title: High-throughput evaluation method for drug association with pregnane X receptor (PXR) using differential scanning fluorometry.
abstract: The pregnane xenobiotic receptor (PXR) is a key transcriptional regulator of cytochrome P450 (CYP) 3A, a crucial enzyme in the metabolism and detoxification of xenobiotics and endobiotics. PXR is activated by a wide variety of chemicals and serves as a master regulator of detoxification in mammals. Here, we report a fast evaluation method for PXR-drug interactions using differential scanning fluorometry (DSF). DSF analysis revealed that PXR associates with a fluorescence dye in the native state as well as in the unfolded state, which prevented precise evaluation of any shift in the transition midpoint (ΔT (m)) due to association with a drug. Hence, we defined a new parameter, (dF/dT)(50), where F is fluorescence intensity and T is temperature, to describe the ligand concentration. (dF/dT)(50) exhibited better correlation with EC(50) (r(2) = 0.84) than with ΔT m (r(2) = 0.71). The correlation of ΔT m measured using differential scanning calorimetry (DSC) with EC(50) (r(2) = 0.86) was similar to the above (dF/dT)(50) correlation. Therefore, the use of (dF/dT)(50) enables DSF to be used for the rapid evaluation of PXR-drug interactions and could provide prescreening to narrow down the collection of candidate ligands that most likely result in transcriptional activation of CYP3A4.
authors.affiliation: 1Analysis and Pharmacokinetics Research Labs, Department of Drug Discovery, Astellas Pharma Inc., Tsukuba, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Sekiguchi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kobashigawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Moriguchi
authors.forename: Masashi
authors.initials: M
authors.lastname: Kawasaki
authors.forename: Masamichi
authors.initials: M
authors.lastname: Yuda
authors.forename: Toshio
authors.initials: T
authors.lastname: Teramura
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D000924:Anticholesteremic Agents / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004164:Diphosphonates / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D056920:Nuclear Receptor Coactivator 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000077297:Pregnane X Receptor; D011485:Protein Binding; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000924:Anticholesteremic Agents; D004164:Diphosphonates; D005456:Fluorescent Dyes; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; C100355:SR 12813; D051544:Cytochrome P-450 CYP3A; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1
keywords: DSC; DSF; PXR; differential scanning calorimeter; differential scanning fluorometry; pregnane X receptor; reporter gene assay