Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Analysis of in vitro activities and modes of action of synthetic antimicrobial peptides derived from an alpha-helical 'sequence template'.

OBJECTIVES: Cationic antimicrobial peptides (AMPs) are indispensable components of innate immune systems and promising candidates for novel anti-infective strategies. We rationally designed a series of peptides based on a template derived from known alpha-helical AMPs, which were then analysed regarding efficacy against clinical isolates and antibiotic mechanisms. METHODS: Efficacy tests included standard MIC and synergy assays. Whole cell assays with staphylococcal strains included killing kinetics, efflux experiments and determination of membrane depolarization. The transcriptional response of AMP-treated Staphylococcus aureus SG511 was analysed using a Scienion genomic microarray covering (approximately 90% of) the S. aureus N315 genome and AMP P16(6|E). RESULTS: The AMPs showed remarkable broad-spectrum activity against bacteria and fungi regardless of any pre-existing antibiotic resistance mechanism. Whole cell assays indicated that the AMPs target the cytoplasmic membrane; however, significant membrane leakage and depolarization was only observed with a standard laboratory test strain. Transcriptional profiling identified up-regulation of putative efflux pumps and of aerobic energy generation mechanisms as major counter activities. Important components of the staphylococcal cell wall stress stimulon were up-regulated and the lipid metabolism was also affected. CONCLUSIONS: The broad spectrum activity of amphiphilic helical AMPs is based on multiple stresses resulting from interactions with microbial membranes; however, rather than killing through formation of pores, the AMPs appear to interfere with the coordinated and highly dynamic functioning of membrane bound multienzyme complexes such as electron transport chains and cell wall or lipid biosynthesis machineries.

Role of biochemical markers of bone remodeling in clinical practice.

Bone tissue is subject to remodeling throughout the lifetime of an individual. Through a continuous remodeling cycle, actuated via the so-called 'bone remodeling units', old bone is resorbed by osteoclasts with the formation of cavities that are subsequently filled by osteoblasts. Bone loss observed in old age and in women after menopause is due to an imbalance between bone resorption and formation. Biochemical markers provide a dynamic view of the remodeling process, which covers rate of turnover and pathogenesis, and should improve fracture risk prediction. Furthermore, they can be used to monitor the short-term effects of therapy, and indicate if an excessive slowing of the remodeling process is occurring. When searching for markers of bone remodeling, biochemists have focused mainly on skeletal molecules that can be dosed in plasma and/or urine, as indicators of osteoblast function (i.e. bone alkaline phosphatase, osteocalcin, procollagene I C- and N-terminal propeptides) or osteoclast function (i.e. pyridinium crosslinks, collagen I C- and N-terminal telopeptides). The clinical significance of any marker for bone remodeling depends on two fundamental characteristics: specificity and variability. If the objective is to monitor therapeutic efficacy, it seems most rational to use a resorption marker for drugs that act principally on osteoclast, such as estrogens or bisphosphonates, while for drugs that act principally on osteoblast, such as PTH-peptides a marker for bone formation would be more appropriate.

Localization of beta-defensin genes in non human primates.

Defensins are a family of host defence peptides that play an important role in the innate immunity of mammalian and avian species. In humans, four beta-defensins have been isolated so far, corresponding to the products of the genes DEFB1 (h-BD1, GenBank accession number NM_005218); DEFB4 (h-Bd2, NM_004942.2), DEFB103 (h-BD3, NM_018661); and DEFB104 (hBD4, NM_080389) mapping on chromosome 8p23.22. We have localized beta-defensin genes on metaphasic chromosomes of great apes and several non-human primate species to determine their physical mapping. Using fluorescent in situ hybridization and BAC probes containing the four beta-defensin genes, we have mapped the homologous regions to the beta-defensin genes on chromosome 8p23-p.22 in non-human primates, while no signals were detected on prosimians chromosomes.

Evolution of the beta defensin 2 gene in primates.

