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Rhieu S.Y.,Epimer LLC | Annalora A.J.,Oregon State University | Laporta E.,University at Albany | Welsh J.,University at Albany | And 6 more authors.
Journal of Cellular Biochemistry | Year: 2014

The potency of 25-hydroxyvitamin D3 (25(OH)D3) is increased by several fold through its metabolism into 1α,25- dihydroxyvitamin D3 (1α,25(OH)2D3) by cytochrome P450 27B1 (CYP27B1). Thus, the pivotal role of 1α-hydroxylation in the activation of vitamin D compounds is well known. Here, we examined the metabolism of 25-hydroxy-16-ene-23-yne-vitamin D3 (25(OH)-16-ene-23-yne-D3), a synthetic analog of 25(OH)D3 in a cell-free system and demonstrated that 25(OH)-16-ene-23-yne-D3 is neither activated by CYP27B1 nor inactivated by cytochrome P450 24A1 (CYP24A1). These findings were also confirmed in immortalized normal human prostate epithelial cells (PZ-HPV-7) which are known to express both CYP27B1 and CYP24A1, indicating that the structural modifications featured in 25(OH)-16-ene-23-yne-D3 enable the analog to resist the actions of both CYP27B1 and CYP24A1. To provide intelligible structure-function information, we also performed molecular docking analysis between the analog and CYP27B1. Furthermore, 25(OH)-16-ene-23-yne-D3 was found to suppress the growth of PZ-HPV-7 cells with a potency equivalent to 1α,25(OH) 2D3. The antiproliferative activity of 25(OH)-16-ene-23-yne-D3 was found to be vitamin D receptor (VDR)-dependent as it failed to inhibit the growth of mammary tumor cells derived from VDR-knockout mice. Furthermore, stable introduction of VDR into VDR-knockout cells restored the growth inhibition by 25(OH)-16-ene-23-yne-D 3. Thus, we identified 25-hydroxy-16-ene-23-yne-vitamin D3 as a novel non-1α-hydroxylated vitamin D analog which is equipotent to 1α,25(OH)2D3 in its antiproliferative activity. We now propose that the low potency of the intrinsic VDR-mediated activities of 25(OH)D3 can be augmented to the level of 1α,25(OH) 2D3 without its activation through 1α-hydroxylation by CYP27B1, but by simply preventing its inactivation by CYP24A1. J. Cell. Biochem. 115: 1392-1402, 2014. © 2014 Wiley Periodicals, Inc. Source

Sawada D.,Teikyo University | Tsukuda Y.,Teikyo University | Yasuda K.,Toyama Prefectural University | Sakaki T.,Toyama Prefectural University | And 7 more authors.
Chemical and Pharmaceutical Bulletin | Year: 2012

A previous report has demonstrated the existence of a C4-hydroxylated vitamin D2 metabolite in serum of rats treated with pharmacological doses of vitamin D2. However, the biological significance and metabolic fate of this metabolite have not been described. To explore its potential biological activities, we therefore synthesized 1α,4 α,25-trihydroxyvitamin D3 and its diastereoisomer, 1α,4β,25-trihydroxyvitamin D3, using Trost Pd-mediated coupling reaction, and studied their vitamin D receptor (VDR) binding affinity, osteocalcin promoter transactivation activity, and their further metabolism by human CYP24A1 as well as by human liver microsomal fraction based on CYP- and UDP-glucuronosyltransferases (UGTs)-reactions. © 2012 The Pharmaceutical Society of Japan. Source

Gathungu R.M.,Northeastern University | Flarakos C.C.,Northeastern University | Satyanarayana Reddy G.,Epimer LLC | Satyanarayana Reddy G.,Brown University | Vouros P.,Northeastern University
Mass Spectrometry Reviews | Year: 2013

This review highlights the superseding role of mass spectrometry in the structural characterization and quantitation of vitamin D compounds in comparison to other analytical methods (e.g., UV, bioassays) that lack the sensitivity and specificity of mass spectrometry. After a short introduction to the biochemistry of vitamin D compounds, an overview of the current techniques to characterize and quantitate vitamin D compounds is given with emphasis on the contribution of mass spectrometry. © 2012 Wiley Periodicals, Inc. Source

Takano M.,Teikyo University | Sawada D.,Teikyo University | Sawada D.,Okayama University | Yasuda K.,Toyama Prefectural University | And 8 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2015

Three different A-ring perhydroxylated trihydroxyvitamin D3 metabolites were synthesized from their appropriate A-ring precursors and CD-ring for their potential therapeutic applications. We first chemically synthesized 1α,2α,25-trihydroxyvitamin D3 [1α,2α,25(OH)3D3] to study its VDR binding affinity because this metabolite is a product of recombinant human CYP3A4 catalysis when 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3 (O2C3), a more potent vitamin D receptor (VDR) binder than 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], is used as the substrate. We found that this metabolite retained 27.3% of the VDR binding affinity compared to 1α,25(OH)2D3. The kcat/Km value of CYP24A1 for 1α,2α,25(OH)3D3 is 60% of that for 1α,25(OH)2D3. Since the biological activity and the metabolic fate of a naturally occurring C4-hydroxylated vitamin D2 metabolite found in the serum of rats treated with pharmacological doses of vitamin D2 have never been described, we next synthesized 1α,4α,25-trihydroxyvitamin D3 and its diastereoisomer, 1α,4β,25-trihydroxyvitamin D3, to study their metabolism and biological activities. Both 4-hydroxylated isomers showed weaker VDR binding affinity than 1α,25(OH)2D3. Although either 4-hydroxylated isomer can be metabolized by CYP24A1 almost at the same level as 1α,25(OH)2D3, their metabolic patterns catalyzed by uridine 5′-diphosphoglucuronosyltransferase (UGT) are different; only the 4α-hydroxylated analog can be metabolized by UGT to produce a glucuronate conjugate. The results provide important information for the synthesis of new novel chemotherapeutic vitamin D analogs which would be less subjective to degradation and therefore more bioavailable than 1α,25(OH)2D3. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. © 2014 Elsevier Ltd. All rights reserved. Source

Rhieu S.Y.,Epimer LLC | Rhieu S.Y.,Brown University | Rhieu S.Y.,U.S. National Institute of Standards and Technology | Annalora A.J.,Scripps Research Institute | And 10 more authors.
Journal of Cellular Biochemistry | Year: 2013

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)2D 3 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)2D 3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid. J. Cell. Biochem. 114: 2293-2305, 2013. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc. Source

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