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...