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(Unpublished)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