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Researchers developed a novel fluorescent assay to rapidly screen for new antibiotics targeting bacterial RNA. This high-throughput method effectively measures aminoglycoside binding affinity in living cells, aiding drug discovery efforts against antibiotic resistance.

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Area of Science:

  • Microbiology
  • Pharmacology
  • Biochemistry

Background:

  • Bacterial antibiotic resistance is a growing global health threat, necessitating new drug development strategies.
  • Current drug discovery methods face challenges in rapidly screening novel antimicrobial compounds in living cells.
  • Fluorescent probes offer a promising avenue for visualizing and quantifying drug-target interactions within microorganisms.

Purpose of the Study:

  • To develop and optimize a cell-based fluorescent assay for rapid screening of aminoglycoside antibiotic candidates.
  • To characterize aminoglycoside binding affinities to bacterial ribosomal RNA (rRNA) in living cells.
  • To evaluate the assay's performance for both planktonic bacteria and biofilms.

Main Methods:

  • A fluorescent aminoglycoside probe was designed to accumulate in *Escherichia coli* cells.
  • Competitive displacement of the fluorescent probe by potential drug candidates was measured via fluorescence intensity.
  • Assay optimization focused on signal-to-background ratio, reliability, and high-throughput screening compatibility.
  • The assay's sensitivity and performance were assessed using various known aminoglycosides and against planktonic cells and biofilms.

Main Results:

  • The fluorescent probe's accumulation and displacement correlated with aminoglycoside binding affinity to the 16S rRNA A-site.
  • A clear hierarchy of binding affinities was established for tested aminoglycosides, with neomycin showing the highest affinity.
  • The assay demonstrated high sensitivity and excellent performance (average Z' factor of 0.80 for planktonic cells, 0.61 for biofilms).

Conclusions:

  • The developed cell-based fluorescent assay enables rapid, direct characterization of aminoglycoside binding in living bacterial cells.
  • This assay holds significant potential for high-throughput screening and identification of novel ribosomal binding antibiotics.
  • The assay's effectiveness in both planktonic and biofilm states highlights its utility in diverse physiological contexts for combating antibiotic resistance.