].

Competitive Binding Assay[1]
Growth and purification of recombinant hGR baculovirus followed the protocol outlined by Summers and Smith . The extract and binding assay buffer consisted of 25 mM sodium phosphate, 10 mM potassium fluoride, 10 mM sodium molybdate, 10% glycerol, 1.5 mM EDTA, 2 mM dithiothreitol, 2 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 1 mM phenylmethylsulfonyl fluoride (pH 7.4), at room temperature. Intracellular receptors produced in this fashion exhibit reproducible interaction with known ligands at the published affinity. These preparations were subjected to extensive quality control experiments before the assays, covering receptor response, specificity, size, and reference ligand affinity. Receptor assays were performed with a final volume of 250 μl containing from 50–75 μg of extract protein, plus 1–2 nM [3H]Dex at 84 Ci/mmol and varying concentrations of competing ligand (0 to 10−5M). Assays were set up using a 96-well minitube system, and incubations were carried out at 4 C for 18 h. Equilibrium under these conditions of buffer and temperature was achieved by 6–8 h. Nonspecific binding was defined as that binding remaining in the presence of 1000 nM unlabeled Dex. At the end of the incubation period, 200 μl of 6.25% hydroxyapatite were added in wash buffer (binding buffer in the absence of dithiothreitol and phenylmethylsulfonyl fluoride). Specific ligand binding to receptor was determined by a hydroxyapatite-binding assay according to the protocol of Wecksler and Norman. Hydroxyapatite absorbs the receptor-ligand complex, allowing for the separation of bound from free radiolabeled ligand. The mixture was vortexed and incubated for 10 min at 4 C and centrifuged, and the supernatant was removed. The hydroxyapatite pellet was washed two times in wash buffer. The amount of receptor-ligand complex was determined by liquid scintillation counting of the hydroxyapatite pellet after the addition of 0.5 mM EcoScint A scintillation cocktail from National Diagnostics.[1]
After correcting for nonspecific binding, IC50 values were determined. The IC50 value is defined as the concentration of competing ligand required to reduce specific binding by 50%; the IC50 values were determined graphically from a log-logit plot of the data. Kd values for the analogs were calculated by application of the Cheng-Prussof equation. Steroid standards are included in each assay, and resulting Kd values are determined by use of a modified Cheng-Prussoff equation.[1]
MR, AR, PR, and ERα expression in the baculovirus system and binding assays was conducted similarly except that labeled ligands were aldosterone [1–2 nM3H-aldosterone from Amersham Pharmacia Biotech (TRK 434), specific activity 60 Ci/mmol], DHT (1–2 nM3H-DHT at 130 Ci/mmol), progesterone [2–3 nM3H -progesterone (93 Ci/mmol], and estradiol [2–3 nM3H-estradiol, 114 Ci/mmol], respectively. Each binding assay point is done in duplicate, and each full experiment is repeated three or more times.

TAT Assay.[1]
TAT activity in H4IIE cells was measured as described previously (60). Preconfluent H4IIE cells in 96-well plates were incubated for 24 h with compound, washed with PBS, and lysed. Extracts were subjected to enzymatic assay as described.[1]
IL-6 was measured in confluent human skin fibroblasts in induction media (1.75% BSA/antibiotics/DMEM) after incubation with induction media for 4–6 h. Media were changed and cells incubated a further 1 h in induction media; compound. IL-1β was then added to a final concentration of 1 ng/ml in induction media, and cells were cultured for 24 h. Media were removed and added to Maxisorp Plate (Nunc) with capture antibody (IL-6-monoclonal mouse antihuman IL-6)-coated wells and incubated at room temperature (RT) overnight. Plate was washed twice in PBS, blocked with 4% BSA/PBS, and incubated 1 h at RT. Secondary antibody-biotinylated monoclonal antihuman IL-6, 500 μg/ml in 4% BSA/PBS, was added and incubated for 2 h at RT, and washed three times in PBS. A 1:5000 diluted ExtrAvidin-horseradish peroxidase solution in 4% BSA/PBS was added and incubated for 30 min at RT. Plates were washed three times in PBS, and substrate solution (One hundred microliters of 3,3′,5,5′ tetramethyl benzidine-hydrogen peroxide was added and incubated 15 min at RT. Reaction was stopped with 50 μl per well of 2 N H2SO4 and OD was read at 450 nm/540 nm. Collagenase was measured in confluent human skin fibroblasts induced in 1.75% BSA-DMEM with compounds for 1 h. IL-1β was added in induction medium (final 1 ng/ml) and the cells were cultured for 24 h.

