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Antimicrobial Agents and Chemotherapy, July 2007, p. 2388-2395, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.00150-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Novel Fluorescence Intensity Screening Assay Identifies New Low-Molecular-Weight Inhibitors of the gp41 Coiled-Coil Domain of Human Immunodeficiency Virus Type 1

Department of Basic Sciences, Touro University—California, Vallejo, California 94592,¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94143²

Received 1 February 2007/ Returned for modification 16 March 2007/ Accepted 13 April 2007

A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophore-labeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z') of 0.88 and can rank order inhibitors at 10 µM concentration with K_is in the range of 0.2 µM to 30 µM, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.

Published ahead of print on 23 April 2007.