Screening of selective inhibitors of 1α,25-dihydroxyvitamin D3 24-hydroxylase using recombinant human enzyme expressed in Escherichia coli.
High-level heterologous expression of human 1α,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1) in Escherichia coli was attained via a fusion construct by appending the mature CYP24A1 without the leader sequence to the maltose binding protein (MBP). Facile purification was achieved efficiently through affinity chromatography and afforded fully functional enzyme of near homogeneity, with a k(cat) of 0.12 min(-1) and a K(M) of 0.19 μM toward 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. A convenient and reliable cell-free assay was established and used to screen vitamin D analogues with potential inhibitory properties toward CYP24A1. Some of the compounds exhibited potent inhibition with K(I) values as low as 0.021 μM. Furthermore, TS17 and CPA1 exhibited superior specificity toward CYP24A1 over 25-hydroxyvitamin D(3) 1α-hydroxylase (CYP27B1), with selectivities of 39 and 80, respectively. Addition of TS17 or CPA1 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3) in the medium. Their activities in vitamin D receptor (VDR) binding, CYP24A1 transcription, and HL-60 cell differentiation were evaluated as well.
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, Wisconsin 53706, United States.
Jinge
J
Zhu
Rafal
R
Barycki
Grazia
G
Chiellini
Hector F
HF
Deluca
D000818:Animals; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D065668:Vitamin D3 24-Hydroxylase
D004791:Enzyme Inhibitors; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; C585550:Cyp24a1 protein, mouse; D065668:Vitamin D3 24-Hydroxylase