Gram-Negative Outer and Inner Membrane Models: Insertion of Cyclic Cationic Lipopeptides Adria ` Clausell, † Maria Garcia-Subirats, ‡ Montserrat Pujol, † M. Antonia Busquets, † Francesc Rabanal, ‡ and Yolanda Cajal* ,† Physical Chemistry Department and Institute of Nanoscience and Nanotechnology, UniVersity of Barcelona, AV. Joan XXIII s/n, 08028 Barcelona, Spain, and Department of Organic Chemistry, UniVersity of Barcelona, Martı ´ i Franque ` s 1, 08028 Barcelona, Spain ReceiVed: July 26, 2006; In Final Form: October 18, 2006 Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram- negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn- 3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/ area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [D-Phe 6 -Leu 7 ] into [D-Phe 6 -Dab 7 ], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where L-R,γ-diaminobutyric acid residues Dab 1 and Dab 8 are replaced by L-R,γ-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB- np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity. Introduction The global emergence of multidrug-resistant bacteria is an important clinical problem that is increasingly limiting the use of currently known antibiotics. There is a great demand to find new drugs to overcome this problem, but experience has proven that the time to develop resistance to new antibiotics is often very short. This is essentially related to the mechanism of action of traditional antibiotics, with five main specific targets in the cell: cell-wall synthesis, DNA gyrase, metabolic enzymes, DNA-directed RNA polymerase, and protein synthesis. 1 Bacteria can develop genetic resistance toward these mechanisms of action at a rate that depends on many factors, such as the number of targets. Antimicrobial peptides (AMPs) are a class of antibiotics that rarely spur the development of resistant microorganisms. AMPs are secreted by many living organisms in response to infection by Gram-negative and Gram-positive bacteria. 2-4 They are a diverse group of molecules, with more than 800 described so far. 5 Despite their diversity, the main target of most AMPs is the lipid bilayer itself, without any stereospecific interaction with chiral receptors or enzymes. 2,6 To develop genetic resis- tance is therefore very costly for the bacteria. Polymyxin B (PxB) is a nonribosomally synthesized AMP produced by Bacillus polymyxa, with selectivity against Gram- negative bacteria, including multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa strains. 7-10 As do most AMPs, PxB has a highly amphipathic nature, an important feature for membrane interaction. A second important charac- teristic shared by most AMPs is a net cationic charge at physiological pH, with five positive charges due to L-R,γ- diaminobutyric acid (Dab) in the case of PxB. The cationic character of PxB facilitates its preferential interaction with the bacterial membranes, rich in anionic phospholipids such as phosphatidylglycerol and cardiolipin. In contrast, the outer leaflet of mammalian cell membranes mainly comprises phosphati- dylcholine and cholesterol, which are charge-neutral at physi- ological pH. The mechanism of action of PxB involves the inner and outer membranes of the Gram-negative bacteria. PxB binds to the anionic surface molecule lipopolysaccharide (LPS) in the outer membrane (OM), leading to self-promoted uptake across the OM 11 and subsequently reaching the periplasmic space and interacting with the cytoplasmic membrane. 12 The mechanism of bacterial killing is clearly not related to membrane perme- ation, which takes place at concentrations well above the minimal inhibitory concentration (MIC). 7,11-13 The actual mech- * Corresponding author: phone 34-93-4035988; fax 34-93-4035987; e-mail ycajal@ub.edu. † Physical Chemistry Department and Institute of Nanoscience and Nanotechnology. ‡ Department of Organic Chemistry. 551 J. Phys. Chem. B 2007, 111, 551-563 10.1021/jp064757+ CCC: $37.00 © 2007 American Chemical Society Published on Web 12/23/2006