Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Xiang Wang

Second Advisor

Amy E. Palmer

Third Advisor

James Goodrich

Fourth Advisor

Shelley Copley

Fifth Advisor

Corrella Detweiler

Abstract

Part I:

Histone demethylases (HDMs) are vital epigenetic regulators that have been implicated in multiple disease states including cancers. Demethylation catalyzed by jumonji-C domain-containing HDMs (JHDMs) is coupled to decarboxylation of α-ketoglutarate (α-KG). Based on this mechanism, we developed a bivalent inhibitor by linking together mimics of the primary substrate (methyllysine) and the cofactor (α-KG) called methylstat. Methylstat is a pan-JHDM inhibitor that is also active in cells.

Using methylstat as a starting point, a fluorescent analog, methylstatfluor, was developed and used as a tracer in fluorescence polarization (FP) - based binding and competition assays to determine quantitatively the binding affinities of JHDM inhibitors and native substrates. The FP assay was miniaturized and adapted for high-throughput screening in order to facilitate future discovery of class-specific JHDM inhibitors.

Part II:

Antibiotic resistance is an urgent global health concern. In the United States alone, antibiotic resistant bacteria caused over 2,000,000 illnesses and 23,000 deaths last year. Resistance-modifying agents (RMAs) offer an approach to address antibiotic resistance. These restore antibiotic sensitivity by targeting resistance conferring genes. Recently, we have used a natural-product-like library of indole alkaloids to discover novel RMAs for a variety of antibiotics.

One compound in particular, known as Of1, showed a dramatic potentiating effect in combination with beta-lactam antibiotics in Methicillin-resistant Staphylococcus aureus (MRSA). Of1 is capable of re-sensitizing multiple MRSA strains to a variety of beta-lactam antibiotics, but possesses no antibiotic activity on its own and has no super-sensitizing effect in non-resistant strains of S. aureus.

Investigations of the mode of action of Of1 revealed that it exerts its effects independently of beta-lactamase inhibition. We further discovered that Of1 is capable of reducing transcription of the inducible β-lactam resistance determinants, β-lactamase and PBP2a. Finally, we found that Of1 stabilizes the binding interaction between the β-lactamase repressor protein, BlaI, and the β-lactamase DNA promoter region. We hypothesize that through this stabilizing action, Of1 reduces transcription of the active resistance determinants, β-lactamase and PBP2a. This discovery would represent an entirely novel mechanism and drug target for combating β-lactam resistance.

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