{"doi":"10.1101/2024.04.19.590252","title":"Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays","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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":488361,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9549,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":395712,"name":"Alice C. Fan","orcid":"0000-0002-4813-5444","position":1,"is_corresponding":false},{"id":1332752,"name":"Zihao Wang","orcid":"0009-0003-7502-3258","position":2,"is_corresponding":false},{"id":681262,"name":"Sai Kiran Naineni","orcid":null,"position":3,"is_corresponding":false},{"id":301116,"name":"Regina Cencic","orcid":"0000-0002-8777-103X","position":4,"is_corresponding":false},{"id":1333066,"name":"Steve B. Garcia-Gutierrez","orcid":null,"position":5,"is_corresponding":false},{"id":1332753,"name":"Kesha Patel","orcid":"0009-0001-5840-9680","position":6,"is_corresponding":false},{"id":791953,"name":"Sidong Huang","orcid":"0000-0002-2838-4726","position":7,"is_corresponding":false},{"id":275156,"name":"Lauren E. Brown","orcid":"0000-0001-9489-484X","position":8,"is_corresponding":false},{"id":253656,"name":"Andrew Emili","orcid":"0000-0001-8995-246X","position":9,"is_corresponding":false},{"id":275161,"name":"John A. Porco","orcid":"0000-0002-2991-5680","position":10,"is_corresponding":false},{"id":817256,"name":"Stanley I. Goldstein","orcid":"0000-0003-2271-7267","position":0,"is_corresponding":true}],"reference_count":94,"raw_metadata":null,"created_at":"2026-07-19T02:08:15.008850Z","pmid":"38659867","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}