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Journal of Biomolecular Screening
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A Time-Resolved Fluorescence Assay to Identify Small-Molecule Inhibitors of HIV-1 Fusion

Géry Dams

Tibotec BVBA, Mechelen, Belgium

Koen Van Acker

Tibotec BVBA Gen De Wittelaan L 11B 3 2800 Mechelen, Belgium E-mail: kvacker{at}tibbe.jnj.com

Emmanuel Gustin

Tibotec BVBA, Mechelen, Belgium

Inge Vereycken

Tibotec BVBA, Mechelen, Belgium

Lieve Bunkens

Tibotec BVBA, Mechelen, Belgium

Pascale Holemans

Tibotec BVBA, Mechelen, Belgium

Liesbet Smeulders

Tibotec BVBA, Mechelen, Belgium

Reginald Clayton

Tibotec BVBA, Mechelen, Belgium

Asa Ohagen

Tibotec BVBA, Mechelen, Belgium

Kurt Hertogs

Tibotec BVBA, Mechelen, Belgium

Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Förster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions. (Journal of Biomolecular Screening 2007:865-874)

Key Words: HIV-1 • gp41 • fusion inhibitor • screening • Förster resonance energy transfer

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 865-874 (2007)
DOI: 10.1177/1087057107304645
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