Antibacterial Assay. Synthetic PLP-53 was tested for activity against the Gram-negative bacterium Escherichia coli K-12 and the Gram-positive bacterium Bacillus subtilis Marburg 168. Bacteria were grown on LB agar plates overnight at 37 °C. One colony was picked from each plate and used to inoculate a 5 mL liquid culture. Each culture was incubated overnight at 37 °C with shaking. Fresh 5 mL aliquots of LB were inoculated with 25 μL of overnight culture and incubated at 37 °C. These cultures were diluted to an
OD600 (optical density at 600 nm) of 0.1 and spread onto LB agar plates with a sterile swab. Synthetic PLP-53 was dissolved to a concentration of 10 μg/μL in DMSO and dispensed onto sterile 8 mm diameter filter paper discs in the following amounts: 3.1, 6.25, 12.5, 25, 50, and 100 μg. A disc containing 50 μg of kanamycin in water was used as a positive control, and a disc with 5 μL water as a negative control. All discs were left to dry completely before placing them onto the inoculated LB agar plates. The plates were incubated overnight at 37 °C and inspected in the morning for bacterial growth.
Antifungal Assay. Synthetic PLP-53 was tested for activity against the fungus Aspergillus fumigatus. The fungus was grown on ayeast extract, peptone, dextrose (YPD) agar plate. Spores were harvested from the plate with 3 mL of 0.1% (v/v) Tween 80 and dispensed into YPD medium. This culture was adjusted to an OD600 of 0.05, and 180 μL of it was dispensed into 20 wells of a 96-well plate. PLP-53 solution (20 μL) was added to 10 of these wells at the following concentrations: 5000, 2500, 1250, 625, 313, 156, 78, 39, 19,
and 9 μg/μL. These solutions were made by serial dilution with water from an initial solution of 5000 μg/μL synthetic PLP-53 in DMSO. Since DMSO is known to be toxic to fungi at high concentrations, in the other 10 wells we made a series of DMSO controls by adding 20 μL of the same dilutions of DMSO without PLP-53. The plate was incubated at 37 °C for 24 h and visually inspected for fungal growth.