Functional analysis of the lipoglycodepsipeptide antibiotic ramoplanin

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.     

Rethinking ramoplanin: the role of substrate binding in inhibition of peptidoglycan biosynthesis

Ramoplanin is a cyclicdepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. It was proposed in 1990 to block the MurG step of peptidoglycan synthesis by binding to the substrate of MurG, Lipid I. The proposed mechanism of MurG inhibition has become widely accepted even though it was never directly tested. In this paper, we disprove the accepted mechanism for how ramoplanin functions, and we present an alternative mechanism. This work has implications for the design of ramoplanin derivatives and may influence how other proposed substrate binding antibiotics are studied.     

Ramoplanin inhibits bacterial transglycosylases by binding as a dimer to lipid II

Ramoplanin is a lipglycodepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. Its mechanism of action has been the subject of debate. It was originally proposed to inhibit the MurG step of peptidoglycan synthesis by binding Lipid I. In this paper, we report that ramoplanin inhibits bacterial transglycosylases by binding to Lipid II, the substrate for these enzymes. The inhibition curves reveal that the inhibitory species has a stoichiometry of 2:1 ramoplanin:Lipid II. A Job titration confirms that ramoplanin binds as a dimer to Lipid II. The apparent dissociation constant is in the nanomolar range, which is unusually low given the nature of the interacting species. We show that Lipid II binding is coupled to the formation of a higher order species, which may explain the tight binding. We also present a testable model for the binding-competent dimeric conformation of ramoplanin.     

Alanine scan of [L-Dap(2)]ramoplanin A2 aglycon: assessment of the importance of each residue

In efforts that define the importance of each residue and that identify key regions of the molecule, an alanine scan of the ramoplanin A2 aglycon, a potent antibiotic that inhibits bacterial cell wall biosynthesis, is detailed. As a consequence of both its increased stability (lactam vs lactone) and its "relative" ease of synthesis, the alanine scan was conducted on [Dap2]ramoplanin A2 aglycon, which possesses antimicrobial activity equal to or slightly more potent than that of ramoplanin A2 or its aglycon. Thus, 14 key analogues of the ramoplanin A2 aglycon, representing a scan of residues 3-13, 15, and 17, were prepared enlisting a convergent solution-phase total synthesis that consolidated the effort to a manageable level. The antimicrobial activity of the resulting library of analogues provides insight into the importance and potential role of each residue of this complex glycopeptide antibiotic.     

Enzymatic Synthesis of Lipid II and Analogues

The emergence of antibiotic resistance has prompted active research in the development of antibiotics with new modes of action. Among all essential bacterial proteins, transglycosylase polymerizes lipid II into peptidoglycan and is one of the most favorable targets because of its vital role in peptidoglycan synthesis. Described in this study is a practical enzymatic method for the synthesis of lipid II, coupled with cofactor regeneration, to give the product in a 50–70 % yield. This development depends on two key steps: the overexpression of MraY for the synthesis of lipid I and the use of undecaprenol kinase for the preparation of polyprenol phosphates. This method was further applied to the synthesis of lipid II analogues. It was found that MraY and undecaprenol kinase can accept a wide range of lipids containing various lengths and configurations. The activity of lipid II analogues for bacterial transglycolase was also evaluated.     

Total synthesis of lysobactin

Antibiotic resistance has become a significant public health concern. Antibiotics that belong to new structural classes and manifest their biological activity via novel mechanisms are urgently needed. Lysobactin, a depsipeptide antibiotic has displayed very strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) as well as vancomycin-resistant enterococci (VRE) with minimum inhibitory concentrations (MICs) ranging from 0.39 to 0.78 microg/mL. The MIC values against VRE were more than 50-fold lower than those reported for vancomycin itself. Lysobactin was found to inhibit nascent peptidoglycan formation; however, this activity was not antagonized in the presence of N-acyl-L-Lys-D-Ala-D-Ala, the binding domain on the cell wall precursors that is utilized by vancomycin. Thus, lysobactin represents a promising agent for the treatment bacterial infections due to resistant pathogens. We describe a convergent synthesis of lysobactin that relies upon a highly efficient macrocyclization reaction to assemble the 28-membered cyclic depsipeptide. This synthesis provides the foundation for further study of the mode of action utilized by lysobactin and its analogues.     

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.     

