Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire‐bellied toad,Bombina orientalis

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian‐derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin‐like peptide (BLP‐7) and a novel bombinin H‐type peptide (named as Bombinin H‐BO) from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor‐encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α‐helical structural features. The synthetic replicate of BLP‐7 displayed more potent antimicrobial activity than Bombinin H‐BO against Gram‐positive and Gram‐negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK‐HEP‐1/Huh7) at the non‐toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti‐infection and anticancer therapy.     

Bioactivities of Jc‐SCRIP,a Type 1 Ribosome‐Inactivating Protein from Jatropha curcas Seed Coat

In this study, a type 1 RIP, designated as Jc‐SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE‐Sephacel? and CM‐cellulose columns. Purification fold of Jc‐SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI‐TOF/MS. It exhibited hemagglutination activity and possessed strong N‐glycosidase activity. The antimicrobial activity of Jc‐SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm . Jc‐SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF‐7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC₅₀ values of 0.15, 0.25, and 0.40 mm , respectively. The results suggested that Jc‐SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.     

Structural Factors Affecting Cytotoxic Activity of (E)‐1‐(Benzo[d ][1,3]oxathiol‐6‐yl)‐3‐phenylprop‐2‐en‐1‐one Derivatives

Derivatives of (E)‐1‐(5‐alkoxybenzo[d][1,3]oxathiol‐6‐yl)‐3‐phenylprop‐2‐en‐1‐one 1 demonstrated exceptionally high in vitro cytotoxic activity, with IC₅₀ values of the most active derivatives in the nanomolar range. To identify structural fragments necessary for the activity, several analogs deprived of selected fragments were prepared, and their cytotoxic activity was tested. It was found that the activity depends on combined effects of (i) the heterocyclic ring, (ii) the alkoxy group at position 5 of the benzoxathiole ring, and (iii) the substituents in the phenyl ring B. Replacement of the sulfur atom by oxygen does not influence the activity. None of the listed structural fragments alone assured high cytotoxic activity.     

Antibacterial activities and molecular mechanism of amino‐terminal fragments from pig nematode antimicrobial peptide CP‐1

High manufacturing costs and weak cell selectivity have limited the clinical application of naturally occurring peptides when faced with an outbreak of drug resistance. To overcome these limitations, a set of antimicrobial peptides was synthesized with the general sequence of (WL)n, where n = 1, 2, 3, and WL was truncated from the N‐terminus of Cecropin P1 without initial serine residues. The antimicrobial peptide WL3 exhibited stronger antimicrobial activity against both Gram‐negative and Gram‐positive microbes than the parental peptide CP‐1. WL3 showed no hemolysis even at the highest test concentrations compared to the parental peptide CP‐1. The condition sensitivity assays (salts, serum, and trypsin) demonstrated that WL3 had high stability in vitro. Fluorescence spectroscopy and electron microscopy indicated that WL3 killed microbes by means of penetrating the membrane and causing cell lysis. In a mouse model, WL3 was able to significantly reduce the bacteria load in major organs and cytokines (TNF‐α, IL‐6, and IL‐1β) levels in serum. In summary, these findings suggest that WL3, which was modified from a natural antimicrobial peptide, has enormous potential for application as a novel antibacterial agent.     

Design,synthesis, and antimicrobial activities of novel functional peptides against Gram‐positive and Gram‐negative bacteria

The extensive use of antibiotics in medicine results in the multidrug resistance of bacteria, making the development of new antimicrobial agents an urgent need. Antimicrobial peptides (AMPs) are considered as a new class of antibiotic with characteristics including an ability to kill target cells rapidly and a broad spectrum of activity. We have developed a potent antimicrobial peptide MAP‐0403 (MIC = 5 μM), but it exhibited a high hemolytic side‐effect (70.7%). To reduce its hemolytic effect and increase antimicrobial activity, three peptides derivatives of MAP‐0403 (J‐1, J‐2, and J‐3) were designed, synthesized by solid phase synthesis, purified by RP‐HPLC, and characterized by MALDI‐TOF MS. Structure–activity relationships of these peptides were studied by using circular dichroism and antimicrobial assays. The percentage of helical structure in J‐1, J‐2, and J‐3 was lower than that of MAP‐0403. The antimicrobial activity of J‐1 was the same as that of MAP‐0403 (MIC = 5 μM), J‐2 exhibited the highest activity (MIC = 2.5 μM), while J‐3 showed decreased activity (MIC = 10 μM). Compared to MAP‐0403, J‐2 showed significantly reduced hemolytic effect (3.4%), while J‐1 and J‐3 showed slightly decreased hemolytic effect (46.2%, 55.6%, respectively). Peptide J‐2 was discovered as a novel and potent antimicrobial agents.     

