Integrating Docking, Dynamics, and Assays to Predict Antimicrobial Peptide Interactions with Mycolic Acid Membranes in Mycobacterium tuberculosis

Abstract

The global burden of multidrug-resistant tuberculosis (MDR-TB) underscores the urgent need for novel therapeutics with distinct mechanisms of action. Here, we report a comparative evaluation of four antimicrobial peptides (AMPs) derived from the amphibian peptide B1CTcu5, integrating experimental validation with molecular modeling to elucidate structure–activity relationships. Among them, W-B1CTcu5, featuring a single N-terminal tryptophan substitution, exhibited the most potent antimycobacterial activity (MIC = 3.2 μg/mL) against Mycobacterium tuberculosis (MTB) combined with high structural stability, persistent membrane interaction, and multitarget affinity against key MTB proteins, including the porin MspA, the transporter CpnT, and the cell wall enzyme Ag85B. In contrast, analogs with reduced hydrophobic anchoring or dynamic instability demonstrated diminished efficacy despite partial membrane insertion or surface affinity. Molecular dynamics simulations revealed that peptides with low root-mean-square deviation and minimal residue fluctuation retained compact, α-helical conformations and maintained productive bilayer engagement, which are traits correlated with antimicrobial performance. However, the hemolytic properties of W-B1CTcu5 highlight a therapeutic trade-off between potency and host toxicity. Together, these findings emphasize the predictive power of dynamic structural descriptors in AMP design, and identify W-B1CTcu5 as a promising, yet optimization-requiring, scaffold for future design of anti-TB AMPs.