{"doi":"10.3390/ijms24119139","title":"A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription","abstract":"Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.","journal":"International Journal of Molecular Sciences","year":2023,"id":371170,"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":6,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9594,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":945051,"name":"Dong‐Eun Kim","orcid":"0000-0001-6545-8387","position":1,"is_corresponding":false},{"id":1131281,"name":"Kyung Lee Yu","orcid":null,"position":2,"is_corresponding":false},{"id":1130806,"name":"Chul Min Park","orcid":"0000-0002-5440-935X","position":3,"is_corresponding":false},{"id":1130807,"name":"Hong Gi Kim","orcid":"0000-0003-4380-3551","position":4,"is_corresponding":false},{"id":505647,"name":"Kyung-Chang Kim","orcid":null,"position":5,"is_corresponding":false},{"id":1130808,"name":"Songmee Bae","orcid":"0000-0002-7423-2360","position":6,"is_corresponding":false},{"id":781390,"name":"Cheol‐Hee Yoon","orcid":"0000-0002-5696-0567","position":7,"is_corresponding":false},{"id":1131280,"name":"Young Hyun Shin","orcid":null,"position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-19T01:15:45.330525Z","pmid":"37298089","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":[]}