TAN-1057A: a translational inhibitor with a specific inhibitory effect on 50S ribosomal subunit formation.

The inhibitory activities of a novel antibiotic compound have been investigated. A synthetic version of the natural product TAN-1057A was examined for its effects on translation and ribosomal subunit formation. The antibiotic at 6 microg/ml reduced the growth rate of wild-type Staphylococcus aureus cells by 50%. The IC50 for inhibition of protein synthesis in these cells was 4.5 microg/ml. Pulse and chase labeling kinetics showed a strong inhibitory effect on 50S ribosomal subunit formation as well. The IC50 for this process was 9 microg/ml, indicating an equivalent inhibitory effect of the antibiotic on translation and 50S synthesis. The post-antibiotic effect of the drug was investigated. Protein synthesis resumed rapidly after removal of the drug from cells, but full recovery of the normal 50S subunit complement in treated cells required 1.5 h. The dual inhibitory effects of this compound are compared with other antimicrobial agents having similar effects on cell growth.

Structure-activity relationships for six ketolide antibiotics.

Six structurally related 3-keto-substituted macrolide antibiotics (ketolides) were compared for concentration-dependent inhibitory effects on growth rate, viable cell number, and protein synthesis rates in Staphylococcus aureus cells. Inhibitory effects on 50S ribosomal subunit formation were also examined, as this is a second target for these antibiotics. A concentration range of 0.01 to 0.1 microg/ml was tested. An IC50 for inhibition of translation and 50S synthesis was measured for each compound, to relate structural features to inhibitory activity. ABT-773 was the most effective of the six compounds tested with an IC50 = 0.035 microg/ml. HMR 3004 was almost as effective with an IC50 = 0.05 microg/ml. Two 2-fluoroketolides (HMR 3562 and HMR 3787) were equivalent in their inhibitory activity with an IC50 = 0.06 microg/ml. Telithromycin (HMR 3647) had an IC50 = 0.08 microg/ml, and HMR 3832 was least effective with an IC50 = 0.11 microg/ml. Each antibiotic had an equivalent inhibitory effect on translation and 50S subunit formation. These results indicate specific structural features of these antimicrobial agents, which contribute to defined inhibitory activities against susceptible organisms.

Specific inhibition of 50S ribosomal subunit formation in Staphylococcus aureus cells by 16-membered macrolide, lincosamide, and streptogramin B antibiotics.

The translational functions of the bacterial ribosome are the target for a large number of antimicrobial agents. The 14- and 16-membered macrolides, the lincosamides, and the streptogramin B type antibiotics are thought to share certain inhibitory properties, based on both biochemical and genetic studies. We have shown previously that the 14-membered macrolides, like erythromycin, have an equivalent inhibitory effect on translation and the formation of the 50S ribosomal subunit in growing bacterial cells. To extend this work, we have now tested the 16-membered macrolides spiramycin and tylosin, the lincosamides lincomycin and clindamycin, and 3 streptogramin B compounds pristinamycin I(A), virginiamycin S, and CP37277. Each of these was a specific inhibitor of 50S subunit formation, in addition to having an inhibitory effect on translation. By contrast, two streptogramin A compounds, virginiamycin M1 and CP36926, as well as chloramphenicol, were effective inhibitors of translation without showing a specific effect on the assembly of the large ribosomal subunit. A combination of an A and B type streptogramin (virginiamycin M1 and pristinamycin I(A)) demonstrated a synergistic inhibition of protein synthesis without exhibiting a specific inhibition of 50S subunit formation. These results extend our observations on 50S assembly inhibition to the entire class of MLS(B) antibiotics and reinforce other suggestions concerning their common ribosome-binding site and inhibitory functions.

Evernimicin (SCH27899) inhibits both translation and 50S ribosomal subunit formation in Staphylococcus aureus cells.

