Multiple activities in lantibiotics--models for the design of novel antibiotics?

Lantibiotics are antibiotic peptides distinguished by the presence of the rare thioether amino acids lanthionine and/or methyllanthionine. They are produced by Gram-positive bacteria as gene-encoded precursor peptides and undergo post-translational modification to generate the mature peptide. The structural gene for the prepeptide and the genes involved in biosynthesis, processing, export as well as regulation and producer strain self-protection are organized in clusters. Based on their structural and functional features lantibiotics are currently divided into two major groups. The flexible amphiphilic type-A lantibiotics act primarily by pore formation in the bacterial membrane, a mechanism which was recently shown, e.g. for nisin and epidermin, to involve the interaction with specific docking molecules such as the membrane precursor lipid II. The rather rigid and globular type-B lantibiotics inhibit enzyme functions through interaction with the respective substrates: mersacidin and actagardine inhibit the cell wall biosynthesis by complexing lipid II, whereas the cinnamycin-like peptides inhibit phospholipases by binding phosphoethanolamine. Lantibiotics have attracted much attention in recent years and undergone extensive characterization. New insights into the mode of action and structure-function relationships as well as the biochemistry and the genetics will be outlined in this review.     

Chemical modification monitored by electrospray mass spectrometry: a rapid and simple method for identifying and studying functional residues in enzymes

A simple method to identify functional amino acids in enzymes is described. This method is based on the mass spectrometric detection of molecular weight changes as the consequence of chemical modification of enzymes with group-specific reagents. Here we report the use of phenylglyoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyrocarbonate to identify functional amino acid residues. Precise information is obtained about the stoichiometry of reaction, and a relationship between the loss of enzyme activity and the amount of chemical modification is easily established. Modification sites are located by proteolytic digestion of the modified enzyme, followed by peptide mapping based on high-pressure liquid chromatography using an electrospray mass spectrometer as an on-line detector. In comparison with more conventional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodology is limited only by the stability of the chemically modified species produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have been confirmed by site-directed mutagenesis and X-ray crystallography.     

Recent advances in the medicinal chemistry of alpha-aminoboronic acids, amine-carboxyboranes and their derivatives

This article describes recent developments in the synthesis and biological activity of alpha-aminoboronic acids, amine-carboxyboranes and their derivatives as potential therapeutic agents. alpha-Amino acid analogues are of considerable interest as inhibitors of enzymes involved in amino acid and peptide metabolism. In particular, alpha-amino alkylphosphonic acids and alpha-amino alkylboronic acids, in which the carboxyl group of amino acids is replaced by a phosphonic acid or boronic acid function, respectively, constitute a unique class of amino acid mimics from which a number of potent enzyme inhibitors have been synthesized. The inhibitory activity mainly stems from the fact that the tetrahedral phosphonic moiety or the tetrahedral adduct of electrophilic boronic acid is a good mimic of the putative tetrahedral transition state or intermediate encountered in the enzymatic hydrolysis or formation of peptides. Since the peptide hydrolysis and formation invariably involves the tetrahedral high energy species in the course of the reaction, these amino acid mimics serve as a general key element for inhibitors of a broad spectrum of proteases and peptide ligases. Serine protease inhibitors provide promising compounds having a P site binding moiety and a boronic acid chelating moiety. The compounds have been shown to have high inhibitory activity.     

Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.     

Discovery and development of lantibiotics; antimicrobial agents that have significant potential for medical application

Introduction: Antimicrobial drug resistance is driving the need for novel therapeutics. Amongst the most promising antibacterial agents that are being investigated as replacements for current therapeutic antibiotics are antibacterial peptides, such as the lanthionine-containing peptide antibiotics (lantibiotics). Areas covered: This review focuses on the current methods used for discovery of potentially exploitable lantibiotics for medical applications and discusses relevant recent innovations that will have a positive impact on the discovery of useful lantibiotics. Expert opinion: Recent technological advances in a number of fields mean that increased research into the identification and characterisation of new lantibiotics is feasible. We need to increase our understanding of the various mechanisms of antibacterial action exhibited by lantibiotics and apply this knowledge to peptide engineering or novel practical applications. The advent of next-generation sequencing approaches now negate the need for extensive reverse genetics and employment of bioinformatics approaches is greatly assisting the identification of potentially useful inhibitors in the genomes of a range of clinically significant bacteria. These advances in genetic analysis and engineering will facilitate increased exploitation of lantibiotics in medical therapy.     

