Metabolic Stability of 3-Epi-1α, 25-Dihydroxyvitamin D3 Over 1 α 25-Dihydroxyvitamin D3: Metabolism and Molecular Docking Studies Using Rat CYP24A1

3-epi-1,25-dihydroxyvitamin D-3 (3-epi-1,25(OH)(2)D-3), a natural metabolite of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), 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-1,25(OH)(2)D-3 needed an explanation as 3-epi-1,25(OH)(2)D-3, unlike 1,25(OH)(2)D-3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1,25(OH)(2)D-3 over 1,25(OH)(2)D-3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1,25(OH)(2)D-3 and 1,25(OH)(2)D-3 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-1,25(OH)(2)D-3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1,25(OH)(2)D-3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1,25(OH)(2)D-3. This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1,25(OH)(2)D-3 by rat CYP24A1 leading to its final inactivation is slower than that of 1,25(OH)(2)D-3. 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-1,25(OH)(2)D-3, unlike 1,25(OH)(2)D-3, 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-1,25(OH)(2)D-3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid. J. Cell. Biochem. 114: 2293-2305, 2013. (c) 2013 Wiley Periodicals, Inc.