The inhibitory activities of new compounds against

The inhibitory activities of new compounds against P. aeruginosa were determined in both DTSB media supplemented with heme or free iron and LB media (Table 1). The MIC50 values of the compounds ranged from 42 to 260 μg/mL in DTSB medium and from 26 to 230 μg/mL in LB medium. Among these new inhibitors, 1423 mg 2e has the smallest MIC50 value in both DTSB and LB medium. The compound 4d has the largest MIC50 value in DTSB medium and the compound 4c has the largest MIC50 value in LB medium. The results showed most compounds had good anti-microbial effects against P. aeruginosa in both DTSB medium supplemented with heme and LB medium. However, the compounds did not inhibit the growth of P. aeruginosa at different times in DTSB + FeCl3 media. The OD600 values of 300 μg/ml compounds at different time in DTSB + FeCl3 media are showed in the session 3 of supplemental materials. The MIC50 values of the compounds in LB medium were lower than those obtained from the experiments in DTSB medium (P < 0.05). The research data of MIC50 values of the compounds on PAO1 are detailed in supplementary material. We have developed new inhibitors that inhibits pa-HemO, a key enzyme for iron acquisition of P. aeruginosa. Conventionally the broth dilution method is commonly applied for measuring the MIC50. It requires the preparation of series dilutions of the compounds. Due to the relatively low water solubility of many therapeutic candidates, polar organic solvents such as methanol, ethanol or DMSO are widely used to enhance the aqueous solubility of compounds. DMSO is the most commonly preferred since they are miscible with water [20]. However in various bioassays, DMSO has been reported for their antimicrobial effect [21]. Thus it becomes essential to ensure that the final concentration of DMSO is not interfere with the bioassay (MIC determination). We determined the highest possible concentration of DMSO that can be safely used to measure the MIC50 of new compounds without obvious cytotoxicity. The results showed that 1% was the highest concentration that DMSO has no significant effects on bacterial growth. Therefore, the concentration of DMSO used in our MIC50 assay was set at 1%. In this experiment, most of the compounds had good KD and MIC50 measurements. This suggests that these compounds inhibited the uptake of heme by P. aeruginosa and bacteria growth by competitively binding to pa-HemO. When the concentration of heme reaches a certain level, it also has a toxic effect on P. aeruginosa [22]. Reducing heme toxicity can also be accomplished by the heme oxygenase-mediated degradation of heme. Due to the action of pa-HemO inhibitors, the ingested heme cannot be utilized and have a toxic effect on P. aeruginosa, leading to the inhibition of bacterial growth. Compound 7d showed relatively weak binding affinities while indicating the inhibition of P.aeruginosa growth by MIC50 test. We reasoned that this compound may block the growth of P.aeruginosa by a different mechanism. The specific mechanism of action will be further studied in future trials. Compound 7b and 7c demonstrated good KD values but had relatively poor MIC50 values. We reasoned that the presence of the special group in these compounds might prohibit the in vitro effects of inhibitors, as evidenced in a number of previous studies [23,24]. DTSB is an iron-depleted medium [25]. In the culture environment of DTSB plus heme, the compounds mainly inhibited the growth of P. aeruginosa by competitively binding to pa-HemO and inhibiting the utilization of heme. LB medium is a nutrient-rich formulations that provide peptides, peptones, vitamins (including iron) and trace elements [26]. In LB medium, the compounds may interfere with the other iron metabolic pathways of P. aeruginosa or inhibit the growth of P. aeruginosa by other mechanisms than inhibiting the uptake of heme by bacteria. This may lead that compounds show stronger inhibitory effects on P. aeruginosa in the LB medium test group than in DTSB medium test group. The specific mechanism needs further study.