Circular micro-proteins and mechanisms of cyclization

Transpeptidation reactions result in the formation of new peptide bonds and this can occur between two separate peptides or within the one peptide. These reactions are catalyzed by enzymes and when the N- and C-terminus of the one peptide are joined it results in the formation of cyclic proteins. Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins but gives the resultant cyclic proteins exceptional stability. The mechanisms are not well understood and this review documents what is known of the events that lead to cyclization. Gene encoded cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the θ-defensins and retrocyclins. Small cyclic proteins are also found in fungi and a large range of cyclic proteins are expressed in cyanobacteria. Three types of cyclic proteins have been found in plants, the small cyclic proteins of 5-12 amino acids, the cyclic proteins from sunflower which are made up of 12-14 amino acids, and the larger group known as cyclotides which contain 28-37 amino acids. Three classes of enzymes are able to catalyse transpeptidation reactions, these include the cysteine, serine and threonine proteases. However only cysteine and serine proteases have been documented to cyclize proteins. The cyclotides from Oldenlandia affinis, the plant in which cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack of the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.     

Lanthiopeptin, a new peptide antibiotic. Production, isolation and properties of lanthiopeptin

A strain of Streptoverticillium cinnamoneum produced a peptide antibiotic named lanthiopeptin, which contained four unusual amino acids, erythro-beta-hydroxyaspartic acid, mesolanthionine, threo-beta-methyllanthionine and lysinoalanine. Lanthiopeptin showed antiviral activity against herpes simplex virus type 1 KOS strain infection in Vero cells by cytopathic effect reduction assay. The structure of lanthiopeptin is similar to that of ancovenin.     

Structure−function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C‐terminal end to a central position favors selective antibacterial activity

Abstract: Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC , is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide‐bridged forms have relatively broad‐spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKC AANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Isolation and characterization of katanosins A and B

Two peptide antibiotics katanosins A and B were isolated from the culture broth of a strain related to the genus Cytophaga. These antibiotics are basic peptides soluble in aqueous alcohols. The molecular formulae C57H95N15O17 for A and C58H97N15O17 for B were indicated. The constituent amino acids of katanosin A are suggested to be Thr (1), Ser (1), Val (1), Leu (3), Arg (1) and three unusual amino acids. In katanosin B, the Val residue is replaced by Ile. Katanosins A and B are active against Gram-positive bacteria in vitro and in vivo.     

YM-266183 and YM-266184, novel thiopeptide antibiotics produced by Bacillus cereus isolated from a marine sponge II. Structure elucidation

YM-266183 and YM-266184 are new antibacterial substances that have activity against drug-resistant bacteria produced by Bacillus cereus QN03323. These structures were elucidated by MS and NMR spectral analysis. YM-266183 and YM-266184 are the cyclic thiopeptides containing thiazole and pyridine moieties, and several unusual amino acids.     

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metal–chelate chiral capillary electrophoretic method and a cyclodextrin mediated host–guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and β-hydroxyl-N-methy-valine in the proposed structure have been confirmed as d-serine and l-β-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Biophysical studies and anti‐growth activities of a peptide,a certain analog and a fragment peptide derived from α‐fetoprotein

Abstract: A chemically synthesized 34‐amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti‐growth assays were performed using the original cysteine‐containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti‐growth activity of the peptide, while gel‐filtration experiments showed the zinc ions maintained the peptide in its anti‐growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti‐growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti‐cancer protocols.     

Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal

Lipid peroxidation yields the aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Protein adduction by 4HNE is thought to be involved in the pathogenesis of several diseases. Currently, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e., Arg, Cys, His, Met, and Lys) were reacted with 4HNE and 4ONE and analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Post-source decay analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His, and Lys are modified by 4HNE and 4ONE. In addition, Arg was adducted by 4ONE. The predominant adduct resulting from modification of peptides by 4HNE or 4ONE had a mass of 156 or 154 Da (respectively), indicating that adduction occurs via Michael addition. Reactivity of amino acids toward 4HNE and 4ONE was found to have the following order: Cys > His > Lys (> Arg for 4ONE). The presence of an Arg on a Cys-containing peptide increased the reaction rate with 4HNE and 4ONE by a factor of approximately 5-6 compared to the Cys nucleophile alone. Rate constants determined for the modification of Cys by the lipid aldehydes demonstrated a >100-fold difference in reactivity between 4HNE and 4ONE toward Cys. Results of the present study indicate that both 4HNE and 4ONE modify amino acid nucleophiles; however, the reactivity between these two lipid aldehydes differs both qualitatively and quantitatively.     