The effects of the everninomicin antibiotic evernimicin (SCH27899) on growing Staphylococcus aureus cells were investigated. Cellular growth rates and viable cell numbers decreased with increasing antibiotic concentrations. The rate of protein synthesis, measured as (35)S-amino acid incorporation, declined in parallel with the growth rate. Significantly, the formation of the 50S ribosomal subunit was inhibited in a dose-dependent fashion as well. 30S ribosomal subunit synthesis was not affected over the same concentration range. Evernimicin did not stimulate the breakdown of mature ribosomal subunits. Pulse-chase labeling experiments revealed a reduced rate of 50S subunit formation in drug-treated cells. Two erythromycin-resistant strains of S. aureus that carried the ermC gene were as sensitive as wild-type cells to antibiotic inhibition. In addition, two methicillin-resistant S. aureus organisms, one sensitive to erythromycin and one resistant to the macrolide, showed similar sensitivities to evernimicin. These results suggest a use for this novel antimicrobial agent against antibiotic-resistant bacterial infections.

Superiority of 11,12 carbonate macrolide antibiotics as inhibitors of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells.

Three pairs of related macrolide antibiotics, differing at the 11,12 position of the macrolactone ring, were compared for effects on growth rate, cell viability, protein synthesis, and 50S ribosomal subunit formation in Staphylococcus aureus cells. For each parameter measured, the 11,12 carbonate-derivatized compound was more inhibitory compared with the corresponding 11,12-hydroxy antibiotic. Substitution at the 3-position of the ring was also important in the relative inhibition observed. The degree of inhibition found in two different growth media was proportional to the generation time of the cells. Inhibition of both protein synthesis and 50S subunit formation by each drug correlated well with the inhibition of cell viability. The results indicate that closure of the 11,12-hydroxyl groups in macrolide antibiotics with a carbonate substitution generates a more effective antimicrobial agent.

Molecular investigation of the postantibiotic effects of clarithromycin and erythromycin on Staphylococcus aureus cells.

The kinetics of recovery after inhibition of growth by erythromycin and clarithromycin were examined in Staphylococcus aureus cells. After inhibition for one mass doubling by 0.5 microg of the antibiotics/ml, a postantibiotic effect (PAE) of 3 and 4 h duration was observed for the two drugs before growth resumed. Cell viability was reduced by 25% with erythromycin and 45% with clarithromycin compared with control cells. Erythromycin and clarithromycin treatment reduced the number of 50S ribosomal subunits to 24 and 13% of the number found in untreated cells. 30S subunit formation was not affected. Ninety minutes was required for resynthesis to give the control level of 50S particles. Protein synthesis rates were diminished for up to 4 h after the removal of the macrolides. This continuing inhibition of translation was the result of prolonged binding of the antibiotics to the 50S subunit as measured by 14C-erythromycin binding to ribosomes in treated cells. The limiting factors in recovery from macrolide inhibition in these cells, reflected as a PAE, are the time required for the synthesis of new 50S subunits and the slow loss of the antibiotics from ribosomes in inhibited cells.

A comparison of the inhibition of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells by nine different macrolide antibiotics.

Nine structurally similar macrolide antibiotics were tested at a concentration of 0.5 microg/ml for their relative inhibitory effects on ribosome functions in Staphylococcus aureus cells. Eight of the compounds examined inhibited protein synthesis at this concentration. Seven of the nine compounds were also effective in blocking formation of the 50S ribosomal subunit. Roxithromycin and 14-hydroxy clarithromycin inhibited protein synthesis to a greater extent than they affected 50S subunit formation. Conversely, the compound 11, 12-carbonate-3 deoxy-clarithromycin affected 50S assembly more than translation. Only clarithromycin had any effect on 30S ribosomal subunit assembly. The decline in growth rate and cell number was proportional to the effect on ribosome formation or function by each compound. These inhibitory activities can be related to structural differences between these macrolide antibiotics.

Inhibition of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells by 11 different ketolide antibiotics.

Eleven structurally similar ketolide antibiotics were tested at a concentration of 1 microg/ml for their relative inhibitory effects on growth and ribosome activities in Staphylococcus aureus cells. Ten of the compounds examined had an inhibitory effect on protein synthesis at this concentration and eight of the 11 compounds were also effective inhibitors of the formation of the 50S ribosomal subunit. All of the drugs tested inhibited protein synthesis to a greater extent than they affected 50S subunit formation. The decline in growth rate and cell number was proportional to the effect on ribosome formation and function. The growth of an ermC erythromycin-resistant strain of S. aureus was also significantly inhibited by nine ketolide compounds, suggesting that they were not inducers of methylase gene expression. These inhibitory activities can be related to structural differences between these ketolide antibiotics.