title: Metabolic stability of 3-epi-1α,25-dihydroxyvitamin D3 over 1 α 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1.
abstract: 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.
authors.affiliation: Epimer LLC, North Smithfield, Rhode Island 02896, USA.
authors.forename: Steve Y
authors.initials: SY
authors.lastname: Rhieu
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Annalora
authors.forename: Guochun
authors.initials: G
authors.lastname: Wang
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Flarakos
authors.forename: Rose M
authors.initials: RM
authors.lastname: Gathungu
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
authors.forename: Rita
authors.initials: R
authors.lastname: Sigüeiro
authors.forename: Antonio
authors.initials: A
authors.lastname: Mouriño
authors.forename: Inge
authors.initials: I
authors.lastname: Schuster
authors.forename: G Tayhas R
authors.initials: GT
authors.lastname: Palmore
authors.forename: G Satyanarayana
authors.initials: GS
authors.lastname: Reddy
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006887:Hydroxycholecalciferols / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D006887:Hydroxycholecalciferols; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D013250:Steroid Hydroxylases; C585551:Cyp24a1 protein, rat; D065668:Vitamin D3 24-Hydroxylase; C008088:alfacalcidol
keywords: 3-EPI-1α,25-DIHYDROXYVITAMIN D3; 3-EPI-CALCITROIC ACID; 3-EPI-VITAMIN D3; C-3 EPIMERIZATION PATHWAY; CYP24A1; MOLECULAR DOCKING