Two-dimensional alanine scanning mutational analysis of the interaction between the vitamin D receptor and its ligands: studies of A-ring modified 19-norvitamin D analogs.

To clarify the structure-function relationship (SFR) of vitamin D analogs in terms of their interaction with the vitamin D receptor (VDR), we have proposed a new approach, two-dimensional alanine scanning mutational analysis (2D-ASMA). In this paper, attention was focused on the interactions around the A-ring of vitamin D. For this purpose, we synthesized four new 2-substituted 19-norvitamin D derivatives (3-6). The VDR affinity (3-6: 1, 5, 2 and 1/140, respectively) and transcriptional activity (3-6: 10, 30, 2 and 0.3, respectively) of the four compounds were evaluated relative to 1,25-(OH)(2)D(3) (5) (normalized to 1). Then, the transcriptional activities of wild-type and 18 mutant VDRs induced by the four compounds (3-6) were investigated. The results of this 18 x 4 2D-ASMA were presented as a patch table, and the effects of the mutations were analyzed in comparison with the natural hormone (1) and 2-methylene-19-nor-20-epi-1,25-(OH)(2)D(3) (2MD, 2). Of the four A-ring analogs, the 2alpha-hydroxyethoxy derivative (3) showed striking differences in the pattern on the patch table. From the results, we suggest a docking mode of this compound (3) in which the A-ring adopts the alpha conformation.