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Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor γ
Vol.
186,
No.
July
31, 1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
IDENTIFICATION HUMAN
OF SYNTHETIC RETINOIDS WITH SELECTIVITY NUCLEAR RETINOIC ACID RECEPTOR y
977-983
FOR
Bruno A. Bernardl, Jean-Michel Bemardonl, Chantal Delesclusel, Bernard Martini, Marie-Cecile Lenoirl, Jean Maignan2, Bruno Charpentierl, William R. Pilgriml, Uwe Reichertl and Braham Shrootl 1 Centre International de Recherches Dermatologiques Galderma (GIRD BP 87,06902 Sophia Antipolis CCdex, Valbonne, France 2 Laboratoires de Recherche Fondamentale, 93600 Aulnay-sous-Bois, France
Received
June
Galdermal,
L’OrCal,
8, 1992
The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR CL, p and y. Since RARy is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RARy in skin and(ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RARyselectivity. b 1992Academic Press, Inc. Retinoic acid (RA) plays a fundamental role in cell proliferation, differentiation and malignant transformation (1,2). The effects of RA and synthetic derivatives are mediated by two classes of nuclear receptors, the retinoic acid receptors (RARa, RARP, RARy) which belong to the erbA-related steroid/thyroid nuclear receptor superfamily (3,4,5), and the retinoid X receptors (RXRs) (6). The classification into RAR or RXR subfamilies is mainly based on differences in primary structure, sensitivity to certain retinoid ligands and ability to regulate transcription of different target genes by specific interactions with cognate DNA elements (7,8). Moreover, RXRs can serve as coregulators that selectively facilitate element-specific binding of the steroid/thyroid nuclear receptors, by the formation of heterodimeric complexes (9,lO). Although it is not known whether the various RARs exhibit selectivity towards responsive elements of target genes, it has been shown that they differ in their tissuespecific expression patterns during embryogenesis and in adult life (11,12). For example in adult rodents and humans, RARa is ubiquitous, RARP is high in heart, lung and spleen, while RARy is confined to lung and skin (13,14). The discovery of synthetic retinoids that are able to discriminate between the different RARs would thus help to
977
Ail
Copyright 0 1992 righm of reproduction
0006-29 I X/9? $4.00 by Academic Press. Inc. in aryform reserw-Pd.
Vol.
186,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
understand their respective roles in governing the biological effects of RA, and more specifically, whether the biological effects of RA on skin are primarily mediated by RAR. Moreover, a selective retinoid might be a specific drug for given tissues, with a consequently higher therapeutic index. In this respect, a number of synthetic retinoids have been recently identified, but their selectivity in functional assays is either restricted to RARa or indifferently to both RARB and RAR? (15-Z). Since RARy may represent a major target for RA action in adult human skin (13,14), it was of importance to design retinoids specific for this receptor. In this paper, we describe three retinoids which specifically bind to RARy in vitro and retain their RARy-selectivity in a functional transcription assay. MATERIALS AND METHODS Chemicals: Compounds 1, $5 6 2 and 8 have already been described elsewhere (20,21,22), as well as compounds 2 (Am580) and 3 (Am80) (23). Their formulas and chemical names are indicated in fig.1. Synthesis of compound 9 is depicted in fig.2. Analytical data for compound 9 are as follows: mp 137-138°C; EI-MS: m/z 364 (M+), 347 (MOH)+; 1H NMR (DMSOd6) 6 1.03 (s,6H, CH3), 1.05 (s, 6H, CH3), 1.43 (s, 4H, CH2), 5.02 (t,lH, CH), 6.31-6.56 (2H, CH = CH), 6.91-7.69 (7H, Ar-H). The tritiated form of compound I (CD367), used in binding assay, was prepared in our institute (24). RA was purchased from Interchim (Montlucon, France). Binding
assay: All binding assays were performed as previously described (20,21), on nuclear extracts of COS-7 cells transfected with pSG5-derived expression vectors for RARa (4), RARB (25) or RARy (provided by Dr.M.Pfahl, La Jolla Cancer Research Foundation). Functional transactivation assay: This assay was performed as previously described (20). Briefly, HeLa cells were cotransfected with 2 ug of expression vectors for human RARa, RARB or RARr and with 5pg of TRE3-tk-CAT reporter plasmid (26), which responds equally well to RARa, RARJ3 and RARy. They were then grown for twenty-four hours in the presence of different concentrations of the various retinoids tested. CAT activity was determined in lysates and expressed as percentage of maximal induction, after background CAT activity had been substracted. Two to four dose-response experiments were performed. The retinoid concentrations giving half maximal activation (ACsu) were determined from the plots. Accuracy was found to be within a factor of 2. RESULTS AND DISCUSSION By using both an in vitro binding assay and a functional transactivation assay, we had previously been able to identify selective high affinity RARo: and RARBy ligands, but no selective R4Ry ligand (20). The good correlation observed between in vitro binding and the functional transactivation assay was probably due to the fact that the level of endogenous RAR in HeLa cells was below the threshold for activation of the TRES-tk-CAT construct (27). More recently, we have found that compound Z 978
Vol.
