KKL-35 is an inhibitor of trans-translation (IC50: 0.9 μM) with broad-spectrum antibiotic activity.

Trans-translation tagging reaction:0.9 µM

Overnight cultures of S. flexneri 2a 2457T, B. anthracis, M. smegmatis, JW5503, and NSR253 were grown in lysogeny broth (LB). Cultures were diluted to a final inoculum of ～5 × 10^5 cfu/mL in 96-well microtiter plates, compounds were added at appropriate concentrations, and growth was observed after 24-h incubation at 37 °C. The MIC was determined by the concentration of the compound in the last well that showed no bacterial growth. For MBC assays, 10 μL from wells containing the MIC, 2× MIC, and 4× MIC of each compound was diluted 10-fold, spotted on LB plates, and grown overnight at 37 °C. An inhibitor was scored as bactericidal if it resulted in 1,000-fold reduction in colony-forming units per milliliter from the original inoculum [1].