Total synthesis of the ramoplanin A2 and ramoplanose aglycon

A convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon is disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26, and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadepsipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected L-threo-beta-hydroxyasparagine and the development of effective and near-racemization free conditions for esterification of its hindered alcohol (EDCI, DMAP, 0 degrees C). The coupling sites were chosen to maximize the convergency of the synthesis including that of the three subunits, to prevent late stage racemization of carboxylate-activated phenylglycine-derived residues, and to enlist beta-sheet preorganization of an acyclic macrocyclization substrate for 49-membered ring closure. As such, macrocyclization at the chosen Phe(9)-D-Orn(10) site may benefit from both beta-sheet preorganization as well as closure at a D-amine terminus. Deliberate late stage incorporation of the subunit bearing the labile depsipeptide ester and a final stage Asn(1) side chain introduction provides future access to analogues of the aglycons which themselves are reported to be equally potent or more potent than the natural products in antimicrobial assays.     

Total synthesis and examination of three key analogues of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity

The total synthesis and evaluation of three key ramoplanin aglycon analogues are detailed. The first (5a) represents replacement of the labile depsipeptide ester with a stable amide (HAsn2 --> Dap2) with removal of the HAsn pendant carboxamide, and it was found to be slightly more potent than the natural aglycon in antimicrobial assays providing a new lead structure with an improved profile and a more stable and accessible macrocyclic template on which to conduct structure-function studies. In contrast, a second amide analogue 5b which contains a single additional methylene relative to 5a (HAsn2 --> Dab2) was found to be inactive in antimicrobial assays (>100-fold loss in activity). The third key analogue 5c in which the Asn1 lipid side chain was replaced with an acetyl group revealed that it contributes significantly to the antimicrobial activity (16-fold) of the ramoplanins, but is not essential.     

Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC-MS/MS

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high‐performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10–5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays. Copyright © 2010 John Wiley & Sons, Ltd.     

In Vivo Probe of Lipid II‐Interacting Proteins

β‐Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin‐binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP–Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d ‐amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.     

Total synthesis of the ramoplanin A2 and ramoplanose aglycon

Full details of a convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon are disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26 (residues 1, 2 and 15-17), and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadepsipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected l-threo-beta-hydroxyasparagine and the development of effective and near-racemization free conditions for esterification of its hindered alcohol (EDCI, DMAP, 0 degrees C). The coupling sites were chosen to maximize the convergency of the synthesis including that of the three subunits, to prevent late stage racemization of carboxylate-activated phenylglycine-derived residues, and to enlist beta-sheet preorganization of an acyclic macrocyclization substrate for 49-membered ring closure. By altering the order of final couplings, two macrocyclization sites, Phe(9)-d-Orn(10) and Gly(14)-Leu(15), were examined. Macrocyclization at the highly successful Phe(9)-d-Orn(10) site (89%) may benefit from both beta-sheet preorganization as well as closure at a d-amine terminus within the confines of a beta-turn at the end of the H-bonded antiparallel beta-strands. A more modest, but acceptable macrocyclization reaction at the Gly(14)-Leu(15) site (40-50%) found at the other end of the H-bonded antiparallel beta-strands within a small flexible loop may also benefit from preorganization of the cyclization substrate, is conducted on a substrate incapable of competitive racemization, and accommodates the convergent preparation of analogues bearing depsipeptide modifications. Deliberate late-stage incorporation of the subunit bearing the labile depsipeptide ester and a final stage Asn(1) side-chain introduction provides future access to analogues of the aglycons which themselves are equally potent or more potent than the natural products in antimicrobial assays.     

Design, synthesis, and biological evaluation of platensimycin analogues with varying degrees of molecular complexity

The molecular design, chemical synthesis, and biological evaluation of two distinct series of platensimycin analogues with varying degrees of complexity are described. The first series of compounds probes the biological importance of the benzoic acid subunit of the molecule, while the second series explores the tetracyclic cage domain. The biological data obtained reveal that, while the substituted benzoic acid domain of platensimycin is a highly conserved structural motif within the active compounds with strict functional group requirements, the cage domain of the molecule can tolerate considerable structural modifications without losing biological action. These findings refine our present understanding of the platensimycin pharmacophore and establish certain structure-activity relationships from which the next generation of designed analogues of this new antibiotic may emerge.     

Determination of ramoplanin in human urine by high-performance liquid chromatography with automated column switching.

Ramoplanin is a novel glycolipodepsipeptide antibiotic, currently undergoing clinical trials. This method describes the determination of ramoplanin by direct injection of human urine into a coupled-column liquid chromatographic system. An internal-surface reversed-phase column has been used for on-line sample clean-up and enrichment. Analytical separation of ramoplanin and MDL 62,456 used as internal standard, has been achieved on a ABZ+ reversed-phase column with ammonium acetate buffer-acetonitrile-methanol according to a gradient profile. Analytes were detected by their UV absorbance at 270 nm. The limit of quantitation was 0.1 microgram/ml urine and the limit of detection was found to be 0.035 microgram/ml, corresponding to 13.7 pmol/ml. Linearity was determined in the range 0.1-2 micrograms/ml. Precision (relative standard deviation) ranged from 0.71 to 8.75% and the accuracy from -9.9 to 11.6%. Different human sources were tested and no interference between analytes and urine constituents was observed. The method is simple and rapid, requiring a total analysis time of 35 min per sample and reaching greater selectivity and accuracy than microbiological assays.     

