Eelen Guy, Verlinden Lieve, Rochel Natacha, Claessens Frank, De Clercq Pierre, Vandewalle Maurits, Tocchini-Valentini Giuseppe, Moras Dino, Bouillon Roger, Verstuyf Annemieke
Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Belgium.
Mol Pharmacol. 2005 May;67(5):1566-73. doi: 10.1124/mol.104.008730. Epub 2005 Feb 22.
Two 14-epi-analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)(2)D(3)], 19-nor-14-epi-23-yne-1,25-(OH)2D3 (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)2D3 (TX527), show enhanced antiproliferative (at least 10-fold) and markedly lower calcemic effects both in vitro and in vivo, compared with 1,25-(OH)2D3. 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)2D3 (up to 16- and 20-fold stronger induction of VDR-SRC-1 interaction for TX522 and TX527 at 10(-10) M). Similar assays in which metabolism of 1,25-(OH)2D3 was blocked with VID400, a selective inhibitor of the 1,25-(OH)2D3-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 angstroms, 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 - 二羟基维生素D3 [1,25-(OH)₂D₃] 的两种14 - 表位类似物，19 - 去甲 - 14 - 表位 - 23 - 炔 - 1,25-(OH)₂D₃（TX522）和19 - 去甲 - 14,20 - 双表位 - 23 - 炔 - 1,25-(OH)₂D₃（TX527），与1,25-(OH)₂D₃相比，在体外和体内均显示出增强的抗增殖作用（至少强10倍）且血钙升高作用明显更低。本研究旨在评估它们在维生素D受体（VDR）与共激活因子相互作用水平上的超激动效应。用与VP16融合的VDR和与GAL4 - DNA结合结构域融合的类固醇受体共激活因子1（SRC - 1）、转录中介因子2（Tif2）或DRIP205进行的哺乳动物双杂交试验表明，14 - 表位类似物比1,25-(OH)₂D₃更有效地诱导VDR - 共激活因子相互作用（在10⁻¹⁰ M时，TX522和TX527对VDR - SRC - 1相互作用的诱导强度分别比1,25-(OH)₂D₃强16倍和20倍）。用1,25-(OH)₂D₃代谢酶CYP24的选择性抑制剂VID400阻断1,25-(OH)₂D₃代谢的类似试验表明，这些类似物在建立VDR - 共激活因子相互作用方面增强的效力只能部分归因于它们对代谢降解的抗性增加。与人类VDR配体结合结构域复合的TX522的晶体结构表明，C14的表位构型导致配体的CD环移动了0.5埃，从而使C12原子与Val300的接触更紧密。此外，由于含三键侧链的刚性，TX522的C22与Ile268的CD1原子有额外接触。VDR激活螺旋H12的位置和构象严格保持。总之，本研究对TX522在配体结合口袋中的对接有了更深入的了解，并表明更强的VDR - 共激活因子相互作用是这两种14 - 表位类似物超激动活性的基础。