BIOCHEMICAL
186, No. 2, 1992
AND BIOPHYSICAL
RESEARCtI COMMUNICATIONS COOH
COOH
tP
!P 0 CooH P0 0
O 0 CooH 0 xJ3
30
2(Am58@
cH&J-cooHc$IQ-cooH 5(CD2019)
4fCD417)
YC;“’
WCOOH
tXCD564)
WD
&,L
Structure
7(CD437)
1530)
and chemical
9(CD6661
nomenclature
of the retinoids
used.
1 (CD367):4-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoicacid; 2 (Am580): 4-((5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido) benzoic acid; 3 (Am80): 4-((5,6,7,8-Tetrahydro-5,5,8,8-tetramethyI-Znaphthalenyl) carbamoyl)benzoic acid; 4 (CD417):6-(3-tert-Butyl-4-methoxyphenyl)-2-naphthoic acid; 5 (CD2019):6-(3-(1-Methylcyclohexyl)-4-methoxyphenyl)-2-naphthoic acid; 6 (CD564): 6-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthoyl)-2-naphthoicacid; 2 (CD437): 6-(3-(l-Adamantyl)-4-hydroxyphenyl)-2-naphthoic acid; 8 (CD1530): 4-(6-Hydroxy-7(I-adamantyl)-2-naphthyl)benzoic acid; 9 (CD666):(E)-4-(1-Hydroxy-1-(5,6,7,8tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-2-propenyl)benzoic acid.
exhibited RARr selectivity
in in vitro binding studies (21) (Table 1). Compound 2 is
derived from compound CD271 (adapalene) in that the methoxy
group (28,29) is
replaced by a hydroxyl group. This simple chemical modification was apparently sufficient to shift the RAR selectivity towards the gamma subtype, since adapalene initially exhibited a RARP selectivity in in vitro binding assay, with Kd values of 1100 nM, 34 nM and 130 nM, for RARa, RARP and RARy, respectively (29). We therefore investigated whether compounds 8 and 3 which also carry a hydroxyl group 979
Vol.
186,
No.
2, 1992
&$&
BIOCHEMICAL
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COMMUNICATIONS
Scheme for the synthesis of compound 9 (CD666). a) NaOH(lN)/MeOH, (48%); b) NaBHq/CeC13/MeOH, (61%).
on the left lipophilic part of the molecule, would also exhibit RARr binding selectivity. Table 1 shows that this is indeed the case, the affinity of all three compounds for RARy being at least 35 times greater than for RARa or RARP. However, if selectivity was observed in this in vitro binding assay, it remained to be established that this selectivity was maintained in a functional assay, where cellular context, the set of nuclear transcription factors and/or biotransformation of the compounds could possibly result in the loss of specificity. To make sure that our transactivation assay would discriminate for selective RARa, RARP and RARy ligands,
COMPOUND RA 1 2 3 4 5 6 7 8 9
RARa 15.5 5.3 8.0 62.0 6500.0 920.0 530.0 6500.0 2750.0 2240.0
RARP
RARy
4.5
3.0 1.3 450.0 816.0 426.0 160.0 7.0 77.0 150.0 68.0
3.0 131.0 280.0 36.0 26.0 9.0 2480.0 1500.0 2300.0
Kd (nM) values for RARa, RARB, and RARy obtained in saturation experiments with reference compound 1 (CD367) and in competition experiments with the other synthetic retinoids. Values represent the mean of two or three determinations. 980
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No.
