Inhibitors of difficult protein-protein interactions identified by high-throughput screening of multiprotein complexes

ACS Chem Biol. 2013 Sep 20;8(9):1988-1997. doi: 10.1021/cb400356m. Epub 2013 Jul 9.

Abstract

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Drug Discovery
  • Drug Evaluation, Preclinical / methods
  • Escherichia coli / drug effects*
  • Escherichia coli / metabolism
  • Escherichia coli Infections / drug therapy
  • Escherichia coli Proteins / antagonists & inhibitors*
  • Escherichia coli Proteins / metabolism*
  • HSP40 Heat-Shock Proteins / antagonists & inhibitors
  • HSP40 Heat-Shock Proteins / metabolism*
  • HSP70 Heat-Shock Proteins / antagonists & inhibitors
  • HSP70 Heat-Shock Proteins / metabolism*
  • Heat-Shock Proteins / antagonists & inhibitors
  • Heat-Shock Proteins / metabolism*
  • High-Throughput Screening Assays / methods
  • Humans
  • Models, Molecular
  • Protein Interaction Maps / drug effects*

Substances

  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • GrpE protein, E coli
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • dnaK protein, E coli