Effect of Chain Elongation on Biological Properties of the Toxin Paralysin β‐Alanyl‐tyrosine

In hemolymph of insect species, compounds with remarkable properties for pharmaceutical industry are present. At the first line, there were found compounds of low molecular mass, less than 1 kDa. One of such compounds, β‐alanyl‐tyrosine (252 Da), was isolated from larval hemolymph of some species of holometabolous insects (e.g. Neobellieria bullata). Its paralytic activity and antimicrobial properties were described until now. In this study, we present the effect of elongation of β‐alanyl‐tyrosine by repeating of this motive on the biological and physical properties of prepared analogues. For assessment of antimicrobial properties of these new compounds strains of Gram‐positive, Gram‐negative bacteria and fungi were used, we also followed the haemolytic activity and toxic effect on human cell culture HepG2. On the base of ECD spectroscopy measurement, subsequent molecular modelling and known secondary structure of original β‐alanyl‐tyrosine dipeptide, the secondary structures of repeating sequences of β‐AY were specified. The repeating structures of β‐alanyl‐tyrosine show increase in antimicrobial activity; for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, minimal inhibitory concentration was decreased from 30 to 15 mm for 2xβ‐AY, 0.4 mm for 4xβ‐AY and 0.25 mm for 6xβ‐AY.     

Isolation,functional characterization,and biological properties of MCh‐AMP1, a novel antifungal peptide from Matricaria chamomilla L.

The antimicrobial activities of natural products have attracted much attention due to the increasing incidence of pathogens that have become resistant to drugs. Thus, it has been attempted to promisingly manage infectious diseases via a new group of therapeutic agents called antimicrobial peptides. In this study, a novel antifungal peptide, MCh‐AMP1, was purified by reverse phase HPLC and sequenced by de novo sequencing and Edman degradation. The antifungal activity, safety, thermal, and pH stability of MCh‐AMP1 were determined. This peptide demonstrated an antifungal activity against the tested Candida and Aspergillus species with MIC values in the range of 3.33–6.66 μM and 6.66–13.32 μM, respectively. Further, physicochemical properties and molecular modeling of MCh‐AMP1 were evaluated. MCh‐AMP1 demonstrated 3.65% hemolytic activity at the concentration of 13.32 μM on human red blood cells and 10% toxicity after 48 hr at the same concentration on HEK293 cell lines. The antifungal activity of MCh‐AMP1 against Candida albicans was stable at a temperature range of 30–50°C and at the pH level of 7–11. The present study indicates that MCh‐AMP1 may be considered as a new antifungal agent with therapeutic potential against major human pathogenic fungi.     

Antimicrobial activity of arginine‐ and tryptophan‐rich hexapeptides: the effects of aromatic clusters,d‐amino acid substitution and cyclization

Abstract: Many antimicrobial peptides bear arginine (R)‐ and tryptophan (W)‐rich sequence motifs. Based on the sequence Ac‐RRWWRF‐NH₂, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, l ‐d ‐amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW‐peptides displayed moderate activity towards Gram‐positive Bacillus subtilis (15 < MIC < 31 μm ) and were inactive against Gram‐negative Escherichia coli at peptide concentrations <100 μm . Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d ‐amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW‐peptides at 100 μm concentration was low (<7% lysis for linear R/W‐rich peptides and <28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high‐performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.     

Synthesis,in vitro evaluation,and 68Ga‐radiolabeling of CDP1 toward PET/CT imaging of bacterial infection

Bacterial infections are a major concern in the human health sector due to poor diagnosis and development of multidrug‐resistant strains. PET/CT provides a means for the non‐invasive detection and localization of the infectious foci; however, the radiotracers available are either cumbersome to prepare or their exact contribution toward the imaging is not yet established. Human antimicrobial peptides are of interest for development as PET radiotracers as they are an integral component of the immune system, non‐immunogenic toward the recipient, and show selectivity toward pathogens such as bacteria. Herein we report on the potential of LL37, a human cathelicidin antimicrobial peptide, as a radiotracer for bacterial imaging. Bifunctional chelator 1,4,7‐triazacyclononane,1‐glutaric acid‐4,7‐acetic acid was utilized to functionalize the antimicrobial peptide, which in turn was capable of chelating gallium. The synthesized ^natGa‐CDP1 showed bacterial selectivity and low affinity toward hepatic cells, which are favorable characteristics for further preclinical application.     

