Ketolide Antimicrobial Activity Persists after Disruption of Interactions with Domain II of 23S rRNA

Abstract

Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis. An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities. Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752. We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding. Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides. Mutations at position 748 confer no resistance. Substitution of guanine at A2058 gives rise to the MLS B (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs. However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA. In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin. Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin. The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance.

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

“…However, the conclusions about the contribution of the 11,12 side chain to the drug activity should be drawn only cautiously. Although original studies suggested that interactions of the side chain of ketolides with A752 in the loop of helix 35 in 23S rRNA significantly contribute to the drugs' affinity (9,17,49), more recent genetic studies questioned that conclusion because mutations at and around A752 or at U2609, with which the side chains interact, had only a minor effect upon drug inhibitory action (14,21,30,49). In agreement with these observations, the additional interaction afforded by the extended side chains of telithromycin and CEM-101 or, for that matter, the fluorine atom at the C-2 position of the CEM-101 lactone did not translate in our study to an improved ability of either of the ketolides compared with that of azithromycin in inhibiting erythromycin binding to the ribosome (Table 1).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…Similarly, the role of position G745 in the tylosin-ribosome interactions in E. coli has been described (Liu & Douthwaite, 2002b), while positions G748 and A752 are reportedly involved in the interactions between ketolides and ribosomes (Novotny et al, 2004). Additionally, both L4 and L22 proteins are implicated in this ribosome-macrolide interaction (Chittum & Champney, 1994;Gabashvili et al, 2001).…”

“…Alterations present in domain II of the 23S rRNA affect specific macrolides like tylosin and ketolide antibiotics but not erythromycin or azithromycin. Thus, amino acid changes, deletions or insertions at position A752 result in low levels of telithromycin resistance (Novotny et al, 2004). Curiously, the concomitant presence of any A752 alteration in combination with the presence of A2058G results in the loss of telithromycin resistance but with an increase in tylosin resistance (Novotny et al, 2004).…”

“…Additionally, other alterations have been described in Enterobacteriaceae, such as those affecting positions A745, A752, U754, G2057, A2032, A2062, A2503, U2609, C2610 or C2611, which are able to affect the activity of macrolides either by increasing the levels of resistance, or resulting in enhanced activity. Moreover, an association has been described between a specific 23S rRNA deletion (D1219-1230) and the development of erythromycin resistance (Douthwaite et al, 1985;Krokidis et al, 2014;Novotny et al, 2004;V azquez-Laslop et al, 2008V azquez-Laslop et al, , 2010V azquez-Laslop et al, , 2011Vannuffel et al, 1992, Weisblum, 1995 (Table 3). Alterations present in domain II of the 23S rRNA affect specific macrolides like tylosin and ketolide antibiotics but not erythromycin or azithromycin.…”

“…Thus, amino acid changes, deletions or insertions at position A752 result in low levels of telithromycin resistance (Novotny et al, 2004). Curiously, the concomitant presence of any A752 alteration in combination with the presence of A2058G results in the loss of telithromycin resistance but with an increase in tylosin resistance (Novotny et al, 2004). Meanwhile, the U754A alteration has a slight affect on the MIC of some ketolides, such as telithromycin, but not on that of others as in the case of the new ketolide K1835 (Krokidis et al, 2014).…”