{"doi":"10.1016/j.aspetd.2025.100003","title":"Evaluation of molecular glue–induced neomorphic protein-protein interaction with a cell lysate–based time-resolved fluorescence resonance energy transfer assay","abstract":"Neomorphic protein-protein interactions (neoPPIs) induced by mutated amino acid residues present a promising class of therapeutic targets for proximity pharmacology-enabled drug discovery. Small molecule compounds, such as cyclosporin and FK506, have the capability to mimic the mutational effect to create new interaction interfaces to enhance the interaction of a protein with an unrelated or low-affinity protein. Such small molecules can function as molecular glues to purposefully generate neoPPIs for physiological reprogramming and therapeutic intervention. Thus, an unbiased approach to the systematic discovery of neoPPI-inducing compounds is much needed to accelerate the proximity pharmacology field. With the demonstrated small molecule–induced neoPPI of oncogenic KRAS with cyclophilin A (CYPA), here, we report the development of a robust cell lysate–based time-resolved fluorescence resonance energy transfer assay to characterize KRAS-CYPA neoPPI-inducing molecular glues. We show that the developed time-resolved fluorescence resonance energy transfer assay can monitor the induced interaction of CYPA with oncogenic KRAS variants, KRAS-G12C, G12D, and G12V. The assay was optimized to achieve a robust performance in a miniaturized format, which was utilized to demonstrate the potent effect of RMC-7977, a known molecular glue, in inducing the KRAS-CYPA interaction. This validated assay platform allows for the characterization of molecular glue–induced KRAS-CYPA neoPPI, which is readily applicable to explore the vast chemical space for the discovery of novel KRAS-CYPA neoPPI inducers and can be generally applicable for unbiased discovery of proximity-inducing molecular glues. Molecular glues offer a promising strategy to target “undruggable” proteins by inducing neomorphic protein-protein interactions. This study develops a high-throughput, cell lysate–based time-resolved fluorescence resonance energy transfer assay to detect molecular glue–induced KRAS–cyclophilin A neomorphic protein-protein interaction in a physiologically relevant environment, enabling the discovery of small molecules that could expand therapeutic options for KRAS-driven cancers.","journal":"ASPET Discovery","year":2025,"id":525383,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9556,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":273149,"name":"Yuhong Du","orcid":null,"position":1,"is_corresponding":false},{"id":254877,"name":"Haian Fu","orcid":"0000-0002-2362-7979","position":2,"is_corresponding":false},{"id":271941,"name":"Xiulei Mo","orcid":"0000-0002-7558-3453","position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":null,"created_at":"2026-07-19T02:50:21.274116Z","pmid":"41918840","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":[]}