Influence of 1 alpha, 25-dihydroxyvitamin D3 analogs on coactivator recruitment to the vitamin D receptor transcriptional complex .

摘要

1alpha,25-Dihydroxyvitamin D3 (1,25(OH) 2D3) analogs are potential therapeutic agents for a wide range of disorders, including osteoporosis, psoriasis, inflammatory autoimmune disease, and certain cancers. It is unclear whether tissue selectivity and potency of 1,25(OH)2D3 analogs are determined by pharmacokinetic phenomena or cellular processes. It has been hypothesized that tissue selectivity and potency is determined at the molecular level through differential coactivator recruitment to the vitamin D receptor (VDR) transcriptional complex. To test this hypothesis, we developed a method of isolating the transcriptional complex that associates with the VDR bound to 1,25(OH)2D3 or analog. This method utilizes a biotinylated vitamin D response element (VDRE) and avidin sepharose to isolate complex-bound VDR from nuclear extract. Under the described conditions, the assay allows for the formation of a ligand-dependent transcriptional complex. This assay allows for binding of 95% of the VDR input to the VDRE and recovery of 98% of this VDR during elution, with no significant protein loss detected during the wash steps.; We used the coactivator binding assay to characterize the transcriptional complex that associates with the VDR in the presence of various 1,25(OH) 2D3 analogs. 2-Methylene-19-nor-(20S)-1alpha,25(OH) 2D3 (2MD) and 2-methylene-19-nor-1alpha-hydroxy bishomopregnacalciferol (2MbisP) enhanced the binding of several proteins to the VDR transcriptional complex, relative to 1,25(OH)2D3 or the 20R isomers. 2MD, which unlike 2MbisP contains a full-length side chain and a 25-hydroxyl, also recruits VDR-interacting protein 205 (DRIP205) and DRIP240. When the flexibility of the side chain was reduced by a 17-20 double bond, the enhanced binding was eliminated. Compounds containing modifications to carbon 2 were also tested and had no effect on coactivator binding to the VDR transcriptional complex. Although the results do not explain tissue selectivity, they establish a relationship between analog structure and coactivator recruitment. 20S analogs enhance the binding of select coactivators to the VDR transcriptional complex relative to 1,25(OH)2D3 or corresponding R isomer. Coactivator binding is further enhanced by the presence of a full-length side chain, while modifications at carbon 2 alone do not impact coactivator binding. The developed assay should be a useful tool in the selective modification of 1,25(OH)2D 3 to improve potency or selectivity.