Design, synthesis and preliminary evaluation of peptidomimetic inhibitorsof HIV aspartic protease with an epoxyalcohol core

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The dolphin proline-rich antimicrobial peptide Tur1A inhibits protein synthesis by targeting the bacterial ribosome

Proline-rich antimicrobial peptides (PrAMPs) internalize into susceptible bacteria using specific transporters and interfere with protein synthesis and folding. To date, mammalian PrAMPs have so far only been identified in artiodactyls. Since cetaceans are co-phyletic with artiodactyls, we mined the genome of the bottlenose dolphin Tursiops truncates, leading to the identification of two PrAMPs, Tur1A and Tur1B. Tur1A, which is orthologous to the bovine PrAMP Bac7, is internalized into E. coli without damaging the membranes using the inner membrane transporters SbmA and YjiL/MdM. Furthermore, like Bac7, Tur1A also inhibits bacterial protein synthesis by binding to the ribosome and blocking the transition from the initiation to the elongation phase. By contrast, Tur1B is a poor inhibitor of protein synthesis and may utilize another mechanism of action. An X-ray structure of Tur1A bound within the ribosomal exit tunnel provides a basis to develop these peptides as novel antimicrobial agents

DAMPD: a manually curated antimicrobial peptide database

The demand for antimicrobial peptides (AMPs) is rising because of the increased occurrence of pathogens that are tolerant or resistant to conventional antibiotics. Since naturally occurring AMPs could serve as templates for the development of new anti-infectious agents to which pathogens are not resistant, a resource that contains relevant information on AMP is of great interest. To that extent, we developed the Dragon Antimicrobial Peptide Database (DAMPD, http://apps.sanbi.ac.za/dampd) that contains 1232 manually curated AMPs. DAMPD is an update and a replacement of the ANTIMIC database. In DAMPD an integrated interface allows in a simple fashion querying based on taxonomy, species, AMP family, citation, keywords and a combination of search terms and fields (Advanced Search). A number of tools such as Blast, ClustalW, HMMER, Hydrocalculator, SignalP, AMP predictor, as well as a number of other resources that provide additional information about the results are also provided and integrated into DAMPD to augment biological analysis of AMPs

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

Background: Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. Methodology and Principal Findings: Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various "superbugs'' including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. Conclusions/Significance: Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise "tuning'' of toxicity and proteolytic stability may be achieved by changing tag-length and adding W-or F-amino acid tags

Prediction of Antibacterial Activity from Physicochemical Properties of Antimicrobial Peptides

Consensus is gathering that antimicrobial peptides that exert their antibacterial action at the membrane level must reach a local concentration threshold to become active. Studies of peptide interaction with model membranes do identify such disruptive thresholds but demonstrations of the possible correlation of these with the in vivo onset of activity have only recently been proposed. In addition, such thresholds observed in model membranes occur at local peptide concentrations close to full membrane coverage. In this work we fully develop an interaction model of antimicrobial peptides with biological membranes; by exploring the consequences of the underlying partition formalism we arrive at a relationship that provides antibacterial activity prediction from two biophysical parameters: the affinity of the peptide to the membrane and the critical bound peptide to lipid ratio. A straightforward and robust method to implement this relationship, with potential application to high-throughput screening approaches, is presented and tested. In addition, disruptive thresholds in model membranes and the onset of antibacterial peptide activity are shown to occur over the same range of locally bound peptide concentrations (10 to 100 mM), which conciliates the two types of observations

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion

End-Tagging of Ultra-Short Antimicrobial Peptides by W/F Stretches to Facilitate Bacterial Killing

BACKGROUND: Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility. METHODOLOGY AND PRINCIPAL FINDINGS: A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli. CONCLUSIONS/SIGNIFICANCE: End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications

Optically pure, water-stable metallo-helical ‘flexicate’ assemblies with antibiotic activity

The helicates—chiral assemblies of two or more metal atoms linked by short or relatively rigid multidentate organic ligands—may be regarded as non-peptide mimetics of α-helices because they are of comparable size and have shown some relevant biological activity. Unfortunately, these beautiful helical compounds have remained difficult to use in the medicinal arena because they contain mixtures of isomers, cannot be optimized for specific purposes, are insoluble, or are too difficult to synthesize. Instead, we have now prepared thermodynamically stable single enantiomers of monometallic units connected by organic linkers. Our highly adaptable self-assembly approach enables the rapid preparation of ranges of water-stable, helicate-like compounds with high stereochemical purity. One such iron(II) ‘flexicate’ system exhibits specific interactions with DNA, promising antimicrobial activity against a Gram-positive bacterium (methicillin-resistant Staphylococcus aureus, MRSA252), but also, unusually, a Gram-negative bacterium (Escherichia coli, MC4100), as well as low toxicity towards a non-mammalian model organism (Caenorhabditis elegans)

iTRAQ-Coupled 2-D LC-MS/MS Analysis of Membrane Protein Profile in Escherichia coli Incubated with Apidaecin IB

Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the Gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins—apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides

Expression and Activity of a Novel Cathelicidin from Domestic Cats

Cathelicidins are small cationic antimicrobial peptides found in many species including primates, mammals, marsupials, birds and even more primitive vertebrates, such as the hagfish. Some animals encode multiple cathelicidins in their genome, whereas others have only one. This report identifies and characterizes feline cathelicidin (feCath) as the sole cathelicidin in domestic cats (Felis catus). Expression of feCath is predominantly found in the bone marrow, with lower levels of expression in the gastrointestinal tract and skin. By immunocytochemistry, feCath localizes to the cytoplasm of neutrophils in feline peripheral blood. Structurally, the mature feCath sequence is most similar to a subgroup of cathelicidins that form linear α-helices. feCath possesses antimicrobial activity against E. coli D31, Salmonella enterica serovar Typhimurium (IR715), Listeria monocytogenes and Staphylococcus pseudintermedius (clinical isolate) similar to that of the human ortholog, LL-37. In contrast, feCath lacks the DNA binding activity seen with LL-37. Given its similarity in sequence, structure, tissue expression, and antimicrobial activity, the cathelicidin encoded by cats, feCath, belongs to the subgroup of linear cathelicidins found not only in humans, but also non-human primates, dogs, mice, and rats