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Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
Published on: April 9, 2014
Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface
Manuel Tsiang1, Gregg S Jones, Magdeleine Hung
1Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States. MTsiang@gilead.com
Biochemistry
|January 13, 2011
Summary
A new assay detects HIV-1 integrase (IN) dimers. Surprisingly, dithiothreitol (DTT) weakens IN dimerization, while LEDGF peptides and small molecules stabilize IN dimers, impacting HIV-1 replication.
Area of Science:
- Biochemistry
- Virology
- Molecular Biology
Background:
- HIV-1 integrase (IN) is a critical enzyme for viral replication.
- Understanding IN dimerization is essential for developing antiviral strategies.
- The interaction between IN and its cofactor LEDGF is a key target for drug development.
Purpose of the Study:
- To develop a homogeneous time-resolved FRET-based assay for detecting HIV-1 IN dimers.
- To investigate the effect of dithiothreitol (DTT) on IN dimerization.
- To evaluate the impact of LEDGF-derived peptides and small molecules on IN dimerization.
Main Methods:
- Homogeneous time-resolved fluorescence resonance energy transfer (FRET) assay.
- Utilized epitope-tagged IN monomers coupled to distinct fluorophores.
- Studied IN mutants and peptides derived from LEDGF.
- Mathematical modeling of binding kinetics.
Main Results:
- Dithiothreitol (DTT), an essential reducing agent, unexpectedly weakened IN monomer interaction.
- Threitol, lacking thiol groups, did not affect IN dimer formation, indicating DTT's thiol groups are responsible.
- LEDGF-derived peptides and a small molecule inhibitor of LEDGF binding promoted IN dimerization and blocked subunit exchange.
- Cysteine residues in IN were found to be dispensable for DTT's dimer dissociation effect.
Conclusions:
- A novel FRET assay effectively detects HIV-1 IN dimerization.
- DTT's thiol groups destabilize IN dimers, contrary to its role in enzymatic activity.
- Peptide and small molecule inhibitors targeting the IN-LEDGF interaction stabilize IN dimers, suggesting a new therapeutic avenue.
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