Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands

We designed and synthesized vitamin
D analogues with an electrophile
as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin
D analogues 1–4 have an electrophilic
enone group at the side chain for conjugate addition to His301 or
His393 in the VDR. All compounds showed specific VDR-binding potency
and agonistic activity. Covalent bond formations of 1–4 with the ligand-binding domain (LBD) of VDR
were evaluated by electrospray ionization mass spectrometry. All compounds
were shown to covalently bind to the VDR-LBD, and the abundance of
VDR-LBD corresponding conjugate adducts of 1–4 increased with incubation time. Enone compounds 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compounds. Furthermore, we successfully
obtained cocrystals of VDR-LBD with analogues 1–4. X-ray crystallographic analysis showed a covalent bond
with His301 in VDR-LBD. We successfully synthesized vitamin D analogues
that form a covalent bond with VDR-LBD.