With the aim of further investigating the molecular evolution of beta defensin genes, after having analysed beta defensin 1 (DEFB1) in humans and several nonhuman primate species, we have studied the evolution of the beta defensin 2 gene (DEFB2), which codifies for a peptide with antimicrobial and chemoattractant activity, in humans and 16 primate species. We have found evidence of positive selection during the evolution of orthologous DEFB2 genes at two points on a phylogenetic tree relating these primates: during the divergence of the platyrrhines from the catarrhines and during the divergence of the Cercopithecidae from the Hylobatidae, Great Apes and humans. Furthermore, amino acid variations in Old World Monkeys seem to centre either on residues that are involved in oligomerisation in the human molecule, or that are conserved (40-80%) in beta-defensins in general. It is thus likely that these variations affect the biological function of the molecules and suggest that their synthesis and functional analysis might reveal interesting new information as to their role in innate immunity.

Molecular diversity in gene-encoded, cationic antimicrobial polypeptides.

Gene-encoded, ribosomally synthesised antimicrobial peptides (AMPs) are an ancient and pervasive component of the innate defence mechanisms used by multicellular organisms to control the natural flora and combat pathogens. Bacteria also produce such AMPs to maintain ecological niches free of rival strains. Several hundred different peptides have been characterised to date, and they show a marked degree of variability in both sequence and structure, having evolved to act against distinct microbial targets in different physiological contexts. Many of these peptides appear to function via a selective, but not receptor-mediated, permeabilisation of microbial membranes, while others interact with specific membrane associated or intracellular targets. This review presents a broad survey of different amp structural classes, emphasising both their molecular diversity and underlying similarities. The mode of action of these peptides and potential for biomedical and other application is also briefly discussed.

Amphipathic alpha helical antimicrobial peptides.

Antimicrobial peptides (AMPs) that assume an amphipathic alpha helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic alpha helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram-positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram-negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time-killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti-infective agents.

A novel [60]fullerene amino acid for use in solid-phase peptide synthesis.

[see structure]. A fullerene derivative containing a free amino group has been condensed with N-Fmoc-L-glutamic acid alpha-tert-butyl ester to give a C60-functionalized amino acid. The carboxylic end of this amino acid has been deprotected in acidic conditions, and the resulting acid has been used for solid-phase peptide synthesis. The final peptide, cleaved from the resin, was very soluble in water solutions and showed antimicrobial activity against two representative bacteria.

Amphipathic, alpha-helical antimicrobial peptides.

Gene-encoded antimicrobial peptides are an important component of host defense in animals ranging from insects to mammals. They do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. They are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. A particularly abundant and widespread class of antimicrobial peptides are those with amphipathic, alpha-helical domains. Due to their relatively small size and synthetic accessibility, these peptides have been extensively studied and have generated a substantial amount of structure-activity relationship (SAR) data. In this review, alpha-helical antimicrobial peptides are considered from the point of view of six interrelated structural and physicochemical parameters that modulate their activity and specificity: sequence, size, structuring, charge, amphipathicity, and hydrophobicity. It begins by providing an overview of how these vary in peptides from different natural sources. It then analyzes how they relate to the currently accepted model for the mode of action of alpha-helical peptides, and discusses what the numerous SAR studies that have been carried out on these compounds and their analogues can tell us. A comparative analysis of the many alpha-helical, antimicrobial peptide sequences that are now available then provides further information on how these parameters are distributed and interrelated. Finally, the systematic variation of parameters in short model peptides is used to throw light on their role in antimicrobial potency and specificity. The review concludes with some considerations on the potentials and limitations for the development of alpha-helical, antimicrobial peptides as antiinfective agents.

Aspartic protease inhibitors. An integrated approach for the design andsynthesis of diaminodiol-based peptidomimetics.

