No abstract

The retinoic acid receptors (RARs) transduce retinoid dependant gene regulation, and many biological effects of retinoids are mediated through binding and activation of three closely related receptor subtypes (RAR alpha, RAR beta, and RAR gamma). In order to investigate the role of receptor subtypes, we have carried out a chemical synthesis program to seek selective retinoids for these receptors. We measured receptor binding affinity using recombinant RAR alpha, -beta, and -gamma proteins and assessed cellular differentiating activity in F9 murine teratocarcinoma cells (F9 cells). This research has identified the 4-substituted-3-(1-adamantyl)phenyl moiety as a new pharmacophore which can replace the beta-cyclogeranylidene ring of the naturally occurring all-trans-retinoic acid. Two chemical series derived from the general structures 6-(3-tertioalkylphenyl)-2-naphthoic acid (series I) and 4-[(E)-2-(3-tertioalkylphenyl)propenyl]benzoic acid (series II) were developed. In particular, we have obtained the RAR gamma selective derivatives 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (7) [Ki(RAR alpha) = 6500 nM, Ki(RAR beta) = 2480 nM, Ki(RAR gamma) = 77 nM] and 4-[(E)-2-[3-(1-adamantyl)-4-hydroxyphenyl]propenyl]benzoic acid (19) [Ki(RAR alpha) = 1,144 nM, Ki(RAR beta) = 1245 nM, Ki(RAR gamma) = 53 nM]. In series I, the presence of a phenol group, irrespective of the nature of tertioalkyl group, imparted at least partial RAR gamma selectivity, whereas in series II, the presence of both adamantyl and phenol groups is needed to confer RAR gamma selectivity. The RAR gamma selective ligands induce differentiation in F9 cells (7, AC50 = 33 nM; 19, AC50 = 66 nM). From series I, a mixed RAR beta-gamma agonist with potent cellular differentiating activity was selected for development as a topical antiacne agent, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid (5, CD 271) [Ki(RAR alpha) = 1100 nM, Ki-(RAR beta) = 34 nM, Ki(RAR gamma) = 130 nM, AC50(F9) = 37 nM]. Finally, from series II, we have obtained a weak antagonist in the F9 cellular differentiation assay, 4-[(E)-2-(3-tert-butyl-4-hydroxyphenyl)propenyl]benzoic acid (15, IC50 = 700 nM).

Retinoic acids are morphogenic signaling molecules that are derived from vitamin A and involved in a variety of tissue functions. Two groups of their nuclear receptors have been identified: retinoic acid receptors (RARs) and retinoic acid X receptors (RXRs). All-trans retinoic acid is the high affinity ligand for RARs, and 9-cis retinoic acid also binds to RXRs with high affinity. In cells at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid via unknown mechanisms. It was previously shown that retinoic acids prevents activation-induced death of thymocytes. Here, we report that both all-trans and 9-cis retinoic acid induce apoptosis of mouse thymocytes and purified CD4+CD8+ cells in ex vivo cultures, with 9-cis retinoic acid being 50 times more effective. The induction of apoptosis by retinoic acids is mediated by RARgamma because (a) the phenomenon can be reproduced only by RARgamma-selective retinoic acid analogs, (b) the cell death induced by either retinoic acids or RARgamma analogs can be inhibited by RARgamma-specific antagonists, and (c) CD4+CD8+ thymocytes express RARgamma. In vivo administration of an RARgamma analog resulted in thymus involution with the concomitant activation of the apoptosis-related endonuclease and induction of tissue transglutaminase. The RARgamma pathway of apoptosis is RNA and protein synthesis dependent, affects the CD4+CD8+ double positive thymocytes, and can be inhibited by the addition of either Ca2+ chelators or protease inhibitors. Using various RAR- and RXR-specific analogs and antagonists, it was demonstrated that stimulation of RAR alpha inhibits the RARgamma-specific death pathway (which explains the lack of apoptosis stimulatory effects of all-trans retinoic acid at physiological concentrations) and that costimulation of the RXR receptors (in the case of 9-cis retinoic acid) can neutralize this inhibitory effect. It is suggested that formation of 9-cis retinoic acid may be a critical element in regulating both the positive selection and the "default cell death pathway" of thymocytes.

From a series of naphthalene and benzoic acid derivatives we have identified synthetic retinoic acid analogues exhibiting high selectivity for the nuclear retinoic acid receptors RARα (Am 580), RARβ (CD 2019) and RARγ (CD 437) as well as ligands sharing high affinities for all RAR subtypes (CD 367). The compounds were evaluated in two complementary screening systems: (1) binding to nuclear proteins extracted from COS-7 cells after transfection with the appropriate expression vectors, and (2) induction of plasminogen activator in the embryonic mouse teratocarcinoma cell line F9. All compounds behaved as retinoic acid agonists in the F9 test.

Thymocytes can be induced to undergo apoptotic cell death by activation through the T-cell receptor (TCR). This process requires macromolecular synthesis and has been shown to be inhibited by retinoic acids (RAs). Two groups of nuclear receptors for RAs have been identified: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). All-trans-RA is the high-affinity ligand for RARs, and 9-cis-RA additionally binds to RXRs with high affinity. Because 9-cis-RA is much more potent in inhibiting TCR-mediated death than all-trans-RA, it was suggested that RXRs participate in the process. In the present study various synthetic retinoid analogues were used to address this question further. The results presented suggest that the inhibitory effect of RAs on activation-induced death of thymocytes is mediated via RARalpha, because (1) it can be reproduced by various RARalpha analogues both in vitro and in vivo, (2) the effect of RAs can be inhibited by the addition of an RARalpha antagonist, (3) CD4+CD8+thymocytes, which die on TCR stimulation, express RARalpha. Stimulation of RARgamma, in contrast, enhances the activation-induced death of thymocytes and inhibits its prevention by RARalpha stimulation. RXR co-stimulation suspends this inhibitory effect of RARgamma and permits the preventive function of RARalpha on activation-induced death. Our results suggest a complex interaction between the various isoforms of retinoid receptors and demonstrate that low (physiological) concentrations of all-trans-RA do not affect the activation-induced death of thymocytes because the RARalpha-mediated inhibitory and the RARgamma-mediated enhancing pathways are in balance, whereas if 9-cis-RA is formed, additional stimulation of RXRs permits the inhibitory action of RARalpha.