BIOCHEMICAL
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AND
TABLE
COMPOUND RA 1 2 3 4 5 6 7 8 9
RARa
BIOPHYSICAL
COMMUNICATIONS
2
RARP
2.12 0.23 0.36 1.46 71.10 19.80 18.00 140.00 37.50 149.00
RESEARCH
3.62 0.37 24.56 6.87 3.56 3.80 0.28 28.40 28.50 50.80
RARy 2.46 0.25 27.86 148.66 69.10 46.90 1.23 7.30 1.80 1.40
ACSO (nM) values of transcriptional activation by RA and synthetic retinoids. These values are the mean of two to six determinations, obtained graphically from induction curves.
we also included in our study known non-specific reference ligands (RA and compound lJ, reference ligands with partial RARa specificity (compounds 2. and 3J (20), a ligand with RARP-RARy specificity (compound 6J (20), as well as compounds 5 and 2, recently described as RARP and RARy selective, respectively, on the basis of in vitro binding studies (21). As shown in table 2, the specificity of all ligands was conserved in the transactivation assay. As previously observed, compounds 2 and 3 exhibited a RARa specificity, whereas compound 6 retained RARP-RARy selectivity (20). Compound 5 exhibited RARP selectivity, as observed in in vitro binding assay (21). The new compound 4 was also found to be RARP selective, in both in vitro binding and transactivation assays. Similarly, the RARy selectivity of compound 2 (21) was maintained in the transactivation assay. Moreover two new compounds, S and 2 were found to be RARy selective, in both assays. It is interesting to note that compounds 4 and ?5,exhibiting RARP selectivity, belong to the same chemical series. Starting from this series, replacement of the methoxy group of the parent compound (CD271) by a hydroxyl group led to compound z behaving as a RARy selective compound. Moreover, introduction of a hydroxyl group in other chemical series also led to gamma selective compounds (8 and 9. The data reported in this paper thus provide a basis to further investigation of structure / RAR-affinity relationships among retinoids. In conclusion, we have identified retinoids with RARP (compounds 4 and 5J and RARy specificity (compounds 2, 8 and 9J, which could be used, along with RARa specific compounds 2 and 5 to further elucidate the biological roles of RAR in skin; the structure-activity insights from this work will help in the design of therapeutically active, receptor selective retinoids. The next step will be the comparison of the effects of RARa, RARP and RARy specific compounds on the differentiation process in pharmacological and toxicological test systems strongly modulated by RA. 981
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No.
