MSI-594

Lipopolysaccharide (LPS) provides a well-organized permeability barrier at the outer membrane of Gram-negative bacteria. Host defense cationic antimicrobial peptides (AMPs) need to disrupt the outer membrane before gaining access to the inner cytoplasmic membrane or intracellular targets. Several AMPs are largely inactive against Gram-negative pathogens due to the restricted permeation through the LPS layer of the outer membrane. MSI-594 (GIGKFLKKAKKGIGAVLKVLTTG) is a highly active AMP with a broad-spectrum of activities against bacteria, fungi, and virus. In the context of LPS, MSI-594 assumes a hairpin helical structure dictated by packing interactions between two helical segments. Residue Phe5 of MSI-594 has been found to be engaged in important interhelical interactions. In order to understand plausible structural and functional inter-relationship of the helical hairpin structure of MSI-594 with outer membrane permeabilization, a mutant peptide, termed MSI-594F5A, containing a replacement of Phe5 with Ala has been prepared. We have compared antibacterial activities, outer and inner membrane permeabilizations, LPS binding affinity, perturbation of LPS micelles structures by MSI-594 and MSI-594F5A peptides. Our results demonstrated that the MSI-594F5A has lower activities against Gram-negative bacteria, due to limited permeabilization through the LPS layer, however, retains Gram-positive activity, akin to MSI-594. The atomic-resolution structure of MSI-594F5A has been determined in LPS micelles by NMR spectroscopy showing an amphipathic curved helix without any packing interactions. The 3D structures, interactions, and activities of MSI-594 and its mutant MSI-594F5A in LPS provide important mechanistic insights toward the requirements of LPS specific conformations and outer membrane permeabilization by broad-spectrum antimicrobial peptides.

Multidrug resistance against the existing antibiotics is one of the most challenging threats across the globe. Antimicrobial peptides (AMPs), in this regard, are considered to be one of the effective alternatives that can overcome bacterial resistance. MSI-594, a 24-residue linear alpha-helical cationic AMP, has been shown to function via the carpet mechanism to disrupt bacterial membrane systems. To better understand the role of lipid composition in the function of MSI-594, in the present study, eight different model membrane systems have been studied using accelerated molecular dynamics (aMD) simulations. The simulated results are helpful in discriminating the particular effects of cationic MSI-594 against zwitterionic POPC, anionic POPG and POPS, and neutral POPE lipid moieties. Additionally, the effects of various heterogeneous POPC/POPG (7 : 3), POPC/POPS (7 : 3), and POPG/POPE (1 : 3 and 3 : 1) bilayer systems on the dynamic interaction of MSI-594 have also been investigated. The effect on the lipid bilayer due to the interaction with the peptide is characterized by lipid acyl-chain order, membrane thickness, and acyl-chain dynamics. Our simulation results show that the lipid composition affects the membrane interaction of MSI-594, suggesting that membrane selectivity is crucial to its mechanism of action. The results reported in this study are helpful to obtain accurate atomistic-level information governing MSI-594 and its membrane disruptive antimicrobial mechanism of action, and to design next generation potent antimicrobial peptides.

Multidrug resistance against the existing antibiotics is becoming a global threat, and any potential drug that can be designed using cationic antimicrobial peptides (AMP) could be an alternate solution to alleviate this existing problem. The mechanism of action of killing bacteria by an AMP differs drastically in comparison to that of small molecule antibiotics. The main target of AMPs is to interact with the lipid bilayer of the cell membrane and disrupt it to kill bacteria. Consequently, the modes of membrane interaction that lead to the selectivity of an AMP are very important to understand. Here, we have used different membrane compositions, such as negatively charged, zwitterionic, or mixed large unilamellar vesicles (LUVs), to study the interaction of four different synthetically designed cationic, linear antimicrobial peptides: MSI-78 (commercially known as pexiganan), MSI-367, MSI-594, and MSI-843. Our solid-state nuclear magnetic resonance (NMR) experiments confirmed that the MSI peptides fragmented LUVs through a detergent-like carpet mechanism depending on the amino acid sequence of the MSI peptide and/or the membrane composition of LUVs. Interestingly, the fragmented lipid aggregates such as SUVs or micelles are sufficiently small to produce an isotropic peak in the (31)P NMR spectrum. These fragmented lipid aggregates contain only MSI peptides bestowed with lipid molecules as confirmed by NMR in conjunction with circular dichroism spectroscopy. Our results also demonstrate that cholesterol, which is present only in the eukaryotic cell membrane, inhibits the MSI-induced fragmentation of LUVs, suggesting that the MSI peptides can discriminate the bacteria and the eukaryotic cell membranes, and this selectivity could be used for further development of novel antibiotics.