Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens.

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low µM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D-11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.

Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell-Wall Precursor Lipid†II.

The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered "resistance-proof" antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.

Developing Equipotent Teixobactin Analogues against Drug-Resistant Bacteria and Discovering a Hydrophobic Interaction between Lipid II and Teixobactin.

Teixobactin, targeting lipid II, represents a new class of antibiotics with novel structures and has excellent activity against Gram-positive pathogens. We developed a new convergent method to synthesize a series of teixobactin analogues and explored structure-activity relationships. We obtained equipotent and simplified teixobactin analogues, replacing the l- allo-enduracididine with lysine, substituting oxygen to nitrogen on threonine, and adding a phenyl group on the d-phenylalanine. On the basis of the antibacterial activities that resulted from corresponding modifications of the d-phenylalanine, we propose a hydrophobic interaction between lipid II and the N-terminal of teixobactin analogues, which we map out with our analogue 35. Finally, a representative analogue from our series showed high efficiency in a mouse model of Streptococcus pneumoniae septicemia.

Effect of 4-methoxy 1-methyl 2-oxopyridine 3-carbamide on Staphylococcus aureus by inhibiting UDP-MurNAc-pentapeptide, peptidyl deformylase and uridine monophosphate kinase.

AIMS: The present study aimed to investigate the anti-Staphylococcus aureus and anti-biofilm properties of 4-methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC) on S. aureus UDP-MurNAc-pentapeptide (MurF), peptidyl deformylase (PDF) and uridine monophosphate kinase (UMPK). METHODS AND RESULTS: The in vitro efficacy of MMOXC was evaluated using quantitative polymerase chain reaction, in vitro assays and broth microdilution methods. Further, the minimum inhibitory concentration (MIC), IC50 and zone of inhibition were recorded in addition to the anti-biofilm property. MMOXC inhibited pure recombinant UMPK and PDF enzymes with a Ki of 0·37 and 0·49 µmol l-1 . However Ki was altered for MurF with varying substrates. The MurF Ki for UMT, d-Ala-d-Ala and ATP as substrates was 0·3, 0·25 and 1·4 µmol l-1 , respectively. Real-time PCR analysis showed a significant reduction in PDF and MurF expression which correlated with the MIC90 at 100 µmol l-1 and IC50 in the range 42 ± 1·5 to 50 ± 1 µmol l-1 against all strains tested. At 5 µmol l-1 MMOXC was able completely to remove preformed biofilms of S. aureus and other drug resistant strains. CONCLUSIONS: MMOXC was able to kill S. aureus and drug resistant strains tested by inhibiting MurF, UMPK and PDF enzymes and completely obliterated preformed biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth reduction and biofilm removal are prerequisites for controlling S. aureus infections. In this study MMOXC exhibited prominent anti-S. aureus and anti-biofilm properties by blocking cell wall formation, RNA biosynthesis and protein maturation.

Targeting bactoprenol-coupled cell envelope precursors.

Targeting the bactoprenol-coupled cell wall precursor lipid II is a validated antibacterial strategy. In this review, selected prototype lipid II-binding antibiotics of different chemical classes are discussed. Although these compounds attack the same molecular target, they trigger nuanced and diverse cellular effects. Consequently, the mechanisms of antibacterial resistance and the likelihood of resistance development may vary substantially.

New Found Hope for Antibiotic Discovery: Lipid II Inhibitors.

Research into antibacterial agents has recently gathered pace in light of the disturbing crisis of antimicrobial resistance. The development of modern tools offers the opportunity of reviving the fallen era of antibacterial discovery through uncovering novel lead compounds that target vital bacterial cell components, such as lipid II. This paper provides a summary of the role of lipid II as well as an overview and insight into the structural features of macrocyclic peptides that inhibit this bacterial cell wall component. The recent discovery of teixobactin, a new class of lipid II inhibitor has generated substantial research interests. As such, the significant progress that has been achieved towards its development as a promising antibacterial agent is discussed.

Total Synthesis of Laspartomycin C and Characterization of Its Antibacterial Mechanism of Action.

Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to specifically bind undecaprenyl phosphate (C55-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically used antibiotics target the lipid II pathway, there are no approved drugs that act on its C55-P precursor.

Bioanalytical LC/MS study of potential bacterial transglycosylation inhibitors.

Bacterial transglycosylation is an interesting target in antibiotic drug development. An in vitro transglycosylation assay was developed and used to search for possible inhibitors of Staphylococcus aureus Penicillin Binding Protein 2-mediated transglycosylation. Since the substrate, Lipid II, has no UV-chromophore, the assay relies on LC coupled to MS for analysis of the incubation mixtures. Extracts from Thymus sipyleus, Salvia verticillata, Salvia virgata and Oolong tea were tested, as well as epigallocatechin gallate and ursolic acid, which are chemical compounds derived from plants. Matrix effects hampered Lipid II quantification in samples treated with very high concentrations of extracts. None of these extracts or isolated compounds appeared to have inhibitory activities towards the transglycosylation function of Penicillin Binding Protein 2.

Hit 'em where it hurts: The growing and structurally diverse family of peptides that target lipid-II.

Understanding the mode of action of antibiotics is becoming more and more important in the time that microorganisms start to develop resistance. One very well validated target of several classes of antibiotics is the peptidoglycan precursor lipid II. In this review different classes of lipid II targeting antibiotics will be discussed in detail, including the lantibiotics, human invertebrate defensins and the recently discovered teixobactin. By hitting bacteria where it hurts, at the level of lipid II, we expect to be able to develop efficient antibacterial agents in the future. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Structure-activity exploration of a small-molecule Lipid II inhibitor.

We have recently identified low-molecular weight compounds that act as inhibitors of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprises specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetyl glucosamine [GlcNAc]-N-acetyl muramic acid [MurNAc] disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. One of our lead compounds, a diphenyl-trimethyl indolene pyrylium, termed BAS00127538, interacts with the MurNAc moiety and the isoprenyl tail of Lipid II. Here, we report on the structure-activity relationship of BAS00127538 derivatives obtained by in silico analyses and de novo chemical synthesis. Our results indicate that Lipid II binding and bacterial killing are related to three features: the diphenyl moiety, the indolene moiety, and the positive charge of the pyrylium. Replacement of the pyrylium moiety with an N-methyl pyridinium, which may have importance in stability of the molecule, did not alter Lipid II binding or antibacterial potency.

Self-resistance mechanisms of actinomycetes producing lipid II-targeting antibiotics.

Glycopeptides and several lantibiotics are lipid II-targeting antibiotics produced by actinomycetes. To protect themselves from their own product, antibiotic producers developed self-resistance mechanisms. Inspection of different producer strains revealed that their resistance is not only based on a single determinant but on the synergistic action of different factors. Glycopeptide producers possess different ways to synthesize a modified peptidoglycan to prevent the binding of the glycopeptide antibiotic. One possible modification is the synthesis of peptidoglycan precursors terminating with a D-alanyl-D-lactate (D-Ala-D-Lac) rather than with a D-alanyl-D-alanine (D-Ala-D-Ala) resulting in a 1000-fold decreased binding affinity of the glycopeptide to its target. The reprogramming of the peptidoglycan precursor biosynthesis is based on the action of VanHAX or paralogous enzymes as it was shown for Amycolatopsis balhimycina. A second peptidoglycan modification resulting in glycopeptide resistance was investigated in the glycopeptide A40926 producer Nonomuraea ATCC 39727. Nonomuraea eliminates the glycopeptide target by synthesizing a peptidoglycan with 3-3 cross-linked peptide stems. The carboxypeptidase VanYn provides tetrapeptides which serve as substrates for the L,D-transpeptidase catalyzing the formation of 3-3 cross-links. The occurrence of 3-3 cross-linked dimers is also an important feature of the lantibiotic NAI-107 producer Microbispora ATCC PTA-5024. Moreover, the D-Ala in the fourth position in the acceptor peptide of muropeptides is exchanged to glycine or serine in Microbispora, a side reaction of the L,D-transpeptidase. Together with the lipoprotein MlbQ, the ABC transporter MlbYZ and the transmembrane protein MlbJ it might contribute to the self-resistance in Microbispora ATCC PTA-5024.

