Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

Goldstein, S. I.; Fan, A. C.; Wang, Z.; Naineni, S. K.; Lengqvist, J.; Chernobrovkin, A.; Garcia-Gutierrez, S. B.; Cencic, R.; Patel, K.; Huang, S.; Brown, L. E.; Emili, A.; Porco, J. A.

doi:10.1101/2024.04.19.590252

bioRxiv · 10.1101/2024.04.19.590252

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

Goldstein, S. I.·Fan, A. C.·Wang, Z.·Naineni, S. K.·Lengqvist, J.·Chernobrovkin, A.·Garcia-Gutierrez, S. B.·Cencic, R.·Patel, K.·Huang, S.·Brown, L. E.·Emili, A.·Porco, J. A.

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Abstract

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 and validate novel DDX targets of high-profile rocaglates - including the clinical candidate Zotatifin - 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 towards characterization of novel molecular clamps and associated RNA helicases.

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Goldstein, S. I., Fan, A. C., Wang, Z., Naineni, S. K., Lengqvist, J., Chernobrovkin, A., Garcia-Gutierrez, S. B., Cencic, R., Patel, K., Huang, S., Brown, L. E., Emili, A., Porco, J. A.. 2024-04-19. Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR). https://doi.org/10.1101/2024.04.19.590252

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