Probing a water channel near the A-ring of receptor-bound 1α,25-dihydroxyvitamin D3 with selected 2α-substituted analogues

The crystal structure of the vitamin D receptor (VDR) in complex with 1α,25(OH)₂D₃ revealed the presence of several water molecules near the A-ring linking the ligand C-2 position to the protein surface. Here, we report the crystal structures of the human VDR ligand binding domain bound to selected C-2α substituted analogues, namely, methyl, propyl, propoxy, hydroxypropyl, and hydroxypropoxy. These specific replacements do not modify the structure of the protein or the ligand, but with the exception of the methyl substituent, all analogues affect the presence and/or the location of the above water molecules. The integrity of the channel interactions and specific C-2α analogue directed additional interactions correlate with the binding affinity of the ligands. In contrast, the resulting loss or gain of H-bonds does not reflect the magnitude of HL60 cell differentiation. Our overall findings highlight a rational approach to the design of more potent ligands by building in features revealed in the crystal structures.