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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
PubMed

Insights

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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