Rational design of peptides with enhanced antimicrobial and anti‐biofilm activities against cariogenic bacterium Streptococcus mutans

Streptococcus mutans (S. mutans) is known to be a leading cariogenic pathogen in the oral cavity. Antimicrobial peptides possess excellent properties to combat such pathogens. In this study, we compared the antimicrobial activity of novel linear reutericin 6‐ and/or gassericin A‐inspired peptides and identified LR‐10 as the leading peptide. Antibacterial assays demonstrate that LR‐10 is more active against S. mutans (3.3 μM) than many peptide‐based agents without resistance selection, capable of killing many oral pathogens, and tolerant of physiological conditions. LR‐10 also presented a faster killing rate than chlorhexidine and erythromycin, and appeared to display selective activity against S. mutans within 10 s. S. mutans is usually encased in plaque biofilms. Biofilm inhibitory assays indicated that LR‐10 had excellent inhibitory effect on the biofilm formation of S. mutans and biofilm‐encased cells in vitro at low concentrations (6.5 μM). Consistent with most peptides, LR‐10 kills S. mutans mainly by disrupting the cell membranes. Notably, both hemolytic activity assays and cytotoxicity tests indicated that LR‐10 could keep biocompatible at the effective concentrations. Hence, LR‐10 could be a good candidate for clinical treatment of dental caries.     

Role of positively charged residues on the polar and non‐polar faces of amphipathic α‐helical antimicrobial peptides on specificity and selectivity for Gram‐negative pathogens

We have designed de novo and synthesized eight 26‐residue all D‐conformation amphipathic α‐helical cationic antimicrobial peptides (AMPs), four with “specificity determinants” which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin‐sensitive, and eight of which were methicillin‐resistant. The four AMPs without specificity determinants are extremely hemolytic. In contrast, the four AMPs with specificity determinants had dramatic improvements in therapeutic indices showing the importance of specificity determinants in removing eukaryotic cell toxicity. The specificity determinants combined with the location of positively charged residues on the polar face provide Gram‐negative pathogen selectivity between A. baumannii and S. aureus. Specificity determinants maintain excellent antimicrobial activity in the presence of human sera, whereas the AMPs without specificity determinants were inactive. This study clearly shows the potential of amphipathic α‐helical AMPs with specificity determinants as therapeutics to replace existing antibiotics.     

Novel bioactive peptides demonstrating anti‐dengue virus activity isolated from the Asian medicinal plant Acacia Catechu

The therapeutic activities of food‐derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti‐DENV activity. Half maximal inhibitory concentration of 0.18 μg/ml effectively inhibited DENV foci formation. Treatment with 1.25 μg/ml crude peptide extract could reduce virus production less than 100‐fold with no observable cell toxicity. Peptide sequences were determined by high‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 μM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Effects of mastoparan and related peptides on phosphoinositide breakdown in HL-60 cells and cell-free preparations

In differentiated HL-60 cells the amphiphilic peptide mastoparan induces a dose-dependent stimulation of phosphoinositide breakdown with an EC₅₀ value of 9μM. Such stimulation can be markedly reduced by pretreatment of the cells with pertussis toxin (100 ng/ml, 2h). In membranes obtained from differentiated HL-60 cells, guanine nucleotides stimulate the formation of IP2 and IP3. Calcium ions also induce phosphoinositide breakdown in this preparation independent of the presence of guanine nucleotides. In HL-60 cell membranes, mastoparan inhibited GTPγS-stimulation of phosphoinositide breakdown with an IC₅₀ value of 3 μM. Such inhibitory activity of mastoparan also was present in membranes from cells pretreated with pertussis toxin. Calcium-induced stimulation of phosphoinositide breakdown was not significantly inhibited by mastoparan. The analogs mastoparan-X and polistes mastoparan had similar inhibitory activity, whereas the analog des-Ile¹-Asn²-mastoparan was inactive. In permeabilized HL-60 cells mastoparan also inhibited phosphoinositide breakdown. Another amphiphilic peptide, melittin, was inactive in HL-60 intact cells, but similar to mastoparan, inhibited guanine nucleotide-induced phosphoinositide breakdown in HL-60 cell membranes and permeabilized cells. Thus, mastoparan peptides can stimulate phosphoinositide breakdown in intact HL-60 cells, probably through the interaction with a guanine nucleotide binding protein. In permeabilized cells an in cell membranes, mastoparan induces inhibition of guanine nucleotide-mediated phosphoinositide breakdown presumably through an interaction with an intracellular site. The inhibitory action of mastoparan and melittin is probably related to the amphiphilic character of these peptides.     