Aspartic proteases play key roles in a variety of pathologies, including acquired immunodeficiency syndrome. Peptidomimetic inhibitors can act as drugs to combat these pathologies. We have developed an integrated methodology for preparing human immunodeficiency virus (HIV)-1 aspartic protease diaminodiol inhibitors, based on a computational method that predicts the potential inhibitory activity of the designed structures in terms of calculated enzyme-inhibitor complexation energies. This is combined with a versatile synthetic strategy that couples a high degree of stereochemical control in the central diaminodiol module with complete flexibility in the choice of side chains in the core and in flanking residues. A series of 23 tetrameric, pentameric and hexameric inhibitors, with a wide range of calculated relative complexation energies (-47.2 to +117 kJ.mol-1) and predicted hydrophobicities (logPo/w = 1.8-8.4) was thus assembled from readily available amino acids and carboxylic acids. The IC50 values for these compounds ranged from 3.2 nM to 90 microM, allowing study of correlations between structure and activity, and individuation of factors other than calculated complexation energies that determine the inhibition potency. Multivariable regression analysis revealed the importance of side-chain bulkiness and rigidity at the P2, P2' positions, suggesting possible improvements for the prediction process used to select candidate structures.

Stereoselective synthesis of non symmetric dihydroxyethylene dipeptide isosteres via epoxy alcohols derived from alpha-amino acids.

(1R,2R,3S,4S)-4-Amino-3-hydroxy-1,2-epoxybutanes, accessible in four steps from L-aminoesters, react regio- and stereoselectively with diethyl aluminum cyanide to give (1R,2S,3S,4S)-4-amino-2,3-dihydroxynitriles. Hydrolysis yields hydroxylactones equivalent to 2,3-dihydroxy-4-aminoacids. The sequence provides a novel approach to dihydroxyethylene isosteres potentially useful for new HIV-protease inhibitors.

Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity.

Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.

Wide-spectrum antibiotic activity of synthetic, amphipathic peptides.

PGYa and PGAa are synthetic, amphipathic, alpha-helical peptides that were designed using a novel "sequence template" approach. Their antimicrobial activity was tested against several pathogenic clinical isolates, most of which were multiply resistant to conventional antibiotics. PGYa appeared to be more active towards Gram-positive species (MIC = 0.5-4 microM), towards such Gram negative species as P. aeruginosa, X. maltophilia, E. coli, K. pneumoniae and S. enteritidis (MIC = 1.4 microM), and towards the filamentous fungus A. niger (MIC = 8 microM). Conversely, PGAa showed the greater activity towards three Candida species (MIC = 2.16 microM). The peptides were shown to have a bactericidal activity, resulting in a decrease of viability for both Gram-positive and -negative bacteria of 3-6 logs within 60 min. Scanning electron microscopy of S. aureus and E. coli treated with PGYa shows considerable roughening and blebbing of the bacterial surfaces providing conclusive evidence that the peptide is membrane active.

A computational study of the resistance of HIV-1 aspartic protease to the inhibitors ABT-538 and VX-478 and design of new analogues.

Recent experimental findings with HIV-1 protease (HIV-1 PR) mutants containing variations at four residues, M46I, L63P, V82T and I84V, have shown that only mutants containing the latter two exhibit cross resistance to the inhibitors ABT-538 and VX-478. The V82T and I84V modifications in fact concern residues in the active site while the other two are in the flap (M46I) and hinge (L63P) domains of the enzyme. We have modelled the M46I/L63P, V82T/I84V and M46I/L63P/ V82T/I84V (4X) mutants of HIV-PR and computed their complexation energies with these two inhibitors. A good correlation was found between these complexation energies and the trend in published inhibition constants for these inhibitors. Reasons for the decrease in binding affinities with the two critical mutants (V82T/I84V and 4X) have also been elucidated in detail. Based on these findings, we have designed several analogues of ABT-538 and VX-478, some of which show a better calculated binding affinity towards both mutant and wild type PR.

Rational design of inhibitors for drug-resistant HIV-1 aspartic protease mutants.

