Epimer LLC, North Smithfield, Rhode Island 02896, USA.
J Cell Biochem. 2013 Oct;114(10):2293-305. doi: 10.1002/jcb.24576.
3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), a natural metabolite of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exhibits potent vitamin D receptor (VDR)-mediated actions such as inhibition of keratinocyte growth or suppression of parathyroid hormone secretion. These VDR-mediated actions of 3-epi-1α,25(OH)2D3 needed an explanation as 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1α,25(OH)2D3 over 1α,25(OH)2D3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1α,25(OH)2D3 and 1α,25(OH)2D3 using both the technique of isolated rat kidney perfusion and purified rat CYP24A1 in a cell-free reconstituted system. For the first time, these studies resulted in the isolation and identification of 3-epi-calcitroic acid as the final inactive metabolite of 3-epi-1α,25(OH)2D3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1α,25(OH)2D3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1α,25(OH)2D3 . This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1α,25(OH)2D3 by rat CYP24A1 leading to its final inactivation is slower than that of 1α,25(OH)2D3. To elucidate the mechanism responsible for this important finding, we performed a molecular docking analysis using the crystal structure of rat CYP24A1. Docking results suggest that 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, binds to CYP24A1 in an alternate configuration that destabilizes the formation of the enzyme-substrate complex sufficiently to slow the rate at which 3-epi-1α,25(OH)2D3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid.
3-epi-1α,25-二羟维生素 D3（3-epi-1α,25(OH)2D3）是 1α,25-二羟维生素 D3（1α,25(OH)2D3）的天然代谢产物，具有抑制角质形成细胞生长或抑制甲状旁腺激素分泌等有效的维生素 D 受体（VDR）介导作用。3-epi-1α,25(OH)2D3 的这些 VDR 介导作用需要解释，因为与 1α,25(OH)2D3 不同，3-epi-1α,25(OH)2D3 对 VDR 的亲和力较低。3-epi-1α,25(OH)2D3 的代谢稳定性被认为是一种可能的解释。为了进一步支持这一假设，我们现在使用大鼠肾灌注的技术和无细胞重建系统中的纯化大鼠 CYP24A1 进行了 3-epi-1α,25(OH)2D3 和 1α,25(OH)2D3 之间的比较代谢研究。首次从大鼠 CYP24A1 产生的 3-epi-1α,25(OH)2D3 的最终无活性代谢物中分离和鉴定了 3-epi-降钙素酸。此外，在相同的实验条件下，我们注意到从 3-epi-1α,25(OH)2D3 产生的 3-epi-降钙素酸的量是从 1α,25(OH)2D3 产生的类似最终无活性代谢物 calcitroic 酸的三倍少。这一关键观察最终使我们得出结论，大鼠 CYP24A1 对 3-epi-1α,25(OH)2D3 的侧链整体氧化速率导致其最终失活的速率比 1α,25(OH)2D3 慢。为了阐明导致这一重要发现的机制，我们使用大鼠 CYP24A1 的晶体结构进行了分子对接分析。对接结果表明，与 1α,25(OH)2D3 不同，3-epi-1α,25(OH)2D3 以一种不稳定的构象与 CYP24A1 结合，足以使酶-底物复合物的形成失稳，从而减缓 3-epi-1α,25(OH)2D3 通过代谢成 3-epi-降钙素酸而被 CYP24A1 失活的速率。
