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

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 12, 2025

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

Language: Английский

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 19, 2024

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies substrate-dependent engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP RNA (TSAR) as a template proteome-wide ligand binding. Using thermal shift assays, show that simple biochemical additives can facilitate detection native cell lysates. We apply our approach to rocaglates, family molecules specifically clamp eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) helicases. To identify unexpected interactions, optimized class-specific denaturation window evaluated analog probe dependencies key rocaglate-DDX interactions. report novel DDX targets rocaglate clamping spectrum, confirm DDX3X common several widely studied analogs, provide insights into divergent affinities between synthetic rocaglates. independently validate high-profile including clinical candidate Zotatifin (

Language: Английский

Exploring Butein’s anticancer potential in colorectal cancer: Insights into proliferation inhibition, apoptosis induction, and metastasis suppression via p38 signaling

Journal of Functional Foods, Journal Year: 2024, Volume and Issue: 119, P. 106293 - 106293

Published: June 13, 2024

Language: Английский