Date of Award

5-2008

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

John Berger

Committee Member

Johannes Schelvis

Committee Member

Jefffrey Toney

Abstract

The vast overuse of antibiotics along with extrachromosomal gene insertion between different bacterial species has led to the Darwinian consequence of antibiotic resistance. Amongst the contributing factors to the rise of bacterial resistances are the production of ß-lactamases, which pose a major impediment to treatment with ß-lactam drugs such as penicillins and carbapenems. SPM-1 is a metallo-ß-lactamase (MBL) that confers antibiotic resistance to bacteria through zinc-mediated hydrolysis of ß-lactams. Bacteria that express the SPM-1 metalloenzyme show reduced sensitivity to ß-lactam drugs. One method to defeat this form of resistance is to administer a ß-lactam antibiotic with a potent ß-lactamase inhibitor as a combination therapy. The administration of clavulanic acid with amoxicillin, marketed as AugmentinTM, has proven to be clinically successful against serine-based ß-lactamases. The introduction of new inhibitors can reinstall the use of now currently ineffective antibiotics.

Natural products are a source of structurally diverse therapeutics preselected by evolution for potent activity. The coevolution of metallo-ß-lactamases and bacteria has already been shown to have produced several natural product inhibitors of the enzymes and the screening of natural product extracts is a promising source of metallo-ß-lactamase inhibitors. Due to the complexity and highly absorptive properties of natural product extracts, many screening techniques including UV-VIS spectrophotometry are incapable of detecting inhibitors in extract with any selectivity or sensitivity. Following the separation of extract components by techniques such as high performance liquid chromatography (HPLC), metallo-ß-lactamase inhibitors can be screened for higher selectively at micromolar concentrations.

A coupled high-performance liquid chromatography mass spectrometry method for the detection of MBL inhibition by monitoring degradation of penicillin in the presence of SPM-1 has been developed and evaluated against 12 compounds, which previously displayed over 50% inhibition from the National Cancer Institute Chemical Diversity Set. Assays of penicillin G in the presence of SPM-1 and active NCI diversity set compounds show reduced rates of penicillin G hydrolysis over a 3-hour period as determined by LC-MS analysis. LC-MS analysis of SPM-1 inhibition allows for the detection of a specific substrate and eliminates false positives while running at concentrations that are biologically significant. Following validation of the LC-MS technique, natural product extracts Phoradendron californicum and Strophostyles helvola were analyzed for inhibitive MBL properties. While P. californicum exhibited little inhibition, S. helvola prevented ß-lactam hydrolysis at levels equal to and exceeding those NCI chemical diversity set compounds with predetermined inhibitive properties. Further separation of the S. helvola extract and continued inhibitive screening may lead to a potent novel métallo-ß-lactamase inhibitor.

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