Macrolide resistance in Streptococcus pneumoniae has been associated with two main mechanisms: target modification by Erm methyltransferases and efflux by macrolide pumps. The ketolide ABT-773, which has a 3-keto group and no L-cladinose sugar, represents a new class of drugs with in vitro activity against a variety of resistant bacteria. Several approaches were undertaken to understand how ABT-773 was able to defeat resistance mechanisms. We demonstrated tighter ribosome binding of ABT-773 than erythromycin. We also showed that ABT-773 (i) accumulated in macrolide-sensitive S. pneumoniae at a higher rate than erythromycin, (ii) was able to bind with methylated ribosomes, though at lower affinities than with wild-type ribosomes, and (iii) accumulated in S. pneumoniae strains with the efflux-resistant phenotype.Macrolide resistance in Streptococcus pneumoniae has been associated with two main mechanisms. The first resistant strains described were of the macrolide-lincosamide-streptogramin B (MLS) phenotype (18). These strains have a posttranslational modification of 23S rRNA due to the methylation of A2058 (Escherichia coli numbering system) by Erm methyltransferase, resulting in a conformational change in the ribosomes. Resistance to macrolides may be constitutively expressed or induced by sub-MICs of the macrolide (18,20). The second mechanism of resistance recently found in S. pneumoniae, and referred to as the M phenotype, results from the active efflux of 14-and 15-membered macrolides (16). The macrolide efflux pump protein in S. pneumoniae is encoded by the gene mefE (17). The prevalence of M phenotype resistance in clinical isolates of S. pneumoniae has been reported to be a significant portion (60 to 75%) of the resistance population in the United States (8, 14,15,16).The ketolide ABT-773, which has a 3-keto group and no L-cladinose sugar, represents a new class of drugs with in vitro activity against a variety of resistant phenotypes (1). The purpose of this study was to understand how ABT-773 is able to defeat these resistance mechanisms. Several approaches were undertaken, including the measurement of drug-ribosome binding kinetics, drug uptake and efflux rates, and drug effect on cell-free protein translation. MATERIALS AND METHODSChemicals and radioisotopes. Todd-Hewitt broth and yeast extract were obtained from Difco Laboratories (Detroit, Mich.) Miller's M9 medium was prepared with M9 salts purchased from Life Technologies LTD (Paisley, Scotland) but without vitamins or amino acids, as described elsewhere (10). Luciferin reagents (GenLux kit) were purchased from Wallac (Turku, Finland). All other chemicals, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), were purchased from Sigma Chemical Co. (St. Louis, Mo.). [N-methyl-14 C]erythromycin (55 mCi/mmol) was obtained from NEN Life Science Products (Boston, Mass.), and unlabeled erythromycin was made in-house. [N-methyl-14 C]ABT-773 (27 mCi/mmol) was prepared in-house by coupling 6-0-allyl ketolide and 3-[U-14 C]bromoquinoline according to a...

