Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria

Abstract

Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNA are used to prepare two key components of this assay: (1) Cy5-EF-Tu:GTP and (2) Cy3-Phe-tRNA. When mixed and Cy3 is excited at 532 nm, increased Cy5 fluorescence intensity is observed at 665 nm due to ternary complex formation and FRET. If the same assay is carried out in presence of an inhibitor, such as GE2270A (a known inhibitor of the EF-Tu-tRNA interaction), fluorescence intensity is significantly diminished. To establish proof of principle and to show the adaptability of this assay to high throughput screening (HTS), we analyzed the effect of different classes of antibiotics, including beta-lactams, quinolone compounds, and protein synthesis inhibitors, on fluorescence. The assay was done in a 96-well microplate. We observed inhibition by GE2270A, and no effect of nineteen other tested antibiotics, confirming the ability of this FRET assay to serve as a screen for potential inhibitor molecules of ternary complex formation from libraries of compounds.

Efficiency of protein synthesis inhibition depends on tRNA and codon compositions

“…Our finding, that the efficiency of protein synthesis inhibition mediated by EF-Tu depends on the tRNA/codon composition, hints towards a potential use of EF-Tu targeting drugs for tissue- or pathogen-specific treatments. It thus may encourage further studies for the identification of novel compounds directed against EF-Tu [41].…”

Efficiency of protein synthesis inhibition depends on tRNA and codon compositions

“…Our finding, that the efficiency of protein synthesis inhibition mediated by EF-Tu depends on the tRNA/codon composition, hints towards a potential use of EF-Tu targeting drugs for tissue- or pathogen-specific treatments. It thus may encourage further studies for the identification of novel compounds directed against EF-Tu [38].…”