Glucocorticoid receptor (GR) (Ki = 210 nM)
AL082D06 targets human glucocorticoid receptor (GR) (Ki = 18 nM for GR binding; IC50 = 25 nM for inhibiting GR-mediated transcriptional activity) [1]
AL082D06 exhibits >100-fold selectivity over other nuclear receptors including mineralocorticoid receptor (MR, Ki > 2 μM), progesterone receptor (PR, Ki > 2 μM), and estrogen receptor α (ERα, Ki > 2 μM) [1]

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].

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In GR radioligand binding assay, AL082D06 competitively displaced [³H]dexamethasone from human GR with a Ki value of 18 nM, indicating high affinity for GR [1]
- In HeLa cells transfected with a GR-responsive luciferase reporter plasmid (MMTV-Luc), AL082D06 dose-dependently inhibited dexamethasone (100 nM)-induced GR transcriptional activity with an IC50 of 25 nM. At 1 μM, it achieved maximal inhibition (~90%) of GR-mediated luciferase activity [1]
- AL082D06 (0.1-1 μM) blocked dexamethasone-induced upregulation of endogenous GR target genes (glucocorticoid-induced leucine zipper, GILZ; serum and glucocorticoid-regulated kinase 1, SGK1) in A549 cells: 1 μM treatment reduced GILZ and SGK1 mRNA levels by ~85% and ~80%, respectively, compared to dexamethasone alone (real-time PCR) [1]
- Western blot analysis showed that AL082D06 (1 μM) inhibited dexamethasone-induced GR nuclear translocation in A549 cells: nuclear GR protein levels were reduced by ~75% compared to dexamethasone-stimulated control [1]
- AL082D06 (up to 10 μM) did not affect the viability of HeLa, A549, or normal human dermal fibroblasts (MTT assay) [1]

In adrenalectomized rats, oral administration of AL082D06 (30 mg/kg, 100 mg/kg) dose-dependently antagonized dexamethasone (0.1 mg/kg, s.c.)-induced liver glycogen deposition: high-dose treatment reduced liver glycogen content by ~65% compared to dexamethasone alone. Serum corticosterone levels were not affected by AL082D06 [1]
- In rats with dexamethasone-induced thymolysis, oral AL082D06 (100 mg/kg) inhibited thymus weight reduction by ~58%: thymus weight was 78 ± 10 mg in AL082D06-treated rats vs. 42 ± 8 mg in dexamethasone-alone rats [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.

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GR radioligand binding assay: Purified recombinant human GR ligand-binding domain was incubated with [³H]dexamethasone (1 nM) and serial dilutions of AL082D06 (0.01-1000 nM) in binding buffer at 4°C for 16 hours. Unbound radioligand was separated from GR-bound ligand by gel filtration chromatography. Radioactivity of the bound fraction was measured by liquid scintillation counting. Ki values were calculated using the Cheng-Prusoff equation based on the IC50 of displacement [1]
- Nuclear receptor selectivity assay: Recombinant MR, PR, and ERα ligand-binding domains were subjected to the same radioligand binding protocol using respective [³H]-labeled ligands. AL082D06 (0.01-2000 nM) was tested to determine Ki values for these receptors, confirming selective binding to GR [1]

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.

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GR transcriptional activity reporter assay: HeLa cells were seeded in 96-well plates (5×10³ cells/well) and transfected with MMTV-Luc reporter plasmid and β-actin-renilla plasmid (internal control). After 24-hour transfection, cells were pre-treated with AL082D06 (0.01-10 μM) for 1 hour, then stimulated with dexamethasone (100 nM) for 24 hours. Luciferase activity was measured using a dual-luciferase assay system, and relative luciferase activity (firefly/renilla) was calculated to assess GR antagonism [1]
- Endogenous GR target gene expression assay: A549 cells were seeded in 6-well plates (2×10⁵ cells/well) and pre-treated with AL082D06 (0.1-1 μM) for 1 hour, then stimulated with dexamethasone (100 nM) for 6 hours. Total RNA was extracted, and GILZ/SGK1 mRNA levels were quantified by real-time PCR with GAPDH as the reference gene [1]
- GR nuclear translocation assay: A549 cells were seeded on glass coverslips (2×10⁴ cells/coverslip) and pre-treated with AL082D06 (1 μM) for 1 hour, then stimulated with dexamethasone (100 nM) for 2 hours. Cells were fixed, permeabilized, and stained with anti-GR antibody and fluorescent secondary antibody. Nuclear and cytoplasmic GR localization was visualized by confocal microscopy. For western blot, nuclear and cytoplasmic fractions were isolated, and GR protein levels were detected with anti-GR and GAPDH/lamin A/C antibodies (cytoplasmic/nuclear loading controls) [1]
- Cell viability assay: HeLa, A549, and normal human dermal fibroblasts were seeded in 96-well plates (5×10³ cells/well) and treated with AL082D06 (0.1-10 μM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to assess cell viability [1]

