Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS-TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate-TE interaction during the macrocyclization step are still unknown. To understand the TE-mediated macrocyclization, here we report the development of a substrate-based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p-nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)-TE containing tyrocidine synthetase.

Polymyxin B-inspired non-hemolytic tyrocidine A analogues with significantly enhanced activity against gram-negative bacteria: How cationicity impacts cell specificity and antibacterial mechanism.

Naturally occurring cyclic antimicrobial peptides (AMPs) such as tyrocidine A (Tyrc A) and gramicidin S (GS) are appealing targets for the development of novel antibiotics. However, their therapeutic potentials are limited by undesired hemolytic activity and relatively poor activity against Gram-negative bacteria. Inspired by polycationic lipopeptide polymyxin B (PMB), the so called 'last-resort' antibiotic for the treatment of infections caused by multidrug-resistant Gram-negative bacteria, we synthesized and biologically evaluated a series of polycationic analogues derived from Tyrc A. We were able to obtain peptide 8 that possesses 5 positive charges exhibiting potent activities against both Gram-negative and Gram-positive bacteria along with totally diminished hemolytic activity. Intriguingly, antibacterial mechanism studies revealed that, rather than the 'pore forming' model that possessed by Tyrc A, peptide 8 likely diffuses membrane in a 'detergent-like' manner. Furthermore, when treating mice with peritonitis-sepsis, peptide 8 showed excellent antibacterial and anti-inflammatory activities in vivo.

Oligomerisation of tryptocidine C, a Trp-rich cyclodecapeptide from the antimicrobial tyrothricin complex.

Tryptocidine C (TpcC, cyclo[D-Phe1-Pro2-Trp3-D-Trp4-Asn5-Gln6-Trp7-Val8-Orn9-Leu10]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp7 replaced by Tyr7). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 µM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 µM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres.

Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification.

The group of bacterial non-ribosomally produced peptides (NRPs) forms a rich source of antibiotics, such as daptomycin, vancomycin, and teixobactin. The difficulty of functionally expressing and engineering the corresponding large biosynthetic complexes is a bottleneck in developing variants of such peptides. Here, we apply a strategy to synthesize mimics of the recently discovered antimicrobial NRP brevicidine. We mimicked the molecular structure of brevicidine by ribosomally synthesized, post-translationally modified peptide (RiPP) synthesis, introducing several relevant modifications, such as dehydration and thioether ring formation. Following this strategy, in two rounds peptides were engineered in vivo, which showed antibacterial activity against Gram-negative pathogenic bacteria susceptible to wild-type brevicidine. This study demonstrates the feasibility of a strategy to structurally and functionally mimic NRPs by employing the synthesis and post-translational modifications typical for RiPPs. This enables the future generation of large genetically encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens.

Tyrocidine A interactions with saccharides investigated by CD and NMR spectroscopies.

Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by ß-sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H-bonding, possibly ß-sheet structures. The amides involved in intramolecular H-bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1 H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA-glucose interactions characterized by a dissociation constant around 200 µM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible.

Stabilizing the monomeric amyloid-ß peptide by tyrocidine A prevents and reverses amyloidogenesis without the accumulation of oligomers.

Amyloid-ß (Aß) oligomers are causative agents triggering AD pathogenesis, but their elimination remains challenging. We herein reported a natural cyclopeptide tyrocidine A prevents and reverses amyloidogenesis without Aß oligomer accumulation by stabilizing the monomeric, but not the oligomeric state of Aß peptides.

Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi.

The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe1-Pro2-X3-x4-Asn5-Gln6-X7-Val8-X9-Leu10) with Trp3,4/Phe3,4 in the aromatic dipeptide unit, Lys9/Orn9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe3-D-Phe4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. ß-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the ß-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall ß-glucans that could modulate their activity.

Insights into the chemical logic and enzymatic machinery of NRPS assembly lines.

Appreciation that some cyclic peptide antibiotics such as gramicidin S and tyrocidine were nonribosomally synthesized has been known for 50 years. The past two decades of research including advances in bacterial genetics, genomics, protein biochemistry and mass spectrometry have codified the principles of assembly line enzymology for hundreds of nonribosomal peptides and in parallel for thousands of polyketides. The advances in understanding the strategies used for chain initiation, elongation and termination from these assembly lines have revitalized natural product biosynthetic communities.

Detailed SAR and PCA of the tyrocidines and analogues towards leucocin A-sensitive and leucocin A-resistant Listeria monocytogenes.

The tyrocidines, antimicrobial cyclic decapeptides from Bacillus aneurinolyticus, have potent activity with drug/disinfectant potential, specifically against Listeria monocytogenes. The tyrocidine activity is dependent on an amphipathic balance. Structure-activity relationship (SAR) analysis combined with principal component analysis showed the best activity correlation with hydropathy and solvent accessible volume (hydrophobicity parameters), Mr and molecular volume (steric/size parameters), coupled with rigid sequence and charge prerequisites. For potent activity against L. monocytogenes strains, there is a prerequisite for a Tyr or Phe in the (W/F)(w/f)NQ(Y/F/W) sequence of the variable pentapeptide and ornithine (Orn, O) as cationic residue in the conserved V(K/O)LfP pentapeptide, particularly with Trp in the aromatic dipeptide moiety of the variable pentapeptide. The roles of Trp and Orn in the tyrocidines were confirmed with most active peptide, tyrocidine B (TrcB) containing Orn and a Trp-D-Phe in the aromatic dipeptide moiety. However, a novel analogue with a trimethylated ornithine and Phe-D-Phe showed an activity rivalling that of TrcB. Our results emphasized that activity is dictated by interplay between the character of the aromatic residues in the variable pentapeptide and the cationic residue. Any residue change resulting in tighter membrane/cell wall interaction is likely to trap tyrocidines and impede their mechanism of action.

