Antimicrobial benzodiazepine‐based short cationic peptidomimetics

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic ‘back’, and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well‐known antimicrobial peptide MSI‐78. In contrast to MSI‐78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A novel cysteine‐free venom peptide with strong antimicrobial activity against antibiotics‐resistant pathogens from the scorpion Opistophthalmus glabrifrons

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic‐resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α‐helical molecule. Protein sequence homology search revealed that Opisin shares 42.1–5.3% sequence identities to the 17/18‐mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram‐positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0–10.0 μM; in contrast, it possesses much lower activity against the tested Gram‐negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin‐ and vancomycin‐resistant pathogens with the MICs ranging from 2.0 to 4.0 μM and from 4.0 to 6.0 μM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin‐resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic‐resistant bacterial infections. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

IL‐18 Cytokine Levels Modulate Innate Immune Responses and Cryptosporidiosis in Mice

IL‐18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL‐18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment with recombinant IL‐18 (rIL‐18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN‐γ levels as well as the up‐regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 (Defb3) were observed in the intestinal mucosa of mice treated with rIL‐18. In addition, C. parvum infection significantly increased mRNA expression levels (> 50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in mRNA expression of IL‐33 (a recently identified cytokine in the same family as IL‐18) in the small intestinal tissue from mice treated with rIL‐18. In comparison, the respective genes were induced by IL‐18 depletion. Our findings suggest that IL‐18 can mediate its protective effects via different routes such as IFN‐γ induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity.     

Microwave‐assisted solid‐phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C‐terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80‐amino acid peptide sequence of rat NPGL by microwave‐assisted solid‐phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione‐containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

N‐terminal guanidinylation of the cyclic 1,4‐ureido‐deltorphin analogues: the synthesis,receptor binding studies,and resistance to proteolytic digestion

The synthesis of a series of N‐guanidinylated cyclic ureidopeptides, analogues of 1,4‐ureido‐deltorphin/dermorphine tetrapeptide is described. The δ‐ and μ‐opioid receptor affinity of new guanidinylated analogues and their non‐guanidinylated precursors was determined by the displacement radioligand binding experiments. Our results indicate that the guanidinylation of cyclic 1,4‐ureidodeltorphin peptide analogues does not exhibit a uniform influence on the opioid receptor binding properties, similarly as reported earlier for some linear peptides. All analogues were also tested for their in vitro resistance to proteolysis during incubation with large excess of chymotrypsin, pepsin, and papain by means of mass spectroscopy. Guanidinylated ureidopeptides 1G–4G showed mixed μ agonist/δ agonist properties and high enzymatic stability indicating their potential as therapeutic agents for treatment of pain. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

β‐Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β‐hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template‐based approach was used to create therapeutically valuable analogs of arenicin‐1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin‐1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non‐polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50‐fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin‐1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin‐1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Towards broadly protective polyvalent vaccines against hand,foot and mouth disease

Hand, foot, and mouth disease (HFMD) caused by multiple enterovirus infections is a serious health threat to children in the Asia–Pacific region. This article reviews progresses in the development of vaccines for HFMD and discusses the need for polyvalent HFMD vaccines for conferring broad-spectrum protection.