Bacterial Cell Wall Biosynthesis Research Articles

Effect of a small molecule Lipid II binder on bacterial cell wall stress.

We have recently identified small molecule compounds that act as binders of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprised a hydrophilic head group that includes a peptidoglycan subunit composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) coupled to a short pentapeptide moiety. This headgroup is coupled to a long bactoprenol chain via a pyrophosphate group. Here, we report on the cell wall activity relationship of dimethyl-3-methyl(phenyl)amino-ethenylcyclohexylidene-propenyl-3-ethyl-1,3-benzothiazolium iodide (compound 5107930) obtained by functional and genetic analyses. Our results indicate that compounds bind to Lipid II and cause specific upregulation of the vancomycin-resistance associated gene vraX. vraX is implicated in the cell wall stress stimulon that confers glycopeptide resistance. Our small molecule Lipid II inhibitor retained activity against strains of Staphylococcus aureus mutated in genes encoding the cell wall stress stimulon. This suggests the feasibility of developing this new scaffold as a therapeutic agent in view of increasing glycopeptide resistance.

Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED50 ∼0.15 μg mL-1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition.

Antimicrobial peptides of invertebrates. Part 2. biological functions and mechanisms of action

Antimicrobial peptides (AMPs) of invertebrates are characterized by a wide variety of mechanisms of their action that involve not only disruption of the barrier function of a membrane of a target cell, but specific inhibition of metabolism processes via ligand-receptor interactions with specific molecules on the surface or within the cell as well. Endogenous AMPs can play a role of mediators of the immune system (immunomodulators) by activation of phagocytosis and chemotaxis, and by stimulation of production of cytokines. The second part of the review focuses on biological functions and mechanisms of action of AMPs of invertebrates. A problem of biological significance of antimicrobial properties in vitro is reviewed. The main mechanisms of the AMP action on a membrane (models of the barrel-stave channel and the toroidal pore and the “carpet” mechanism) are described, and the problems of selectivity of interaction with a microbial membrane are analyzed. The data on alternative mechanisms of the antimicrobial action, such as inhibition of transcription and translation, sequestration of metal ions, and inhibition of biosynthesis of bacterial and fungal cell walls, are presented. A number of examples demonstrate the AMP regulatory activities of invertebrates.

Improving the N-terminal diversity of sansanmycin through mutasynthesis

BackgroundSansanmycins are uridyl peptide antibiotics (UPAs), which are inhibitors of translocase I (MraY) and block the bacterial cell wall biosynthesis. They have good antibacterial activity against Pseudomonas aeruginosa and Mycobacterium tuberculosis strains. The biosynthetic gene cluster of sansanmycins has been characterized and the main biosynthetic pathway elucidated according to that of pacidamycins which were catalyzed by nonribosomal peptide synthetases (NRPSs). Sananmycin A is the major compound of Streptomyces sp. SS (wild type strain) and it bears a non-proteinogenic amino acid, meta-tyrosine (m-Tyr), at the N-terminus of tetrapeptide chain.ResultsssaX deletion mutant SS/XKO was constructed by the λ-RED mediated PCR targeting method and confirmed by PCR and southern blot. The disruption of ssaX completely abolished the production of sansanmycin A. Complementation in vivo and in vitro could both recover the production of sansanmycin A, and the overexpression of SsaX apparently increased the production of sansanmycin A by 20 %. Six new compounds were identified in the fermentation culture of ssaX deletion mutant. Some more novel sansanmycin analogues were obtained by mutasynthesis, and totally ten sansanmycin analogues, MX-1 to MX-10, were purified and identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The bioassay of these sansanmycin analogues showed that sansanmycin MX-1, MX-2, MX-4, MX-6 and MX-7 exhibited comparable potency to sansanmycin A against M. tuberculosis H37Rv, as well as multi-drug-resistant (MDR) and extensive-drug-resistant (XDR) strains. Moreover, sansanmycin MX-2 and MX-4 displayed much better stability than sansanmycin A.ConclusionsWe demonstrated that SsaX is responsible for the biosynthesis of m-Tyr in vivo by gene deletion and complementation. About twenty novel sansanmycin analogues were obtained by mutasynthesis in ssaX deletion mutant SS/XKO and ten of them were purified and structurally identified. Among them, MX-2 and MX-4 showed promising anti-MDR and anti-XDR tuberculosis activity and greater stability than sansanmycin A. These results indicated that ssaX deletion mutant SS/XKO was a suitable host to expand the diversity of the N-terminus of UPAs, with potential to yield more novel compounds with improved activity and/or other properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0471-1) contains supplementary material, which is available to authorized users.

Production of the Ramoplanin Activity Analogue by Double Gene Inactivation.

Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin.

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

Three-dimensional QSAR analysis and design of new 1,2,4-oxadiazole antibacterials.

