Goldstein, Stanley I.Fan, Alice C.Wang, ZihaoNaineni, Sai K.Lengqvist, JohanChernobrovkin, AlexeyGarcia-Gutierrez, Steve B.Cencic, ReginaPatel, KeshaHuang, SidongBrown, Lauren E.Emili, AndrewPorco, John A.2024-06-062024-06-062024-04-19S.I. Goldstein, A.C. Fan, Z. Wang, S.K. Naineni, J. Lengqvist, A. Chernobrovkin, S.B. Garcia-Gutierrez, R. Cencic, K. Patel, S. Huang, L.E. Brown, A. Emili, J.A. Porco. 2024. "Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)." bioRxiv. https://doi.org/10.1101/2024.04.19.590252https://hdl.handle.net/2144/48987Uncompetitive 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 the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of 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) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (eFT226), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.enThe copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.Article2024-06-0410.1101/2024.04.19.5902520000-0003-2271-7267 (Goldstein, Stanley I)916353