Superagonistic Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained by Vitamin D Receptor-Coactivator Interaction

Abstract

Two 14-epi-analogs of 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], 19-nor-14-epi-23-yne-1,25-(OH) 2 D 3 (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH) 2 D 3 (TX527), show enhanced antiproliferative (at least 10-fold) and markedly lower calcemic effects both in vitro and in vivo, compared with 1,25-(OH) 2 D 3 . This study aimed to evaluate their superagonistic effect at the level of interaction between the Vitamin D receptor (VDR) and coactivators. Mammalian two-hybrid assays with VP16-fused VDR and GAL4-DNA-binding-domain-fused steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor 2 (Tif2), or DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH) 2 D 3 (up to 16-and 20-fold stronger induction of VDR-SRC-1 interaction for TX522 and TX527 at 10 Ϫ10 M). Similar assays in which metabolism of 1,25-(OH) 2 D 3 was blocked with VID400, a selective inhibitor of the 1,25-(OH) 2 D 3 -metabolizing enzyme CYP24, showed that the enhanced potency of these analogs in establishing VDR-coactivator interactions can only partially be accounted for by their increased resistance to metabolic degradation. Crystallization of TX522 complexed to the ligand binding domain of the human VDR demonstrated that the epi-configuration of C14 caused the CD ring of the ligand to shift by 0.5 Å, thereby bringing the C12 atom into closer contact with Val300. Moreover, C22 of TX522 made an additional contact with the CD1 atom of Ile268 because of the rigidity of the triple bond-containing side chain. The position and conformation of the activation helix H12 of VDR was strictly maintained. In conclusion, this study provides deeper insight into the docking of TX522 in the LBP and shows that stronger VDR-coactivator interactions underlie the superagonistic activity of the two 14-epi-analogs.1,25-Dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], the biologically active form of vitamin D, plays a major role in bone metabolism and in calcium and phosphate homeostasis. In addition to this classic effect, 1,25-(OH) 2 D 3 also has an antiproliferative and prodifferentiating effect on various normal as well as malignant cells (Bouillon et al., 1995

New insights into Vitamin D sterol-VDR proteolysis, allostery, structure–function from the perspective of a conformational ensemble model

“…Only H-bonds formed by KH are depicted as black dotted lines. This model is in strong agreement with the x-ray crystal structure [13], which was used Conversion of MK into a superagonist or superantagonist Representative MK dose-response curves for hVDRwt and single and double point mutants that turn MK into a significantly more potent agonist (H305F and H305F/H397F) or significantly more potent antagonist (R158F and R402E). All assays were performed in CV1 cells transiently transfected with the specified full length hVDR construct and an osteocalcin-VDRE driven SEAP-reporter (see methods).…”

“…1C, yellow circles). Residues N424, E425, I426, and S427 are unresolved in the 1,25D-VDR x-ray structures [11,12,13,14,15], but when added in an α-helical orientation to the x-ray construct (pdb code: 1DB1), prior to assembly minimization, it was observed that R402 (H11) formed Coulombic interactions with either E425 and S398 or S427 and S398 (see methods; Fig. 1C, pink ribbon in yellow circle).…”

“…1B), have shown that agonists occupy the same relative steric space in the VDR LBD, termed the genomic pocket (G-pocket), and stabilize only the closed helix-12 conformation (VDR-c1, Fig. 1C) [11,12,13,14,15]. Analysis of these VDR structures and molecular models with complete AF2-domains, reveal that unlike any other hormonally regulated NR, H12 is held in the closed conformation (c1) by three salt-bridges: 1) K264---E420 2) R154---K413/ R154---L414/ R154---D232 and 3) R402---S427/ S398/ E425 (Fig.…”

Inecalcitol, an analog of 1,25D₃, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system

“…These effects contribute to their superagonistic action. 4 Both molar dose and exposure time, inecalcitol-modulated mRNA expression was significantly stronger than 1,25D 3 for all the target genes ( Fig. 1C-H).…”

“…3 In addition, inecalcitol and TX527 each are more resistant to metabolic degradation through 24-hydroxylase (CYP24A1). 4 Together, these biochemical characteristics contribute to their enhanced in vitro activity compared with 1,25D 3 .…”