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ACKNOWLEDGMENTS We thank I’. Chambon, A. Dejean and M. Petkovich for the generous gift of TRES-tk-CAT, and RARa, R4RJ3 expression vectors. We also wish to thank I. Carlavan for technical assistance in preparing COS-cell extracts, Dr. M. Pfahl for critical reading of the manuscript, and Prof. Hans Schaefer for stimulating discussions and continuous support.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Sporn, M.B., Roberts, A.B., and Goodman, D.S. (Eds.1 (1984) The Retinoids, Vol.l-2. Academic Press, New York. Darmon, M. (1991) Sem. Dev. Biol. 2,219-228. Giguere,V., Ong,E.S., Segui,P., and Evans, R.M. (1987) Nature 330,624-629. Petkovich, M., Brand, N.J., Krust, A., and Chambon, P. (1987) Nature 330, 444450. Glass, C.K., Devary, O.V., and Rosenfeld, M.G. (1990) Cell 63,729-738. Mangelsdorf, D.J., Ong, ES., Dyck, J.A., and Evans, R.M. (1990) Nature 345, 224229. Umesomo, K., Murakami, K.K., Thompson, C.C., and Evans, R.M. (1991) Cell 65, 1255-1266. Naar, A.M., Boutin, J.-M., Lipkin, S.M., Yu, V.C., Holloway, J.M., Glass, C.K., and Rosenfeld, M.G. (1991) Cell 65,1267-1279. Yu, V.C., Delsert, C., Andersen, B., Holloway, J.M., Devary, O.V., NaEr, A.M., Kim, A.Y., Boutin, J.-M., Glass, C.K., and Rosenfeld, M. (1991) Cell 67,1251-1266. Zhang, X.-K., Hoffmann, B., Tran, P.B.-V., Graupner, G., and Pfahl, M. (1992) Na hue 355,441-446. Doll&, I’., Ruberte, E.,Kastner,P., Petkovich, M., Stoner, C.M.,Gudas, L.J., and Chambon, I’. (1989) Nature 342,702-705. Ruberte, E., Doll& I’., Krust, A., Zelent, A., Morriss-Kay, G., and Chambon, I’. (1990) Development 108,213-222. Elder, J.T., Fisher, G.J., Zhang Q.-Y., Eisen, D., Krust, A., Kastner, I’., Chambon, P., and Voorhees, J.J. (1991) J.Invest.Dermatol96,425-433. Rees, J. (1992) Br.J.Dermatol. 126,97-104. Hashimoto, Y., Kagechika, H., and Shudo, K. (1990) Bioch.Bioph.Res.Commun. 166,1300-1307. Astrom, A., Pettersson, U., Krust, A., and Voorhees, J.J. (1990) Bioch.Biophys.Res.Commun. 173,339-345. Crettaz, M., Baron, A., Siegenthaler, G., and Hunziker, W. (1990) Bi0chem.J. 272, 391-397. Graupner, G., Malle, G., Maignan, J., Lang, G., Prunieras, M, and Pfahl, M. (1991) Bioch.Biophys.Res.Commun. 179,1554-1561. Lehmann, J., Dawson, MI., Hobbs, P.D., Husmann, M., and Pfahl, M. (1991) Cancer Res.51,4804-4809. Delescluse, C., Cavey, M.-T., Martin, B., Bernard, B.A., Reichert, U., Maignan, J., Darmon, and Shroot, B. (1991) MolPharmacol. 40,556-562. Martin, B., Bernardon, J.-M., Cavey, M.-T., Bernard, B.A., Carlavan, I., Charpentier, B., Pilgrim, W.R., Shroot, B., and Reichert, U. (1992) Skin Pharmacol. 5,57-65. Shroot, B., Bernardon, J.-M., and Nedoncelle, I’. (1987) Eur.Patent 210.929. 982
Vol.
23. 24. 25. 26. 27. 28. 29.
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No.
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Kagechika, H., Kawachi, E., Hashimoto, Y., Himi, T., and Shudo, K. (1988) J.Med.Chem. 31,2182-2192. Darmon, M., Rocher, M., Cavey, M.-T., Martin, B., Rabilloud, T., Delescluse, C., and Shroot, B. (1988) Skin Pharmacol. 1,161-175. Brand, N., Petkovich, M., Krust, A., Chambon, P., De The, H., Marchio, A., Tiollais, I’., and Dejean, A. (1988) Nature 332,850-853. Zelent, A., Krust, A., Petkovich, M., Kastner, I”., and Chambon, P. (1989) Nature 339,714-717. Sucov, H.M., Murakami, K.K., and Evans, R.M. (1990) Proc.Natl.Acad.Sci.USA 87,5392-5396. Bailly, J., Delescluse, C., Bernardon, J.-M., Charpentier, B., Martin, B., Pilgrim, W.R., Shroot, B., and Darmon, M. (1990) Skin Pharmacol. 3,256-267. Shroot, B., Bernardon, J.-M., Charpentier, B., Martin, B., and Cavey, M.-T. (1991) J. Invest. Dermatol. 96,577A.
