In vitro activity: KL-10 is a small-molecule ribosome rescue inhibitor that possesses a broad-spectrum of antimicrobial activity against bacteria. KKL-10 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 reduced intracellular bacteria by>99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. KKL-10 was not cytotoxic to eukaryotic cells in culture. Therefore, KKL-10 is a good lead compound for antibiotic development.

Kinase Assay: For MIC assays, triplicate 2-fold serial dilutions of each compound were made in cation-adjusted Mueller-Hinton broth (CAMHB) and added to a 96-well microtiter plate. Stocks of each compound were prepared in 100% dimethyl sulfoxide (DMSO). Overnight cultures of LVS or Schu S4 were diluted to an OD600 of 0.05 in CAMHB to a final volume of 0.1 ml and added directly to the diluted compounds. The microtiter plates were incubated overnight (~18 h) at 37°C in a humidified incubator with 5% CO2. Bacterial growth was monitored by measuring the optical density at 600 nm. The MIC was determined by observing the lowest concentration at which the compound prevented a significant increase in the optical density. To enumerate F. tularensis after exposure to various concentrations of KKL-10 or KKL-40, the contents of the MIC assay microtiter plate were removed and plated on chocolate agar at appropriate dilutions. After incubation for 48 h at 37°C and 5% CO2, colonies were counted to calculate CFU per milliliter.

Cell Assay: Cytotoxicity assays were performed using RAW 264.7 cells and a lactate dehydrogenase (LDH) release assay kit (Pierce Biochemicals, USA) following the manufacturer's instructions.