Design,synthesis, and in vitro antimicrobial activity of hydrazide–hydrazones of 2‐substituted acetic acid

In this study, 30 hydrazide–hydrazones of phenylacetic ( 3 – 10 ) and hydroxyacetic acid ( 11 – 32 ) were synthesized by the condensation reaction of appropriate 2‐substituted acetic acid hydrazide with different aromatic aldehydes. The obtained compounds were characterized by spectral data and evaluated in vitro for their potential antimicrobial activities against a panel of reference strains of micro‐organisms, including Gram‐positive bacteria, Gram‐negative bacteria, and fungi belonging to the Candida spp. The results from our antimicrobial assays indicated that among synthesized compounds 3 – 32 , especially compounds 6 , 14, and 26 showed high bactericidal activity (MIC = 0.488–7.81 μg/ml) against reference Gram‐positive bacteria, and in some cases, their activity was even better than that of commonly used antibiotics, such as cefuroxime or ampicillin.     

Antibacterial activity of 15‐residue lactoferricin derivatives

Abstract: Lactoferricins are a class of antibacterial peptides isolated after gastric‐pepsin digest of the mammalian iron‐chelating‐protein lactoferrin. For investigation of antibacterial activity, we prepared short synthetic derivatives of bovine, human, caprine, murine and porcine lactoferricins with 15‐amino‐acid residues of high sequence homology. The peptides corresponded to amino‐acid residues 17–31 of the mature bovine lactoferrin. Only the bovine and caprine derivatives displayed measurable antibacterial activity, with the bovine one having a minimal inhibitory concentration of 24 µm and being 10 times more active than the caprine one against Escherichia coli. An alanine‐scan of the bovine lactoferricin derivative was performed to identify specific amino acids that were important for the antibacterial activity. We found that neither of the two tryptophan residues (Trp 6 and Trp 8) present in the bovine lactoferricin derivative could be replaced by alanine without a major loss of antibacterial activity. The other lactoferricin derivatives tested contained only one tryptophan residue (Trp 6). Modified human, caprine and porcine lactoferricin derivatives containing two tryptophan residues (Trp 6 and Trp 8) displayed minimal inhibitory concentrations of 74, 174 and 219 µm , respectively, which represented up to a six‐fold increase in antibacterial activity. The alanine‐scan also revealed that the antibacterial activity was increased when acetamidomethyl‐protected cysteine and unprotected glutamine (Cys 3 and Gln 7) were replaced with alanine. Only the bovine lactoferricin derivative and a few of its alanine‐modified derivatives displayed measurable activity against Staphylococcus aureus.     

Susceptibility of human and murine Chlamydia trachomatis serovars to granulocyte‐ and epithelium‐derived antimicrobial peptides

Abstract: Four antimicrobial peptides, protegrin‐1, RTD‐1, cryptdin‐4, and indolicidin, were tested for their ability to inhibit the in vitro growth of Chlamydia trachomatis serovars E, L2, and mouse pneumonitis (MoPn). In general, protegrin‐1 was found to have the strongest anti‐chlamydial activity. Overall, of the three serovars tested, L2 was the most susceptible while MoPn was the most resistant to these peptides.     

Anticancer activity of VmCT1 analogs against MCF‐7 cells

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe–Leu–Gly–Ala–Leu–Trp–Asn–Val–Ala–Lys–Ser–Val–Phe–NH₂) is an α‐helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF‐7 mammary cancer cells. The results indicated that [Glu]⁷‐VmCT1‐NH₂, [Lys]³‐VmCT1‐NH₂, and [Lys]⁷‐VmCT1‐NH₂ analogs presented moderated helical tendency (0.23–0.61) and tendency of anticancer activity at 25 μmol/L in 24 hr of experiment; and [Trp]⁹‐VmCT1‐NH₂ analog that presented low helical tendency and moderated anticancer activity at 50 μmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.