Retinoic Acid Receptors (RARs) - α, β, and γ subtypes (antagonist) [1]

Kd: 2 nM (RARα), 2 nM (RARβ), 3 nM (RARγ)[1]

AGN 193109 is a very potent retinoic acid receptor antagonist, with Kds of 2 nM, 2 nM, and 3 nM for RARα, RARβ, and RARγ, to name a few. Because it does not bind to or transactivate through any RXRs, AGN 193109 is entirely RAR specific[1]. In ECE16-1 cells, AGN 193109 (100 nM) prevents the morphological alteration that is dependent on TTNPB (a retinoic acid receptor agonist). In ECE16-1 cells, AGN193109 totally demonstrates the effect of retinoid-dependent growth suppression at 100 nM, but at 10 nM it partially reverses it. Additionally, TTNPB-induced decreases in K5, K6, K14, K16, and K17 and increases in K7, K8, and K19 levels are eliminated by AGN193109 (100 nM)[2].

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Retinoids are important physiological agents that regulate epithelial cell differentiation and proliferation. The importance of these agents in regulating growth, development, and differentiation has led to a search for new retinoid agonists and antagonists. In the present manuscript we show that AGN193109, a retinoid analog, is an efficient antagonist of retinoid action in human cervical epithelial cells. Treatment of ECE16-1 cells with natural or synthetic retinoids reduces cytokeratin K5, K6, K14, K16, and K17 levels, increases cytokeratin K7, K8, and K19 levels, increases retinoic acid receptor-beta (RAR beta) mRNA levels, suppresses proliferation, and alters cell morphology. Co-treatment with AGN193109 prevents these responses. Half-maximal and maximal antagonism is observed at a molar ratio of AGN193109: retinoid agonist of 1:1 and 10:1, respectively. When administered alone AGN193109 has no agonist activity. Thus, AGN193109, which binds to RAR alpha, RAR beta, and RAR gamma with Kd values = 2,2, and 3 nm, respectively, but is unable to bind to the retinoid X receptors, is a highly active antagonist of retinoid action in ECE16-1 cells[2].

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In receptor binding assays using baculovirus-expressed RARs, AGN 193109 bound with very high affinity to all three RAR subtypes, with Kd values of 2 nM for RARα, 2 nM for RARβ, and 3 nM for RARγ. This represents approximately 4-6 fold higher affinity than the natural hormone, all-trans-retinoic acid (RA). [1]
- AGN 193109 was completely RAR-specific, as it did not bind to or transactivate through any of the retinoid X receptors (RXRα, β, or γ). [1]
- In transactivation assays using CV-1 cells co-transfected with RAR holoreceptors and a TREpɑl-luciferase reporter gene, AGN 193109 showed absolutely no agonist activity at any of the three RAR subtypes, even at receptor-saturating concentrations. [1]
- AGN 193109 potently antagonized RA-induced transcriptional activity. At equimolar concentrations (10⁻⁸ M), it inhibited RA activity by approximately 85% at RARα, 62% at RARβ, and 100% at RARγ. A 10-fold excess of AGN 193109 (10⁻⁷ M) completely abrogated the transactivation activity of RA at all three RARs. [1]
- The antagonist activity of AGN 193109 was further confirmed in other assays, including dose-dependent inhibition of RA activity in a chimeric estrogen receptor-RAR transactivation assay and suppression of transglutaminase induction by RAR agonists in 3T3 cells stably transfected with RARβ and RARγ. [1]

AGN 193109 (1.15 μmol/kg) reduces the increase in spleen weight of mice induced by TTNPB, but does not cause overt toxicity or affect spleen weight in animals. Additionally, AGN 193109 dramatically lessens the cutaneous toxicity that ATRA causes. Topical administration of AGN 193109 (0.30 or 1.20 μmol/kg) effectively lowers cutaneous toxicity and weight loss brought on by oral TTNPB cotreatment[3].