This report describes a method for the assessment of inhibitor binding affinities to wild type HIV-1 aspartic protease and to its drug-resistant mutant forms. We have elaborated a refined method for molecular modeling of the 3D structures of mutant enzymes and enzyme-inhibitor complexes based on the crystal structure of the wild type form, which employs a full thermodynamic cycle. Model complexes of four HIV-1 aspartic protease mutants with ten analogs of the A77003 inhibitor were considered. Predictions of inhibition efficiency, resistance potential, and hydrophilicity of the redesigned A77003 analogs were obtained by employing molecular mechanics for the evaluation of enzyme-inhibitor complexation energy and the polarizable continuum model for the estimation of solvent effects. Simple qualitative indicators for structural modifications aimed at overcoming the emergence of HIV resistance to protease inhibitors and at increasing the bioavailability of pseudopeptide inhibitors are examined. A semi-quantitative method for the description of enzyme-ligand binding and its implications for the rational design of inhibitors with higher binding affinity towards emerging HIV PR mutants is presented.

Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity.

Novel alpha-helical antimicrobial peptides have been devised by comparing the N-terminal sequences of many of these peptides from insect, frog and mammalian families, extracting common features, and creating sequence templates with which to design active peptides. Determination of the most frequent amino acids in the first 20 positions for over 80 different natural sequences allowed the design of one peptide, while a further three were based on the comparison of the sequences of alpha-helical antimicrobial peptides derived from the mammalian cathelicidin family of precursors. These peptides were predicted to assume a highly amphipathic alpha-helical conformation, as indicated by high mean hydrophobic moments. In fact, circular dichroism experiments showed clear transitions from random coil in aqueous solution to an alpha-helical conformation on addition of trifluoroethanol. All four peptides displayed a potent antibacterial activity against selected gram-positive and gram-negative bacteria (minimum inhibitory concentrations in the range 1-8 microM), including some antibiotic resistant strains. Permeabilization of both the outer and cytoplasmic membranes of the gram-negative bacterium, Escherichia coli, by selected peptides was quite rapid and a dramatic drop in colony forming units was observed within 5 min in time-killing experiments. Permeabilization of the cytoplasmic membrane of the gram-positive bacterium, Staphylococcus aureus, was instead initially quite slow, gathering speed after 45 min, which corresponds to the time required for significant inactivation in time-killing studies. The cytotoxic activity of the peptides, determined on several normal and transformed cell lines, was generally low at values within the minimum inhibitory concentration range.

Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides.

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N-terminal region while the C-terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their primary sequence, such as the presence of N-terminal 5-oxoproline (pyroglutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangement. The N-terminal domains of the cathelicidins present one or two Asp-Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30-50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-terminus. Finally, similarity searching and topology-based structure prediction underline a significant sequential and structural similarity between the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki = 0.6-3 microM).

Development of pseudopeptide inhibitors of HIV-1 aspartic protease: analysis and tuning of the subsite specificity.

HIV-1 aspartic protease (PR) is a promising target for acquired immunodeficiency syndrome (AIDS) therapy, and the development of PR inhibitors can be accelerated by computer-aided design methods. We describe an approach for the design of new inhibitors, based on the modification of a known reference inhibitor, and the calculation of relative binding energies, taking into account contributions from all species in the binding equilibrium (inhibitor, PR and inhibitor/PR complex), as well as their solvation. This allows for a rational selection of new structures that are likely to have increased inhibition potency. We have analyzed reduced amide bond hexapeptides (Ac-P3-P2-P1-phi[CH2-NH]-P1'-P2-P3'-NH2), based on the structure of the known inhibitor MVT-101. A maximum gain in binding energy (approximately -55 kcal/mol) is observed when Phe or Tyr are present in positions P1 and P1', Glu in position P2' and aromatic residues (Phe, Trp or Tyr) in positions P3 and P3', while, in general, the presence of positively charged residues is destabilizing. This specificity is explained in terms of the interaction of individual inhibitor residues with proximal and distal PR residues. The validity of this computational approach has been confirmed by solid-phase synthesis of several of the designed pseudopeptides, followed by in vitro enzyme inhibition assaying. The best candidate structures show IC50 values in the low nanomolar range.