School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
Drug Metab Dispos. 2013 May;41(5):1112-24. doi: 10.1124/dmd.113.050955. Epub 2013 Mar 1.
CYP11A1 can hydroxylate vitamin D3 at carbons 17, 20, 22, and 23, producing a range of secosteroids which are biologically active with respect to their ability to inhibit proliferation and stimulate differentiation of various cell types, including cancer cells. As 1α-hydroxylation of the primary metabolite of CYP11A1 action, 20S-hydroxyvitamin D3 [20(OH)D3], greatly influences its properties, we examined the ability of both human and mouse CYP27B1 to 1α-hydroxylate six secosteroids generated by CYP11A1. Based on their kcat/Km values, all CYP11A1-derived metabolites are poor substrates for CYP27B1 from both species compared with 25-hydroxyvitamin D3. No hydroxylation of metabolites with a 17α-hydroxyl group was observed. 17α,20-Dihydroxyvitamin D3 acted as an inhibitor on human CYP27B1 but not the mouse enzyme. We also tested CYP27B1 activity on 20,24-, 20,25-, and 20,26-dihydroxyvitamin D3, which are products of CYP24A1 or CYP27A1 activity on 20(OH)D3. All three compounds were metabolized with higher catalytic efficiency (kcat/Km) by both mouse and human CYP27B1 than 25-hydroxyvitamin D3. CYP27B1 action on these new dihydroxy derivatives was confirmed to be 1α-hydroxylation by mass spectrometry and nuclear magnetic resonance analyses. Both 1,20,25- and 1,20,26- trihydroxyvitamin D3 were tested for their ability to inhibit melanoma (SKMEL-188) colony formation, and were significantly more active than 20(OH)D3. This study shows that CYP11A1-derived secosteroids are 1α-hydroxylated by both human and mouse CYP27B1 with low catalytic efficiency, and that the presence of a 17α-hydroxyl group completely blocks 1α-hydroxylation. In contrast, the secondary metabolites produced by subsequent hydroxylation of 20(OH)D3 at C24, C25, or C26 are very good substrates for CYP27B1.
CYP11A1 可以在碳 17、20、22 和 23 位羟基化维生素 D3，生成一系列具有生物活性的甾体化合物，能够抑制多种细胞类型的增殖并刺激其分化，包括癌细胞。由于 CYP11A1 作用的初级代谢物 1α-羟化，20S-羟基维生素 D3[20(OH)D3]极大地影响了其性质，我们研究了人源和鼠源 CYP27B1 对 CYP11A1 生成的六种甾体化合物的 1α-羟化能力。基于它们的 kcat/Km 值，与 25-羟基维生素 D3 相比，所有 CYP11A1 衍生的代谢物都是两种物种 CYP27B1 的较差底物。没有观察到带有 17α-羟基的代谢物的羟化。17α，20-二羟基维生素 D3 是人源 CYP27B1 的抑制剂，但对鼠源酶没有作用。我们还测试了 CYP27B1 对 20，24-、20，25-和 20，26-二羟基维生素 D3 的活性，它们是 CYP24A1 或 CYP27A1 对 20(OH)D3 作用的产物。与 25-羟基维生素 D3 相比，这三种化合物都被鼠源和人源 CYP27B1 以更高的催化效率（kcat/Km）代谢。通过质谱和核磁共振分析证实，CYP27B1 对这些新的二羟基衍生物的作用是 1α-羟化。测试了 1，20，25-和 1，20，26-三羟基维生素 D3 抑制黑色素瘤（SKMEL-188）集落形成的能力，它们比 20(OH)D3 活性显著更高。这项研究表明，CYP11A1 衍生的甾体化合物被人和鼠源 CYP27B1 以低催化效率 1α-羟化，并且 17α-羟基的存在完全阻止了 1α-羟化。相比之下，20(OH)D3 在 C24、C25 或 C26 位的后续羟化产生的次级代谢物是 CYP27B1 的很好的底物。
