New Teixobactin Analogues with a Total Lactam Ring.

Teixobactin is a new antibiotic peptide with strong efficacy against several Gram-positive resistant bacteria, the structure of which is extremely difficult to obtain in the laboratory via multistep conventional synthesis. To face the increasing antibiotic resistant bacteria, it is fundamental to introduce new types of antibiotics with innovative mechanisms of action without resistance; thus, many scientists are studying and developing new methods to synthesize teixobactin analogues. In this work, seven Arg10-teixobactin analogues with a total lactam ring have been prepared via solid phase peptide synthesis. In order to obtain the total lactam ring, d-Thr8 was replaced by (2R,3S)-diamino-propionic acid. To verify their antimicrobial activity and efficacy, each analogue was tested with MIC against different resistant pathogens, showing an interesting activity for Nle11 containing compounds.

Development of teixobactin analogues containing hydrophobic, non-proteogenic amino acids that are highly potent against multidrug-resistant bacteria and biofilms.

Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms.

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains.

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.

Mode of action of teixobactins in cellular membranes.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.

Cysteines and Disulfide-Bridged Macrocyclic Mimics of Teixobactin Analogues and Their Antibacterial Activity Evaluation against Methicillin-Resistant Staphylococcus Aureus (MRSA).

Teixobactin is a highly potent cyclic depsipeptide which kills a broad range of multi-drug resistant, Gram-positive bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA) without detectable resistance. In this work, we describe the design and rapid synthesis of novel teixobactin analogues containing two cysteine moieties, and the corresponding disulfide-bridged cyclic analogues. These analogues differ from previously reported analogues, such as an Arg10-teixobactin, in terms of their macrocyclic ring size, and feature a disulfide bridge instead of an ester linkage. The new teixobactin analogues were screened against Methicillin-resistant Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. Interestingly, one teixobactin analogue containing all l-amino acid building blocks showed antibacterial activity against MRSA for the first time. Our data indicates that macrocyclisation of teixobactin analogues with disulfide bridging is important for improved antibacterial activity. In our work, we have demonstrated the unprecedented use of a disulfide bridge in constructing the macrocyclic ring of teixobactin analogues.

Design and Syntheses of Highly Potent Teixobactin Analogues against Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo.

The cyclic depsipeptide, teixobactin, kills a number of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, d-Arg4-Leu10-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo.

Syntheses of potent teixobactin analogues against methicillin-resistant Staphylococcus aureus (MRSA) through the replacement of l-allo-enduracididine with its isosteres.

The recently discovered cyclic depsipeptide, teixobactin, is a highly potent antibiotic against multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobaterium tuberculosis. It comprises of 4 D amino acids and a rare l-allo-enduracididine amino acid. The synthesis of a properly protected l-allo-enduracididine amino acid and its incorporation into teixobactin is time consuming, synthetically challenging and low yielding and is therefore a major bottleneck in the development of potent analogues of teixobactin. In this article, we have synthesised 8 analogues of teixobactin using commercially available building blocks by replacing the l-allo-enduracididine amino acid with its isosteres. Furthermore, we have tested all the compounds against a panel of Gram positive bacteria including MRSA and explained the observed trend in biological activity. Although all the analogues were active, three analogues from this work, showed very promising activity against MRSA (MIC 1 µg mL-1). We can conclude that amino acids which are the closest isosteres of l-allo-enduracididine are the key to synthesising simplified potent analogues of teixobactin using rapid syntheses and improved yields.

Defining the molecular structure of teixobactin analogues and understanding their role in antibacterial activities.

The discovery of the highly potent antibiotic teixobactin, which kills the bacteria without any detectable resistance, has stimulated interest in its structure-activity relationship. However, a molecular structure-activity relationship has not been established so far for teixobactin. Moreover, the importance of the individual amino acids in terms of their l/d configuration and their contribution to the molecular structure and biological activity are still unknown. For the first time, we have defined the molecular structure of seven teixobactin analogues through the variation of the d/l configuration of its key residues, namely N-Me-d-Phe, d-Gln, d-allo-Ile and d-Thr. Furthermore, we have established the role of the individual d amino acids and correlated this with the molecular structure and biological activity. Through extensive NMR and structural calculations, including molecular dynamics simulations, we have revealed the residues for maintaining a reasonably unstructured teixobactin which is imperative for biological activity.

Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding.

Teixobactin is a highly promising antibacterial depsipeptide consisting of four d-amino acids and a rare l-allo-enduracididine amino acid. l-allo-Enduracididine is reported to be important for the highly potent antibacterial activity of teixobactin. However, it is also a key limiting factor in the development of potent teixobactin analogues due to several synthetic challenges such as it is not commercially available, requires a multistep synthesis, long and repetitive couplings (16-30 hours). Due to all these challenges, the total synthesis of teixobactin is laborious and low yielding (3.3%). In this work, we have identified a unique design and developed a rapid synthesis (10 min µwave assisted coupling per amino acid, 30 min cyclisation) of several highly potent analogues of teixobactin with yields of 10-24% by replacing the l-allo-enduracididine with commercially available non-polar residues such as leucine and isoleucine. Most importantly, the Leu10-teixobactin and Ile10-teixobactin analogues have shown highly potent antibacterial activity against a broader panel of MRSA and Enterococcus faecalis (VRE). Furthermore, these synthetic analogues displayed identical antibacterial activity to natural teixobactin (MIC 0.25 µg mL-1) against MRSA ATCC 33591 despite their simpler design and ease of synthesis. We have confirmed lipid II binding and measured the binding affinities of individual amino acid residues of Ala10-teixobactin towards geranyl pyrophosphate by NMR to understand the nature and strength of binding interactions. Contrary to current understanding, we have shown that a cationic amino acid at position 10 is not essential for target (lipid II) binding and potent antibacterial activity of teixobactin. We thus provide strong evidence contrary to the many assumptions made about the mechanism of action of this exciting new antibiotic. Introduction of a non-cationic residue at position 10 allows for tremendous diversification in the design and synthesis of highly potent teixobactin analogues and lays the foundations for the development of teixobactin analogues as new drug-like molecules to target MRSA and Mycobacterium tuberculosis.

Efficient total syntheses and biological activities of two teixobactin analogues.

The discovery of the new antibiotic teixobactin has been timely in the race for unearthing novel antibiotics wherein the emergence of drug resistant bacteria poses a serious threat worldwide. Herein, we present the total syntheses and biological activities of two teixobactin analogues. This approach is simple, efficient and has several advantages: it uses commercially available building blocks (except AllocHN-d-Thr-OH), has a single purification step and a good recovery (22%). By using this approach we have synthesised two teixobactin analogues and established that the d-amino acids are critical for the antimicrobial activity of these analogues. With continuing high expectations from teixobactin, this work can be regarded as a stepping stone towards an in depth study of teixobactin, its analogues and the quest for synthesising similar molecules.