983
186,
No.
July
31, 1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
IDENTIFICATION HUMAN
OF SYNTHETIC RETINOIDS WITH SELECTIVITY NUCLEAR RETINOIC ACID RECEPTOR y
977-983
FOR
Bruno A. Bernardl, Jean-Michel Bemardonl, Chantal Delesclusel, Bernard Martini, Marie-Cecile Lenoirl, Jean Maignan2, Bruno Charpentierl, William R. Pilgriml, Uwe Reichertl and Braham Shrootl 1 Centre International de Recherches Dermatologiques Galderma (GIRD BP 87,06902 Sophia Antipolis CCdex, Valbonne, France 2 Laboratoires de Recherche Fondamentale, 93600 Aulnay-sous-Bois, France
Received
June
Galdermal,
L’OrCal,
8, 1992
The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR CL, p and y. Since RARy is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RARy in skin and(ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RARyselectivity. b 1992Academic Press, Inc. Retinoic acid (RA) plays a fundamental role in cell proliferation, differentiation and malignant transformation (1,2). The effects of RA and synthetic derivatives are mediated by two classes of nuclear receptors, the retinoic acid receptors (RARa, RARP, RARy) which belong to the erbA-related steroid/thyroid nuclear receptor superfamily (3,4,5), and the retinoid X receptors (RXRs) (6). The classification into RAR or RXR subfamilies is mainly based on differences in primary structure, sensitivity to certain retinoid ligands and ability to regulate transcription of different target genes by specific interactions with cognate DNA elements (7,8). Moreover, RXRs can serve as coregulators that selectively facilitate element-specific binding of the steroid/thyroid nuclear receptors, by the formation of heterodimeric complexes (9,lO). Although it is not known whether the various RARs exhibit selectivity towards responsive elements of target genes, it has been shown that they differ in their tissuespecific expression patterns during embryogenesis and in adult life (11,12). For example in adult rodents and humans, RARa is ubiquitous, RARP is high in heart, lung and spleen, while RARy is confined to lung and skin (13,14). The discovery of synthetic retinoids that are able to discriminate between the different RARs would thus help to
977
Ail
Copyright 0 1992 righm of reproduction
0006-29 I X/9? $4.00 by Academic Press. Inc. in aryform reserw-Pd.
Vol.
186,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
understand their respective roles in governing the biological effects of RA, and more specifically, whether the biological effects of RA on skin are primarily mediated by RAR. Moreover, a selective retinoid might be a specific drug for given tissues, with a consequently higher therapeutic index. In this respect, a number of synthetic retinoids have been recently identified, but their selectivity in functional assays is either restricted to RARa or indifferently to both RARB and RAR? (15-Z). Since RARy may represent a major target for RA action in adult human skin (13,14), it was of importance to design retinoids specific for this receptor. In this paper, we describe three retinoids which specifically bind to RARy in vitro and retain their RARy-selectivity in a functional transcription assay. MATERIALS AND METHODS Chemicals: Compounds 1, $5 6 2 and 8 have already been described elsewhere (20,21,22), as well as compounds 2 (Am580) and 3 (Am80) (23). Their formulas and chemical names are indicated in fig.1. Synthesis of compound 9 is depicted in fig.2. Analytical data for compound 9 are as follows: mp 137-138°C; EI-MS: m/z 364 (M+), 347 (MOH)+; 1H NMR (DMSOd6) 6 1.03 (s,6H, CH3), 1.05 (s, 6H, CH3), 1.43 (s, 4H, CH2), 5.02 (t,lH, CH), 6.31-6.56 (2H, CH = CH), 6.91-7.69 (7H, Ar-H). The tritiated form of compound I (CD367), used in binding assay, was prepared in our institute (24). RA was purchased from Interchim (Montlucon, France). Binding
assay: All binding assays were performed as previously described (20,21), on nuclear extracts of COS-7 cells transfected with pSG5-derived expression vectors for RARa (4), RARB (25) or RARy (provided by Dr.M.Pfahl, La Jolla Cancer Research Foundation). Functional transactivation assay: This assay was performed as previously described (20). Briefly, HeLa cells were cotransfected with 2 ug of expression vectors for human RARa, RARB or RARr and with 5pg of TRE3-tk-CAT reporter plasmid (26), which responds equally well to RARa, RARJ3 and RARy. They were then grown for twenty-four hours in the presence of different concentrations of the various retinoids tested. CAT activity was determined in lysates and expressed as percentage of maximal induction, after background CAT activity had been substracted. Two to four dose-response experiments were performed. The retinoid concentrations giving half maximal activation (ACsu) were determined from the plots. Accuracy was found to be within a factor of 2. RESULTS AND DISCUSSION By using both an in vitro binding assay and a functional transactivation assay, we had previously been able to identify selective high affinity RARo: and RARBy ligands, but no selective R4Ry ligand (20). The good correlation observed between in vitro binding and the functional transactivation assay was probably due to the fact that the level of endogenous RAR in HeLa cells was below the threshold for activation of the TRES-tk-CAT construct (27). More recently, we have found that compound Z 978
Vol.
