No abstract

Daptomycin is a clinically important lipopeptide antibiotic that kills Gram-positive bacteria through membrane depolarization. Its activity requires calcium and the presence of phosphatidylglycerol in the target membrane. Calcium and phosphatidylglycerol also promote the formation of daptomycin oligomers, which have been assumed but not proven to be required for the bactericidal effect. Daptomycin shares substantial structural similarity with another lipopeptide antibiotic, A54145; the two have identical amino acid residues in 5 out of 13 positions and similar ones in 4 more positions. We here examined whether these conserved residues are sufficient for oligomer formation. To this end, we used fluorescence energy transfer and excimer fluorescence to detect hybrid oligomers of daptomycin and CB-182,462, a semisynthetic derivative of A54145. Mixtures of the two compounds indeed produced hybrid oligomers, but at the same time displayed a significantly less than additive antibacterial activity against Bacillus subtilis. The existence of functionally impaired oligomers indicates that oligomer formation is indeed important for antibacterial function. However, it also shows that oligomerization is not sufficient; once formed, the oligomers must take another step in order to acquire antibacterial activity. Thus, the amino acid residues shared between daptomycin and CB-182,462 suffice for formation of the oligomer, but not for its subsequent activation.

The accumulated evidence has shown that lipids and polymers each have distinct advantages as carriers for siRNA delivery. Composite materials comprising both lipids and polymers may present improved properties that combine the advantage of each. Cationic amphiphilic macromolecules (CAMs) containing a hydrophobic alkylated mucic acid segment and a hydrophilic poly(ethylene glycol) (PEG) tail were non-covalently complexed with two lipids.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), to serve as a siRNA delivery vehicle. By varying the weight ratio of CAM to lipid, cationic complexes with varying compositions were obtained in aqueous media and their properties evaluated. CAM-lipid complex sizes were relatively independent of composition, ranging from 100 to 200 nm, and zeta potentials varied from 10 to 30 mV. Transmission electron microscopy confirmed the spherical morphology of the complexes. The optimal N/P ratio was 50 as determined by electrophoretic mobility shift assay. The ability to achieve gene silencing was evaluated by anti-luciferase siRNA delivery to a U87-luciferase cell line. Several weight ratios of CAM-lipid complexes were found to have similar delivery efficiency compared to the gold standard, Lipofectamine. Isothermal titration calorimetry revealed that siRNA binds more tightly at pH = 7.4 than pH = 5 to CAM-lipid (1:10 w/w). Further intracellular trafficking studies monitored the siRNA escape from the endosomes at 24 h following transfection of cells. The findings in the paper indicate that CAM-lipid complexes can serve as a novel and efficient siRNA delivery vehicle.

Cation-π interactions of methylated ammonium ions play a key role in a broad range of biochemical systems. These include methyl-lysine binding proteins which bind to methylated sites on histone proteins, lysine demethylase enzymes which demethylate these sites, and neurotransmitter receptor complexes which bind choline derived ligands. Recognition in these systems is achieved through an ‘aromatic cage’ motif in the binding site. Here we use high level quantum mechanical calculations to address how cation-π interactions of methylated ammonium ions are modulated by a change in methylation state and interaction geometry. We survey methyl-lysine and choline derived complexes in the Protein Databank to validate our results against available structural data. A quantitative description of cation-π interactions of methylated ammonium systems is critical to structure-based efforts to target methyl-lysine binding proteins and demethylase enzymes in the treatment of unregulated transcriptional control, and neurotransmitter receptors in the treatment of neurological disease. It is our hope that our work will serve as a benchmark for the development of physical chemistry based force fields that can accurately model the contribution of cation-π interactions to binding and specificity in these systems.

The effects of three acidic hexapeptides on in vitro hydroxyapatite growth were characterized by pH-stat kinetic studies, adsorption isotherms, and molecular modeling. The three peptides, pSDEpSDE, SDESDE, and DDDDDD, are equal-length model compounds for the acidic sequences in osteopontin, a protein that inhibits mineral formation in both calcified and noncalcified tissues. Growth rates from 1.67 mM calcium and 1.00 mM phosphate solution were measured at pH 7.4 and 37 degrees C in 150 mM NaCl. pSDEpSDE was a strong growth inhibitor when preadsorbed onto hydroxyapatite (HA) seeds from > or = 0.67 mM solutions, concentrations where adsorption isotherms showed relatively complete surface coverage. The nonphosphorylated SDESDE control showed no growth inhibition. Although it adsorbed to almost the same extent as pSDEpSDE, it rapidly desorbed under the pH-stat growth conditions while pSDEpSDE did not. DDDDDD exhibited weak inhibition as its concentration was increased and similar adsorption/desorption behavior to pSDEpSDE. Molecular modeling yielded binding energy trends based on simple adsorption of peptides on the [100] surface that were consistent with observed inhibition, but not for the [001] surface. The relatively unfavorable binding energies for peptides on the [001] surface suggest that their absorption will be primarily on the [100] face. The kinetic and adsorption data are consistent with phosphorylation of osteopontin acting to control mineral formation.