The oxadiazole antibacterials, a class of newly discovered compounds that are active against Gram-positive bacteria, target bacterial cell-wall biosynthesis by inhibition of a family of essential enzymes, the penicillin-binding proteins. Ligand-based 3D-QSAR analyses by comparative molecular field analysis (CoMFA), comparative molecular shape indices analysis (CoMSIA) and Field-Based 3D-QSAR evaluated a series of 102 members of this class. This series included inactive compounds as well as compounds that were moderately to strongly antibacterial against Staphylococcus aureus. Multiple models were constructed using different types of energy minimization and charge calculations. CoMFA derived contour maps successfully defined favored and disfavored regions of the molecules in terms of steric and electrostatic properties for substitution.

Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic Level.

Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of l- to d-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered molecular dynamics (MD), docking, and experimental studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-associated changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-associated phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly associated with each of the GRY53/7HC–ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC–ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-associated GR changes are consistent with the experimentally observed quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and high throughput screening are discussed.

Ceftolozane/Tazobactam: A Novel Cephalosporin/β-Lactamase Inhibitor Combination.

Ceftolozane/tazobactam is a novel antipseudomonal β-lactam/β-lactamase inhibitor combination that is currently approved by the United States Food and Drug Administration for the treatment of complicated intraabdominal infections (cIAI) and complicated urinary tract infections (cUTI). It exhibits bactericidal properties through inhibition of bacterial cell wall biosynthesis, which is mediated through penicillin-binding proteins (PBPs). Ceftolozane is a potent PBP3 inhibitor and has a higher affinity for PBP1b compared with other β-lactam agents. Ceftolozane/tazobactam differs from other cephalosporins due to its increased activity against some AmpC β-lactamases and Pseudomonas aeruginosa. The addition of tazobactam provides enhanced activity against extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and certain anaerobic organisms. Population pharmacokinetic studies for ceftolozane and ceftolozane/tazobactam are best described by a two-compartment model with zero-order input and linear elimination. Similar to other cephalosporins, the best pharmacodynamic property to predict efficacy for ceftolozane/tazobactam is a concentration that remains above the minimum inhibitory concentration (MIC) for 40-50% of the dosing interval. For Enterobacteriaceae and P. aeruginosa strains, the time above the MIC (T > MIC) needed to produce bactericidal activity was much less with ceftolozane than other cephalosporins, with T > MIC requirements of approximately 30%. For currently approved indications, the dose of ceftolozane/tazobactam is 1.5 g (ceftolozane 1 g/tazobactam 0.5 g) intravenously every 8 hours given as a 1-hour infusion. Ceftolozane has low plasma protein binding (20%) and is predominantly excreted unchanged in the urine (≥ 92%). Dosage adjustments are required for moderate-to-severe renal impairment and in patients receiving hemodialysis. Based on data from clinical trials, adverse effects due to ceftolozane/tazobactam do not differ considerably from other cephalosporins, with the most common being nausea, diarrhea, headache, and pyrexia. Ceftolozane/tazobactam is a promising new agent for the treatment of cIAI and cUTI, including those caused by multidrug-resistant gram-negative organisms.

Essential protein SepF of mycobacteria interacts with FtsZ and MurG to regulate cell growth and division.

Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we showed that Rv2147c (or SepF) of Mycobacterium tuberculosis interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in vivo. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. Using Mycobacterium smegmatis as a surrogate system, we confirmed that sepFMSMEG is essential. Knockdown of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of division. Furthermore, SepF interacted with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo, suggesting that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential component of the cell division complex in mycobacteria.

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.

Affinities and in-plane stress forces between glycopeptide antibiotics and biomimetic bacterial membranes

Understanding the molecular basis of interactions between antibiotics affecting bacterial cell wall biosynthesis and cellular membranes is important in rational drug design of new drugs to overcome resistance. However, a precise understanding of how bacteriostatic antibiotics effect action often neglects the effect of biophysical forces involved following antibiotic-receptor binding events. We have employed a combination of a label-free binding biosensor (surface plasmon resonance, SPR) and a force biosensor (in-plane stress cantilever), together with model membrane systems to study the complex interplay between glycopeptide antibiotics, their cognate ligands and different model membranes. Bacterial cell wall precursor analogue N-α-Docosanoyl-ε-acetyl-Lys-d-Alanine-d-Alanine (doc-KAA) was inserted into lipid layers comprised of zwitterionic or anionic lipids then exposed to either vancomycin or the membrane-anchored glycopeptide antibiotic teicoplanin. Binding affinities and kinetics of the antibiotics to these model membranes were influenced by electrostatic interactions with the different lipid backgrounds, in addition to ligand affinities. In addition, cantilever sensors coated with model membranes showed that planar surface stress changes were induced by glycopeptide antibiotics adsorption and caused compressive surface stress generation in a ligand-dependent manner.

