Bacterial ribosome is a preferred target for both natural and clinical antibiotics. However, treatment of bacterial infections is becoming problematic due to the development of drug resistance that severely impairs the efficacy of currently available antibiotics. Most of known translation inhibitors act upon very few sites in th ribosome and these sites are subject to chemical modification by specific pre-existing natural resistance enzymes circulating within microbial communities. Identification of compounds interfering with translation by binding to new ribosomal sites will lead to development of novel antibiotics active against the resistant strains. We designed an experimental strategy to identify compounds that inhibit translation upon binding to four principally new target sites in the ribosome. Pairs of E. coli strains were prepared such that they express either the normal bacterial ribosome or a hybrid ribosome in which the site of interest was mutated to mimic the counterpart sequence in the human cytoplasmic ribosome. Each strain expresses a different fluorescent protein that allows for monitoring each type of cells in a mixed culture. Chemical libraries will be screened for compounds that preferentially inhibit growth of the wild-type cells while allowing growth of cells carrying "humanized" ribosome. The primary hits will be validated in dose-dependent bacterial growth inhibition assays, and the site of action will be verified with combination of in vitro translation experiments and chemical RNA footprinting. Because of the nature of the whole-cell screening protocol, the identified compounds should be able to inhibit bacterial growth by selectively inhibiting bacterial translation via binding to the predefined ribosomal sites.
Bacterial resistance to antibiotics is in rapid expansion and is causing a severe threat to public health. The rate of bacteria acquiring resistance is much faster than the rate at which new antibiotics are discovered. We devised a strategy to identify compounds that target new binding sites on the bacterial ribosome, thereby avoiding cross-resistance with other antibiotics that inhibit protein synthesis.
|Orelle, Cedric; Carlson, Skylar; Kaushal, Bindiya et al. (2013) Tools for characterizing bacterial protein synthesis inhibitors. Antimicrob Agents Chemother 57:5994-6004|
|Orelle, Cedric; Szal, Teresa; Klepacki, Dorota et al. (2013) Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition. Nucleic Acids Res 41:e144|