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Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes
journal contribution
posted on 2013-09-20, 00:00 authored by Laura
C. Cesa, Srikanth Patury, Tomoko Komiyama, Atta Ahmad, Erik R. P. Zuiderweg, Jason E. GestwickiProtein–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.