Fig. 3: Validation of AMP candidates.

a The AlphaFold 3 predicted structure and net charge (at pH 7) of the 38 validated AMPs. AMP-1 to AMP-20 were randomly selected from the top 100 candidates targeting S. aureus, and AMP-21 to AMP-40 were randomly selected from the top 100 candidates targeting E. coli. Among them, the chemical synthesis of AMP-5 and 18 failed. The color of the ellipse in the lower right corner of each 3D structure diagram represents the minimum inhibitory concentration (MIC) value against E. coli K88 (left) and S. aureus ATCC 29213 (right). b Comparison of antimicrobial activity of AMP candidates against Gram-positive and Gram-negative bacteria. b displays the log10-transformed MIC values (μM) of AMP candidates against Gram-positive S. aureus ATCC 29213 (y-axis) and Gram-negative E. coli K88 (x-axis). Each dot represents a different AMP, color-coded by its designed target: yellow-green for AMPs with a top 100 score against Gram-positive bacteria (G+) and green for those with a top 100 score against Gram-negative bacteria (G-). The control of antibiotics Polymyxin B and Ampicillin is highlighted in red. AMPs clustered near the origin show broad-spectrum efficacy, while those positioned at the extremes indicate selective efficacy against either Gram-positive or Gram-negative bacteria. c Nine AMPs with MIC values less than 5 μM for both targets in a were selected for further determination of MIC against E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), S. aureus (ATCC25923), and E. faecalis (ATCC 29212). The figure shows the MIC values of the nine AMPs against each target. Ampicillin and Polymyxin B were used as positive controls. The MIC was determined by averaging the results from triplicate assays across three independent experiments (n = 3).