The activity of a new ketolide, ABT-773, was compared to the activity of the ketolide telithromycin (HMR-3647) against over 600 gram-positive clinical isolates, including 356 Streptococcus pneumoniae, 167 Staphylococcus aureus, and 136 Streptococcus pyogenes isolates. Macrolide-susceptible isolates as well as macrolide-resistant isolates with ribosomal methylase (Erm), macrolide efflux (Mef), and ribosomal mutations were tested using the NCCLS reference broth microdilution method. Both compounds were extremely active against macrolide-susceptible isolates, with the minimum inhibitory concentrations at which 90% of the isolates tested were inhibited (MIC 90 s) for susceptible streptococci and staphylococci ranging from 0.002 to 0.03 g/ml for ABT-773 and 0.008 to 0.06 g/ml for telithromycin. ABT-773 had increased activities against macrolide-resistant S. pneumoniae (Erm MIC 90 , 0.015 g/ ml; Mef MIC 90 , 0.12 g/ml) compared to those of telithromycin (Erm MIC 90 , 0.12 g/ml; Mef MIC 90 , 1 g/ml). Both compounds were active against strains with rRNA or ribosomal protein mutations (MIC 90 , 0.12 g/ml). ABT-773 was also more active against macrolide-resistant S. pyogenes (ABT-773 Erm MIC 90 , 0.5 g/ml; ABT-773 Mef MIC 90 , 0.12 g/ml; telithromycin Erm MIC 90 , >8 g/ml; telithromycin Mef MIC 90 , 1.0 g/ml). Both compounds lacked activity against constitutive macrolide-resistant Staphylococcus aureus but had good activities against inducibly resistant Staphylococcus aureus (ABT-773 MIC 90 , 0.06 g/ml; telithromycin MIC 90 , 0.5 g/ml). ABT-773 has superior activity against macrolide-resistant streptococci compared to that of telithromycin.Increasing antimicrobial resistance, particularly in Streptococcus pneumoniae, has increased the need for drugs effective against resistant isolates. ABT-773 (A-195773.0) is a novel compound in the ketolide class with significant advantages over the general antimicrobial profile of macrolide antibiotics. Ketolide antibiotics are extremely active against macrolideresistant streptococci with either low-level (macrolide efflux [Mef]) or high-level (ribosomal methylase [Erm]) resistance to macrolides (1). Ketolides also have excellent in vitro activities against macrolide-resistant organisms with inducible methylase expression, as these compounds appear to be noninducers of methylase (2, 15). ABT-773 has excellent in vitro potency against macrolide-resistant gram-positive pathogens and has entered phase III clinical trials for the treatment of bacterial upper and lower respiratory tract infections (3). In this study, the in vitro activities of ABT-773 against macrolideresistant and -susceptible respiratory pathogens are compared to those of telithromycin, another ketolide antibiotic, as well as those of a representative of the macrolide class, erythromycin.(This work was previously presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 1999). MATERIALS AND METHODS Susceptibility testing.Testing was performed using the broth mic...

A novel series of erythromycin derivatives has been discovered with potent activity against key respiratory pathogens, including those resistant to erythromycin. These compounds are characterized by having an aryl group tethered to the C-6 position of the erythronolide skeleton. Extensive structural modification of the C-6 moiety led to the discovery of several promising compounds with potent activity against both mef- and erm-mediated resistant Streptoccoccus pneumoniae. Preliminary mechanistic studies indicated that the new macrolides are potent protein synthesis inhibitors, which interact with methylated ribosomes isolated from resistant organisms. In experimental animal models, these compounds exhibited excellent in vivo efficacy and balanced pharmacokinetic profiles.

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We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.Respiratory tract infections are the number 1 killer worldwide, responsible for over 50 million deaths each year. Although antibacterial therapy has successfully stemmed the tide against infection since the middle of the last century, antibacterial resistance of Streptococcus pneumoniae, the most commonly identified pathogen associated with community-acquired pneumonia, is on the rise (2,4,8,22). A recent worldwide study documents that a significant fraction of S. pneumoniae isolates have reduced susceptibility to penicillin (36%) and macrolides (31%) (5). Although overall rates of resistance to fluoroquinolones are low, these rates were found to be increasing rapidly in Canada (1). In general, pathogenic bacteria continuously evolve mechanisms of resistance to currently used antibacterial agents. The discovery of novel antibacterial classes would be the most powerful way to generate new therapy against these resistant pathogens. Unfortunately, novel antibacterial classes have been difficult to discover, with the oxazolidinones, identified in 1980, being the last example to successfully reach the clinic.The bacterial ribosome is a proven target for antibacterial chemotherapy (6,17,20,21). Since the 1940s, small-molecule ribosome inhibitors such as chloramphenicol, tetracyclines, macrolides, aminoglycosides, and, more recently, oxazolidinones have been used to combat bacterial infections in humans (11). These diverse chemical classes of ribosome inhibitors each bind to a different site on the ribosome, which is not surprising given its large size and complexity. Accordingly, drug resistance to each class generally develops separately, such that resista...

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.