Cytotoxicity Assays. We evaluated the cytotoxicity of the three cyclohexylalanine analogues toward HeLa cells to further assess the potential utility of these antibiotics, and we compared the analogues to D-Thr5-clovibactin to further evaluate the effect of their increased hydrophobicity. The cytoxicity was assessed using the Promega Cytotox-Glo assay, with 24-hour exposure of the HeLa cells to the antibiotics. The cyclohexylalanine analogues showed no significant cytoxicity at concentrations as high as 50 μg/mL (Figure ). At 100 μg/mL, the analogues showed moderate cytotoxicity. In comparison, D-Thr5-clovibactin showed no cytotoxicity at concentrations as high 100 μg/mL. These studies further suggest that the cyclohexylalanine analogues could be administered at concentrations well above the MIC values. Nevertheless, the antibiotic activity occurs at concentrations substantially below those that are damaging to mammalian cells. For these reasons, D-Cha2, Cha7, and Cha8,D-Thr5-clovibactin warrant further investigation through preclinical studies in rodents or other animal models.