Summary
While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, “universal” model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this “universal” mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation.
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Bottger, E. C., Springer, B., Prammananan, T., Kidan, Y., and Sander, P. (2001) Structural basis for selectivity and toxicity of ribosomal antibiotics. EMBO Rep. 2, 318–323.
Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz ,T.A. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289,905–920.
Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107, 679–688.
Nissen, P., Hansen, J., Ban, N., Moore, P. B., and Steitz, T.A. (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930.
Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M., Janell, D., Bashan, A., Bartels, H., Agmon, I., Franceschi, F., and Yonath, A. (2000) Structure of functionally activated small ribosomal subunit at 3.3 Angstroms resolution. Cell 102, 615–623.
Schuwirth, B. S., Borovinskaya, M. A., Hau, C. W., Zhang, W., Vila-Sanjurjo, A., Holton, J. M., and Cate, J. H. (2005) Structures of the bacterial ribosome at 3.5 A resolution. Science 310, 827–834.
Wimberly, B. T., Brodersen, D. E., Clemons, W. M. Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339.
Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Earnest,T. N., Cate, J H, and Noller,H F. (2001) Crystal structure of the ribosome at 5.5 Aresolution. Science 292, 883–896.
Poehlsgaard, J., and Douthwaite, S. (2005) The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3, 870–881.
Bottger, E. C. (2006) The ribosome as a drug target. Trends Biotechnol. 24, 145–147.
Harms, J. M., Bartels, H., Schlunzen, F., and Yonath, A. (2003) Antibiotics acting on the translational machinery. J. CellSci. 116, 1391–1393.
Tenson, T., and Mankin, A. (2006) Antibiotics and the ribosome. Mol. Microbiol. 59, 1664–1677.
Brandi, L., Lazzarini, A., Cavaletti, L., Abbondi, M., Corti, E.,Ciciliato, I., Gastaldo, L., Marazzi, A., Feroggio, M., Fabbretti,A., Maio, A., Colombo, L., Donadio, S., Marinelli, F., Losi, F., Gualerzi, C. O., and Selva, E. (2006) Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp. Biochemistry 45, 3692–3702.
Brandi, L., Fabbretti, A., La Teana, A., Abbondi, M., Losi, D., Donadio, S., etal. (2006) Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiatic. Proc Natl Acad Sci USA 103, 39–44.
Brandi, L., Fabbretti, A., Di Stefano, M., Lazzarini, A., Abbondi,M., and Gualerzi, C. O. (2006) Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits. RNA 12, 1262–1270
McCarthy, J. E., and Gualerzi, C. (1990) Translational control of prokaryotic gene expression. Trends Genet. 6, 78–85.
Cigan, A. M., and Donahue, T. F. (1987) Sequence and structural features associated with translational initiator regions in yeast–A review. Gene 59, 1–18.
Iizuka, N., Najita, L., Franzusoff, A., and Sarnow, P. (1994) Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol. Cell Biol. 14, 7322–7330.
Calogero, R. A., Pon, C. L., Canonaco, M. A., and Gualerzi, C. O. (1988) Selection of the mRNA translation initiation region by Escherichia coli ribosomes. Proc. Natl. Acad. Sci. USA 85, 6427–6431.
Carroll, R., and Lucas-Lenard, J. (1993) Preparation of a cell-free translation system with minimal loss of initiation factor eIF-2/eIF-2B activity. Anal. Biochem. 212, 17–23.
Acknowledgments
The authors would like to thank Prof. Fabrizio Loreni forthe kind gift of theHeLa S3 cell line.
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Brandi, L., Dresios, J., Gualerzi, C.O. (2008). Assays for the Identification of Inhibitors Targeting Specific Translational Steps. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_8
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DOI: https://doi.org/10.1007/978-1-59745-246-5_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-915-4
Online ISBN: 978-1-59745-246-5
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