eprintid: 34411 rev_number: 15 userid: 7429 dir: disk0/00/03/44/11 datestamp: 2018-06-28 20:12:35 lastmod: 2018-06-28 20:12:35 status_changed: 2018-06-28 20:12:35 type: thesis_degree metadata_visibility: show contact_email: jfs378@nyu.edu item_issues_id: similar_title_34143 item_issues_type: similar_title item_issues_description: Similar title to Shaw, JoAnna F. IDENTIFICATION OF NOVEL BACTERIAL MurA INHIBITORS. Master's Thesis, University of Pittsburgh. (Unpublished) item_issues_timestamp: 2018-04-25 06:02:18 item_issues_status: autoresolved item_issues_count: 0 eprint_status: archive creators_name: Shaw, JoAnna F. creators_email: jfs58@pitt.edu creators_id: jfs58 title: Identification of novel bacterial MurA inhibitors ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: mura inhibitor, antibiotic resistance, fosfomycin, drug discovery, high-throughput screening, enzyme kinetics abstract: Antibiotic resistance is a persistent and serious public health issue which causes many illnesses and deaths per year. In contrast to the rapid increase and spread of drug-resistant bacteria, antibiotic development has slowed, and there is a clear need to identify and develop antibiotics with new scaffolds and mechanisms of action. In particular, there is a specific need for novel antimicrobial agents which are active against both gram negative and gram positive pathogens. The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. Currently, the only antibiotic targeted toward MurA is fosfomycin, which inhibits MurA by forming a covalent bond with MurA’s active site residue, Cys115. However, MurA variants which lack the cysteine residue in the active site (e.g. M. tuberculosis MurA and some vancomycin resistant Enterococcus (VRE) strains) are resistant to fosfomycin. The goal of this study was to identify novel MurA inhibitors with a different mechanism of action than fosfomycin, and which are active against a range of gram negative and gram positive bacteria. To this end, we have developed and optimized an in vivo high-throughput screening assay to test recombinant MurA enzyme against the TimTec ApexScreen library, a drug library with 5,040 structurally diverse compounds. The hits identified from this screen were further validated using different assays, including bacterial growth curves. Minimum Inhibitory Concentration (MIC) values were determined for the three most promising hits. Toxicity assays for these hits were also conducted in two cell lines, and selectivity indexes for each inhibitor were calculated. Future directions include further characterizing the mechanisms of action and testing for toxicity in other cell lines than HeLa and 293T, including primary cells. date: 2018-06-28 date_type: published pages: 59 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: thesis_advisor etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Sluis-Cremer, Nicolas etdcommittee_name: Doi, Yohei etdcommittee_name: Martinson, Jeremy etdcommittee_email: nps2@pitt.edu etdcommittee_email: yod4@pitt.edu etdcommittee_email: jmartins@pitt.edu etd_defense_date: 2018-04-18 etd_approval_date: 2018-06-28 etd_submission_date: 2018-04-24 etd_release_date: 2018-06-28 etd_access_restriction: immediate etd_patent_pending: FALSE thesis_type: thesis degree: MS citation: Shaw, JoAnna F. (2018) Identification of novel bacterial MurA inhibitors. Master's Thesis, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/34411/1/ShawJoAnna_MSthesis_April2018.pdf