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Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening
Richard L Chapman1, Thomas B Stanley, Richard Hazen
1Department of Molecular Screening, GlaxoSmithKline, PO Box 1-3398, Research Triangle Park, NC 27709-3398, USA.
Antiviral Research
|June 14, 2002
Summary
Researchers screened over 500,000 small molecules to find HIV Rev protein-RNA interaction modulators. While several inhibitors and enhancers were identified in vitro, none showed antiviral activity in cell-based assays.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- The human immunodeficiency virus (HIV) Rev protein is essential for viral replication, mediating the export of unspliced viral RNA from the nucleus.
- Modulating the interaction between the Rev protein and its RNA target (Rev response element) is a potential antiviral strategy.
Purpose of the Study:
- To identify small molecules that modulate the protein-RNA interaction of the HIV Rev system using a high-throughput screening approach.
- To characterize the identified modulators and investigate their mechanism of action.
Main Methods:
- A high-throughput scintillation proximity assay was employed to screen over 500,000 small molecules for their effect on the binding of biotinylated HIV Rev protein to tritiated Rev response element RNA.
- Hit validation was performed using gel shift assays and surface plasmon resonance (SPR) analysis.
- Mechanism of inhibition was investigated by comparing binding constants with IC50 values.
Main Results:
- Several chemical classes of small molecule inhibitors and enhancers of the Rev protein-RNA interaction were identified, with molecular weights between 400-600 Da.
- The most common inhibitor motif featured an acidic moiety on a linear aromatic system.
- Many modulators exhibited EC50 values in the low micromolar range, with some below 1 microM, and structure-activity relationships suggested specific molecular interactions.
- In vitro validation confirmed inhibition for several compounds, with one inhibitor demonstrating a mechanism consistent with direct binding to the Rev protein.
Conclusions:
- Small molecules can effectively modulate the essential HIV Rev protein-RNA interaction in vitro.
- Despite promising in vitro activity, none of the identified compounds demonstrated significant HIV antiviral activity in relevant cell-based assays, highlighting the challenges in translating in vitro findings to in vivo efficacy.