Synthesis and membrane action of polymyxin B analogues

Abstract

We have designed synthetic peptides that mimic the primary and secondary structure of the cationic lipopeptide antibiotic polymyxin B (PxB) in order to determine the structural requirements for membrane action and to assess possible therapeutic potential. Two analogues with related sequences to that of PxB, but including synthetic simplifications (disulphide bridge between two cysteines in positions 4 and 10, N-terminal nonanoic acid), have been synthesized. Peptide-lipid interactions have been studied by fluorescence resonance energy transfer between pyrene and 4,4-difluoro-5-methyl-4-bora-3alpha,4alpha-diaza-s-indacene-3-dodecanoyl (BODIPY)probes covalently linked to phospholipids, and the possibility of membrane disruption or permeabilization has been assessed by light scattering and fluorescence quenching assays. The synthetic peptide sP-B, which closely mimics the primary and secondary structures of PxB, binds to vesicles of anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or of lipids extracted from Escherichia coli membranes, and induces apposition of the vesicles and selective lipid exchange without permeabilization of the membrane. We conclude that sP-B forms functional vesicle-vesicle contacts that are selective, as previously described for PxB. The second analogue, sP-C, has a permutation of two amino acids that breaks the hydrophobic patch formed by D-Phe and Leu residues on the cyclic part of the sequence. sP-C lipopeptide is more effective than sP-B in inducing lipid mixing, but shows no selectivity for the lipids that exchange through the vesicle-vesicle contacts, and at high concentrations has a membrane-permeabilizing effect. The deacylated and non-antibiotic derivative PxB-nonapeptide (PxB-NP) does not induce the formation of functional intervesicle contacts in the range of concentrations studied.

Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity

“…In fact, the hydrophobic moiety of CAAs is responsible for their insertion into the hydrophobic core of the bacterial lipid bilayer, leading to membrane permeabilisation and eventually to CAAs-mediated bacterial killing as convincingly indicated by the lack of membrane permeabilisation of a deacylated polymyxin analogue (Vaara and Vaara, 1983;Tsubery et al, 2000Tsubery et al, , 2001Tsubery et al, , 2002Katz et al, 2003;Clausell et al, 2005Clausell et al, , 2007.…”

“…Whereas the interaction of other CAAs with biological membranes has been the subject of numerous studies, particularly with polymyxin B (PMB) (Chen and Feingold, 1972;Clausell et al, 2004Clausell et al, , 2005Clausell et al, , 2007Hartmann et al, 1978;Katsu et al, 1984Katsu et al, , 2002Katz et al, 2003;Galla, 1981, 1982;Tsubery et al, 2000Tsubery et al, , 2001Tsubery et al, , 2002Wiese et al, 1998Wiese et al, , 2003, relatively little information is available regarding the interaction of SQ with cellular membranes (Chen et al, 2006;Salmi et al, 2008a;Selinsky et al, 1998Selinsky et al, , 2000.…”

“…CAAs include cationic surfactants, cyclic lipopeptides, ␤-sheet and ␣-helix-forming peptides and cholesterol derivatives (Savage et al, 2002). The antibacterial activity of CAAs relies on their ability to interact with bacterial lipids, especially lipopolysaccharide (LPS) and phosphatidylglycerol (PG), resulting in: (i) the release of divalent cations bridging LPS molecules together, (ii) a change in the outer membrane permeability barrier, (iii) the release of membrane vesicle, (iv) membrane lipid exchange, which in turn causes membrane permeabilisation and bacteria killing (Katsu et al, 1984(Katsu et al, , 2002Savage et al, 2002;Clausell et al, 2004Clausell et al, , 2005Clausell et al, , 2007McBroom and Kuehn, 2007). Binding of CAAs to bacterial membranes involves both electrostatic and hydrophobic interactions (Savage et al, 2002).…”

Insights into the structure of the PmrD protein with molecular dynamics simulations

“…(1), where x m , y m , z m are the Cartesian coordinates found at the NMR structure and x t , y t , z t are the Cartesian coordinates of trajectory frame t. N is the number of atoms:…”

“…Two separate two-component regulatory systems (TCRs), PmrA-PmrB and PhoP-PhoQ are known to be involved in the resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human neutrophils [1][2][3]. Transcription of PmrA-activated genes is promoted by either of two pathways: (i) growth in low extracellular magnesium in a process that requires PhoP-PhoQ, the second two-component regulatory system [4] and (ii) growth in the presence of high iron or acidic pH.…”