Surfactant amphiphilic macromolecules (AMs) were complexed with a 1:1 ratio of 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), either by a coevaporation (CE) or postaddition (PA) method, to form AM–lipid complexes with enhanced drug delivery applications. By characterizing the surfactant–lipid interactions, these heterogeneous drug delivery systems can be better controlled and engineered for optimal therapeutic outcomes. In this study, the physical interactions between DOPE:DOTAP liposomes and AM surfactants were investigated. Langmuir fllm balance and isothermal calorimetry studies showed cooperative intermolecular interactions between pure lipids and AM in monolayers and high thermostability of structure formed by the addition of AM micelles to DOTAP: DOPE vesicles in buffer solution respectively. Increasing the AM weight ratio in the complexes via the CE method led to complete vesicle solubilization—from lamellar aggregates, to a mixture of coexisting vesicles and micelles, to mixed micelles. Isothermal calorimetry evaluation of AM-lipid complexes shows that, at higher AM weight ratios, PA-produced complexes exhibit greater stability than complexes at lower AM weight ratios. Similar studies show that AM-lipid complexes produced by the CE methods display stronger interactions between AM-lipid components than complexes produced by the PA method. The results suggest that the PA method produces vesicles with AM molecules associated with its outer leaflet only (i.e., an AM-coated vesicle), while the CE method produces complexes ranging from mixed vesicles to mixed micelle in which the AM-lipid components are more intimately associated. These results will be helpful in the design of AM-lipid complexes as structurally defined, stable, and effective drug delivery systems.

Daptomycin is an anionic membrane active antimicrobial lipopeptide used to treat serious infections caused by gram-positive bacteria. It causes target membrane depolarization by forming oligomeric pores that allow leakage of potassium ions, but the complete mechanism of action is unresolved. Antibiotic function is calcium dependant and requires the presence of anionic phospholipids like phosphatidylglycerol (PG) in the target membrane. Because of its efficacy in treating infections resistant to many front-line antibiotics, the recent emergence of strains displaying reduced daptomycin susceptibility is troubling. These quasi-resistant strains have modified membrane lipid content, including an increased presence of cardiolipin (CL) and lysyl-PG, the latter possibly due to the observed up-regulation of mprF, a gene that codes for a protein with lysyl-PG synthase and flippase activity. Using monolayer and bilayer model systems, we sought here to study the effects of the presence of CL on daptomycin binding to PG-containing membranes. Surprisingly, isothermal titration calorimetry (ITC) revealed that daptomycin-membrane affinity increased when small amounts of CL was present and continued to increase until 10 mol%, above which the trend reversed drastically. Results from Langmuir monolayer insertion experiments also show that CL affects the degree of drug insertion into PG-containing lipid films as determined by greater increases in surface pressure after daptomycin was injected into the aqueous subphase when CL was present. Preliminary results from monolayer insertion experiments in which lysyl-PG was included in the films indicate that this lipid also significantly affects drug-monolayer interactions. We hypothesize from these results that alteration of the lipid content of bacterial membranes represents an important component of the potential resistance mechanism and should be considered in the development and formulation of the next generation of membrane targeting antibiotics.

that peptides in the I-state form pores, while peptides that remain in the S-state use the carpet model of membrane disruption. Here, OCD is used to investigate the orientation and threshold concentration of Piscidin 1 and Piscidin 3 (P3) in bacterial and mammalian-mimicking lipid systems and thereby determine the mechanism of action of P1 and P3 in these lipid systems. Both peptides are a 22-residue alpha-helical AMPs isolated from the mast cells of hybrid striped sea bass. P1 is both more antimicrobial and hemolytic than P3. We hypothesize that the two peptides behave differently in bacterial versus human cells due to the differences in membrane composition and that P1 initiates its activity at a lower threshold concentration. Mammalian and bacterial membrane mimics have been made using 4:1 PC/CHL (phosphocholine and cholesterol, respectively) and 3:1 PC/PG (phosphoglycerol). The bilayer orientations of piscidin have been investigated over a large range of P/L ratios using OCD. Membrane thinning was studied by x-ray. These studies provide insight into the mechanism of action of an important class of AMPs and may help provide design principles for new drug candidates. 1236-Pos Board B128Surface and Membrane Binding Properties of the Lipopeptide Daptomycin Evan Mintzer, Nasim Tishbi. Stern College for Women, New York, NY, USA. Daptomycin, an antimicrobial lipopeptide used to treat infections caused by Gram-positive bacteria that are resistant to many conventional therapies, acts through calcium-mediated binding to and rapid depolarization of the target bacterial membrane. Convincing evidence has recently been reported suggesting that small daptomycin oligomers form at the membrane surface and that these complexes represent the active state of the drug. Daptomycin's activity is closely correlated with the presence of phosphatidylglycerol (PG) in the target membrane. Although there have no cases of clinical resistance to daptomycin reported, troubling signs are emerging indicating that changes in lipid composition of bacterial membranes cause decreased susceptibility to the drug. It is therefore of interest to gain a more profound understanding of the details of daptomycin's mechanism of activity at the membrane level and the possible causes of potential resistance and their relationship to lipid composition. In the current study, we report on our investigation into the surface and membrane binding properties of daptomycin. From the Gibb's adsorption isotherm, we estimate the molecular area of daptomycin at the air-aqueous interface. Using Langmuir monolayers as membrane models, we also report limiting surface pressures and kinetics for daptomycin insertion to lipid films comprised of pure PG or PG-phosphatidylcholine mixtures. Finally, we attempt to correlate daptomycin's binding behavior in monolayers to that in bilayers, in the form of unilamellar vesicles, by presenting results from isothermal titration experiments. The results represent, for the first time, thermodynamic binding parameters for daptomycin-membr...