Sylvie E. Blondelle

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity.

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.

Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies.

In the race for supremacy, microbes are sprinting ahead. This warning by the World Health Organization clearly demonstrates that the spread of antibiotic-resistant bacteria leads to a global health problem and that antibiotics never seen before by bacteria are urgently needed. Antimicrobial peptides represent such a source for novel antibiotics due to their rapid lytic activity (within minutes) through disruption of cell membranes. However, due to the similarities between bacterial, fungal, and mammalian plasma cell membranes, a large number of antimicrobial peptides have low lytic specificities and exhibit a broad activity spectrum and/or significant toxic effect toward mammalian cells. Mutation strategies have allowed the development of analogues of existing antimicrobial peptides having greater lytic specificities, although such methods are lengthy and would be more efficient if the molecular mechanisms of action of antimicrobial peptides were clearly elucidated. Synthetic combinatorial library approaches have brought a new dimension to the design of novel biologically active compounds. Thus, a set of peptide analogues were generated based on the screening of a library built around an existing lytic peptide, and on a deconvolution strategy directed toward activity specificity. These peptide analogues also served as model systems to further study the effect of biomembrane mimetic systems on the peptides structural behavior relevant to their biological activities.

Novel antimicrobial compounds identified using synthetic combinatorial library technology

The recent emergence of combinatorial chemistry has greatly advanced the development of biologically active lead compounds. It is anticipated that combinatorial library technology will add great value to the fight against drug-resistant bacterial strains, which pose increasingly serious health hazards. Owing to the need to use complex cell-based assays and, in turn, to screen free compounds in solution, the potential use of combinatorial libraries in the field of infectious diseases has not yet been fully explored. Despite these limitations, a number of new antimicrobial and/or antifungal compounds have been successfully identified from pools of millions of other compounds.

Induced conformational states of amphipathic peptides in aqueous/lipid environments

Specific conformational effects have been reported for amphipathic model peptides upon binding of defined hydrophobic domains to nonpolar stationary phases during reversed-phase high performance liquid chromatography (RP-HPLC). Such induced conformations are found to be especially pronounced for peptides that are amphipathic in an alpha-helical conformation. Such induced amphipathic conformations resulted in substantially later elution than predicted using amino acid-based retention coefficients. In the present study, the induced conformational behavior of model peptides observed during RP-HPLC was correlated with their secondary structure as determined by circular dichroism (CD) spectroscopy in both aqueous solution and C18-mimetic environments. The experimental retention times of the peptides studied were found to correlate with their CD spectra in the presence of lipids, whereas a poor correlation was observed with their CD spectra in the presence of trifluoroethanol. A new approach was developed to evaluate the induction of secondary structure in peptides due to interactions at aqueous/lipid interfaces, which involves the measurement of the CD ellipticities of peptides bound to a set of C18-coated quartz plates. An excellent correlation was found in this environment between the RP-HPLC retention times and CD ellipticities of the bound peptides.

Poly‐(L‐alanine) expansions form core β‐sheets that nucleate amyloid assembly

Expansion to a total of 11–17 sequential alanine residues from the normal number of 10 in the polyadenine‐binding protein nuclear‐1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult‐onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self‐assembly of synthetic poly‐(L‐alanine) peptides having 3–20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length‐dependent but also are determined by concentration, temperature, and incubation time. No β‐sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the β‐sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple‐green birefringence with Congo red; however, like amyloid, X‐ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross‐β arrangement). Neighboring β‐sheets are quarter‐staggered in the hydrogen‐bonding direction such that the alanine side‐chains were closely packed in the intersheet space. Strong van der Waals contacts between side‐chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5–85°C), and pH (2–10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core β‐sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies. Proteins 2005.

Identification of antimicrobial peptides by using combinatorial libraries made up of unnatural amino acids.

The use of water-soluble synthetic peptide combinatorial libraries permits the systematic examination of tens to hundreds of millions of peptides in existing microdilution assays. In the present study, we prepared and determined the antistaphylococcal activities of two new synthetic peptide combinatorial libraries (one N-acetylated, the other not) composed of tetrapeptides having one position defined and the remaining three positions made up of mixtures of L-, D-, and unnatural amino acids (a total of 58 different amino acids). These libraries, when used in conjunction with an iterative selection process, allowed for the development of a series of individually defined tetrapeptides with high levels of activity against Staphylococcus aureus. The activities of the final individual peptides against two additional strains of gram-positive bacteria (methicillin-resistant S. aureus and Streptococcus sanguis), a gram-negative bacterium (Escherichia coli), as well as the yeast Candida albicans were also determined. Cell viability assays showed that the identified peptides are bacteriostatic against both S. aureus and E. coli.

Secondary structure induction in aqueous vs membrane-like environments

The conformational propensity of the 20 naturally occurring amino acids was determined in aqueous 3‐[N‐morpholino]propane‐sulfonic acid (MOPS) buffer, protein interior‐like [nonmicellar sodium dodecylsulfate (SDS)] and membrane‐like environments (micellar SDS and lysophosphatidylglycerol/lysophosphatidylcholine micelles) using a single “guest” position in a polyalanine‐based model host peptide (Ac‐KYA13K‐NH2). This model system allows the intrinsic α‐helical or β‐sheet propensity of the amino acids to be determined without intra‐ and interchain side chain interactions. The overall environment dependence observed for the conformational propensity for the amino acids studied confirms the importance of determining propensity in lipidic environments to better elucidate the biological functions of proteins. The hydrophobic interactions between peptide side chains and lipids appeared to be the primary forces driving the conformational induction in lipidic environments of the model peptides studied. Finally, when comparing the results of these studies with those reported in the literature, the local environment was found to highly influence 65% of the 20 naturally occurring amino acids.

GENERATION AND USE OF NONSUPPORT-BOUND PEPTIDE AND PEPTIDOMIMETIC COMBINATORIAL LIBRARIES

Publisher Summary The practical use of nonsupport-bound combinatorial libraries represents an important breakthrough in all the areas of basic research and drug discovery. The use of a wide variety of chemical transformations permits a range of peptidomimetic libraries to be generated that greatly expands the chemical diversity available. The results described in this chapter demonstrate that an existing peptide positional scanning- synthetic combinatorial libraries (PS-SCL) can be chemically transformed to generate a peptidomimetic SCL, from which highly active individual compounds can be identified. The synthesis and deconvolution methods developed for peptide libraries are easily applied to other types of chemical pharmacophores. The soluble nature of the nonsupport-bound combinatorial libraries is a distinct advantage over the other methods in that membrane-bound and whole cell assays can also be used. In addition, the deconvolution methods used allow the chemical structure of peptidic, peptidomimetic, and organic compounds to be determined based solely on the structural similarities of compounds, within each active pool or sublibrary.

Structural Features Governing the Activity of Lactoferricin-Derived Peptides That Act in Synergy with Antibiotics against Pseudomonas aeruginosa In Vitro and In Vivo

ABSTRACT Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearmans ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives.

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.