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AGN 193109 was recently identified as a potent retinoic acid receptor (RAR) antagonist in vitro. The purpose of the present study was to determine if AGN 193109 functions as an RAR antagonist in vivo and thus could prevent and/or treat retinoid toxicity. Female hairless mice were treated topically for 5 consecutive days with the synthetic retinoic acid receptor agonist (E)-4-[2-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen-1-yl]benzoic acid (TTNPB) alone or in the presence of a 1-, 4-, or 16-fold molar excess of AGN 193109. TTNPB caused skin flaking, skin abrasions, and splenomegaly, and these effects were blocked in a dose-dependent fashion by AGN 193109 cotreatment. In the same model, AGN 193109 also decreased topical irritation induced by the natural RAR agonist, all-trans-retinoic acid. To determine if topical AGN 193109 could block toxicity induced by an oral retinoid, mice were treated by gavage with TTNPB (0.75 mumol/kg/day) and topically with 0, 0.3, or 1.2 mumol/kg/day of AGN 193109 for 4 days. TTNPB treatment alone caused cutaneous irritation and weight loss, and these effects were inhibited by AGN 193109 cotreatment. To determine if AGN 193109 could be used to treat preexisting retinoid toxicity, mice were pretreated topically on Days 1-2 with TTNPB (0.72 mumol/kg/day) and then treated topically on Days 3-5 with 0, 1.44, 7.2, or 36.0 mumol/kg of AGN 193109. TTNPB pretreatment caused precipitous weight loss and, in the absence of AGN 193109 intervention, 60% mortality. AGN 193109 treatment at all dose levels significantly accelerated recovery of body weight and prevented death in TTNPB-intoxicated mice. These data demonstrate that AGN 193109 is a potent RAR antagonist and a potential antidote of retinoid intoxication in vivo. In addition to potential clinical applications in the prevention and treatment of retinoid toxicity, AGN 193109 should provide a powerful experimental tool for the elucidation of retinoid biology[3].

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In female hairless mice, topical co-treatment with AGN 193109 dose-dependently blocked cutaneous toxicity (skin flaking and abrasions) and splenomegaly induced by the RAR-specific agonist TTNPB. An equimolar dose of AGN 193109 drastically reduced topical irritation, and a 16-fold molar excess completely blocked it. It also reversed TTNPB-induced weight loss. [3]
- AGN 193109 significantly reduced topical irritation (cutaneous toxicity score from 6 ± 1 to 2 ± 1) induced by the natural RAR agonist, all-trans-retinoic acid (ATRA), in the same mouse model. [3]
- When TTNPB was administered orally (0.75 μmol/kg/day for 4 days) to induce systemic toxicity, topical application of AGN 193109 (0.30 or 1.20 μmol/kg/day) significantly reduced both the resulting weight loss and cutaneous toxicity in a dose-dependent manner. The higher dose of AGN 193109 almost completely blocked these effects. [3]
- In a pre-existing toxicity model, mice were pretreated with a high dose of topical TTNPB (0.72 μmol/kg/day) for 2 days, causing severe weight loss and 60% mortality. Subsequent treatment with topical AGN 193109 (1.44, 7.2, or 36.0 μmol/kg/day) on days 3-5 significantly accelerated body weight recovery and prevented death (0% mortality) in all treatment groups, demonstrating its potential as an antidote for retinoid intoxication. [3]

Protein and Nucleic Acid Methods [2]
Poly(A)+ RNA was prepared, electrophoresed, transferred to nylon membrane, and hybridized with 32P-labeled cDNAs encoding glyceraldehyde-3-phosphate dehydrogenase, cytokeratin K6, or RARβas described previously. For detection of cytokeratins, ECE16-1 cells were labeled with [35S]methionine and cytokeratins were prepared and electrophoresed in two dimensions exactly as described previously.

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Cells (10,000/cm2) are seeded in complete medium and allowed to attach overnight. The cells are then shifted to defined medium (DM), allowed to equilibrate for 24 h, and treatment is initiated by addition of fresh DM or DM containing epidermal growth factor (EGF) or retinoid. After 3 days of daily treatment with retinoid, the cells are harvested with 0.025% trypsin, 1 mM EDTA, fixed in isotonic buffer containing 4% formaldehyde, and counted using a counter[2].

