Metabolic stability of 3-Epi-1α,25-dihydroxyvitamin D₃over 1α, 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.

Calcitroic Acid–A Review

“…38 Finally, a novel metabolism pathway of 1,25(OH) 2 D 3 that includes the formation of epi-1,25(OH) 2 D 3 has shown the formation of epi-calcitroic acid with the 3β-OH configuration. 39 …”

The vitamin D metabolome: An update on analysis and function

“…Biological effects have been described for 3‐epi‐1α,25(OH) 2 D3 including having similar PTH suppression compared with 1α,25(OH) 2 D3 . This metabolite also has anti proliferative effects of approximately 30% compared with 1α,25(OH) 2 D3 along with low calcaemic activity . Circulating levels of 3‐epi‐25OHD3 have been quantified that correlate with 25OHD3 levels and are generally between 4% and 10% of 25OHD3 concentration and tracks well over time .…”

“…All major vitamin D metabolites have been reported to be epimerized. Following epimerization, the epimers can be further metabolised by the same enzymes involved in the hydroxylation pathways of 25OHD3 . 25OHD3, 1α,25(OH) 2 D3, and 24R,25(OH) 2 D3 can all be metabolised directly to their respective epimer metabolites 3‐epi‐25OHD3, 3‐epi‐1α,25(OH) 2 D3, and 3‐epi‐24R,25(OH) 2 D3 (Figure ) .…”

“…Following epimerization, the epimers can be further metabolised by the same enzymes involved in the hydroxylation pathways of 25OHD3 . 25OHD3, 1α,25(OH) 2 D3, and 24R,25(OH) 2 D3 can all be metabolised directly to their respective epimer metabolites 3‐epi‐25OHD3, 3‐epi‐1α,25(OH) 2 D3, and 3‐epi‐24R,25(OH) 2 D3 (Figure ) . The epimerized product 3‐epi‐25OHD3 can be hydroxylated at C1α and C24 to directly form 3‐epi‐1α,25(OH) 2 D3 and 3‐epi‐24,25(OH) 2 D3 by CYP27B1 and CYP24A1 .…”

“…Following epimerization, the epimers can be further metabolised by the same enzymes involved in the hydroxylation pathways of 25OHD3. [109][110][111] 25OHD3, 1α,25(OH) 2 D3, and 24R,25(OH) 2 D3 can all be metabolised directly to their respective epimer metabolites 3-epi-25OHD3, 3-epi-1α,25(OH) 2 D3, and 3-epi-24R,25(OH) 2 D3 (Figure 7). 109 This metabolite also has anti proliferative effects of approximately 30% compared with 1α,25(OH) 2 D3 109,110 along with low calcaemic activity.…”