Correction: Synthetic ramoplanin analogues are accessible by effective incorporation of arylglycines in solid-phase peptide synthesis.

[This corrects the article DOI: 10.1039/D3SC01944F.].

Synthetic ramoplanin analogues are accessible by effective incorporation of arylglycines in solid-phase peptide synthesis.

The threat of antimicrobial resistance to antibiotics requires a continual effort to develop alternative treatments. Arylglycines (or phenylglycines) are one of the signature amino acids found in many natural peptide antibiotics, but their propensity for epimerization in solid-phase peptide synthesis (SPPS) has prevented their use in long peptide sequences. We have now identified an optimized protocol that allows the synthesis of challenging non-ribosomal peptides including precursors of the glycopeptide antibiotics and an analogue of feglymycin (1 analogue, 20%). We have exploited this protocol to synthesize analogues of the peptide antibiotic ramoplanin using native chemical ligation/desulfurization (1 analogue, 6.5%) and head-to-tail macrocyclization in excellent yield (6 analogues, 3-9%), with these compounds extensively characterized by NMR (U-shaped structure) and antimicrobial activity assays (two clinical isolates). This method significantly reduces synthesis time (6-9 days) when compared with total syntheses (2-3 months) and enables drug discovery programs to include arylglycines in structure-activity relationship studies and drug development.

Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term 'antibiotic-chemoattractants', consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development.

Staphylococcus aureus entanglement in self-assembling ß-peptide nanofibres decorated with vancomycin.

The increasing resistance of pathogenic microbes to antimicrobials and the shortage of antibiotic drug discovery programs threaten the clinical use of antibiotics. This threat calls for the development of new methods for control of drug-resistant microbial pathogens. We have designed, synthesised and characterised an antimicrobial material formed via the self-assembly of a population of two distinct ß-peptide monomers, a lipidated tri-ß-peptide (ß3-peptide) and a novel ß3-peptide conjugated to a glycopeptide antibiotic, vancomycin. The combination of these two building blocks resulted in fibrous assemblies with distinctive structures determined by atomic force microscopy and electron microscopy. These fibres inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and associated directly with the bacteria, acting as a peptide nanonet with fibre nucleation sites on the bacteria observed by electron microscopy and confocal microscopy. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity and establish an innovative strategy to develop self-assembled antimicrobial materials for future biomedical application.

The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins.

The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and ß-glucan with NaD1 and other plant defensins. We discovered that the ß-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

Examination of the Interaction between a Membrane Active Peptideand Artificial Bilayers by Dual Polarisation Interferometry.

Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid order of the bilayer.

Diversity of nature's assembly lines - recent discoveries in non-ribosomal peptide synthesis.

The biosynthesis of complex natural products by non-ribosomal peptide synthetases (NRPSs) and the related polyketide synthases (PKSs) represents a major source of important bioactive compounds. These large, multi-domain machineries are able to produce a fascinating range of molecules due to the nature of their modular architectures, which allows natural products to be assembled and tailored in a modular, step-wise fashion. In recent years there has been significant progress in characterising the important domains and underlying mechanisms of non-ribosomal peptide synthesis. More significantly, several studies have uncovered important examples of novel activity in many NRPS domains. These discoveries not only greatly increase the structural diversity of the possible products of NRPS machineries but - possibly more importantly - they improve our understanding of what is a highly important, yet complex, biosynthetic apparatus. In this review, several recent examples of novel NRPS function will be introduced, which highlight the range of previously uncharacterised activities that have now been detected in the biosynthesis of important natural products by these mega-enzyme synthetases.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-ß motif which consists of an α helix and a triple-stranded ß-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

The plant defensin NaD1 introduces membrane disorder through a specific interaction with the lipid, phosphatidylinositol 4,5 bisphosphate.

Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.