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bioRxiv · 10.1101/2023.06.09.544387

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA

Marathe, N.·Nguyen, H. A.·Alumasa, J. N.·Kuzmishin Nagy, A. B.·Vazquez, M. Y.·Dunham, C. M.·Keiler, K. C.
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Abstract

Trans-Translation is conserved throughout bacteria and is essential in many species. High-throughput screening identified a tetrazole-based trans-translation inhibitor, KKL-55, that has broad-spectrum antibiotic activity. A biotinylated version of KKL-55 pulled down Elongation Factor Thermo-unstable (EF-Tu) from bacterial lysates. Purified EF-Tu bound KKL-55 in vitro with a Kd = 2 {micro}M, confirming a high-affinity interaction. An X-ray crystal structure showed KKL-55 binds in domain 3 of EF-Tu, and mutation of residues in the binding pocket abolished KKL-55 binding. RNA binding assays in vitro showed that KKL-55 inhibits binding between EF-Tu and tmRNA, but not between EF-Tu and tRNA. These data demonstrate a new mechanism for inhibition of EF-Tu function and suggest that this specific inhibition of EF-Tu*tmRNA binding is a viable target for antibiotic development. IMPORTANCEEF-Tu is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers tmRNA-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

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Marathe, N., Nguyen, H. A., Alumasa, J. N., Kuzmishin Nagy, A. B., Vazquez, M. Y., Dunham, C. M., Keiler, K. C.. 2023-06-09. Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA. https://doi.org/10.1101/2023.06.09.544387

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