Efficacy of the small molecule inhibitor of Lipid II BAS00127538 against Acinetobacter baumannii.

OBJECTIVE: To test the activity of a small molecule compound that targets Lipid II against Acinetobacter baumannii. METHODS: Susceptibility to small molecule Lipid II inhibitor BAS00127538 was assessed using carbapenem- and colistin-resistant clinical isolates of A. baumannii. In addition, synergy between colisitin and this compound was assessed. RESULTS: Small molecule Lipid II inhibitor BAS00127538 potently acts against A. baumannii and acts synergistically with colistin. CONCLUSION: For the first time, a compound that targets Lipid II is described that acts against multi-drug resistant isolates of A. baumannii. The synergy with colistin warrants further lead development of BAS00127538.

Turning defense into offense: defensin mimetics as novel antibiotics targeting lipid II.

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.

Interaction of type A lantibiotics with undecaprenol-bound cell envelope precursors.

Lantibiotics are a unique group within the antimicrobial peptides characterized by the presence of thioether amino acids (lanthionine and methyllanthionine). These peptides are produced by and primarily act on Gram-positive bacteria exerting multiple activities at the cytoplasmic membrane of susceptible strains. Previously, the cell wall precursor lipid II was identified as the molecular target for the prototype lantibiotic nisin. Binding and sequestration of lipid II blocks the incorporation of the central cell wall precursor into the growing peptidoglycan network, thereby inhibiting the formation of a functional cell wall. Additionally, nisin combines this activity with a unique target-mediated pore formation, using lipid II as a docking molecule. The interaction with the pyrophosphate moiety of lipid II is crucial for nisin binding. We show that, besides binding to lipid II, nisin interacts with the lipid intermediates lipid III (undecaprenol-pyrophosphate-N-acetyl-glucosamine) and lipid IV (undecaprenol-pyrophosphate-N-acetyl-glucosamine-N-acetyl-mannosamine) of the wall teichoic acid (WTA) biosynthesis pathway. Binding of nisin to the precursors was observed at a stoichiometry of 2:1. The specific interaction with WTA precursors further promoted target-mediated pore formation in artificial lipid bilayers. Specific interactions with lipid III and lipid IV could also be demonstrated for related type A lantibiotics, for example, gallidermin, containing the conserved lipid-II-binding motif.

An oldie but a goodie - cell wall biosynthesis as antibiotic target pathway.

Bacterial cell wall biosynthesis represents the target pathway for penicillin, the first antibiotic that was clinically applied on a large scale. Penicillin, by means of its beta-lactam ring, inhibits a number of enzymes which participate in inserting monomeric cell wall building blocks into the cell wall polymer and which have been termed penicillin-binding proteins (PBPs). Ever since the introduction of penicillin, hundreds of beta-lactam antibiotics have been developed and details of their molecular activities elaborated. Meanwhile, various additional classes of antibiotics have been described, which inhibit the same pathway, yet use target molecules others than the PBPs. Such classes include the glycopeptide antibiotics, lipopeptide and lipodepsipeptide antibiotics, the lantibiotics and various other natural product antibiotics with comparatively complex structures. They usually target the membrane-bound steps of the biosynthesis pathway and the highly conserved lipid-bound intermediates of the building block such as lipid II, which represents a particular "Achilles' heel" for antibiotic attack. With in-depth analysis of the activity of more recently identified inhibitors and with the availability of novel techniques for studying prokaryotic cell biology, new insights were obtained into the molecular organisation of the cell wall biosynthesis machinery and its interconnections with other vital cellular processes such as cell division. This, in turn, provides hints for new targets to be exploited and for the development of novel cell wall biosynthesis inhibitors.

Adenosine phosphonate inhibitors of lipid II: alanyl tRNA ligase MurM from Streptococcus pneumoniae.

Adenosine and 2'-deoxyadenosine phosphonate transition state analogues act as the first inhibitors for the MurMN/FemABX family of tRNA-dependent ligases implicated in high-level penicillin resistance in gram-positive bacteria.