{"doi":"10.3390/ijms22020594","title":"Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D","abstract":"<jats:p>N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.</jats:p>","journal":"International Journal of Molecular Sciences","year":2021,"id":14950,"datarank":0.674615780558639,"base_score":2.8903717578961645,"endowment":2.8903717578961645,"self_citation_contribution":0.4335557636844247,"citation_network_contribution":0.24106001687421436,"self_endowment_contribution":0.4335557636844247,"citer_contribution":0.24106001687421436,"corpus_percentile":null,"corpus_rank":null,"citation_count":17,"citer_count":12,"citers_with_citation_signal":9,"citers_with_endowment":9,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":114877,"name":"Lan Chen","orcid":null,"position":1,"is_corresponding":false},{"id":114878,"name":"Rong Huang","orcid":"0000-0002-1477-3165","position":2,"is_corresponding":false},{"id":114876,"name":"Yi-Hsun Ho","orcid":"0000-0002-8592-8347","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"base_score":2.8903717578961645,"endowment":2.8903717578961645,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"33435607","pmcid":"PMC7827481","openalex_id":"https://openalex.org/W3120143791","authors":[],"funders":[{"funder_name":"Purdue University","grant_id":"Institute for Drug Discovery","title":null}],"total_grants":1,"fwci":0.994,"citation_percentile":0.78053153,"influential_citations":0,"citation_trend":[{"year":2021,"count":3},{"year":2022,"count":2},{"year":2023,"count":3},{"year":2024,"count":3},{"year":2025,"count":6}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://www.mdpi.com/1422-0067/22/2/594/pdf?version=1610358923","host_type":"journal"},{"url":"https://www.biorxiv.org/content/biorxiv/early/2020/12/24/2020.12.24.424328.full.pdf","host_type":"GREEN"},{"url":"https://www.mdpi.com/1422-0067/22/2/594/pdf?version=1610358923","host_type":"publisher"},{"url":"https://www.mdpi.com/1422-0067/22/2/594/pdf","host_type":"publisher"},{"url":"https://doi.org/10.3390/ijms22020594","host_type":"journal"},{"url":"https://doaj.org/article/084f7621e9d64b13abceaaa70211b56e","host_type":"repository"},{"url":"http://europepmc.org/pmc/articles/PMC7827481","host_type":"repository"},{"url":"https://dx.doi.org/10.3390/ijms22020594","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/7827481","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC7827481","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC7827481?pdf=render","host_type":"Europe_PMC"}],"fields_of_study":["Peptidase Inhibition and Analysis","Neuropeptides and Animal Physiology","Chemical Synthesis and Analysis","Medicine","Biology","Chemistry","Acetylation","Enzyme Assays","Fluorescence","Fluorescent Dyes","High-Throughput Screening Assays","Histones","Humans","N-Terminal Acetyltransferase D","Pilot Projects","Reproducibility of Results"],"mesh_terms":["Humans","Histones","Fluorescent Dyes","Pilot Projects","Reproducibility of Results","Acetylation","Fluorescence","Enzyme Assays","High-Throughput Screening Assays","N-Terminal Acetyltransferase D"],"keywords":["Acetyltransferases","Acetyltransferase","Acetylation","Histone acetyltransferase","Histone Acetyltransferases","Fluorescence","High-throughput screening","Nat","Biochemistry","Chemistry","Small molecule","Adduct","Arylamine N-acetyltransferase","Molecular biology","Biology","Gene","Fluorescence assay","Acetyltransferase Inhibitors","N-terminal Acetyltransferase D"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Good health and well-being"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-06-01T15:42:46.188697Z","pmid":null,"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":[]}