Cationic amphipathic peptide analogs of cathelicidin LL‐37 as a probe in the development of antimicrobial/anticancer agents

Cathelicidin LL‐37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL‐37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13‐amino acid cathelicidin 17‐29 amide segment F¹⁷KRIV²¹QR²³IK²⁵DF²⁷LR‐NH₂ were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R²³ and K²⁵ in the hydrophilic side, V²¹and F²⁷ in the hydrophobic side of the interphase, and F¹⁷ that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17‐29 fragment to investigate how they affect activity. Substitution of the amino‐terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac‐FKRIVQRIL²⁵DFLR‐NH₂ resulted in significant cytotoxicity against A549 cancer cells with an IC₅₀ value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac‐FKRIVQI²³IKK²⁶FLR‐NH₂, which incorporates the IIKK motif and the peptides FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂ and Ac‐FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂, which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram‐negative bacteria (MIC 3–7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC₅₀ 12.9–9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17‐29 segment, may lead to the preparation of effective biological compounds.     

Antimicrobial function of short amidated peptide fragments from the tick‐derived OsDef2 defensin

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram‐positive and Gram‐negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3–12) and Os(11–22) exhibit activity when screened against Gram‐positive and Gram‐negative bacteria. Carboxyamidation of both peptides increased membrane‐mediated activity, although carboxyamidation of Os(11–22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3–12)NH₂ and Os(11–22)NH₂, have minimum bactericidal concentrations of 3.3 μM against Escherichia coli. Killing was reached within 10 minutes for Os(3–12)NH₂ and only during the second hour for Os(11–22)NH₂. In an E. coli membrane liposome system, both Os and Os(3–12)NH₂ were identified as membrane disrupting while Os(11–22)NH₂ was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3–12)NH₂ did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3–12 fragment relies on a membrane disruptive mechanism while the 11–22 fragment involves additional target mechanisms. The salt‐resistant potency of Os(3–12)NH₂ identifies it as a promising candidate for further development.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Modification of imidazothiazole derivatives gives promising activity in B-Raf kinase enzyme inhibition; synthesis,in vitro studies and molecular docking

B-Raf mutation was identified as a key target in cancer treatment. Based on structural features of dabrafenib (potent FDA approved B-Raf inhibitor), the design of new NH₂-based imidazothiazole derivatives was carried out affording new highly potent derivatives of imidazothiazole-based scaffold with amino substitution on the terminal phenyl ring as well as side chain with sulfonamide group and terminal substituted phenyl ring. In vitro enzyme assay was investigated against V600E B-Raf kinase. Compounds 10l, 10n and 10o showed higher inhibitory activities (IC₅₀ = 1.20, 4.31 and 6.21 nM, respectively). In vitro cytotoxicity evaluation was assessed against NCI-60 cell lines. Most of tested derivatives showed cytotoxic activities against melanoma cell line. Compound 10k exhibited most potent activity (IC₅₀ = 2.68 µM). Molecular docking study revealed that the new designed derivatives preserved the same binding mode of dabrafenib with V600E B-Raf active site. It was investigated that the new modification in the synthesized derivatives (substituted with NH₂) had a significant inhibitory activity towards V600E B-Raf. This core scaffold is considered a key compound for further structural and molecular optimization.     

Cn‐AMP2 from green coconut water is an anionic anticancer peptide

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn‐AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti‐proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC₅₀ values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β‐type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N‐terminal sequence (TES) of Cn‐AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C‐terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn‐AMP2 across the cancer cell membrane to attack intracellular targets and induce anti‐proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

Covalent structure and some pharmacological features of native and cleaved α‐KTx12−1, a four disulfide‐bridged toxin from Tityus serrulatus venom

A toxin with four disulfide bridges from Tityus serrulatus venom was able to compete with ¹²⁵I‐kaliotoxin on rat brain synaptosomal preparations, with an IC₅₀ of 46 nM . The obtained amino acid sequence and molecular mass are identical to the previously described butantoxin. Enzymatic cleavages in the native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridge pattern of α‐KTx_12?1. Also, after the cleavage of the first six N‐terminal residues, including the unusual disulfide bridge which forms an N‐terminus ring, the potency of the cleaved peptide was found to decrease about 100 fold compared with the native protein. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.     

Fe2+ binding on amyloid β‐peptide promotes aggregation

The metal ions Zn²⁺, Cu²⁺, and Fe²⁺ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ_1‐42‐Zn²⁺, Aβ_1‐42‐Cu²⁺, and Aβ_1‐42‐Fe²⁺ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ_1‐42‐Zn²⁺ system possess three turn conformations separated by coil structure. Zn²⁺ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn²⁺ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu²⁺ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe²⁺ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe²⁺ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe²⁺ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc.     

Antihypertensive Effect of Boysenberry Seed Polyphenols on Spontaneously Hypertensive Rats and Identification of Orally Absorbable Proanthocyanidins with Vasorelaxant Activity

An E. coli expression clone coding for human proinsulin, which was fused to NH₂-terminal β-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH₂ terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)_n, (His)_n, (Trp)_n, and (Ser)_n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH₂-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G¹LLKR⁵IKT⁸LL‐NH₂, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly¹, Arg⁵, and Thr⁸ and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF⁵IKK⁸LL‐NH₂ exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.     

Hyperglucosylated adhesin‐derived peptides as antigenic probes in multiple sclerosis: Structure optimization and immunological evaluation

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide–antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N‐linked β‐d ‐glucopyranosyl moieties (N‐Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C‐terminal portion HMW1(1205–1526) were essential for high‐affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347–1354) fragment bearing one or two N‐Glc respectively on Asn‐1349 and/or Asn‐1352. The N‐glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC₅₀ in the range 10^?8–10^?10 M by competitive indirect enzyme‐linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di‐N‐glucosylated adhesin peptide Ac‐KAN (Glc)VTLN (Glc)TT‐NH₂ as the shortest sequence able to inhibit high‐avidity interaction with N‐Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)‐ and circular dichroism (CD)‐based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N‐glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.     

Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (K _D =20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsale, J. N., and Harris, R. B.,J. Prot. Chem. 9, 647–659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25–90°C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unoredered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

IsCT‐based analogs intending better biological activity

IsCT1‐NH₂ is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α‐helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)‐based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out. Peptides with a substitution at position 3 or 5 ([L]³‐IsCT1‐NH₂, [K]³‐IsCT1‐NH₂, or [F]⁵‐IsCT1‐NH₂) showed no significant change in an activity relative to IsCT1‐NH₂. The addition of a proline residue at position 8 ([P]⁸‐IsCT1‐NH₂) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13‐50 μmol L^?1), and the addition of a tryptophan residue at position 1 ([W]¹‐IsCT1‐NH₂) increased the hemolytic activity (MHC = 1.56 μmol L^?1) without an improvement in antimicrobial activity. The analog [A]¹[F]⁵[K]⁸‐IsCT1‐NH₂, which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 μmol L^?1) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.     

Cyclodimerization of immunosuppressive fragment of HLA‐DR molecule. Design,synthesis and ESI‐MS/MS analysis

The nonapeptide fragment of the HLA‐DR molecule, located in the exposed loop of the alpha‐chain (164–172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three‐dimensional structure of the HLA‐DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C‐termini by spacer of (Gly₅)₂‐Lys‐NH₂ and the N‐termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG₅)₂K‐resin analog was synthesized using solid‐phase peptide synthesis protocols. The cyclization was achieved by cross‐linking the N‐terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega‐bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).