title: Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses.
abstract: The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus-firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z' values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.
authors.affiliation: Assay Development and Compound Profiling, GlaxoSmithKline, Stevenage, Hertfordshire, UK.
authors.forename: Roy M
authors.initials: RM
authors.lastname: Katso
authors.forename: Janet H
authors.initials: JH
authors.lastname: Parham
authors.forename: Matilde
authors.initials: M
authors.lastname: Caivano
authors.forename: William C
authors.initials: WC
authors.lastname: Clay
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Condreay
authors.forename: David W
authors.initials: DW
authors.lastname: Gray
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Lindley
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Mason
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rieger
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Wakes
authors.forename: William J
authors.initials: WJ
authors.lastname: Cairns
authors.forename: Raymond V
authors.initials: RV
authors.lastname: Merrihew
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant / Q000235:genetics*; D006801:Humans; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D020033:Protein Isoforms; D011980:Receptors, Progesterone