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

Worldwide, bacterial antibiotic resistance continues to outpace the level of drug development. One way to counteract this threat to society is to identify novel ways to rapidly screen and identify drug candidates in living cells. Developing fluorescent antibiotics that can enter microorganisms and be displaced by potential antimicrobial compounds is an important but challenging endeavor due to the difficulty in entering bacterial cells. We developed a cell-based assay using a fluorescent aminoglycoside molecule that allows for the rapid and direct characterization of aminoglycoside binding in a population of bacterial cells. The assay involves the accumulation and competitive displacement of a fluorescent aminoglycoside binding probe in Escherichia coli as a Gram-negative bacterial model. The assay was optimized for high signal-to-background ratios, ease of performance for reliable outcomes, and amenability to high-throughput screening. We demonstrate that the fluorescent binding probe shows a decrease in fluorescence with cellular uptake, consistent with RNA binding, and also shows a subsequent increase upon the addition of the positive control neomycin. Fluorescence intensity increase with aminoglycosides was indicative of their relative binding affinities for A-site rRNA, with neomycin having the highest affinity, followed by paromomycin, tobramycin, sisomicin, and netilmicin. Intermediate fluorescence was found with plazomicin, neamine, apramycin, ribostamicin, gentamicin, and amikacin. Weak fluorescence was observed with kanamycin, hygromycin, streptomycin, and spectinomycin. A high degree of sensitivity was observed with aminoglycosides known to be strong binders for the 16S rRNA A-site compared with antibiotics that target other biosynthetic pathways. The quality of the optimized assay was excellent for planktonic cells, with an average Z' factor value of 0.80. In contrast to planktonic cells, established biofilms yielded an average Z' factor of 0.61. The high sensitivity of this cell-based assay in a physiological context demonstrates significant potential for identifying potent new ribosomal binding antibiotics.