BIOCHEMICAL
186, No. 2, 1992
AND BIOPHYSICAL
RESEARCtI COMMUNICATIONS COOH
COOH
tP
!P 0 CooH P0 0
O 0 CooH 0 xJ3
30
2(Am58@
cH&J-cooHc$IQ-cooH 5(CD2019)
4fCD417)
YC;“’
WCOOH
tXCD564)
WD
&,L
Structure
7(CD437)
1530)
and chemical
9(CD6661
nomenclature
of the retinoids
used.
1 (CD367):4-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoicacid; 2 (Am580): 4-((5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido) benzoic acid; 3 (Am80): 4-((5,6,7,8-Tetrahydro-5,5,8,8-tetramethyI-Znaphthalenyl) carbamoyl)benzoic acid; 4 (CD417):6-(3-tert-Butyl-4-methoxyphenyl)-2-naphthoic acid; 5 (CD2019):6-(3-(1-Methylcyclohexyl)-4-methoxyphenyl)-2-naphthoic acid; 6 (CD564): 6-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthoyl)-2-naphthoicacid; 2 (CD437): 6-(3-(l-Adamantyl)-4-hydroxyphenyl)-2-naphthoic acid; 8 (CD1530): 4-(6-Hydroxy-7(I-adamantyl)-2-naphthyl)benzoic acid; 9 (CD666):(E)-4-(1-Hydroxy-1-(5,6,7,8tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-2-propenyl)benzoic acid.
exhibited RARr selectivity
in in vitro binding studies (21) (Table 1). Compound 2 is
derived from compound CD271 (adapalene) in that the methoxy
group (28,29) is
replaced by a hydroxyl group. This simple chemical modification was apparently sufficient to shift the RAR selectivity towards the gamma subtype, since adapalene initially exhibited a RARP selectivity in in vitro binding assay, with Kd values of 1100 nM, 34 nM and 130 nM, for RARa, RARP and RARy, respectively (29). We therefore investigated whether compounds 8 and 3 which also carry a hydroxyl group 979
Vol.
186,
No.
2, 1992
&$&
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Scheme for the synthesis of compound 9 (CD666). a) NaOH(lN)/MeOH, (48%); b) NaBHq/CeC13/MeOH, (61%).
on the left lipophilic part of the molecule, would also exhibit RARr binding selectivity. Table 1 shows that this is indeed the case, the affinity of all three compounds for RARy being at least 35 times greater than for RARa or RARP. However, if selectivity was observed in this in vitro binding assay, it remained to be established that this selectivity was maintained in a functional assay, where cellular context, the set of nuclear transcription factors and/or biotransformation of the compounds could possibly result in the loss of specificity. To make sure that our transactivation assay would discriminate for selective RARa, RARP and RARy ligands,
COMPOUND RA 1 2 3 4 5 6 7 8 9
RARa 15.5 5.3 8.0 62.0 6500.0 920.0 530.0 6500.0 2750.0 2240.0
RARP
RARy
4.5
3.0 1.3 450.0 816.0 426.0 160.0 7.0 77.0 150.0 68.0
3.0 131.0 280.0 36.0 26.0 9.0 2480.0 1500.0 2300.0
Kd (nM) values for RARa, RARB, and RARy obtained in saturation experiments with reference compound 1 (CD367) and in competition experiments with the other synthetic retinoids. Values represent the mean of two or three determinations. 980
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No.