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RAR Transactivation Assay: CV-1 cells were co-transfected with a luciferase reporter plasmid (ΔMTV-TREp-LUC) and an expression vector for the indicated retinoid receptor (RARα, RARβ, or RARγ). Cells were then treated with various concentrations of all-trans-retinoic acid (RA) or AGN 193109 to assess agonist activity. For antagonist assays, cells were treated with a fixed concentration of RA (10⁻⁸ M) that induces significant transcriptional activity, along with increasing concentrations of AGN 193109. Luciferase activity was measured to quantify transcriptional activation. [1]
- Receptor Binding Assay: The binding affinity (Kd) of AGN 193109 for the RARs was determined via competition binding assays. Baculovirus-expressed RARs (α, β, and γ) were incubated with 5 nM of [³H]-(all-E)-retinoic acid and increasing concentrations of unlabeled test retinoid (AGN 193109). Kd values were calculated from the competition curves using the equation of Cheng and Prusoff. [1]

Animals:** Female hairless mice (Crl:SKH1-hrBR), 5-11 weeks old, were used. They were housed individually during experiments with free access to food and water. [3]
- **Drug Formulation and Administration:** Retinoids were dissolved in vehicles containing acetone and DMSO (various ratios, e.g., 97.6% acetone/2.4% DMSO, 92.5% acetone/7.5% DMSO, or 70% acetone/30% DMSO) for topical application. For oral administration, TTNPB was dissolved in corn oil. Topical treatments were applied over the entire dorsal skin surface in a volume of 4 or 6 mL/kg. Oral treatments were administered by gavage. All treatments were given once daily. [3]
- **Experiment 1 (Topical Co-treatment):** Mice (n=5-6/group) were treated topically for 5 days with vehicle, TTNPB (0.072 μmol/kg), AGN 193109 (1.15 μmol/kg), or TTNPB (0.072 μmol/kg) plus increasing doses of AGN 193109 (0.072, 0.288, or 1.15 μmol/kg). Mice were euthanized on Day 8. Body weight, cutaneous toxicity (flaking and abrasions scored daily), and spleen weight were measured. [3]
- **Experiment 2 (Oral TTNPB + Topical AGN 193109):** Mice (n=5-6/group) were treated for 4 days with oral TTNPB (0.75 μmol/kg/day) or vehicle (corn oil), and concurrently with topical AGN 193109 (0.3 or 1.20 μmol/kg/day) or vehicle. Mice were euthanized on Day 5. Body weight change and cutaneous toxicity were assessed. [3]
- **Experiment 3 (Treatment of Pre-existing Toxicity):** Mice (n=5/group) were pretreated topically on Days 1-2 with a high dose of TTNPB (0.72 μmol/kg/day). On Days 3-5, they were treated topically with vehicle or AGN 193109 (1.44, 7.2, or 36.0 μmol/kg/day). A control group received vehicle throughout. Body weight was monitored daily, and mortality was recorded. Mice were euthanized on Day 8. [3]
- **Cutaneous Toxicity Scoring:** A semi-quantitative scale was used to score skin flaking (0-5) and abrasions (0-4) daily. These scores were combined to calculate a composite "cutaneous toxicity score" (range 0-17) that accounted for maximal severity, mean severity, and time of onset. [3]

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Animals: Female hairless mice (Crl:SKH1-hrBR), 5-11 weeks old, were used. They were housed individually during experiments with free access to food and water. [3]
- Drug Formulation and Administration: Retinoids were dissolved in vehicles containing acetone and DMSO (various ratios, e.g., 97.6% acetone/2.4% DMSO, 92.5% acetone/7.5% DMSO, or 70% acetone/30% DMSO) for topical application. For oral administration, TTNPB was dissolved in corn oil. Topical treatments were applied over the entire dorsal skin surface in a volume of 4 or 6 mL/kg. Oral treatments were administered by gavage. All treatments were given once daily. [3]
- Experiment 1 (Topical Co-treatment): Mice (n=5-6/group) were treated topically for 5 days with vehicle, TTNPB (0.072 μmol/kg), AGN 193109 (1.15 μmol/kg), or TTNPB (0.072 μmol/kg) plus increasing doses of AGN 193109 (0.072, 0.288, or 1.15 μmol/kg). Mice were euthanized on Day 8. Body weight, cutaneous toxicity (flaking and abrasions scored daily), and spleen weight were measured. [3]
- Experiment 2 (Oral TTNPB + Topical AGN 193109): Mice (n=5-6/group) were treated for 4 days with oral TTNPB (0.75 μmol/kg/day) or vehicle (corn oil), and concurrently with topical AGN 193109 (0.3 or 1.20 μmol/kg/day) or vehicle. Mice were euthanized on Day 5. Body weight change and cutaneous toxicity were assessed. [3]
- Experiment 3 (Treatment of Pre-existing Toxicity): Mice (n=5/group) were pretreated topically on Days 1-2 with a high dose of TTNPB (0.72 μmol/kg/day). On Days 3-5, they were treated topically with vehicle or AGN 193109 (1.44, 7.2, or 36.0 μmol/kg/day). A control group received vehicle throughout. Body weight was monitored daily, and mortality was recorded. Mice were euthanized on Day 8. [3]
- Cutaneous Toxicity Scoring: A semi-quantitative scale was used to score skin flaking (0-5) and abrasions (0-4) daily. These scores were combined to calculate a composite "cutaneous toxicity score" (range 0-17) that accounted for maximal severity, mean severity, and time of onset. [3]

