Novel biosensor using split-luciferase for detecting vitamin D receptor ligands based on the interaction between vitamin D receptor and coactivator
Highlights: • A novel split-luciferase-based biosensor was developed to detect VDR ligands. • LXXLL-motif peptide and ligand-bound LBD formed a functional complex. • The light intensity was increased by 10-fold compared to our previous versions. • The binding affinity of vitamin D analogs was measured in a short time. Vitamin D receptor (VDR) ligands, such as 1α,25-dihydroxyvitamin D{sub 3} [1α,25(OH){sub 2}D{sub 3}] and its analogs, have been investigated for their potential clinical use in the treatment of various diseases such as type I rickets, osteoporosis, psoriasis, leukemia, and cancer. Previously, we reported a split-luciferase-based biosensor that can detect VDR ligands and assess their affinity for the ligand binding domain (LBD) of the VDR in a short time. However, a further increase in its sensitivity was required to detect plasma levels of 1α,25(OH){sub 2}D{sub 3} and its analogs. In this study, a novel type of biosensor called LXXLL + LBD was successfully developed. Here, the split luciferase forms a functional complex based on the intermolecular interaction between the LXXLL motif and the ligand-bound form of the LBD. This biosensor has an approximately 10-fold increase in the light intensity compared to the previous versions. Additionally, the binding affinity of the vitamin D analogs for the wild-type and the rickets-associated mutant R274L of VDR was evaluated.