A Cell-Based\nScreening Assay for rRNA-Targeted Drug\nDiscovery

Worldwide, bacterial antibiotic resistance continues\nto outpace\nthe level of drug development. One way to counteract this threat to\nsociety is to identify novel ways to rapidly screen and identify drug\ncandidates in living cells. Developing fluorescent antibiotics that\ncan enter microorganisms and be displaced by potential antimicrobial\ncompounds is an important but challenging endeavor due to the difficulty\nin entering bacterial cells. We developed a cell-based assay using\na fluorescent aminoglycoside molecule that allows for the rapid and\ndirect characterization of aminoglycoside binding in a population\nof bacterial cells. The assay involves the accumulation and competitive\ndisplacement of a fluorescent aminoglycoside binding probe in Escherichia coli as a Gram-negative bacterial model.\nThe assay was optimized for high signal-to-background ratios, ease\nof performance for reliable outcomes, and amenability to high-throughput\nscreening. We demonstrate that the fluorescent binding probe shows\na decrease in fluorescence with cellular uptake, consistent with RNA\nbinding, and also shows a subsequent increase upon the addition of\nthe positive control neomycin. Fluorescence intensity increase with\naminoglycosides was indicative of their relative binding affinities\nfor A-site rRNA, with neomycin having the highest affinity, followed\nby paromomycin, tobramycin, sisomicin, and netilmicin. Intermediate\nfluorescence was found with plazomicin, neamine, apramycin, ribostamicin,\ngentamicin, and amikacin. Weak fluorescence was observed with kanamycin,\nhygromycin, streptomycin, and spectinomycin. A high degree of sensitivity\nwas observed with aminoglycosides known to be strong binders for the\n16S rRNA A-site compared with antibiotics that target other biosynthetic\npathways. The quality of the optimized assay was excellent for planktonic\ncells, with an average <i>Z</i>′ factor value of\n0.80. In contrast to planktonic cells, established biofilms yielded\nan average <i>Z</i>′ factor of 0.61. The high sensitivity\nof this cell-based assay in a physiological context demonstrates significant\npotential for identifying potent new ribosomal binding antibiotics.