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Identification of novel bacterial MurA inhibitors

Shaw, JoAnna F. (2018) Identification of novel bacterial MurA inhibitors. Master's Thesis, University of Pittsburgh. (Unpublished)

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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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Shaw, JoAnna F.jfs58@pitt.edujfs58
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorSluis-Cremer, Nicolasnps2@pitt.edu
Committee MemberDoi, Yoheiyod4@pitt.edu
Committee MemberMartinson, Jeremyjmartins@pitt.edu
Date: 28 June 2018
Date Type: Publication
Defense Date: 18 April 2018
Approval Date: 28 June 2018
Submission Date: 24 April 2018
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 59
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: mura inhibitor, antibiotic resistance, fosfomycin, drug discovery, high-throughput screening, enzyme kinetics
Date Deposited: 28 Jun 2018 20:12
Last Modified: 28 Jun 2018 20:12
URI: http://d-scholarship.pitt.edu/id/eprint/34411

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