Development of a novel dual CCR5-dependent and CXCR4-dependent cell-cell fusion assay system with inducible gp160 expression.
In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC50 data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSO on this assay system were assessed, and a 2- to 3-fold increase in luciferase activity was observed in the presence of 0.05% to 2% DMSO. Although cell-cell fusion efficiency was enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.
Ji
Changhua
C
Roche Palo Alto, 3411 Hillview Avenue, Palo Alto, CA 94304, USA. Changhua.ji@roche.com
Zhang
Jun
J
Cammack
Nick
N
Sankuratri
Surya
S
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D065100:CCR5 Receptor Antagonists; D002459:Cell Fusion; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015726:Giant Cells; D019302:HIV Envelope Protein gp160 / Q000235:genetics*; D023581:HIV Fusion Inhibitors; D006801:Humans; D020128:Inhibitory Concentration 50; D019713:Receptors, CCR5 / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013997:Time Factors; D015854:Up-Regulation
D065100:CCR5 Receptor Antagonists; D019302:HIV Envelope Protein gp160; D023581:HIV Fusion Inhibitors; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D004121:Dimethyl Sulfoxide