RESUMO
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.
Assuntos
Baculoviridae/genética , DNA Recombinante/genética , Receptores de Progesterona/análise , Receptores de Progesterona/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/metabolismo , Transdução Genética , TransfecçãoRESUMO
The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.
Assuntos
Baculoviridae/genética , Antígenos CD4/metabolismo , Fusão Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV , Piperidinas , Receptores CCR5/metabolismo , Amidas/farmacologia , Ácido Butírico/farmacologia , Antagonistas dos Receptores CCR5 , Linhagem Celular , Linhagem Celular Tumoral , Óxidos N-Cíclicos/farmacologia , Dimetil Sulfóxido/farmacologia , Produtos do Gene env/metabolismo , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV/genética , Humanos , Oximas , Plasmídeos , Piridinas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Transdução Genética , Transfecção , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.
Assuntos
Baculoviridae/genética , Neoplasias Ósseas/genética , Osteossarcoma/genética , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/fisiologia , Transfecção/métodos , Animais , Baculoviridae/metabolismo , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Osteossarcoma/metabolismo , Receptores de Estrogênio/fisiologia , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Xenopus laevisRESUMO
BACKGROUND AND PURPOSE: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X(7) receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. EXPERIMENTAL APPROACH: Antagonist effects at recombinant and chimeric P2X(7) receptors were assessed by ethidium accumulation and radioligand-binding studies. KEY RESULTS: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X(7) receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X(7) receptors and, for each compound, their potency estimates at human and dog P2X(7) receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X(7) receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X(7) receptor. N(2)-(3,4-difluorophenyl)-N(1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373. CONCLUSIONS: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X(7) receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X(7) receptor at which antagonists can bind and affect receptor function.