Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays

Uncompetitive
inhibition is an effective strategy for suppressing
dysregulated enzymes and their substrates, but discovery of suitable
ligands depends on often-unavailable structural knowledge and serendipity.
Hence, despite surging interest in mass spectrometry-based target
identification, proteomic studies of substrate-dependent target engagement
remain sparse. Herein, we describe a strategy for the discovery of
substrate-dependent ligand binding. Using proteome integral solubility
alteration (PISA) assays, we show that simple biochemical additives
can enable detection of RNA-protein-small molecule complexes in native
cell lysates. We apply our approach to rocaglates, molecules that
specifically clamp RNA to eukaryotic translation initiation factor
4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members
of the DEAD-box (DDX) helicase family. To identify unexpected interactions,
we used a target class-specific thermal window and compared ATP analog
and RNA base dependencies for key rocaglate-DDX interactions. We report
novel DDX targets of high-profile rocaglates−including the
clinical candidate Zotatifin−and validate our findings using
limited proteolysis-mass spectrometry and fluorescence polarization
(FP) experiments. We also provide structural insight into divergent
DDX3X affinities between synthetic rocaglates. Taken together, our
study provides a model for screening uncompetitive inhibitors using
a chemical proteomics approach and uncovers actionable DDX clamping
targets, clearing a path toward characterization of novel molecular
clamps and associated RNA helicases.