A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer‐aided Drug Discovery

With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis-farnesyl diphosphate synthase (cis-FPPS) and cis-decaprenyl diphosphate synthase (cis-DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis-FPPS, cis-DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis-DPPS, finding ~1 μm inhibitors, and used the receiver operating characteristic-area under the curve (ROC-AUC) method to test the predictive power of X-ray and MD-derived cis-DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC(50) ~0.5- to 20-μm inhibitor (depending on substrate) of cis-DPPS, a ~660-nm inhibitor of Rv3378c as well as a 4.8-μm inhibitor of cis-FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation.

Tyrosine 110 plays a critical role in regulating the allosteric inhibition of Campylobacter jejuni dihydrodipicolinate synthase by lysine.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-β-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

Tackling vancomycin-resistant bacteria with 'lipophilic-vancomycin-carbohydrate conjugates'.

Vancomycin, a glycopeptide antibiotic, has long been a drug of choice for life-threatening Gram-positive bacterial infections. Vancomycin confers its antibacterial activity by inhibiting bacterial cell wall biosynthesis. However, over the time, vancomycin has also been rendered ineffective by vancomycin-resistant bacteria (VRB). These bacteria developed resistance to it by alteration of cell wall precursor from D-Ala-D-Ala to D-Ala-D-Lac (vancomycin-resistant Enterococci, VRE), which leads to manifold reduction in the binding constant and results in the loss of antibacterial activity. Herein, we report various vancomycin-sugar analogs, based on a simple design rationale, which exhibit increased binding affinity to VRB, thereby resensitizing VRB to vancomycin. Optimized vancomycin-sugar conjugate exhibited 150-fold increase in affinity for N,N'-diacetyl-Lys-D-Ala-D-Lac compared with vancomycin. This improved binding affinity was also reflected in its antibacterial activity, wherein the MIC value was brought down from 750 to 36 μM against VRE (VanA phenotype). To further sensitize against VRE, we appended lipophilic alkyl chain to optimized vancomycin-sugar conjugate. This lipophilic-vancomycin-sugar conjugate was >1000-fold (MIC=0.7 μM) and 250-fold (MIC=1 μM) more effective against VanA and VanB strains of VRE, respectively, compared with vancomycin. Therefore, this synthetically simple approach could lead to the development of new generation of glycopeptide antibiotics, which can be clinically used to tackle VRB infections.

The tRNA-dependent biosynthesis of modified cyclic dipeptides.

In recent years it has become apparent that aminoacyl-tRNAs are not only crucial components involved in protein biosynthesis, but are also used as substrates and amino acid donors in a variety of other important cellular processes, ranging from bacterial cell wall biosynthesis and lipid modification to protein turnover and secondary metabolite assembly. In this review, we focus on tRNA-dependent biosynthetic pathways that generate modified cyclic dipeptides (CDPs). The essential peptide bond-forming catalysts responsible for the initial generation of a CDP-scaffold are referred to as cyclodipeptide synthases (CDPSs) and use loaded tRNAs as their substrates. After initially discussing the phylogenetic distribution and organization of CDPS gene clusters, we will focus on structural and catalytic properties of CDPSs before turning to two recently characterized CDPS-dependent pathways that assemble modified CDPs. Finally, possible applications of CDPSs in the rational design of structural diversity using combinatorial biosynthesis will be discussed before concluding with a short outlook.

Structure-based design, synthesis, and biological evaluation of isatin derivatives as potential glycosyltransferase inhibitors.

Peptidoglycan glycosyltransferase (PGT) has been shown to be an important pharmacological target for the inhibition of bacterial cell wall biosynthesis. Structure-based virtual screening of about 3,000,000 commercially available compounds against the crystal structure of the glycosyltransferase (GT) domain of the Staphylococcus aureus penicillin-binding protein 2 (S. aureus PBP2) resulted in identification of an isatin derivative, 2-(3-(2-carbamimidoylhydrazono)-2-oxoindolin-1-yl)-N-(m-tolyl)acetamide (4) as a novel potential GT inhibitor. A series of 4 derivatives were synthesized. Several compounds showed more active antimicrobial activity than the initial hit compound 4, in particular 2-(3-(2-carbamimidoylhydrazono)-2-oxoindolin-1-yl)-N-(3-nitrophenyl)acetamide (4l), against Gram-positive Bacillus subtilis and S. aureus with MIC values of 24 and 48 μg/mL, respectively. Saturation transfer difference (STD) NMR study revealed that there is a binding contact between 4l and the GT domain of S. aureus PBP2. Competitive STD-NMR further proved that 4l and moenomycin A bind to GT domain in a competitive manner. Molecular docking study suggests a potential binding pocket of 4l in the GT domain of S. aureus PBP2. Taken together, compound 4l would provide a new scaffold for further development of potent GT inhibitors.

Ion-pairing liquid chromatography–tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway

Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-l-Ala, UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ala-d-Glu-l-Lys, and UDP-MurNAc-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3mg dry weight analyzed) treated with fosfomycin, d-boroAla, d-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway.

Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25–4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.