Helix12-Stabilization Antagonist of Vitamin D Receptor

Abstract

To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and synthesized vitamin D analogues with bulky side chains based on the "active antagonist" concept in which antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds 3a and 3b showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and static X-ray crystal structure analyses indicated that compound 3a stabilizes H11-H12 but displaces H6-H7 so that 3a is a novel rather than "active" or "passive" type of antagonist. We classified 3a as a third type of antagonist and called it "H11-H12 stabilization antagonist". HDX-MS analysis indicated that antagonist 3b is an "active" antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse depending on each antagonist structure. Additionally, HDX-MS was proven to be very useful for investigations of protein structure alterations resulting from ligand binding.

Structural Analysis of VDR Complex with ZK168281 Antagonist

“…Due to weak affinity for corepressor peptides 13 or non-optimal crystal packing, only crystals of the complex in the presence of the SRC1 coactivator peptide NR2 (RHKILHRLLQEGS) were obtained and analyzed (Supplementary Table 1). These results are in agreement with previous reports characterizing X-ray structures of VDR complexes in presence of antagonist ligands [14][15][16][17][18][19] . ZK168281 binds to the VDR LBP unambiguously as shown by the POLDER omit map (Figure 1B).…”

Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays

“…Comparatively, passive antagonists tend to fit into the ligand-binding pocket but facilitate repositioning of H12 to a stable but non-active configuration. A third mechanism of receptor antagonism has also been proposed where antagonists can facilitate H12 stabilization but destabilize other regions of the receptor including the dimerization interface, impeding the ability to form productive heterodimers with RXR 50 . It is worth noting however that non-competitive VDR antagonists have also been identified that function through disruption of VDR-coregulator interactions 51 .…”

Regulation of the vitamin D receptor by vitamin D lactam derivatives

“…It was reported that several ligands also barely regulate VDR function . The bulky group in the adamantane compounds protrudes into the ligand‐binding pocket formed by the N‐terminal helix H3, the H6–H7 loop, helix H11, and the H11–H12 loop, leading to the expansion of the pocket (Fig.…”