{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:07:36Z","timestamp":1785370056499,"version":"3.55.0"},"reference-count":43,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,27]],"date-time":"2023-01-27T00:00:00Z","timestamp":1674777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000897","name":"PTC Therapeutics","doi-asserted-by":"publisher","award":["COOPER21G0"],"award-info":[{"award-number":["COOPER21G0"]}],"id":[{"id":"10.13039\/100000897","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000897","name":"PTC Therapeutics","doi-asserted-by":"publisher","award":["NIH GM127374"],"award-info":[{"award-number":["NIH GM127374"]}],"id":[{"id":"10.13039\/100000897","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Cystic Fibrosis Foundation","award":["COOPER21G0"],"award-info":[{"award-number":["COOPER21G0"]}]},{"name":"Cystic Fibrosis Foundation","award":["NIH GM127374"],"award-info":[{"award-number":["NIH GM127374"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren\u2019s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren\u2019s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren\u2019s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.<\/jats:p>","DOI":"10.3390\/biom13020242","type":"journal-article","created":{"date-parts":[[2023,1,30]],"date-time":"2023-01-30T04:31:27Z","timestamp":1675053087000},"page":"242","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy\u2014Application to Nonsense Suppressor Screening and Mechanistic Studies"],"prefix":"10.3390","volume":"13","author":[{"given":"Mikel D.","family":"Ghelfi","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3744-003X","authenticated-orcid":false,"given":"Saleem Y.","family":"Bhat","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6989-9788","authenticated-orcid":false,"given":"Barry S.","family":"Cooperman","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1038\/206994a0","article-title":"Genetic code: The \u2018nonsense\u2019triplets for chain termination and their suppression","volume":"206","author":"Brenner","year":"1965","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.1039\/C4MD00081A","article-title":"When proteins start to make sense: Fine-tuning of aminoglycosides for PTC suppression therapy","volume":"5","author":"Shalev","year":"2014","journal-title":"MedChemComm"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1146\/annurev-genom-091212-153527","article-title":"Therapeutics based on stop codon readthrough","volume":"15","author":"Keeling","year":"2014","journal-title":"Annu. Rev. Genom. Hum. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/bs.apcsb.2022.06.001","article-title":"Translation termination codons in protein synthesis and disease","volume":"132","author":"Lombardi","year":"2022","journal-title":"Adv. Protein Chem. Struct. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1002\/ajh.21539","article-title":"Production of \u03b2-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous \u03b2039 thalassemia patients","volume":"84","author":"Salvatori","year":"2009","journal-title":"Am. J. Hematol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.1002\/emmm.201201438","article-title":"A comparative evaluation of NB30, NB54 and PTC124 in translational read-through efficacy for treatment of an USH1C nonsense mutation","volume":"4","author":"Goldmann","year":"2012","journal-title":"EMBO Mol. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1007\/s00439-017-1779-6","article-title":"The Human Gene Mutation Database: Towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies","volume":"136","author":"Stenson","year":"2017","journal-title":"Hum. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1002\/humu.20763","article-title":"A meta-analysis of nonsense mutations causing human genetic disease","volume":"29","author":"Mort","year":"2008","journal-title":"Hum. Mutat."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4677","DOI":"10.1007\/s00018-021-03809-7","article-title":"Nonsense suppression therapies in human genetic diseases","volume":"78","year":"2021","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e13429","DOI":"10.7554\/eLife.13429","article-title":"Kinetics of initiating polypeptide elongation in an IRES-dependent system","volume":"5","author":"Zhang","year":"2016","journal-title":"Elife"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1285","DOI":"10.1021\/acsmedchemlett.8b00472","article-title":"New in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery","volume":"9","author":"Ng","year":"2018","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e2020599118","DOI":"10.1073\/pnas.2020599118","article-title":"Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms","volume":"118","author":"Ng","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","first-page":"1","article-title":"Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination","volume":"13","author":"Huang","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1038\/nature05756","article-title":"PTC124 targets genetic disorders caused by nonsense mutations","volume":"447","author":"Welch","year":"2007","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1146\/annurev-med-120611-144851","article-title":"Ataluren as an agent for therapeutic nonsense suppression","volume":"64","author":"Peltz","year":"2013","journal-title":"Annu. Rev. