A Vinblastine Fluorescent Probe for Pregnane X Receptor in a Time-resolved Fluorescence Resonance Energy Transfer Assay

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 2013 Sep 19

PMID 24044991

Citations 11

Authors

Wenwei Lin

Taosheng Chen

Affiliations

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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.

Citing Articles

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Lin W, Li Y, Yang L, Chen T ACS Omega. 2021; 6(1):680-695.

PMID: 33458521 PMC: 7807814. DOI: 10.1021/acsomega.0c05221.

Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay.

Lin W, Li Y, Min J, Liu J, Yang L, Lee R Bioconjug Chem. 2020; 31(11):2564-2575.

PMID: 33070611 PMC: 7677184. DOI: 10.1021/acs.bioconjchem.0c00507.

Drug discovery technologies to identify and characterize modulators of the pregnane X receptor and the constitutive androstane receptor.

Chai S, Lin W, Li Y, Chen T Drug Discov Today. 2019; 24(3):906-915.

PMID: 30731240 PMC: 6421094. DOI: 10.1016/j.drudis.2019.01.021.

References

Venkatesh M, Wang H, Cayer J, LeRoux M, Salvail D, Das B . In vivo and in vitro characterization of a first-in-class novel azole analog that targets pregnane X receptor activation. Mol Pharmacol. 2011; 80(1):124-35. PMC: 3127530. DOI: 10.1124/mol.111.071787. View

Lau A, Yang G, Rajaraman G, Baucom C, Chang T . Differential effect of meclizine on the activity of human pregnane X receptor and constitutive androstane receptor. J Pharmacol Exp Ther. 2010; 336(3):816-26. DOI: 10.1124/jpet.110.175927. View

Zhu Z, Kim S, Chen T, Lin J, Bell A, Bryson J . Correlation of high-throughput pregnane X receptor (PXR) transactivation and binding assays. J Biomol Screen. 2004; 9(6):533-40. DOI: 10.1177/1087057104264902. View

Xue Y, Chao E, Zuercher W, Willson T, Collins J, Redinbo M . Crystal structure of the PXR-T1317 complex provides a scaffold to examine the potential for receptor antagonism. Bioorg Med Chem. 2007; 15(5):2156-66. PMC: 1839856. DOI: 10.1016/j.bmc.2006.12.026. View

Dong H, Lin W, Wu J, Chen T . Flavonoids activate pregnane x receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells. BMC Biochem. 2010; 11:23. PMC: 2901217. DOI: 10.1186/1471-2091-11-23. View

Watkins R, Wisely G, Moore L, Collins J, Lambert M, Williams S . The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science. 2001; 292(5525):2329-33. DOI: 10.1126/science.1060762. View

Lau A, Yang G, Rajaraman G, Baucom C, Chang T . Human pregnane X receptor agonism by Ginkgo biloba extract: assessment of the role of individual ginkgolides. J Pharmacol Exp Ther. 2010; 335(3):771-80. DOI: 10.1124/jpet.110.172338. View

Duniec-Dmuchowski Z, Fang H, Strom S, Ellis E, Runge-Morris M, Kocarek T . Human pregnane X receptor activation and CYP3A4/CYP2B6 induction by 2,3-oxidosqualene:lanosterol cyclase inhibition. Drug Metab Dispos. 2009; 37(4):900-8. PMC: 2680536. DOI: 10.1124/dmd.108.025130. View

Xue Y, Moore L, Orans J, Peng L, Bencharit S, Kliewer S . Crystal structure of the pregnane X receptor-estradiol complex provides insights into endobiotic recognition. Mol Endocrinol. 2007; 21(5):1028-38. DOI: 10.1210/me.2006-0323. View

10.

Timsit Y, Negishi M . CAR and PXR: the xenobiotic-sensing receptors. Steroids. 2007; 72(3):231-46. PMC: 1950246. DOI: 10.1016/j.steroids.2006.12.006. View

11.

Teotico D, Bischof J, Peng L, Kliewer S, Redinbo M . Structural basis of human pregnane X receptor activation by the hops constituent colupulone. Mol Pharmacol. 2008; 74(6):1512-20. PMC: 2584763. DOI: 10.1124/mol.108.050732. View

12.

Wang H, Li H, Moore L, Johnson M, Maglich J, Goodwin B . The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor. Mol Endocrinol. 2007; 22(4):838-57. PMC: 5419551. DOI: 10.1210/me.2007-0218. View

13.

Watkins R, Maglich J, Moore L, Wisely G, Noble S, Davis-Searles P . 2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin. Biochemistry. 2003; 42(6):1430-8. DOI: 10.1021/bi0268753. View

14.

Zhang , Chung , OLDENBURG . A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 2000; 4(2):67-73. DOI: 10.1177/108705719900400206. View

15.

Shukla S, Nguyen D, Macarthur R, Simeonov A, Frazee W, Hallis T . Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening. Assay Drug Dev Technol. 2009; 7(2):143-69. PMC: 3116688. DOI: 10.1089/adt.2009.193. View

16.

Kim J, Felts S, Llauger L, He H, Huezo H, Rosen N . Development of a fluorescence polarization assay for the molecular chaperone Hsp90. J Biomol Screen. 2004; 9(5):375-81. DOI: 10.1177/1087057104265995. View

17.

Wang Y, Ong S, Chai S, Chen T . Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol. 2012; 8(7):803-17. PMC: 3593589. DOI: 10.1517/17425255.2012.685237. View

18.

Harmsen S, Meijerman I, Febus C, Maas-Bakker R, Beijnen J, Schellens J . PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line. Cancer Chemother Pharmacol. 2009; 66(4):765-71. PMC: 2904455. DOI: 10.1007/s00280-009-1221-4. View

19.

Lau A, Yang G, Yap C, Chang T . Selective agonism of human pregnane X receptor by individual ginkgolides. Drug Metab Dispos. 2012; 40(6):1113-21. DOI: 10.1124/dmd.112.045013. View

20.

Chrencik J, Orans J, Moore L, Xue Y, Peng L, Collins J . Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Mol Endocrinol. 2005; 19(5):1125-34. DOI: 10.1210/me.2004-0346. View