A Cell-Based Screening Assay for rRNA-Targeted Drug Discovery

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.