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AND
TABLE
COMPOUND RA 1 2 3 4 5 6 7 8 9
RARa
BIOPHYSICAL
COMMUNICATIONS
2
RARP
2.12 0.23 0.36 1.46 71.10 19.80 18.00 140.00 37.50 149.00
RESEARCH
3.62 0.37 24.56 6.87 3.56 3.80 0.28 28.40 28.50 50.80
RARy 2.46 0.25 27.86 148.66 69.10 46.90 1.23 7.30 1.80 1.40
ACSO (nM) values of transcriptional activation by RA and synthetic retinoids. These values are the mean of two to six determinations, obtained graphically from induction curves.
we also included in our study known non-specific reference ligands (RA and compound lJ, reference ligands with partial RARa specificity (compounds 2. and 3J (20), a ligand with RARP-RARy specificity (compound 6J (20), as well as compounds 5 and 2, recently described as RARP and RARy selective, respectively, on the basis of in vitro binding studies (21). As shown in table 2, the specificity of all ligands was conserved in the transactivation assay. As previously observed, compounds 2 and 3 exhibited a RARa specificity, whereas compound 6 retained RARP-RARy selectivity (20). Compound 5 exhibited RARP selectivity, as observed in in vitro binding assay (21). The new compound 4 was also found to be RARP selective, in both in vitro binding and transactivation assays. Similarly, the RARy selectivity of compound 2 (21) was maintained in the transactivation assay. Moreover two new compounds, S and 2 were found to be RARy selective, in both assays. It is interesting to note that compounds 4 and ?5,exhibiting RARP selectivity, belong to the same chemical series. Starting from this series, replacement of the methoxy group of the parent compound (CD271) by a hydroxyl group led to compound z behaving as a RARy selective compound. Moreover, introduction of a hydroxyl group in other chemical series also led to gamma selective compounds (8 and 9. The data reported in this paper thus provide a basis to further investigation of structure / RAR-affinity relationships among retinoids. In conclusion, we have identified retinoids with RARP (compounds 4 and 5J and RARy specificity (compounds 2, 8 and 9J, which could be used, along with RARa specific compounds 2 and 5 to further elucidate the biological roles of RAR in skin; the structure-activity insights from this work will help in the design of therapeutically active, receptor selective retinoids. The next step will be the comparison of the effects of RARa, RARP and RARy specific compounds on the differentiation process in pharmacological and toxicological test systems strongly modulated by RA. 981
Vol.
