title: Novel screening system for high-affinity ligand of heredity vitamin D-resistant rickets-associated vitamin D receptor mutant R274L using bioluminescent sensor.
abstract: Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)2D3.The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)2D3, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR. The chimeric protein LucC-LBD-LucN, which displays the C-terminal domain of luciferase (LucC) at its N-terminus, can detect and discriminate between VDR agonists and antagonists. The LucC-LBD (R274L)-LucN was constructed to screen high-affinity ligands for the mutant VDR (R274L). Of the 33 vitamin D analogs, 5 showed much higher affinities for the mutant VDR (R274L) than 1α,25(OH)2D3, and 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-(OH)2D3showed the highest affinity. These compounds might be potential therapeutics for HVDRR caused by the mutant VDR (R274L).
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mano
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; Imizu Institute, Topu Bio Research Co., Ltd, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Miyu
authors.initials: M
authors.lastname: Nishikawa
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Yasuda
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Ikushiro
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Saito
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sawada
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Honzawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Takano
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi, Tokyo 173-8605, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kittaka
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; Imizu Institute, Topu Bio Research Co., Ltd, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Electronic address: tsakaki@pu-toyama.ac.jp.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sakaki
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D049449:Luminescence; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D018167:Receptors, Calcitriol / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D063730:Rickets, Hypophosphatemic / Q000175:diagnosis* / Q000378:metabolism; D014807:Vitamin D / Q000378:metabolism*
chemical_list: D008024:Ligands; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; C579457:VDR protein, human; D014807:Vitamin D; D008156:Luciferases
keywords: Bioluminescent sensor; HVDRR; Rickets; Split luciferase; Vitamin D receptor