title: Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.
abstract: The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
authors: Caitlin
authors: C
authors: Lynch
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
authors: Jinghua
authors: J
authors: Zhao
authors: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA.
authors: Hongbing
authors: H
authors: Wang
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA. mxia@mail.nih.gov.
authors: Menghang
authors: M
authors: Xia
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6 / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D018703:Founder Effect; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D000090702:Constitutive Androstane Receptor; D007546:Isoquinolines; C000716327:NR1I3 protein, human; D010091:Oximes; D018160:Receptors, Cytoplasmic and Nuclear; D013844:Thiazoles; C475093:6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; D008156:Luciferases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; C037850:PK 11195
keywords: Constitutive Androstane Receptor (CAR); Cytochrome P450 2B6 (CYP2B6); Luciferase; Quantitative high-throughput screening (qHTS)