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OVERCOMING INHIBITOR RESISTANCE IN THE SHV BETA-LACTAMASE

Thomson, Jodi Michelle

Abstract Details

2007, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Beta-Lactam antibiotics have been the cornerstone of antimicrobial chemotherapy since the discovery of penicillin. Unfortunately, resistance to these life-saving agents developed as a result of drug hydrolyzing beta-lactamase enzymes. To circumvent this problem, beta-lactamase inhibitors (clavulanate, sulbactam and tazobactam) were produced to target the prevalent class A beta-lactamases, SHV and TEM. Single amino acid substitutions have arisen in TEM and SHV family beta-lactamases that give rise to inhibitor resistance. One amino acid position in the TEM beta-lactamase that is a “hotspot” for inhibitor resistance is Arg244. Fifteen unique isolates with 6 different amino acids at this residue were identified from clinical isolates possessing TEM. Despite this, the highly similar SHV enzyme remained unchanged at this position. To anticipate the likelihood of inhibitor resistance developing in SHV, and to probe whether this is indicative of a mechanistic difference between these beta-lactamase families, we performed extensive analysis of variants at Arg244 in SHV. Our studies revealed unique properties of inhibitor resistance in SHV. As in TEM, most substitutions at Arg244 lead to the clavulanate resistant phenotype. However, the mechanism of resistance is unique and based solely on a reduction in inhibitor affinity. In contrast, all mutants exhibited increased susceptibility to sulbactam; this occurs despite an equal elevation in KI values. Further studies revealed the mechanism to be highly reduced sulbactam turnover. It is interesting that the phenotype of inhibitor resistance or susceptibility is dictated largely by the properties of the inhibitor, despite the similarity in these compounds. We also extended our studies to novel beta-lactamase inhibitors. We first investigated penem inhibitors, which show clinical promise as broad spectrum inactivators, but hadn’t been tested against inhibitor resistant strains. We also tested boronic acid inhibitors, which are designed with the side chains of currently marketed antibiotics. Not only are these high affinity transition state inhibitors, but also great probes for active site interactions. Finally, we examined the dynamics of beta-lactamase inhibition and protein-protein interaction of SHV with the beta-lactamase inhibitor protein (BLIP). We performed mutagenesis to construct a tight binding variant of SHV and analyzed the inhibition of the wild type and mutant enzymes.
Robert Bonomo (Advisor)
174 p.

Recommended Citations

Citations

  • Thomson, J. M. (2007). OVERCOMING INHIBITOR RESISTANCE IN THE SHV BETA-LACTAMASE [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1181237967

    APA Style (7th edition)

  • Thomson, Jodi. OVERCOMING INHIBITOR RESISTANCE IN THE SHV BETA-LACTAMASE. 2007. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1181237967.

    MLA Style (8th edition)

  • Thomson, Jodi. "OVERCOMING INHIBITOR RESISTANCE IN THE SHV BETA-LACTAMASE." Doctoral dissertation, Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1181237967

    Chicago Manual of Style (17th edition)