Fluorescence Polarization Method To Characterize Macrolide-Ribosome Interactions

Abstract

A fluorescence polarization assay is described that measures the binding of fluorescently labeled erythromycin to 70S ribosomes from Escherichia coli and the displacement of the erythromycin from these ribosomes. The assay has been validated with several macrolide derivatives and other known antibiotics. We demonstrate that this assay is suitable for determining the dissociation constants of novel compounds that have binding sites overlapping those of macrolides. This homogeneous binding assay provides a valuable tool for defining structure-activity relationships among compounds during the discovery and development of new ribosometargeting drugs.Macrolide antibiotics comprise a large group of clinically useful compounds, characterized by having a 14-, 15-, or 16-membered lactone ring with two or more sugar groups attached. Of particular importance are the 14-membered macrolide erythromycin and its newer-generation derivatives clarithromycin, roxithromycin, and azithromycin (a 15-membered macrolide), which are valuable therapeutic agents for the treatment of community-acquired respiratory tract infections. These antibiotics selectively inhibit bacterial protein synthesis by binding reversibly to the ribosome. They have been shown to interact with domain V of 23S rRNA near the peptidyl transferase center (8). Recent structural work (12, 21) has confirmed that these antibiotics exert their inhibitory effect by blocking the entrance to the polypeptide exit tunnel on the 50S ribosomal subunit and thus preventing the extrusion of nascent polypeptides. Sixteen-membered macrolides, such as tylosin, spiramycin, and carbomycin, also bind so as to block the peptide exit tunnel, but in addition these antibiotics have a mycaminose-mycarose disaccharide side chain that protrudes towards the peptidyl transferase center and more directly inhibit the peptidyl transferase reaction (12).Since the introduction of erythromycin nearly 50 years ago, antibacterial resistance to macrolides has been an increasingly persistent threat to public health. One major resistance mechanism is a form of target modification, wherein the dimethylation of A2058 of the 23S rRNA by erm gene-encoded ribosomal methylases results in cross-resistance to macrolides, to the structurally related lincosamides, and to group B streptogramins (commonly referred to as the MLS B phenotype). The emergence of these resistant bacterial pathogens has been of particular concern and has fueled the search for newer and more potent antibiotics with activity against these organisms.For years the interaction of erythromycin and other macrolides with the bacterial ribosome has been the focus of intense investigation, and a number of experimental approaches have been developed to characterize the equilibria of binding and the kinetics of these drugs with their ribosomal target. In many of these studies, the binding of macrolides to ribosomes was investigated using a radiolabeled antibiotic as a ligand (6,7,10,11,19,22). Since binding was measured by filtration, RNA footprintin...

Biochemical Characterization of the Interactions of the Novel Pleuromutilin Derivative Retapamulin with Bacterial Ribosomes

“…2B). In contrast, sparsomycin, a known nonselective protein synthesis inhibitor, A method using fluorescence polarization to characterize drug interactions with the bacterial ribosome has recently been described (23). We took advantage of this technology to develop a robust and sensitive competitive binding assay using a fluorescently labeled pleuromutilin (SB-452466) to determine the IC 50 s of pleuromutilin displacement by other compounds.…”

“…A fluorescently labeled pleuromutilin derivative (SB-452466) (Fig. 1B), labeled with 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY) by standard N-hydroxysuccinimidyl ester chemistry, was prepared, and its ribosome-binding properties were characterized kinetically to enable its use as a ligand in fluorescence polarization assays (23 ]SB-258781 to the ribosomes was competed with retapamulin at room temperature over a time course (2 h for E. coli ribosomes and 5 h for S. aureus ribosomes). The free and bound ligands were then separated through a filter plate (UniFilter GF/B; PerkinElmer) using a cell harvester.…”

Time-Resolved Binding of Azithromycin to Escherichia coli Ribosomes

“…4,5 Although the superior potency of azithromycin has been attributed to its faster penetration of the outer membranes of Gram-negative bacteria, 6,7 this drug also exhibits better ability than erythromycin to compete for [ 14 C]erythromycinbinding sites in Staphylococcus aureus ribosomes and higher affinity for E. coli ribosomes. [8][9][10][11] Accumulated biochemical and genetic evidence suggests that azithromycin exerts its inhibitory effect on microbial growth by binding in a narrow part of the peptide exit tunnel, at a position that is situated between the peptidyl transferase (PTase) center and a constriction in the tunnel formed by proteins L4 and L22. 5,8,[12][13][14][15][16][17] Binding of the drug at this region occludes the lumen of the tunnel, thus aborting the growth of the nascent peptide chain during the early rounds of translation, eventually leading to peptidyl-tRNA "drop-off".…”

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

“…A radioligand displacement assay using [ 3 H]SB-258781 was used to determine the affinity of SB-571519, SB-280080, and SB-275833 to E. coli ribosomes (21). The binding data were fit to a cubic equation (GraFit; Erithacus Software Ltd., Surrey, U.K.) that solves K d for a single binding site with two competitive ligands (54). …”