title: A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay.
abstract: The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand-PXR interactions. Here we report the characterization of BODIPY FL-vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a Kd value of 673 nM. We provide evidence that BODIPY FL-vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL-vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL-vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Wenwei
authors.initials: W
authors.lastname: Lin
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D001896:Boron Compounds / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D000077297:Pregnane X Receptor; D011485:Protein Binding; D011987:Receptors, Steroid / Q000032:analysis* / Q000378:metabolism; D014747:Vinblastine / Q000737:chemistry* / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D014747:Vinblastine
keywords: BODIPY FL vinblastine; Drug metabolism; High-throughput screening; PXR; Time-resolved fluorescence resonance energy transfer assay; Xenobiotics
references.citation: Mol Pharmacol. 2011 Jul;80(1):124-35
references.pmid: 21464197
references.citation: J Pharmacol Exp Ther. 2011 Mar;336(3):816-26
references.pmid: 21131266
references.citation: J Biomol Screen. 2004 Sep;9(6):533-40
references.pmid: 15452340
references.citation: Bioorg Med Chem. 2007 Mar 1;15(5):2156-66
references.pmid: 17215127
references.citation: BMC Biochem. 2010 Jun 16;11:23
references.pmid: 20553580
references.citation: Science. 2001 Jun 22;292(5525):2329-33
references.pmid: 11408620
references.citation: J Pharmacol Exp Ther. 2010 Dec;335(3):771-80
references.pmid: 20739453
references.citation: Drug Metab Dispos. 2009 Apr;37(4):900-8
references.pmid: 19158313
references.citation: Mol Endocrinol. 2007 May;21(5):1028-38
references.pmid: 17327420
references.citation: Steroids. 2007 Mar;72(3):231-46
references.pmid: 17284330
references.citation: Mol Pharmacol. 2008 Dec;74(6):1512-20
references.pmid: 18768384
references.citation: Mol Endocrinol. 2008 Apr;22(4):838-57
references.pmid: 18096694
references.citation: Biochemistry. 2003 Feb 18;42(6):1430-8
references.pmid: 12578355
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Assay Drug Dev Technol. 2009 Apr;7(2):143-69
references.pmid: 19505231
references.citation: J Biomol Screen. 2004 Aug;9(5):375-81
references.pmid: 15296636
references.citation: Expert Opin Drug Metab Toxicol. 2012 Jul;8(7):803-17
references.pmid: 22554043
references.citation: Cancer Chemother Pharmacol. 2010 Sep;66(4):765-71
references.pmid: 20041327
references.citation: Drug Metab Dispos. 2012 Jun;40(6):1113-21
references.pmid: 22393123
references.citation: Mol Endocrinol. 2005 May;19(5):1125-34
references.pmid: 15705662
references.citation: Curr Drug Metab. 2008 May;9(4):310-22
references.pmid: 18473749
references.citation: J Thromb Haemost. 2010 Dec;8(12):2708-17
references.pmid: 20735727
references.citation: J Biol Chem. 2000 May 19;275(20):15122-7
references.pmid: 10748001
references.citation: J Mol Biol. 2003 Aug 22;331(4):815-28
references.pmid: 12909012
references.citation: Ann Pharmacother. 2010 Nov;44(11):1709-17
references.pmid: 20959500
references.citation: J Pharmacol Exp Ther. 2010 Sep 1;334(3):999-1008
references.pmid: 20504912
references.citation: Methods Enzymol. 2005;400:598-618
references.pmid: 16399373
references.citation: J Biol Chem. 2008 Nov 7;283(45):30650-7
references.pmid: 18784074
references.citation: FEBS Lett. 2007 May 1;581(9):1721-6
references.pmid: 17418145
references.citation: Biochem Pharmacol. 2011 Mar 1;81(5):669-79
references.pmid: 21145880
references.citation: Biochem Pharmacol. 1973 Dec 1;22(23):3099-108
references.pmid: 4202581
references.citation: Cell. 1998 Jan 9;92(1):73-82
references.pmid: 9489701
references.citation: Protein Sci. 2011 Oct;20(10):1713-9
references.pmid: 21805522
references.citation: Mol Endocrinol. 2000 Jan;14(1):27-39
references.pmid: 10628745