In the pre-existing toxicity experiment, mice pretreated with TTNPB (0.72 μmol/kg/day for 2 days) and then given vehicle experienced 60% mortality. Subsequent treatment with AGN 193109 at all dose levels (1.44, 7.2, or 36.0 μmol/kg/day) completely prevented this mortality (0% mortality). [3]
- AGN 193109 alone, when administered topically at doses up to 8 μmol/kg for 5 days or 36 μmol/kg for 3 days, did not cause significant weight loss, mortality, or detectable skin irritation (cutaneous toxicity scores of 0-1), indicating it is well-tolerated in this model. One mouse in a high-dose group (1.15 μmol/kg) died, but this was not considered treatment-related as no other animals showed toxicity and much higher doses in subsequent experiments were non-toxic. [3]

[1]. Synthesis and characterization of a highly potent and effective antagonist of retinoic acid receptors. J Med Chem. 1995 Nov 24;38(24):4764-7.

[2]. AGN193109 is a highly effective antagonist of retinoid action in human ectocervical epithelial cells. J Biol Chem. 1996 May 24;271(21):12209-12.

[3]. Specific antagonist of retinoid toxicity in mice. Toxicol Appl Pharmacol. 1996 May;138(1):169-75.

Based on the known antagonist AGN 193109 (2), a series of high-affinity retinoic acid receptor (RAR) antagonists were prepared. After introducing different phenyl groups, para substitution was found to be more advantageous than meta substitution. The antagonist with the highest affinity for RAR contained a hydrophobic group, but the presence of polar functional groups also showed good tolerance. Bioorg Med Chem Lett. 1999 Feb 22;9(4):573-6. doi: 10.1016/s0960-894x(99)00047-5.