Radamycin,a novel thiopeptide produced by streptomyces sp. RSP9. II. Physico-chemical properties and structure determination

The new cyclic peptide antibiotic, radamycin (1) and the known thiopeptide methylsulfomycin I (2) have been isolated from the fermentation broth of a Streptomyces sp. RSP9. The structure of radamycin was elucidated by NMR, LC-MS and FAB-MS and was established as a thiopeptide with oxazole and thiazole moieties, and several unusual amino acids.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Advances in the synthesis of bioactive unnatural amino acids and peptides

The key role of proteins and amino acids in the structure and function of living matter has stimulated extensive studies. Modified amino acids with enhanced biological activity, proteolitic stability and bioavailability are of increasing interest in protein design and engineering as drug candidates. In the last few years, several efforts have been devoted to the synthesis of amino acids having unusual side chains and unnatural chirality, commonly referred to as "nonproteinogenic" or "unnatural" amino acids, even though some of them can be isolated from natural sources. In this review we describe recent advances in the amino acid side-chain transformations and backbone modifications by oxidative and fluorination procedures.     

Synthesis and biological activities of photoaffinity labeling analogues of substance P

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, SP) is an undecapeptide with important properties as a neurotransmitter and with other functions. No specific antagonists and no long-acting analogues of this peptide hormone are known to date. In order to reach these goals, analogues of SP have been prepared which contain potential affinity, as well as photoaffinity labeling functions, suitable for irreversible attachment to SP receptors. We report here the synthesis of SP analogues which have the Phe residues in positions 7 or 8 replaced with (4'-NO2)Phe, (4'-NH2)Phe, (4'-N2+)Phe, and (4'-N3)Phe. Some of these peptides are used for photoaffinity labeling studies using various bioassays. The synthesis of the (NO2)Phe-containing peptide was carried out on solid phase using Nle instead of Met and the Boc strategy up to residue 4; the remaining amino acids were added using an Fmoc strategy. The protected undecapetide was cleaved by ammonolysis, purified by chromatography on silica gel with chloroform/methanol and deprotected afterwards. The amino, diazonium, and azido peptides were obtained in this sequence by chemical modification of the nitro peptides. On guinea pig ileum the modified peptides in position 8 had close to maximal activity, whereas modifications in position 7 produced some reduced activity, especially the nitro modification. No diazonium peptide produced any irreversible effects on guinea pig ileum. Photoinactivation studies were carried out on strips of guniea pig trachea, but no irreversible effects have been observed, neither permanent stimulation nor permanent inactivation. The biological activities and effects are discussed in view of the molecular properties of the synthesized analogues.     

Influence of N-terminal peptide and oligosaccharide on the clearance of t-PA

We have studied the influence of Gly-Ala-Arg peptide at the N-terminus and the oligosaccharide at Asn184 on the clearance of tissue plasminogen activator (t-PA). In order to intensify the influence of these structural features, Gln117 t-PA, which is a mutant tissue plasminogen activator (mt-PA) expressed in mouse C127 cells, was used for the investigation. It is altered to remove a high mannose type oligosaccharide by the mutation of an amino acid from Asn117 to Gln. We isolated 4 variants of Gln117 t-PA by cation-exchange chromatography, which are abbreviated as S-I, S-II, L-I and L-II. These variants originated from the heterogeneity of the peptide chains (S-chain, 527 amino acids, L-chain, 530 amino acids) and oligosaccharide (Type I, 2 oligosaccharides, Type II, 1 oligosaccharide). Pharmacokinetics of these variants were investigated after single intravenous administration to male rats at a dose of 250 microg/kg. Significant differences in pharmacokinetic parameters were observed among these variants, but there was no considerable difference in fibrin clot lysis time (FCLT) activity. Gly-Ala-Arg peptide at the N-terminus increased the CLt, whereas the oligosaccharide at Asn184 decreased the CLt. Moreover, the effects of the N-terminal peptide and the oligosaccharide on the CLt were independent of each other. Our study with Gln117 t-PA revealed the role of the N-terminal peptide found in the L-chain produced during the processing of t-PA precursor.     

新型内皮素受体六肽拮抗剂的设计合成及其生物活性

以内皮素-1(endothelin-1,ET-1)羟基末端六肽(His-Leu-Asp-Ile-Ile-Trp)为模板,应用D型芳香性非天然氨基酸替代六肽结构中His残基进行结构设计,利用固相肽合成技术合成新型ET受体肽类拮抗剂,得到12个新六肽化合物,对所得化合物进行损坏抗ET-1收缩大鼠胸总动脉的活性研究,其中C-1,C-3,C-5,C-7,C-8,C-11等6个化合物在10^-9mol/L浓度水平表现出明显的拮抗活性.