Molecular Dynamics Studies on the Bacterial Membrane Pore Formation by Small Molecule Antimicrobial Agents

Antimicrobial peptides (AMPs) act on the membrane bilayer of pathogens, causing leakage in the membrane and cell death. Amphiphilic kaempferol derivatives possessing basic functional groups show excellent antibacterial activities, which has been proven through experimental techniques. These compounds are known to target negatively charged bacterial membranes. However, the detailed mechanism of action and their structure–activity relationship are not clear. In this work, we reported theoretical investigation on the mechanism of action of two previously reported kaempferol derivatives on a DMPC/DMPG mixed bilayer. Despite the rigid structure of the compounds when compared to AMPs, spontaneous pore formation in the membrane was not observed in 400 ns molecular dynamics (MD) simulations. MD simulations with biasing forces resulted in the formation of pores in the bilayer for the derivatives and not for kaempferol. The stability of the pores was assessed by pore closure timescales in unbiased MD simulations, which was found to be 5.3 and 17.0 ns for 2 and 3, respectively. Free energy change for the permeation into the bilayer for kaempferol (1), tertiary amine derivative (2), and arginine derivative (3) was calculated to be −1.5, −48.2, and −100.3 kJ/mol, respectively, which correlate with their antibacterial activity. Furthermore, our results indicate that compound 3 forms a stable toroidal pore in the membrane when multiple molecules are oriented in a transmembrane configuration. Our work sheds light on the mechanism of action of small molecule antimicrobial agents, which can be exploited for the rational design of drug candidates.