186,
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ACKNOWLEDGMENTS We thank I’. Chambon, A. Dejean and M. Petkovich for the generous gift of TRES-tk-CAT, and RARa, R4RJ3 expression vectors. We also wish to thank I. Carlavan for technical assistance in preparing COS-cell extracts, Dr. M. Pfahl for critical reading of the manuscript, and Prof. Hans Schaefer for stimulating discussions and continuous support.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Sporn, M.B., Roberts, A.B., and Goodman, D.S. (Eds.1 (1984) The Retinoids, Vol.l-2. Academic Press, New York. Darmon, M. (1991) Sem. Dev. Biol. 2,219-228. Giguere,V., Ong,E.S., Segui,P., and Evans, R.M. (1987) Nature 330,624-629. Petkovich, M., Brand, N.J., Krust, A., and Chambon, P. (1987) Nature 330, 444450. Glass, C.K., Devary, O.V., and Rosenfeld, M.G. (1990) Cell 63,729-738. Mangelsdorf, D.J., Ong, ES., Dyck, J.A., and Evans, R.M. (1990) Nature 345, 224229. Umesomo, K., Murakami, K.K., Thompson, C.C., and Evans, R.M. (1991) Cell 65, 1255-1266. Naar, A.M., Boutin, J.-M., Lipkin, S.M., Yu, V.C., Holloway, J.M., Glass, C.K., and Rosenfeld, M.G. (1991) Cell 65,1267-1279. Yu, V.C., Delsert, C., Andersen, B., Holloway, J.M., Devary, O.V., NaEr, A.M., Kim, A.Y., Boutin, J.-M., Glass, C.K., and Rosenfeld, M. (1991) Cell 67,1251-1266. Zhang, X.-K., Hoffmann, B., Tran, P.B.-V., Graupner, G., and Pfahl, M. (1992) Na hue 355,441-446. Doll&, I’., Ruberte, E.,Kastner,P., Petkovich, M., Stoner, C.M.,Gudas, L.J., and Chambon, I’. (1989) Nature 342,702-705. Ruberte, E., Doll& I’., Krust, A., Zelent, A., Morriss-Kay, G., and Chambon, I’. (1990) Development 108,213-222. Elder, J.T., Fisher, G.J., Zhang Q.-Y., Eisen, D., Krust, A., Kastner, I’., Chambon, P., and Voorhees, J.J. (1991) J.Invest.Dermatol96,425-433. Rees, J. (1992) Br.J.Dermatol. 126,97-104. Hashimoto, Y., Kagechika, H., and Shudo, K. (1990) Bioch.Bioph.Res.Commun. 166,1300-1307. Astrom, A., Pettersson, U., Krust, A., and Voorhees, J.J. (1990) Bioch.Biophys.Res.Commun. 173,339-345. Crettaz, M., Baron, A., Siegenthaler, G., and Hunziker, W. (1990) Bi0chem.J. 272, 391-397. Graupner, G., Malle, G., Maignan, J., Lang, G., Prunieras, M, and Pfahl, M. (1991) Bioch.Biophys.Res.Commun. 179,1554-1561. Lehmann, J., Dawson, MI., Hobbs, P.D., Husmann, M., and Pfahl, M. (1991) Cancer Res.51,4804-4809. Delescluse, C., Cavey, M.-T., Martin, B., Bernard, B.A., Reichert, U., Maignan, J., Darmon, and Shroot, B. (1991) MolPharmacol. 40,556-562. Martin, B., Bernardon, J.-M., Cavey, M.-T., Bernard, B.A., Carlavan, I., Charpentier, B., Pilgrim, W.R., Shroot, B., and Reichert, U. (1992) Skin Pharmacol. 5,57-65. Shroot, B., Bernardon, J.-M., and Nedoncelle, I’. (1987) Eur.Patent 210.929. 982
Vol.
23. 24. 25. 26. 27. 28. 29.
186,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Kagechika, H., Kawachi, E., Hashimoto, Y., Himi, T., and Shudo, K. (1988) J.Med.Chem. 31,2182-2192. Darmon, M., Rocher, M., Cavey, M.-T., Martin, B., Rabilloud, T., Delescluse, C., and Shroot, B. (1988) Skin Pharmacol. 1,161-175. Brand, N., Petkovich, M., Krust, A., Chambon, P., De The, H., Marchio, A., Tiollais, I’., and Dejean, A. (1988) Nature 332,850-853. Zelent, A., Krust, A., Petkovich, M., Kastner, I”., and Chambon, P. (1989) Nature 339,714-717. Sucov, H.M., Murakami, K.K., and Evans, R.M. (1990) Proc.Natl.Acad.Sci.USA 87,5392-5396. Bailly, J., Delescluse, C., Bernardon, J.-M., Charpentier, B., Martin, B., Pilgrim, W.R., Shroot, B., and Darmon, M. (1990) Skin Pharmacol. 3,256-267. Shroot, B., Bernardon, J.-M., Charpentier, B., Martin, B., and Cavey, M.-T. (1991) J. Invest. Dermatol. 96,577A.
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