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Here, we establish a robust high-throughput screening using human lysates that bypasses cellular cytotoxic effects. After screening ~28,000 small molecules, we identified known and a novel translation inhibitor, including NT-2, a trichothecene mycotoxin produced by the pathogenic\n                    <jats:italic>Fusarium sporotrichioides<\/jats:italic>\n                    . NT-2 suppressed protein synthesis in human cells and yeast lysates, while sparing translation in bacteria and intact yeast cells. Cryo-EM at 1.76\u2009\u00c5 revealed NT-2 bound at the peptidyl transferase center of the human 60S ribosome. In addition, cryoEM classification of NT-2 treated cells shows ribosomes in an inactive eEF2\/SERBP1-bound dormant state. Together, these results expose NT-2 as a previously unrecognized environmental inhibitor of mammalian protein synthesis and demonstrate the power of cell-free translation screening to reveal new inhibitors with unexpected ribosome fates.\n                  <\/jats:p>","DOI":"10.1038\/s42003-026-10743-6","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T09:02:44Z","timestamp":1790154164000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A human cell-free translation screen identifies the NT-2 mycotoxin as a ribosomal inhibitor that binds the peptidyl transferase center"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-6044-4810","authenticated-orcid":false,"given":"Nino","family":"Schwaller","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominic","family":"Andenmatten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonas","family":"Luginb\u00fchl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julius","family":"Rabl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-1177-3339","authenticated-orcid":false,"given":"Helena","family":"Baur","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Chambon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0934-2608","authenticated-orcid":false,"given":"Jonathan","family":"Vesin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0139-223X","authenticated-orcid":false,"given":"Gerardo","family":"Turcatti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evangelos D.","family":"Karousis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"key":"10743_CR1","doi-asserted-by":"publisher","first-page":"e3001263","DOI":"10.1371\/journal.pbio.3001263","volume":"19","author":"A Corman","year":"2021","unstructured":"Corman, A. et al. A chemical screen for modulators of mRNA translation identifies a distinct mechanism of toxicity for sphingosine kinase inhibitors. PLoS Biol. 19, e3001263 (2021).","journal-title":"PLoS Biol."},{"key":"10743_CR2","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1038\/nchembio.304","volume":"6","author":"T Schneider-Poetsch","year":"2010","unstructured":"Schneider-Poetsch, T. et al. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat. Chem. Biol. 6, 209\u2013217 (2010).","journal-title":"Nat. Chem. Biol."},{"key":"10743_CR3","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-56151-y","volume":"16","author":"E Molina Molina","year":"2025","unstructured":"Molina Molina, E. et al. Targeting eEF1A reprograms translation and uncovers broad-spectrum antivirals against cap or m6A protein synthesis routes. Nat. Commun. 16, 1087 (2025).","journal-title":"Nat. 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