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Development and Automation of a 384-Well Cell Fusion Assay to Identify Inhibitors of CCR5/CD4-Mediated HIV Virus Entry

Laboratory Automation Group (LAG)

Jasween Gill

Francois Bertelli

Sara Letafat

Hit Discovery Group (HDG)

Romu Corbau

Anti-infectives Biology, Pfizer Global Research and Development, Kent, UK

Paul Hayter

Paula Harrison

Andy Tee

Hit Discovery Group (HDG)

Wilma Keighley

Laboratory Automation Group (LAG)

Manos Perros

Anti-infectives Biology, Pfizer Global Research and Development, Kent, UK

Giuseppe Ciaramella

Hit Discovery Group (HDG)

Andreas Sewing

Laboratory Automation Group (LAG)

Christine Williams

Hit Discovery Group (HDG)chris_williams{at}sandwich.pfizer.com

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/ß-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of ß-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturisation to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen ~650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.

Key Words: HIV • HTS • gp120 • CD4 • CCR5

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