Structure-Activity Relationship of the Diaminobutyric Acid Residues in Polymyxins.

Multidrug-resistant Gram-negative bacteria have caused a serious threat to global health, and polymyxins are an important last-line therapy. As resistance to polymyxins is emerging, understanding the structure-activity relationship (SAR) of the polymyxins can facilitate the discovery of novel antimicrobial lipopeptides with improved antibacterial activity. However, l-2,4-diaminobutyric acid (Dab) is a key amino acid for the antibacterial activity of polymyxins, and the SAR of five Dab residues has not been well studied. Here, we employed an all-atom molecular dynamics simulation approach by integrating a lipidomics-informed outer membrane (OM)-based model and systematically investigated the SAR of the five Dab residues, specifically the length of their side chains. The impact of the length of the Dab side chain on three activity-related aspects, namely, the conformation of polymyxins, OM penetration ability, and membrane deformation, was systematically examined at the atomic level and compared with in vitro antimicrobial activity results. We uncovered that altering the side chain length of the Dab residues at different positions significantly affected the antibacterial activity via distinct mechanisms. Longer side chains of Dab residues in the linear tripeptide segment (Dab1 and Dab3) and shorter side chains in the heptapeptide ring (Dab5, Dab8, and Dab9) resulted in marked changes in OM deformation. Importantly, polymyxin activity is governed by the interplay of multiple structural and functional factors. Our mechanism-based SAR model predicted how these position-specific modifications in Dab side chains modulate polymyxin activity, as supported by experimental validation. Specifically, elongation of the Dab1 and Dab5 side chains led to significantly reduced antibacterial activity, while shortening of the Dab3 side chain enhanced activity. Collectively, the atomic-level SAR of polymyxins centered on the Dab residues will help expedite the rational design of new-generation antimicrobial lipopeptides, and our transferable framework provides broad implications for advancing membrane-active therapeutic agents.