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 1 alpha,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) K-M of 0.19 mu M toward 1 alpha,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-1 values as low as 0.021 mu M. Furthermore, TS17 and CPA1 exhibited superior specificity toward CYP24A1 over 25-hydroxyitamin D-3 1 alpha-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.