Deciphering the Mechanism of Action of a Short, Synthetic Designer <scp>AMP</scp> Against Gram‐Negative Bacteria

Antimicrobial peptides (AMPs), produced in various organisms, including plants, as a first line of defense, are potent, functionally versatile, fast-acting small peptides with a net charge and diverse structures. Most AMPs demonstrate potent antibacterial activity, and AMPs with multimodal actions can potentially delay the development of antimicrobial resistance (AMR), one of the top 10 global public health challenges categorized by the WHO. Notably, the FDA has already approved several AMPs (Mol. Wt. ≤ 2 kDa) as antibiotics; however, there are not enough new-age antibiotics in the current pipeline to combat the looming problem of AMR in the clinic. Nevertheless, despite their potential, natural AMPs have their fair share of shortcomings for straightforward therapeutic applications. Therefore, extensive research on developing designer synthetic AMPs with broad-spectrum antimicrobial activity is currently being undertaken to mitigate the AMR challenge. In this context, we recently demonstrated a short synthetic designer AMP (SR17: ≤ 16 aa, mol. Wt. ≤ 2 kDa) that exhibits broad-spectrum bacteriostatic and bactericidal action against both gram-negative (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) and gram-positive (Staphylococcus aureus) bacteria. Interestingly, in gram-negative bacteria, the outer membrane proteins (OMPs) play a key role in transporting nutrients like iron from their surroundings through siderophores, which play a crucial role in various biochemical processes essential for their survival and growth. In the current study, the ability of SR17 to target the iron-transporting OMPs acting as the siderophore uptake system is investigated through computational techniques. A series of docking and molecular dynamics (MD) simulation studies involving iron transporters of various gram-negative bacteria indicate that SR17 can occupy the binding pocket in the OMPs necessary for binding of the iron-chelated siderophores, which is likely to prevent the further uptake of siderophores, affecting the growth and survival of the bacteria. Additionally, SR17 may potentially reach the bacterial cytoplasm by utilizing the siderophore uptake system and disrupt essential cytoplasmic processes, leading to the death of the bacteria, as observed in experimental studies.