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Summary Branched Lipid II, required for the formation of indirectly crosslinked peptidoglycan, is generated by MurM, a protein essential for high-level penicillin resistance in the human pathogen Streptococcus pneumoniae. We have solved the X-ray crystal structure of Staphylococcus aureus FemX, an isofunctional homolog, and have used this as a template to generate a MurM homology model. Using this model, we perform molecular docking and molecular dynamics to examine the interaction of MurM with the phospholipid bilayer and the membrane-embedded Lipid II substrate. Our model suggests that MurM is associated with the major membrane phospholipid cardiolipin, and experimental evidence confirms that the activity of MurM is enhanced by this phospholipid and inhibited by its direct precursor phosphatidylglycerol. The spatial association of pneumococcal membrane phospholipids and their impact on MurM activity may therefore be critical to the final architecture of peptidoglycan and the expression of clinically relevant penicillin resistance in this pathogen., Graphical abstract, Highlights • S. aureus FemX structure generated an improved homology model of S. pneumoniae MurM • A Lipid II binding site in MurM was identified by docking and molecular dynamics • Modeling indicates cardiolipin is enriched at the MurM protein:membrane interface • Enzyme assays show that phospholipids influence MurM activity, With an improved homology model of MurM, York et al., use docking and molecular dynamics studies to identify a potential Lipid II binding site and simulate how MurM interacts with this membrane-embedded substrate. Cardiolipin, a membrane phospholipid, was also found to associate with MurM and upregulate its enzymatic activity.

Diaryltriazene derivatives were synthesized and evaluated for their antimicrobial properties. Initial experiments showed some of these compounds to have activity against both methicillin-resistant strains of Staphylococus aureus (MRSA) and Mycobacterium smegmatis, with MICs of 0.02 and 0.03 μg/mL respectively. Those compounds with potent anti-staphylococcal and anti-mycobacterial activity were not found to act as growth inhibitors of mammalian cell lines or yeast. Furthermore, we demonstrated that one of the most active anti-MRSA diaryltriazene derivatives was subject to very low frequencies of resistance at

MurM is an aminoacyl-tRNA dependant ligase that aminoacylates the Lipid II peptidoglycan precursor, in the human pathogen Streptococcus pneumoniae. MurM is required for the generation of branched peptidoglycan precursors enabling indirect cross-links in the peptidoglycan and is found to be essential for penicillin resistance. In this study we have solved the X-ray crystal structure of Staphylococcus aureus FemX, an isofunctional homologue of MurM, and used this as a template to generate a homology model of MurM. Using this model, we perform molecular docking and molecular dynamics to examine the interaction of the protein with the phospholipid bilayer and the membrane embedded Lipid II substrate of MurM. Our model suggests that MurM is associated with the major membrane phospholipid cardiolipin, and we confirm this with experimental evidence that the activity of MurM is enhanced by this phospholipid and inhibited by its direct precursor phosphatidylglycerol. This suggests that the spatial association of pneumococcal membrane phospholipids and their impact on MurM activity may be a critical to the final architecture of the peptidoglycan and the expression of clinically relevant penicillin resistance in this pathogen.

Summary Background Many patients with major depressive disorder have treatment-resistant depression, defined as no adequate response to two consecutive courses of antidepressants. Some evidence suggests that antiglucocorticoid augmentation of antidepressants might be efficacious in patients with major depressive disorder. We aimed to test the proof of concept of metyrapone for the augmentation of serotonergic antidepressants in the clinically relevant population of patients with treatment-resistant depression. Methods This double-blind, randomised, placebo-controlled trial recruited patients from seven UK National Health Service (NHS) Mental Health Trusts from three areas (northeast England, northwest England, and the Leeds and Bradford area). Eligible patients were aged 18–65 years with treatment-resistant depression (Hamilton Depression Rating Scale 17-item score of ≥18 and a Massachusetts General Hospital Treatment-Resistant Depression staging score of 2–10) and taking a single-agent or combination antidepressant treatment that included a serotonergic drug. Patients were randomly assigned (1:1) through a centralised web-based system to metyrapone (500 mg twice daily) or placebo, in addition to their existing antidepressant regimen, for 21 days. Permuted block randomisation was done with a block size of two or four, stratified by centre and primary or secondary care setting. The primary outcome was improvement in Montgomery-Asberg Depression Rating Scale (MADRS) score 5 weeks after randomisation, analysed in the modified intention-to-treat population of all randomly assigned patients that completed the MADRS assessment at week 5. The study has an International Standard Randomised Controlled Trial Number (ISRCTN45338259) and is registered with the EU Clinical Trial register, number 2009-015165-31. Findings Between Feb 8, 2011, and Dec 10, 2012, 165 patients were recruited and randomly assigned (83 to metyrapone and 82 to placebo), with 143 (87%) completing the primary outcome assessment (69 [83%] in the metyrapone and 74 [90%] in the placebo group). At 5 weeks, MADRS score did not significantly differ between groups (21·7 points [95% CI 19·2–24·4] in the metyrapone group vs 22·6 points [20·1–24·8] in the placebo group; adjusted mean difference of −0·51 points [95% CI −3·48 to 2·46]; p=0·74). 12 serious adverse events were reported in four (5%) of 83 patients in the metyrapone group and six (7%) of 82 patients in the placebo group, none of which were related to study treatment. 134 adverse events occurred in 58 (70%) patients in the metyrapone group compared with 95 events in 45 (55%) patients in the placebo group, of which 11 (8%) events in the metyrapone group and four (4%) in the placebo group were judged by principle investigators at the time of occurrence to be probably related to the study drug. Interpretation Metyrapone augmentation of antidepressants is not efficacious in a broadly representative population of patients with treatment-resistant depression within the NHS and therefore is not an option for patients with treatment-resistant depression in routine clinical practice at this time. Further research is needed to clarify if such augmentation might benefit subpopulations with demonstrable hypothalamic–pituitary–adrenal axis abnormalities. Funding Efficacy and Mechanism Evaluation (EME) programme, a UK Medical Research Council and National Institute for Health Research partnership.

Background: MurE controls stereochemical incorporation of lysine or diaminopimelate into peptidoglycan stem peptides. Results: The structure of S. aureus MurE reveals an unexpected lack of specificity for lysine within the active site. Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity. Significance: This study provides new perspectives in targeting Gram-positive peptidoglycan assembly for antimicrobial discovery., Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Background Bipolar disorder is associated with both white matter abnormalities and hypothalamo–pituitary–adrenal axis dysfunction. In a post-hoc analysis of diffusion tensor data, the relationship between cortisol levels and white matter structural integrity was explored in healthy controls and in euthymic patients with bipolar disorder. Methods Healthy control subjects and patients with bipolar disorder, prospectively verified as euthymic, underwent diffusion tensor MRI: fractional anisotropy and mean diffusivity data in fifteen regions of interest were obtained. Morning and evening salivary cortisol levels (SCLs) were measured. Results Significant negative partial correlations were found between fractional anisotropy and evening SCLs in control subjects in four periventricular regions. This pattern was absent in bipolar patients, possibly due to the presence of an excess of extracellular fluid manifested as a significant increase in mean diffusivity in those regions. Limitations This is an exploratory, post-hoc analysis of data with relatively small sample sizes. Lithium treatment and past substance abuse in the bipolar group are potentially confounding factors in this study. Conclusions These preliminary data show an inverse relationship between evening cortisol levels and a measure of periventricular white matter integrity in healthy controls. This relationship appears disrupted in bipolar patients, possibly due to periventricular osmoregulatory dysfunction, the effects of medication or past substance use. Future research should further investigate the influences of cortisol on oligodendrocyte function, white matter integrity and brain osmoregulation in bipolar disorder.

The Van enzymes are ATP-dependant ligases responsible for resistance to vancomycin in Staphylococcus aureus and Enteroccoccus species. The de novo molecular design programme SPROUT was used in conjunction with the X-ray crystal structure of Enterococcus faeciumd-alanyl-d-lactate ligase (VanA) to design new putative inhibitors based on a hydroxyethylamine template. The two best ranked structures were selected and efficient syntheses developed. The inhibitory activities of these molecules were determined on E. faecium VanA, and due to structural similarity and a common reaction mechanism, also on d-Ala-d-Ala ligase (DdlB) from Escherichia coli. The phosphate group attached to the hydroxyl moiety of the hydroxyethylamine isostere within these systems is essential for their inhibitory activity against both VanA and DdlB.

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (l-Ala/l-Ser) and second (l-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402–6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-l-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is l-Ala-l-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.

The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.

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.