PXD058802

PXD058802 is an original dataset announced via ProteomeXchange.

Title	Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays
Description	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 proteome-wide discovery of substrate-dependent ligand binding. Using PISA and LiP-MS 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 used a target class-specific assay parameters 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.
HostingRepository	PRIDE
AnnounceDate	2025-05-07
AnnouncementXML	Submission_2025-05-07_06:46:10.952.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Stanley Goldstein
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2024-12-11 23:00:26	ID requested
⏵ 1	2025-05-07 06:46:11	announced

10.1021/acs.jproteome.4c01129;

Goldstein SI, Fan AC, Wang Z, Naineni SK, Cencic R, Garcia-Gutierrez SB, Patel K, Huang S, Brown LE, Emili A, Porco JA, Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays. J Proteome Res, 24(4):2026-2039(2025) [pubmed]

submitter keyword: LiP-MS, CETSA,TMTpro, LCMSMS, PISA

John A. Porco, Jr., PhD
contact affiliation	Department of Chemistry, Boston University, USA
contact email	porco@bu.edu
lab head
Stanley Goldstein
contact affiliation	Boston University
contact email	stangold@bu.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD058802
     1. Label: PRIDE project
     2. Name: Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays