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

• Published in: Journal of biomolecular screening Vol. 12; no. 6; pp. 865 - 874
• Main Authors: Dams, Géry, Van Acker, Koen, Gustin, Emmanuel, Vereycken, Inge, Bunkens, Lieve, Holemans, Pascale, Smeulders, Liesbet, Clayton, Reginald, Ohagen, Asa, Hertogs, Kurt
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2007
• Subjects: Amino Acid Sequence; Antiviral Agents - analysis; Antiviral Agents - pharmacology; Binding, Competitive; Fluorescence Resonance Energy Transfer - methods; fusion inhibitor; Förster resonance energy transfer; gp41; HIV Envelope Protein gp41 - chemistry; HIV-1; HIV-1 - drug effects; Microbial Sensitivity Tests - methods; Models, Molecular; Molecular Sequence Data; Peptide Fragments - analysis; Peptide Fragments - pharmacology; screening; Sequence Homology, Amino Acid; Virus Internalization - drug effects; HIV-1; Förster resonance energy transfer; screening; fusion inhibitor; gp41
• ISSN: 2472-5552; 1087-0571; 2472-5560
• DOI: 10.1177/1087057107304645

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.