KKL-35 exhibits broad-spectrum antibiotic activity. It prevents growth of B. anthracis and M. smegmatis with minimum inhibitory concentration (MIC) values of less than 6 μM. It inhibits trans-translation at some step before proteolysis of tagged proteins. It inhibits tagging of DHFR-ns. A large amount of untagged DHFR is produced in reactions with the highest concentrations of it, indicating that it does not inhibit translation. It prevents growth of WT S. flexneri with a MIC of 6 μM, and addition of it to a growing culture of S. flexneri stops growth. In an S. flexneri strain expressing ArfA and deleted for ssrA, addition of it has little effect on viability or growth rate. It inhibits the growth of E. coli ?tolC, which is deficient in small molecule efflux, with an MIC of 0.3 μM.

Evidence suggest that the in vivo effects of KKL-35 are caused by inhibition of the release of nonstop translation complexes by trans-translation. It inhibits trans-translation and prevents growth of S. flexneri strains that require trans-translation. The correlation between inhibition of trans-translation and growth is supported by genetic and pharmacological experiments showing that alternative mechanisms to release nonstop translation complexes relieve the growth suppression of KKL-35.

IC50: 0.9 μM ( trans -translation tagging reaction)