Screening of Selective Inhibitors of 1α,25-Dihydroxyvitamin D3 24-Hydroxylase Using Recombinant Human Enzyme Expressed in Escherichia coli

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• 作者：Jinge Zhu ; Rafal Barycki ; Grazia Chiellini ; Hector F. DeLuca
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：December 14, 2010
• 年：2010
• 卷：49
• 期：49
• 页码：10403-10411
• 全文大小：906K
• 年卷期：v.49,no.49(December 14, 2010)
• ISSN：1520-4995

文摘

High-level heterologous expression of human 1α,25-dihydroxyvitamin D₃ 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 D₃ [1,25(OH)₂D₃]. 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 D₃ 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)₂D₃ in the medium. Their activities in vitamin D receptor (VDR) binding, CYP24A1 transcription, and HL-60 cell differentiation were evaluated as well.