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1016\/j.omtn.2020.04.012","article-title":"Mutation-directed therapeutics for neurofibromatosis type I","volume":"5","author":"Leier","year":"2020","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e1763","DOI":"10.1038\/cddis.2015.108","article-title":"XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death","volume":"6","author":"Oishi","year":"2015","journal-title":"Cell Death Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12353","DOI":"10.1073\/pnas.1615548113","article-title":"Proposing a mechanism of action for ataluren","volume":"113","author":"Siddiqui","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1080\/15476286.2017.1285480","article-title":"Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells","volume":"14","author":"Bidou","year":"2017","journal-title":"RNA Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"127218","DOI":"10.1016\/j.bmcl.2020.127218","article-title":"The relationship between the structure and toxicity of aminoglycoside antibiotics","volume":"30","author":"Siomin","year":"2020","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e52611","DOI":"10.7554\/eLife.52611","article-title":"Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides","volume":"9","author":"Wangen","year":"2020","journal-title":"Elife"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1124\/jpet.120.265595","article-title":"ELX-02 generates protein via premature stop codon read-through without inducing native stop codon read-through proteins","volume":"374","author":"Crawford","year":"2020","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Michorowska, S. (2021). Ataluren\u2014Promising Therapeutic Premature Termination Codon Readthrough Frontrunner. Pharmaceuticals, 14.","DOI":"10.3390\/ph14080785"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4358","DOI":"10.1038\/s41467-021-24575-x","article-title":"A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion","volume":"12","author":"Sharma","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3692","DOI":"10.1093\/nar\/gkab194","article-title":"Effect of small molecule eRF3 degraders on premature termination codon readthrough","volume":"49","author":"Balgi","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e154571","DOI":"10.1172\/JCI154571","article-title":"Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough","volume":"132","author":"Lee","year":"2022","journal-title":"J. Clin. Investig."},{"key":"ref_27","unstructured":"Gasteiger, E., Hoogland, C., Gattiker, A., Wilkins, M.R., Appel, R.D., and Bairoch, A. (2005). The Proteomics Protocols Handbook, Humana Press."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"12508","DOI":"10.1073\/pnas.1605336113","article-title":"Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression","volume":"113","author":"Roy","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1111\/j.1751-1097.1991.tb02006.x","article-title":"Sites and efficacy of photodamage by tin etiopurpurin in vitro using different delivery systems","volume":"54","author":"Kessel","year":"1991","journal-title":"Photochem. Photobiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e2122004119","DOI":"10.1073\/pnas.2122004119","article-title":"2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases","volume":"119","author":"Bidou","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"128989","DOI":"10.1016\/j.bmcl.2022.128989","article-title":"Guanidino quinazolines and pyrimidines promote readthrough of premature termination codons in cells with native nonsense mutations","volume":"76","author":"Morrill","year":"2022","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1080\/15476286.2015.1068497","article-title":"Translational readthrough potential of natural termination codons in eucaryotes--The impact of RNA sequence","volume":"12","author":"Dabrowski","year":"2015","journal-title":"RNA Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1927","DOI":"10.1093\/nar\/gkx1315","article-title":"Eukaryotic translational termination efficiency is influenced by the 3\u2032 nucleotides within the ribosomal mRNA channel","volume":"46","author":"Cridge","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1615","DOI":"10.1042\/BST20180190","article-title":"\u2018Stop\u2019in protein synthesis is modulated with exquisite subtlety by an extended RNA translation signal","volume":"46","author":"Tate","year":"2018","journal-title":"Biochem. Soc. Trans."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1111\/brv.12657","article-title":"Deciphering the molecular mechanism of stop codon readthrough","volume":"96","author":"Palma","year":"2021","journal-title":"Biol. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"102133","DOI":"10.1016\/j.jbc.2022.102133","article-title":"Recognition of 3\u2032 nucleotide context and stop codon readthrough are determined during mRNA translation elongation","volume":"298","author":"Biziaev","year":"2022","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Balic, Z., and Hubmacher, D. (2022). Promoting translational readthrough to augment fibrillin-1 (FBN1) deposition in Marfan syndrome fibroblasts: A proof-of-concept study. bioRxiv.","DOI":"10.1101\/2022.11.23.517642"},{"key":"ref_38","unstructured":"(2022, May 15). Welcome to the CFTR2. Available online: https:\/\/cftr2.org\/mutations_history."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Lashkevich, K.A., Shlyk, V.I., Kushchenko, A.S., Gladyshev, V.N., Alkalaeva, E.Z., and Dmitriev, S.E. (2020). CTELS: A cell-free system for the analysis of translation termination rate. Biomolecules, 10.","DOI":"10.3390\/biom10060911"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2044","DOI":"10.1261\/rna.076588.120","article-title":"Termi-Luc: A versatile assay to monitor full-protein release from ribosomes","volume":"26","author":"Susorov","year":"2020","journal-title":"RNA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1285","DOI":"10.1261\/rna.061051.117","article-title":"Avoidance of reporter assay distortions from fused dual reporters","volume":"23","author":"Loughran","year":"2017","journal-title":"RNA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1016\/j.omtn.2022.04.033","article-title":"Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs","volume":"28","author":"Ko","year":"2022","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-019-08329-4","article-title":"Engineered transfer RNAs for suppression of premature termination codons","volume":"10","author":"Lueck","year":"2019","journal-title":"Nat. Commun."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/2\/242\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:16:59Z","timestamp":1760120219000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/2\/242"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,27]]},"references-count":43,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["biom13020242"],"URL":"https:\/\/doi.org\/10.3390\/biom13020242","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,27]]}}}