Selectivity Modulation and Structure of α/aza-ß3 Cyclic Antimicrobial Peptides.

Potent and selective antimicrobial cyclic pseudopeptides (ACPPs) mixing α- and aza-ß3 -amino acids were developed. Cyclopseudopeptide sequences were designed to investigate the impact of some intrinsic molecular parameters on their biological activities. Fine changes in the nature of the side chains strongly modulated the selectivity of the ACPPs with regard to hemolysis versus antimicrobial activity. The conformational preference of such compounds in various media was extensively studied, and the typical structure of cyclic α/aza-ß3 -pseudopeptides is described for the first time. Interestingly, such scaffolds are stabilized by successive inverse γ- and N-N turns (hydrazino turns), a unique feature due to the aza-ß3 residues. The α-amino acid side chains form a cluster on one face of the ring, while the aza-ß3 -amino acid side chains are projected around the ring in the equatorial orientation. Such structural data are particularly valuable to fine-tune the bioactivity of these ACPPs by a structure-based approach.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

Human erythrocytes covered with magnetic core-shell nanoparticles for multimodal imaging.

Surface functionalization of human red blood cells (hRBCs) with fluorescent and magnetic silica core-shell nanoparticles is used to design a carrier suitable for multimodal imaging with a long circulating time. The coated magnetic hRBCs show no hemolytic activity, while the advantage of the affinity of proteins for silica allows a further coating.

De novo cyclic pseudopeptides containing aza-ß3-amino acids exhibiting antimicrobial activities.

De novo cyclic pseudopeptides composed of α-amino and aza-ß(3)-amino acids were designed with the aim to obtain potential new antimicrobial agents. Antimicrobial cyclic pseudopeptides (ACPPs) are based on the properties of antimicrobial peptides (AMPs), so they are cationic and amphiphilic. Aza-ß(3)-amino acids enhance the in vivo half-life of these compounds and offer the possibility to incorporate a large variety of side chains. Most of the 13 ACPPs exert antimicrobial activities in rich media with broad spectrum of antibacterial activities. Selectivity for bacterial over mammalian cells was determined by testing the hemolytic activities of ACPPs against sheep red blood cells (sRBC). We examined the ratio of cationic to hydrophobic residues as well as the type of hydrophobic side chains essential for biological activity of this class of ACPPs. These results will be useful for designing potential candidates for a therapeutic application.

From a marine neuropeptide to antimicrobial pseudopeptides containing aza-ß(3)-amino acids: structure and activity.

Incorporation of aza-ß(3)-amino acids into an endogenous neuropeptide from mollusks (ALSGDAFLRF-NH(2)) with weak antimicrobial activity allows the design of new AMPs sequences. Depending on the nature of the substitution, this can render the pseudopeptides inactive or lead to a drastic enhancement of the antimicrobial activity without high cytotoxicity. Structural studies of the pseudopeptides carried out by NMR and circular dichroism show the impact of aza-ß(3)-amino acids on peptide structure. The first three-dimensional structures of pseudopeptides containing aza-ß(3)-amino acids in aqueous micellar SDS were determined and demonstrate that the hydrazino turn can be formed in aqueous solution. Thus, AMP activity can be modulated through structural modifications induced by the nature and the position of such amino acid analogues in the peptide sequences.

Structure and mechanism of action of a de novo antimicrobial detergent-like peptide.

The K4 peptide (KKKKPLFGLFFGLF) was recently demonstrated to display good antimicrobial activities against various bacterial strains and thus represents a candidate for the treatment of multiple-drug resistant infections. In this study, we use various techniques to study K4 behaviour in different media: water, solutions of detergent micelles, phospholipid monolayers and suspension of phospholipid vesicles. First, self-assembly of the peptide in water is observed, leading to the formation of spherical objects around 10nm in diameter. The addition of micelles induces partial peptide folding to an extent depending on the charge of the detergent headgroups. The NMR structure of the peptide in the presence of SDS displays a helical character of the hydrophobic moiety, whereas only partial folding is observed in DPC micelles. This peptide is able to destabilize the organization of monolayer membranes or bilayer liposomes composed of anionic lipids. When added on small unilamellar vesicles it generates larger objects attributed to mixed lipid-peptide vesicles and aggregated vesicles. The absence of calcein leakage from liposomes, when adding K4, underlines the original mechanism of this linear amphipathic peptide. Our results emphasize the importance of the electrostatic effect for K4 folding and lipid destabilization leading to the microorganisms' death with a high selectivity for the eukaryotic cells at the MIC. Interestingly, the micrographs obtained by electronic microscopy after addition of peptide on bacteria are also consistent with the formation of mixed lipid-peptide objects. Overall, this work supports a detergent-like mechanism for the antimicrobial activity of this peptide.

Interactions between giant unilamellar vesicles and charged core-shell magnetic nanoparticles.

This work combined two tools, giant unilamellar vesicles (GUVs) and core-shell magnetic nanoparticles (CSMNs), to develop a simplified model for studying interactions between the cell membrane and nanoparticles. We focused on charged functionalized CSMNs that can be either cationic or anionic. Using optical, electron, and confocal microscopy, we found that giant vesicle-nanoparticle interactions did not result from a simple electrostatic phenomenon because cationic CSMNs tended to bind to positively charged bilayers, whereas anionic CSMNs remained inert.

KKKKPLFGLFFGLF: a cationic peptide designed to exert antibacterial activity.

With 14 residues organized as two domains linked by a single proline, the de novo peptide called K4 was designed, using Antimicrobial Peptide Database, to exert antibacterial activity. The N-terminal domain is composed of four lysines enhancing membrane interactions, and the C-terminal domain is putatively folded into a hydrophobic alpha-helix. Following the synthesis, the purification and the structural checking, antibacterial assays revealed a strong activity against gram-positive and gram-negative bacteria including human pathogenic bacteria such as Staphylococcus aureus and some marine bacteria of the genus Vibrio. Scanning electron microscopy of Escherichia coli confirmed that K4 lyses bacterial cells. The cytotoxicity was tested against rabbit erythrocytes and chinese hamster ovary cells (CHO-K1). These tests revealed that K4 is non-toxic to mammalian cells for bacteriolytic concentrations. The peptide K4 could be a valuable candidate for future therapeutic applications.

Allosteric monofunctional aspartate kinases from Arabidopsis.

Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form.

A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase.

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine-sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III.