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Collagenase Assay.[1]
Culture supernatants were added to 0.1% BSA/PBS and incubated for 2 h at RT; after washing, polyclonal rabbit antihuman MMP-1 in assay buffer was added and incubated for 2 h at RT. After washing, horseradish peroxidase-donkey antirabbit Ig in 0.1% BSA/0.1% Tween 20/PBS was added and incubated for 1 h at RT. One hundred microliters of 3,3′,5,5′ tetramethyl benzidine-hydrogen peroxide were added and incubated for approximately 5–30 min at RT, after which 100 μl per well of stop solution (1 N H2SO4) were added and the OD read at 450/540 nm.

[1]. A nonsteroidal glucocorticoid receptor antagonist. Mol Endocrinol. 2003 Jan;17(1):117-27.

Selective intracellular receptor antagonists are used clinically to ameliorate hormone-dependent disease states. Patients with Cushing's syndrome have high levels of the glucocorticoid, cortisol, and suffer significant consequences from this overexposure. High levels of this hormone are also implicated in exacerbating diabetes and the stress response. Selectively inhibiting this hormone may have clinical benefit in these disease states. To this end, we have identified the first selective, nonsteroidal glucocorticoid receptor (GR) antagonist. This compound is characterized by a tri-aryl methane core chemical structure. This GR-specific antagonist binds with nanomolar affinity to the GR and has no detectable binding affinity for the highly related receptors for mineralocorticoids, androgens, estrogens, and progestins. We demonstrate that this antagonist inhibits glucocorticoid-mediated transcriptional regulation. This compound binds competitively with steroids, likely occupying a similar site within the ligand-binding domain. Once bound, however, the compound fails to induce critical conformational changes in the receptor necessary for agonist activity.[1]

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During the course of screening compound libraries for GR modulators, we discovered an antagonist, designated “AL082D06” (D06) that bound specifically to GR with nanomolar affinity. This antagonist is unlike the other frequently used steroidal antagonists for GR, RU-38486 (RU-486) and ZK-98299 (ZK-299), in that it has no measurable binding affinity for the progesterone receptor. As has been described previously, the three-dimensional structure of the ligand defines not only its affinity for the receptor, but also the conformation of that receptor once it has associated with ligand. This new compound appears to bind directly to receptor without inducing the same conformational changes associated with steroidal ligands. These ligands prevent the occurrence of some of the earliest steps in receptor activation. We report here the molecular and cellular characterization of this antagonist.

C23H24CLN3O2

409.91

409.156

C, 67.39; H, 5.90; Cl, 8.65; N, 10.25; O, 7.81

256925-03-8

9822799

Light yellow to yellow solid powder

6.083

0

4

5

29

495

0

IPICUXHYPAMJNC-UHFFFAOYSA-N

InChI=1S/C23H24ClN3O2/c1-25(2)18-9-5-16(6-10-18)23(17-7-11-19(12-8-17)26(3)4)21-15-20(27(28)29)13-14-22(21)24/h5-15,23H,1-4H3

4,4'-((2-chloro-5-nitrophenyl)methylene)bis(N,N-dimethylaniline)

AL-082D06; AL082D06; AL 082D06; 256925-03-8; AL 082D06; AL082D06; 4,4'-((2-chloro-5-nitrophenyl)methylene)bis(N,N-dimethylaniline); TCMDC-124088; 4-[(2-chloro-5-nitrophenyl)-[4-(dimethylamino)phenyl]methyl]-N,N-dimethylaniline; D-06; AL-082D06; D-06; D06; D 06

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)