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].

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].

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.

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].

[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.

A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated. 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 Range of Responses in ECE16-1 Cells[2]
Retinoids produce multiple effects on tissues and cells and different classes of retinoids can produce differing effects within and among cell lines. In ECE16-1 cells, RAR-specific ligands are more active retinoids than RXR-specific ligands. In the present study, we examined the effects of a novel synthetic retinoid, AGN193109, on a variety of retinoid-dependent responses in ECE16-1 cells. We found that AGN193109 is able to eliminate retinoid-dependent changes in cell morphology and prevent retinoid-dependent suppression of proliferation. Moreover, at the biochemical level, AGN193109 prevents retinoid-dependent changes in marker gene expression. We examined genes that are increased (K7, K8, K19, RARβ), as well as those that are suppressed (K5, K6, K14, K16, K17) by retinoid treatment. In each case, the retinoid-dependent response was antagonized by AGN193109. Thus, AGN193109 prevents both stimulatory and suppressive responses. Taken together these results suggest that AGN193109 may be a global antagonist of retinoid action in ECE16-1 cells (i.e. it is likely to antagonize most, and perhaps all, retinoid-dependent responses).

AGN193109 Is an Efficient Antagonist with No Agonist Activity[2]
Several problems can be encountered with receptor antagonists. First, they may have partial agonist activity. This can complicate interpretation of their effects in in vivo systems. To determine whether AGN193109 has intrinsic agonist or antagonist activity, we evaluated its effects on ECE16-1 cell function in the absence of added retinoid agonist. Concentrations of AGN193109 ranging from 0.01 to 1000 nM produced no detectable changes in cell morphology, cell proliferation, or retinoid-responsive marker gene expression. Based on these results we conclude (i) that AGN193109 is a highly effective antagonist of retinoid action in these cells and (ii) that it has not intrinsic agonist activity.
Second, antagonists may have a low affinity for the target receptor. Such ligands must be used at extremely high levels to accomplish the antagonism. AGN193109, however, has an affinity for the RARs of 2-3 nM. Considering that the receptor affinities of tRA, 13-cRA, and 9-cRA range from 7 to 141 nM and TTNPB has affinities ranging from 20 to 51 nM, AGN193109 is a high affinity ligand. This is reflected in its potency at preventing biological end responses. The compound is maximally effective at inhibiting retinoid agonist activity when present at a 10-fold molar excess and is half-maximally effective when present in equal molar amounts. Thus, AGN193109 is a high affinity antagonist.

Natural Ligands That Interact with RAR and RXR Receptors[2]
Our initial studies showed that the synthetic retinoid, AGN193109, which interacts solely with RAR receptors, is effective at antagonizing the effects of the RAR-specific retinoid agonist, TTNPB. However, it was unclear whether the activity of natural ligands would be inhibited by AGN193109. We therefore tested the ability of AGN193109 to antagonize the effects of tRA, 13-cRA, and 9-cRA. 13-cRA, 9-cRA, and tRA interact with RARs, while 9-cRA also has a high affinity for RXRs. The results show that AGN193109 is an effective antagonist of the activity of each of these ligands. Thus, although AGN193109 only interacts with the RAR forms, it can also antagonize the effects of an agent (9-cRA) that interacts with relatively high affinity with both RARs and RXRs. Since AGN193109 does not interact with the RXRs, it is not likely that it directly prevents 9-cRA from interacting with RXR receptor. Moreover, RXR-specific ligands are relatively inactive in ECE16-1 cells(37). Since 9-cRA can efficiently bind to RARs, it is likely that AGN193109 is inhibiting 9-cRA effects mediated via RARs.[2]

Receptor antagonists can be extremely useful ligands, both for understanding mechanism of action and for designing new therapies. In the present manuscript, we show in ECE16-1 cells, a human ectocervical epithelial cell line, that AGN193109 is an extremely active high affinity antagonist of retinoid action that appears to have no intrinsic retinoid agonist activity.[2]

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.)