Serine 232 and methionine 272 define the ligand binding pocket in retinoic acid receptor subtypes.

The transcriptional response mediated by retinoic acid involves a complex series of events beginning with ligand recognition by a nuclear receptor. To dissect the amino acid contacts important for receptor-specific ligand recognition, a series of retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser232) in RARalpha and methionine 272 (Met272) in RARgamma are critical residues for the recognition of their respective receptor-selective analogs. The identification of these key amino acids in the ligand binding pocket is confirmed by the reported crystal structure of RARgamma. Interestingly, the serine at position 232 in RARalpha gives an explanation for the observed differences in the affinity of the naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine and the hydrophobic linker of t-RA. Using this model, a molecular mechanism for the transcriptional antagonism of a synthetic analog is suggested that involves an alteration in the structure of the receptor protein in the region around the AF2 domain in helix 12.

Retinoic acid receptor beta,gamma-selective ligands: synthesis and biological activity of 6-substituted 2-naphthoic acid retinoids.

In search for retinoic acid receptor (RAR) selective ligands, a series of 6-substituted 2-naphthoic acid retinoids were synthesized and evaluated in vitro in a transactivation assay and a competition binding assay for all RARs. These derivatives, in general, showed RAR beta,gamma selectivity. Among these naphthoic acids, oxime derivative 12 was identified as a potent RAR gamma-selective retinoid, while olefinic derivative 11 was found to be comparable to retinoic acid and slightly RAR beta,gamma selective. For the bioassays, a general correlation was observed between the binding affinity of the ligand to the receptors and the potency of the compounds in the transactivation assay. The structure-activity relationship of these naphthoic acids will be discussed.

Retinoic acid receptor gamma mediates topical retinoid efficacy and irritation in animal models.

Among retinoic acid receptors (RARs) alpha, beta, and gamma, the messenger RNA level of RAR-gamma is the most readily detectable by Northern blotting in human and mouse skin. This observation suggests that RAR-gamma may play a critical role in the modulation of the therapeutic benefits and side effects of retinoids in skin. To test this hypothesis, 11 RAR-gamma selective retinoids were synthesized based on three related structures. Each compound was found to prefer RAR-gamma when assessed by retinoid-induced transcriptional activity (RAR-gamma > RAR-beta > RAR-alpha). The apparent Kd for binding to recombinant receptor protein was found to follow a similar trend. To correlate this receptor selectivity with in vivo activity, the compounds were tested topically in the Rhino mouse utriculi reduction and rabbit irritation models, two assays widely used to screen retinoids for efficacy and side effects, respectively. The results indicated that for these compounds, both efficacy in the utriculi reduction assay and irritation potential in rabbits correlated positively with the RAR-gamma transactivation activity, with r2 of 0.9 and 0.8, respectively. These data suggest that RAR-gamma is an important regulator of retinoic acid efficacy in skin and further, that the irritation associated with the use of retinoids is most likely a receptor-mediated process.

The N-terminal portion of domain E of retinoic acid receptors alpha and beta is essential for the recognition of retinoic acid and various analogs.

Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response. Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect. In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta. Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif. The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions. This finding may link ligand binding to other processes important for transcriptional activation.

Role of retinoic acid receptor gamma in the Rhino mouse and rabbit irritation models of retinoid activity.

The three retinoic acid receptors (RAR alpha, RAR beta and RAR gamma) are known to modulate the transcription of target genes through interaction of the individual receptors with their naturally occurring ligand, retinoic acid (RA). Since RA has multiple effects in vivo, considerable effort has recently been devoted to finding selective compounds to elucidate the functions of individual receptors and to relate these functions to specific in vivo effects. The racemic synthetic retinoid 6-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)hydroxy-methyl]-2- naphthalene carboxylic acid has recently been identified as an RAR gamma-selective agonist. A synthetic method involving lipase-mediated transformation has been developed to prepare the individual enantiomers. Discrimination between the two enantiomers is seen in both transcriptional activity and binding to recombinant receptors with the (S)-enantiomer being the more active. Differences between the two compounds are also seen in the Rhino mouse utriculi reduction assay and the rabbit irritation model. In both animal models, the (S)-enantiomer consistently gave a greater response. Taken together, these results suggest that the activity and irritation seen with RA and related compounds is receptor mediated. Further, the strong selectivity of the compounds reported here for RAR gamma suggests that this receptor plays an important role in these in vivo biological activities. The discrimination between these enantiomers may be useful in the design of novel retinoids with uniquely defined biological properties.