Differential inhibition of Staphylococcus aureus PBP2 by glycopeptide antibiotics

The glycopeptide antibiotics prevent maturation of the bacterial cell wall by binding to the terminal d-alanyl-d-alanine moiety of peptidoglycan precursors, thereby inhibiting the enzymes involved in the final stages of peptidoglycan synthesis. However, there are significant differences in the biological activity of particular glycopeptide derivatives that are not related to their affinity for d-Ala-d-Ala. We compare the ability of vancomycin and a set of clinically relevant glycopeptides to inhibit Staphylococcus aureus PBP2 (penicillin binding protein), the major transglycosylase in a clinically relevant pathogen, S. aureus. We report experiments suggesting that activity differences between glycopeptides against this organism reflect a combination of substrate binding and secondary interactions with key enzymes involved in peptidoglycan synthesis.     

The small molecule tool (S)-(-)-blebbistatin: novel insights of relevance to myosin inhibitor design

The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors and to help address this we report here on the impact of structural changes in the methyl-substituted aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to rationalise the observed variations in their biological activity. These studies further support the view that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of relevance to its mode of action. A discussion of the role that these observations have on planning the synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of possibilities by computational methods. These studies are ultimately directed at the development of novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself.     

Synthesis of 1‐C‐Glycoside‐Linked Lipid II Analogues Toward Bacterial Transglycosylase Inhibition

Preparation of Lipid II analogues containing an enzymatically uncleavable 1‐C‐glycoside linkage between the disaccharide moiety and the pyrophosphate‐ or pyrophosphonate‐lipid moiety is described. The synthesis of a common 1‐C‐vinyl disaccharide intermediate has been developed that allows easy preparation of both an elongated sugar‐phosphate bond and a sugar‐phosphonate moiety, which are coupled with the polyprenyl phosphate to give the desired molecules. Inhibition studies show how a subtle structural modification results in dramatically different potency toward bacterial transglycosylase (TGase), and the results identify Lipid II‐C‐O‐PP (IC₅₀=25 μM ) as a potential TGase inhibitor.     

Dendritic cells maturation facilitated by group-adjustable lipopolysaccharide analogues synthesized via RAFT polymerization

Transforming immature DCs into mature state to activate cellular immunity is a critical step in initiating immunoprophylaxis and immunotherapy. Lipopolysaccharides (LPS) can promote DCs maturation by binding receptor on DCs surface, but their clinical application is limited due to biological toxicity. Although many LPS analogues have been developed, complex synthesis and purification hinder their practical application. Here, we propose a novel and simple strategy to synthesize LPS analogues with adjustable structural units. Using monomer units similar to the key functional groups of LPS, we synthesize LPS analogues with different group ratios by RAFT polymerization. The obtained analogues have little negative effect on cell viability. Compared with LPS, the analogues show greater promoting effect on DCs maturation. And the analogues can be applied to different scenarios since the degrees of promoting DCs maturation by LPS analogues with different group ratios are different. This strategy provides a new direction for synthesizing LPS analogues, and it has the potential to produce LPS analogues on a large scale with tunable promoting DCs maturation effect.     

Total Synthesis of Everninomicin 13,384-1-Part 1: Synthesis of the A(1)B(A)C Fragment

The powerful antibiotic everninomicin 13,384-1 (1, Ziracin) has been prepared for the first time through a total synthesis. The 1-->1'-disaccharide and the two orthoesters of this target molecule were introduced by new methodologies using a tin acetal and 1,2-phenylseleno migrations. The reaction sequence also relies on stereoselective glycosidations and subtle manipulations of protecting groups. In addition to the introduction of new synthetic methodologies, this total synthesis should allow the preparation of combinatorial libraries of semisynthetic analogues of this highly promising antibiotic for biological screening purposes.     

Staphylococcus aureus Penicillin‐Binding Protein 2 Can Use Depsi‐Lipid II Derived from Vancomycin‐Resistant Strains for Cell Wall Synthesis

Vancomycin‐resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide‐containing modified cell‐wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin‐binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild‐type S. aureus. However, it is unclear whether VRSA processes the depsipeptide‐containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi‐lipid I, a cell‐wall precursor of VRSA. By using this chemistry, we prepared a depsi‐lipid II analogue as substrate for a cell‐free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi‐lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.