Discovery of Nuclear Receptor Ligands and Elucidation of Their Mechanisms of Action

Abstract

To develop potent ligands for the vitamin D receptor (VDR), we designed and synthesized a series of vitamin D analogues with and without 22-alkyl substituents. These analogues exhibited agonistic, partial agonistic, or antagonistic activity. To elucidate the mechanism of action of the analogues, we conducted crystal structure analyses of the ligand-binding domain (LBD) of VDR complexed with the analogues. The VDR-LBD/agonist complex exhibited precise interactions, which clearly explained VDR agonism. The VDR-LBD/partial agonist complex showed two conformers (agonist and antagonist binding conformers) in a single crystal, demonstrating that partial agonism could be explained by the sum of the agonistic and antagonistic activities. Antagonist binding to the VDR-LBD structure was elucidated using both crystal structure analysis and in-solution structural analyses with the small-angle X-ray scattering (SAXS)-molecular dynamics (MD) and hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) methods. Several antagonistbinding structures were detected. We found that the antagonist binding structures differed depending on the structure of the antagonist itself, and those structures clearly explained the VDR antagonism. Furthermore, the apo VDR-LBD structure without the ligand in the ligand-binding pocket was revealed and found to have an entrance to accommodate the ligand. Thus we elucidated the mechanisms of action of agonists, partial agonists, and antagonists based on structural changes (differences) in the receptor protein induced by ligand binding.

Vitamin D Analogs Bearing C-20 Modifications Stabilize the Agonistic Conformation of Non-Responsive Vitamin D Receptor Variants

“…Several thousand 1,25D 3 analogs were synthetized [ 7 ], and the crystal structures of the VDR ligand-binding domain (LBD) in the presence of 1,25D 3 and of more than 150 analogs were solved [ 8 , 9 , 10 ]. The integrated analysis of these structures highlighted that the ligand positioning in the ligand-binding pocket (LBP) is similar, regardless of the modifications of the agonist ligands, but selective contact points between the various ligands and the 40 residues lining the LBP are formed (reviewed in [ 8 , 9 ]).…”

Mechanism of Vitamin D Receptor Ligand-Binding Domain Regulation Studied by gREST Simulations

“…The vitamin D receptor (VDR) is a member of the nuclear receptor (NR) family. − Depending on the binding of 1α,25-dihydroxyvitamin D 3 , VDR regulates the expression of genes related to calcium homeostasis, cell differentiation, proliferation, and immunomodulation. VDR and NRs are well-known drug targets. − For example, VDR and estrogen receptor (ER) agonists are therapeutic agents for osteoporosis, whereas ER antagonists are used to treat breast cancer.…”

“…According to experimental studies such as X-ray crystallography, NRs are commonly regulated by a local conformational change of the helix (H)­12 of the ligand-binding domain (LBD). − ,− In the apo state, H12 fluctuates freely (e.g., retinoid X receptor α, PDBID: 6HN6 ). Upon agonist binding, H10, H11, and, H12 undergo conformational changes, and H12 adopts an active conformation (e.g., ER/estradiol complex, PDBID: 1ERE; Figure A).…”

“…However, in the case of VDR, the antagonistic activity cannot be explained structurally because X-ray crystal structures of LBD, except for the VDR-LBD/5b complex (PDBID: 5XPL ), are essentially identical to the active conformation, regardless of agonist/antagonist binding (Figure C,D). ,,, The antagonists used were JB (PDBID: 2ZXM ), TEI9647 (PDBID: 3A2H ), ADTT (PDBID: 2ZMI ), ADMI4 (PDBID: 2ZMJ ), and 5b (PDBID: 5XPL ). Because these antagonists have different scaffolds, their structural mechanisms for exhibiting antagonist activities may differ; however, the antagonist-bound conformation around H12 is identical to the active conformation.…”