A human cell-free translation screen identifies the NT-2 mycotoxin as a ribosomal peptidyl transferase inhibitor

Summary Translation inhibitors are invaluable for probing ribosome function and therapeutic applications, but systematic discovery in human systems is limited by the lack of scalable, screening-compatible cell-free platforms. 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 novel translation inhibitors, including NT-2, a trichothecene mycotoxin produced by the pathogenic Fusarium sporotrichioides . NT-2 suppressed protein synthesis in human cells and yeast lysates, while sparing translation in bacteria and intact yeast cells. Cryo-EM at 1.72 Å revealed NT-2 bound at the peptidyl transferase center of the human 60S ribosome, confirming NT-2 as a eukaryote-specific elongation inhibitor that engages ribosomes in a dormant, SERBP1-bound 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.