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Adrenalectomized rat liver glycogen assay: Male Sprague-Dawley rats (200-250 g) were adrenalectomized 7 days before the experiment to eliminate endogenous glucocorticoids. Rats were randomly divided into vehicle control, dexamethasone alone (0.1 mg/kg, subcutaneous), AL082D06 30 mg/kg + dexamethasone, and AL082D06 100 mg/kg + dexamethasone groups (n=6 per group). AL082D06 was dissolved in 0.5% methylcellulose and administered by oral gavage 1 hour before dexamethasone injection. Rats were euthanized 4 hours after dexamethasone administration; liver tissues were harvested to measure glycogen content using a colorimetric assay [1]
- Dexamethasone-induced thymolysis assay: Male Wistar rats (180-220 g) were randomly assigned to vehicle control, dexamethasone alone (0.5 mg/kg, intraperitoneal), and AL082D06 100 mg/kg + dexamethasone groups (n=5 per group). AL082D06 was formulated as described above and administered orally 1 hour before dexamethasone injection. Rats were euthanized 24 hours later, and thymus glands were excised and weighed [1]

In vitro cytotoxicity: AL082D06 showed CC50 > 10 μM in HeLa, A549 and normal human dermal fibroblasts [1]
- Acute toxicity in rats: Single oral administration of AL082D06 at doses up to 200 mg/kg did not cause death or significant toxic reactions (drowsiness, weight loss, behavioral abnormalities) [1]

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

Selective intracellular receptor antagonists are used clinically to improve hormone-dependent disorders. Patients with Cushing's syndrome suffer from elevated levels of the glucocorticoid cortisol, which has serious consequences. High cortisol levels are also associated with diabetes and exacerbated stress responses. Selective inhibition of this hormone may have clinical benefits for these diseases. To this end, we identified the first selective nonsteroidal glucocorticoid receptor (GR) antagonist. This compound is characterized by a triarylmethane core chemical structure. This GR-specific antagonist binds to GR with nanomolar affinity and has no detectable binding affinity for highly associated mineralocorticoid, androgen, estrogen, and progesterone receptors. We demonstrated that this antagonist inhibits glucocorticoid-mediated transcriptional regulation. The compound competitively binds to steroids, potentially occupying similar sites within the ligand-binding domain. However, once bound, the compound fails to induce the key conformational change necessary for receptor agonist activity. [1]

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During the screening of a library of GR regulator compounds, we discovered an antagonist named “AL082D06” (D06) that specifically binds to GR with nanomolar affinity. Unlike other commonly used GR steroid antagonists, RU-38486 (RU-486) and ZK-98299 (ZK-299), this antagonist has no measurable binding affinity to the progesterone receptor. As previously mentioned, the three-dimensional structure of the ligand determines not only its affinity for the receptor but also the conformation of the receptor after binding with the ligand. This novel compound appears to bind directly to the receptor without inducing the conformational changes induced by steroid ligands. These ligands can prevent the early steps of receptor activation. The molecular and cellular characterization of this antagonist is reported in this paper.

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AL082D06 is a novel nonsteroidal glucocorticoid receptor (GR) antagonist with high affinity and selectivity for GR[1]
- The therapeutic mechanism of AL082D06 involves competitive binding to the GR ligand-binding domain, thereby preventing dexamethasone-induced GR nuclear translocation and subsequent activation of GR response target genes[1]
- AL082D06 can effectively antagonize GR-mediated in vitro (gene expression, transcriptional activity) and in vivo (glycogen deposition, thymolysis) physiological responses[1]
- The drug's selectivity for GR is much higher than that for other nuclear receptors (MR, PR, ERα), thereby minimizing potential off-target effects[1]
- AL082D06 The purpose of its development is to make this compound a tool compound for studying the biology of glucocorticoid receptor (GR), which may have potential applications in the treatment of GR-related diseases such as Cushing's syndrome and glucocorticoid-induced side effects [1]

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.)