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.

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Covalent ligands are generally filtered out of chemical libraries used for high-throughput screening, because electrophilic functional groups are considered to be pan-assay interference compounds (PAINS). Therefore, screening strategies that can distinguish true covalent ligands from PAINS are required. Hydrogen/deuterium−exchange mass spectrometry (HDX−MS) is a powerful tool for evaluating protein stability. Here, we report a covalent modifier screening approach using HDX−MS. In this study, HDX−MS was used to classify peroxisome proliferatoractivated receptor γ (PPARγ) and vitamin D receptor ligands. HDX−MS could discriminate the strength of ligand−protein interactions. Our HDX−MS screening method identified LT175 and nTZDpa, which can bind concurrently to the PPARγ ligand-binding domain (PPARγ−LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPARγ−LBD.

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