title: A time-resolved fluorescence assay to identify small-molecule inhibitors of HIV-1 fusion.
abstract: 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.
authors.affiliation: Tibotec BVBA, Mechelen, Belgium.
authors.forename: Géry
authors.initials: G
authors.lastname: Dams
authors.forename: Koen
authors.initials: K
authors.lastname: Van Acker
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gustin
authors.forename: Inge
authors.initials: I
authors.lastname: Vereycken
authors.forename: Lieve
authors.initials: L
authors.lastname: Bunkens
authors.forename: Pascale
authors.initials: P
authors.lastname: Holemans
authors.forename: Liesbet
authors.initials: L
authors.lastname: Smeulders
authors.forename: Reginald
authors.initials: R
authors.lastname: Clayton
authors.forename: Asa
authors.initials: A
authors.lastname: Ohagen
authors.forename: Kurt
authors.initials: K
authors.lastname: Hertogs
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000032:analysis* / Q000494:pharmacology*; D001667:Binding, Competitive; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry; D015497:HIV-1 / Q000187:drug effects*; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments