G.s research study. Transparency declarations None to declare. Supplementary data The spectroscopic data of the quinolones 1C5 and Figure S1 are available as Supplementary data at Online (http://jac.oxfordjournals.org/). Supplementary Data: Click here to view. Acknowledgements We thank Dr Philip Lowden (Birkbeck College, London, UK) for support in the HPLC analysis.. despite the approval of agents such as linezolid, quinupristin/dalfopristin and daptomycin over the last decade. Infection with drug-resistant strains is extremely serious, prolonging treatment time, decreasing the probability of cure and increasing the cost of treatment.5 The current anti-TB chemotherapy must be administered for 6 months for drug-susceptible strains and for 2 years for multidrug-resistant (MDR) or extensively drug-resistant (XDR) infections. Outbreaks of drug-resistant pathogens are more and more frequent everywhere, and it would be catastrophic if these pathogens develop total drug resistance.6,7 Novel chemical entities are therefore required for treating drug-resistant strains. They must be potent enough to reduce the length of treatment and to prevent the emergence of resistance, but they must also be safer than second-line drugs and not interfere with antiretroviral therapy.8 Screening for novel mechanisms of action seems a reasonable strategy to develop inhibitors against MDR, XDR and totally drug-resistant strains of preferentially adds species.19 H37Rv, BCG, MurE ligase activity when assayed colorimetrically. This finding was further confirmed by HPLC quantification of UDP-MurNAc-tripeptide, the product of the MurE reaction. The quinolones were computationally modelled and docked into the published MurE protein X-ray structure (PDB:2wtz) to propose a probable binding site. The docking results and the competition experiments of quinolone 2 with MurE ligands suggest that they bind to a specific hydrophobic pocket close to the uracil-binding site that could be exploited to generate a novel class of antimycobacterials. Materials and methods Reagents Isoniazid, norfloxacin, kanamycin, resazurin, Tween 80, glycerol, Online. Bacterial strains and cells H37Rv (ATCC 27294), BCG (ATCC 35734), mc2155 (ATCC 700084), (ATCC 6841), (ATCC 11758), JM109 (ATCC 53323), (ATCC 25668) and murine RAW264.7 macrophages (ATCC TIB71) were used in this study. EMRSA-15 and -16 were gifts from CFM 4 Dr Paul Stapleton (School of Pharmacy, University of London, UK). Competent BL21(DE3)pLysS cells (New England Biolabs, UK) were used for overproducing MurE ligase of H37Rv. Drug susceptibility The spot culture susceptibility assay for H37Rv, BCG and mc2155 species was performed as described previously.20 Briefly, Middlebrook 7H9 mycobacterial cultures were serially diluted to 105 cfu/mL. A 5 L aliquot of the diluted culture (500 viable cells) was CFM 4 CFM 4 spotted onto 5 mL of solidified Middlebrook 7H10 agar medium, supplemented with 10% Rabbit polyclonal to Myc.Myc a proto-oncogenic transcription factor that plays a role in cell proliferation, apoptosis and in the development of human tumors..Seems to activate the transcription of growth-related genes. (v/v) OADC in a six-well plate containing various concentrations of compounds 1C5. A negative control containing only DMSO was included in each plate. A six-well plate containing various concentrations of isoniazid was also used as a positive control. Following incubation at 37C for 2weeks for slow growers and 3days for and were employed for the MIC determination of the quinolones.18 Kanamycin and norfloxacin were used as positive controls. Susceptibilities of and were assessed as reported previously21 in a microdilution assay in cation-adjusted MuellerCHinton broth using isoniazid as a positive control. Cytotoxicity towards RAW264.7 macrophages RAW264.7 macrophages (National Collection of Type Cultures) were maintained in RPMI-1640 medium supplemented with 2 mM l-glutamine and 10% heat-inactivated fetal calf serum, in a humidified incubator containing 5% CO2, at 37C, and passaged twice before the assay. The cell suspension was adjusted to 5??105 cells/mL and the assay was performed in 96-well cell culture flat-bottom plates (Costar 3596; VWR) in triplicate. Firstly, 2 L of the 10 g/L stock solution of compounds 1C5 was added to 200 L of RPMI-1640 medium in the first row and then 2-fold serially diluted. In each well, 100 L CFM 4 of diluted macrophage cells was added. After 48 h of incubation, the monocytes were washed twice with PBS and fresh RPMI-1640 medium was added. The plates were then revealed with 30 L of a freshly prepared and filter-sterilized aqueous 0.01% resazurin solution, and incubated overnight at 37C. The following day, fluorescence was measured at 590 nm with excitation at 560 nm using a Fluostar Optima microplate reader (BMG LABTECH). MurE ligase inhibition assay The MurE protein of was overexpressed in.