The high resolution structure of tyrocidine A reveals an amphipathic dimer.

Tyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (K(D)) of 10 µM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the K(D). Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase.

ß-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed ß-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the ß-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds.

Freestanding nonproteinogenic amino acids have long been recognized for their antimetabolite properties and tendency to be uncovered to reactive functionalities by the catalytic action of target enzymes. By installing them regiospecifically into biogenic peptides and proteins, it may be possible to usher a new era at the interface between small molecule and large molecule medicinal chemistry. Site-selective protein functionalization offers uniquely attractive strategies for posttranslational modification of proteins. Last, but not least, many of the amino acids not selected by nature for protein incorporation offer rich architectural possibilities in the context of ribosomally derived polypeptides. This Review summarizes the biosynthetic routes to and metabolic logic for the major classes of the noncanonical amino acid building blocks that end up in both nonribosomal peptide frameworks and in hybrid nonribosomal peptide-polyketide scaffolds.

Multiplex de novo sequencing of peptide antibiotics.

Proliferation of drug-resistant diseases raises the challenge of searching for new, more efficient antibiotics. Currently, some of the most effective antibiotics (i.e., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. The isolation and sequencing of cyclic peptide antibiotics, unlike the same activity with linear peptides, is time-consuming and error-prone. The dominant technique for sequencing cyclic peptides is nuclear magnetic resonance (NMR)-based and requires large amounts (milligrams) of purified materials that, for most compounds, are not possible to obtain. Given these facts, there is a need for new tools to sequence cyclic non-ribosomal peptides (NRPs) using picograms of material. Since nearly all cyclic NRPs are produced along with related analogs, we develop a mass spectrometry approach for sequencing all related peptides at once (in contrast to the existing approach that analyzes individual peptides). Our results suggest that instead of attempting to isolate and NMR-sequence the most abundant compound, one should acquire spectra of many related compounds and sequence all of them simultaneously using tandem mass spectrometry. We illustrate applications of this approach by sequencing new variants of cyclic peptide antibiotics from Bacillus brevis, as well as sequencing a previously unknown family of cyclic NRPs produced by marine bacteria. Supplementary Material is available online at www.liebertonline.com/cmb.

Sequencing cyclic peptides by multistage mass spectrometry.

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria.

Bead diffusion assay for discovering antimicrobial cyclic peptides.

A combinatorial library of 15,536 cyclic decapeptide analogues of tyrocidine A and gramicidin S was prepared on photocleavable TentaGel beads. The beads were photolyzed without solvent, and spread onto an agar plate inoculated with bacterial lawn. Clear zones were formed around beads carrying antimicrobially active peptides such as E18 c(kVOrnLfThiYOrnLq), which inhibited growth of B. subtilis and selected MRSA strains.

The reversible macrocyclization of Tyrocidine A aldehyde: a hemiaminal reminiscent of the tetrahedral intermediate of macrolactamization.

In spite of the important role of peptide macrocyclizations for the generation of conformationally constrained biological ligands, our knowledge of factors that determine the inclination of a substrate to cyclize is low. Therefore, methods that give access to the thermodynamic characterization of these processes are desirable. In this work, we present the isosteric substitution of the amide ligation site of a cyclopeptide by an imine. Applied to the decapeptide antibiotic Tyrocidine A (TycA), the reversible cyclization of the linear aldehyde TycA-CHO resulted in the unexpectedly stable hemiaminal Psi[CH(OH)NH]-TycA, which is equivalent to the tetrahedral intermediate of macrolactamization, and which is observed for the first time in a peptidic structure. On the basis of NMR spectroscopy and molecular modeling, we discuss the observed high stereoselectivity of hemiaminal formation, as well as its reluctance to be dehydrated to the imine. As required for thermodynamic analysis, it is possible to establish a pH- and temperature-dependent cyclization equilibrium, which allows determination of the entropy loss of the peptide chain, and quantification of the extent of preorientation of the cyclization precursor.

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis.

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry.

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

Impact of epimerization domains on the intermodular transfer of enzyme-bound intermediates in nonribosomal peptide synthesis.

Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.

Chimeric streptogramin-tyrocidine antibiotics that overcome streptogramin resistance.

Streptogramin antibiotics are comprised of two distinct chemical components: the type A polyketides and the type B cyclic depsipeptides. Clinical resistance to the type B streptogramins can occur via enzymatic degradation catalyzed by the lyase Vgb or by target modification through the action of Erm ribosomal RNA methyltransferases. We have prepared through chemical and chemo-enzymatic approaches a series of chimeric antibiotics composed of elements of type B streptogramins and the membrane-active antibiotic tyrocidine that evade these resistance mechanisms. These new compounds show broad antibiotic activity against gram-positive bacteria including a number of important pathogens, and chimeras appear to function by a mechanism that is distinct from their parent antibiotics. These results allow for the development of a brand new class of antibiotics with the ability to evade type B streptogramin-resistance mechanisms.