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AGN193109 antagonizes a series of responses in ECE16-1 cells[2]
Retinols produce a variety of effects on tissues and cells, and different types of retinols may produce different effects within and between cell lines. In ECE16-1 cells, RAR-specific ligands have stronger retinol activity than RXR-specific ligands. This study investigated the effects of a novel synthetic retinoid, AGN193109, on multiple retinoid-dependent responses in ECE16-1 cells. We found that AGN193109 could eliminate retinoid-induced cell morphological changes and prevent the inhibitory effect of retinoids on cell proliferation. Furthermore, at the biochemical level, AGN193109 also prevented retinoid-induced changes in marker gene expression. We examined genes upregulated (K7, K8, K19, RARβ) and downregulated (K5, K6, K14, K16, K17) after retinoid treatment. The results indicate that AGN193109 can antagonize retinoid-dependent responses. Therefore, AGN193109 can inhibit both stimulative and inhibitory responses. In summary, these results suggest that AGN193109 may be a global antagonist of retinoid action in ECE16-1 cells (i.e., it may antagonize most or even all retinoid-dependent responses).
AGN193109 is a highly potent antagonist without agonist activity [2]
Receptor antagonists may present some problems. First, they may have partial agonist activity. This complicates the interpretation of their role in the in vivo system. To determine whether AGN193109 has intrinsic agonist or antagonist activity, we evaluated its effects on ECE16-1 cell function without the addition of retinoid agonists. AGN193109 at concentrations ranging from 0.01 to 1000 nM did not cause any detectable changes in cell morphology, cell proliferation, or expression of retinoid response marker genes. Based on these results, we conclude that: (i) AGN193109 is a highly potent antagonist of retinoid activity in these cells; and (ii) it does not possess agonist activity itself. Secondly, antagonists may have low affinity for the target receptor. Such ligands must be used at extremely high concentrations to exert their antagonistic effect. However, AGN193109 has an affinity of 2–3 nM for RAR. Considering that the receptor affinities for tRA, 13-cRA, and 9-cRA range from 7 to 141 nM, while the affinity range for TTNPB is 20 to 51 nM, AGN193109 is a high-affinity ligand. This is reflected in its efficacy in inhibiting biological end-effector reactions. The compound exhibits the best inhibitory effect on retinoid agonist activity at a 10-molar excess concentration; at equimolar concentrations, its inhibitory effect is half of the maximum inhibitory effect. Therefore, AGN193109 is a high-affinity antagonist.
Natural ligands interacting with RAR and RXR receptors[2]
Our initial studies showed that the synthetic retinoid AGN193109 interacts only with RAR receptors and is effective in antagonizing the RAR-specific retinoid agonist TTNPB. However, it was unclear whether AGN193109 would inhibit the activity of the natural ligands. Therefore, we tested the ability of AGN193109 to antagonize the effects of tRA, 13-cRA, and 9-cRA. 13-cRA, 9-cRA, and tRA all interact with RAR, while 9-cRA also has a high affinity for RXR. The results showed that AGN193109 is an effective antagonist of the activity of these ligands. Therefore, although AGN193109 interacts only with the RAR form, it can also antagonize the effects of a drug (9-cRA) that has a high affinity for both RAR and RXR. Since AGN193109 does not interact with RXR, it is unlikely to directly block the interaction between 9-cRA and RXR receptors. Furthermore, RXR-specific ligands have relatively low activity in ECE16-1 cells (37). Since 9-cRA can effectively bind to RAR, AGN193109 likely works by inhibiting RAR-mediated 9-cRA effects. [2]
Receptor antagonists are extremely useful ligands, both for understanding mechanisms of action and for designing new therapeutic approaches. In this study, we demonstrated using the human cervical extraepithelial cell line ECE16-1 that AGN193109 is a highly active, highly affinity retinoid antagonist that does not appear to have intrinsic retinoid agonist activity. [2]

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AGN 193109 (Compound 4) is a diarylacetylene-based retinoid that represents a novel class of RAR antagonists. It is described as the most potent and effective antagonist of all three RAR subtypes known at the time of publication (1995). [1]
- Its mechanism of action involves high-affinity binding to the RARs, but unlike agonists such as all-trans-retinoic acid, it induces different conformational changes in the receptors. This prevents the recruitment of coactivator proteins and blocks signal transduction to the transcriptional machinery, thereby inhibiting gene transcription. [1]
- The compound was developed as a powerful tool for research to define the precise physiological functions of RAR hormonal pathways in development and in adult animals, and to elucidate the molecular mechanisms of RAR-mediated transcriptional regulation. [1]
- AGN 193109 was also considered an invaluable lead compound for exploring potential pharmacological applications of RAR antagonists in disease models. A proposed therapeutic application is the prevention and treatment of side effects associated with systemic retinoid therapy, such as mucocutaneous toxicity. [1]

C28H24O2

392.498

392.178

C, 85.68; H, 6.16; O, 8.15

171746-21-7

AGN 193109;171746-21-7;AGN 193109-d7;1216429-25-2

177238

Off-white to light yellow solid powder

1.21 g/cm3

564.5ºC at 760 mmHg

257.1ºC

6.206

1

2

4

30

716

0

NCEQLLNVRRTCKJ-UHFFFAOYSA-N

InChI=1S/C28H24O2/c1-19-4-11-22(12-5-19)24-16-17-28(2,3)26-15-10-21(18-25(24)26)7-6-20-8-13-23(14-9-20)27(29)30/h4-5,8-16,18H,17H2,1-3H3,(H,29,30)

4-[2-[5,5-dimethyl-8-(4-methylphenyl)-6H-naphthalen-2-yl]ethynyl]benzoic acid

AGN-193109; AGN 193109; 171746-21-7; AGN193109; AGN-193109; 4-[2-[5,5-dimethyl-8-(4-methylphenyl)-6H-naphthalen-2-yl]ethynyl]benzoic acid; CHEMBL358145; ZC6062V1O9; AGN 193109 (GMP); AGN193109

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)

DMSO : ~2 mg/mL (~5.10 mM)

Solubility in Formulation 1: 12.5 mg/mL (31.85 mM) in 15% Cremophor EL 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (12.74 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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