On the Mechanism of Action of 9-O-Arylalkyloxime Derivatives of 6-O-Mycaminosyltylonolide, a New Class of 16-Membered Macrolide Antibiotics

Abstract

New 16-membered 9-aryl-alkyl oxime derivatives of 5-O-mycaminosyl-tylonolid (OMT) have recently been prepared and were found to exhibit high activity against macrolide-resistant strains. In this study, we show that these compounds do not affect the binding of tRNAs to ribosomes in a cell-free system derived from Escherichia coli and that they cannot inhibit peptidyltransferase, peptidyl-tRNA translocation, or poly(U)-dependent poly(Phe) synthesis. However, they severely inhibit poly(A)-dependent poly(Lys) synthesis and compete with erythromycin or tylosin for binding to common or partially overlapping sites in the ribosome. According to footprinting analysis, the lactone ring of these compounds seems to occupy the classic binding site of macrolides that is located at the entrance of the exit tunnel, whereas the extending alkyl-aryl side chain seems to penetrate deeper in the tunnel, where it protects nucleoside A752 in domain II of 23S rRNA. In addition, this side chain causes an increased affinity for mutant ribosomes that may be responsible for their effectiveness against macrolide resistant strains. As revealed by detailed kinetic analysis, these compounds behave as slow-binding ligands interacting with functional ribosomal complexes through a one-step mechanism. This type of inhibitor has several attractive features and offers many chances in designing new potent drugs.

Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis

“…In saturation binding experiments, [ 14 C]erythromycin readily bound to ribosomes from both bacteria, exhibiting dissociation constants (K d s) of 66 Ϯ 11 nM and 11 Ϯ 1 nM for the E. coli and S. aureus ribosomes, respectively (data not shown). These values were comparable to those previously published (10 Ϫ8 to 10 Ϫ7 M) (8,19). Binding of erythromycin saturated close to 1 pmol of the drug per 1 pmol of E. coli or S. aureus ribosomes, indicating that the majority of the ribosomes in the preparation were competent for binding.…”

Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity

“…We have previously demonstrated that 16-membered macrolides, like tylosin, inhibit AcPhe-puromycin synthesis, whereas 14-membered macrolides, such as erythromycin, do not (7,16). This is due to the fact that the C-5-disaccharide of tylosin reaches into the peptidyl transferase (PTase) center of the ribosome, whereas the C-5-monosaccharide of 14-membered macrolides is too short (3,13,32,37).…”

“…The initiation inhibitor edeine inhibited both poly(Phe) and poly(Lys) synthesis, as reported previously (8). The differential effects of macrolides on poly(Phe) and poly(Lys) synthesis have been observed previously (16,36). One possible explanation for the lack of inhibitory effect of macrolides on poly(Phe) synthesis is that the highly hydrophobic nascent poly(Phe) chain may displace K-1325 from its binding site on 50S subunits as it passes through the tunnel.…”

The slow dissociation rate of K-1602 contributes to the enhanced inhibitory activity of this novel alkyl–aryl-bearing fluoroketolide

“…This type of competition can follow two alternative mechanisms, as depicted in Schemes 2 and 3. Kinetic equations concerning Schemes 2 and 3 have been derived in previous publications 19,20 , and the main functions are presented in Table 1. Plotting our data to these equations gives us the opportunity to distinguish, which of the two mechanisms is followed by K-1602.…”

“…Following detailed kinetic analysis, we determined the association and dissociation constants of K-1602 with the ribosome. Calculations were based on a previously developed kinetic model [19][20][21] , which allowed us to conclude that K-1602 behaves as a slow binding slowly dissociating inhibitor 22 , thus explaining the unique characteristics and effectiveness of this compound. Slow binding inhibitors represent a large kinetic class, mainly including transition state and reaction intermediate analogs, and as such show promising features for future drug development 23 .…”