A time-resolved fluorescence assay to identify small-molecule inhibitors of HIV-1 fusion
| Autor: | Emmanuel Gustin, Asa Ohagen, Lieve Elisabeth Louis Bunkens, Inge Vereycken, Reginald Clayton, Liesbet Smeulders, Koen Van Acker, Kurt Hertogs, Géry Dams, Pascale Holemans |
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| Rok vydání: | 2007 |
| Předmět: |
Models
Molecular viruses Molecular Sequence Data Microbial Sensitivity Tests Biology Gp41 Biochemistry Antiviral Agents Binding Competitive Analytical Chemistry Viral entry Fluorescence Resonance Energy Transfer Amino Acid Sequence Allophycocyanin Quenching (fluorescence) Sequence Homology Amino Acid Lipid bilayer fusion Virus Internalization Small molecule HIV Envelope Protein gp41 Peptide Fragments Förster resonance energy transfer Membrane HIV-1 Molecular Medicine Biotechnology |
| Zdroj: | Journal of biomolecular screening. 12(6) |
| ISSN: | 1087-0571 |
| Popis: | 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 (Eu 3+ ). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Forster 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 timeresolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions. (Journal of Biomolecular Screening XXXX:xx-xx) |
| Databáze: | OpenAIRE |
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