New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Abstract

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

“…We used the competition-binding assay, exploiting BODIPY-labeled erythromycin (BODIPYERY) to assess the affinity of the synthesized compounds for the ribosome [26, 27]. While CLM binds in the A site of the PTC [6, 28], ERY binds in the upper part of the NPET [7] but their binding sites sufficiently overlap so that even unmodified CLM and ERY compete for their binding to the ribosome [29] (Figure S2).…”

“…Binding affinities of CAM-derivatives to E. coli ribosomes was analyzed by competition binding assay using fluorescently-labeled BODIPY-ERY as described before [26, 27, 37]. BODIPY-ERY (4 nM) was incubated with ribosomes (25 nM) for 30 min at 25°C in the buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM NH 4 Cl, 10 mM Mg(CH 3 COO) 2 , and 0.05% Tween-20.…”

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome

“…To assess whether the synthesized CAM-C4-TPP compound exhibits a higher affinity for the bacterial 70S ribosome due to the presence of the hydrophobic and charged TPP tail, we used the competition-binding assay exploiting either BODIPY-labeled CHL (BODIPY-CAM, Figure S1) or erythromycin (BODIPY-ERY) [13,25,26]. The amphenicol moieties of CAM-C4-TPP, its parent CHL, and the fluorescent derivative BODIPY-CAM are expected to have identical ribosome binding sites and, therefore, these compounds are expected to compete with each other for binding to the ribosome.…”

Single-cell microfluidics facilitates the rapid quantification of antibiotic accumulation in Gram-negative bacteria

“…On the drug development side, the assay also has the advantage of needing only micrograms of chemicals for testing, which is important when evaluating novel, candidate drugs that are typically expensive to manufacture. Our readout is based on fluorescence, and can be used to test the cell permeation properties of new fluorescent antibiotic probes that have been developed for a range of antibiotics including penicillins, glycopeptides, polymixins, oxazolidinones, trimethoprim, macrolides and fluoroquinolones; [50][51][52][53][54] similar probes can be developed for newly discovered antimicrobials as well. The use of more sensitive fluorescent probes could also let us potentially correlate cell survival to reduced accumulation in longer-term experiments in the future.…”