Bacterial drug resistance is an increasingly serious problem that threatens public health and researchers need to develop new drugs. The biosynthetic pathway of the bacterial peptidoglycan is a known and good target for the development of novel antibacterial agents. This research project focused on the first lipid-linked step of peptidoglycan biosynthesis. The enzyme required for this step is Phospho-MurNAc-pentapeptide Translocase MraY. Our aim was to screen for novel inhibitors of MraY. A continuous fluorescence MraY assay was developed and optimised to test known and potential inhibitors such as nucleoside natural products, antimicrobial peptides and structurally new small molecule potential inhibitors. The fluorescence assay was adapted to a high-throughput fluorescence assay in microtitre plate format and around 2,000 compounds were screened from the diversity set of the National Cancer Institute (NCI) against MraY in order to identify novel inhibitors. However, around 22 % of the test compounds from the diversity set interfered with fluorescence. Therefore, a radiochemical MraY assay was developed as an independent method. We eventually identified one potential MraY inhibitor from the diversity set, the naphthylisoquinoline alkaloid, michellamine B, with IC50 values of 400 and 340 μg/ml against E. coli and B. subtilis MraY respectively. The compound showed antibacterial activity against B. subtilis with an MIC value of 16 μg/ml. It was established that MraY inhibition from the pacidamycin producer, S. coeruleorubidus, was detectable directly from culture supernatants by the fluorescence and by the radiochemical MraY assays. Therefore, we tested culture supernatants and cell extracts from various Streptomyces strains. MraY inhibition was observed using cell extracts from Streptomyces venezuelae, and higher levels of inhibition were observed from a gbnB/gbnR S. venezuelae mutant, though it was not possible to identify the active species present. Following an earlier report of halogenated fluoresceins identified from a combined MraY/MurG screen, we also tested several halogenated fluoresceins, and found that phloxine B, a tetra-bromo fluorescein analogue, was an inhibitor of E. coli MraY with an IC50 value of 32 μM, and also inhibited MraY from P. aeruginosa, B. subtilis, M. flavus and S. aureus with IC50 values ranging between 100 and 210 μg/ml.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:618954 |
| Date | January 2014 |
| Creators | Mihalyi, Agnes |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/62707/ |
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