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Inhibition of Class A and C β-Lactamases: Challenges and Promise

Drawz, Sarah Michel

Abstract Details

2010, Doctor of Philosophy, Case Western Reserve University, Pathology.

Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agent of choice for the treatment of many infections. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamase enzymes. To overcome β-lactamase-mediated resistance, β-lactamase inhibitors were introduced (clavulanate, sulbactam, and tazobactam). These inhibitors greatly enhance the efficacy of their partner β-lactams in the treatment of Gram-negative infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam/β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases that are intrinsically resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams.

Here, we demonstrate that the Asn276Asp substitution confers resistance to clavulanate in the class A SHV β-lactamase. Unlike the Asn276Asp substitution in the related TEM enzyme, and inhibitor-resistant β-lactamases in general, the SHV variant maintains a high level of catalytic efficiency for penicillins. This “fine-tuning” of the inhibitor-resistant phenotype may represent a significant evolutionary advance, as the enzyme maintains a balance of desired catalytic properties. By probing Asn276 with selectively designed inhibitors, we explored how the configuration of the conserved β-lactam carboxylate impacts binding. Despite relative distance from the active site, this second-shell residue exerts important effects on enzyme-ligand interactions.

Our work also addresses the class C β-lactamases from Acinetobacter spp. and Pseudomonas aeruginosa, pathogens of increasing clinical concern for which few effective therapeutic options remain. The currently available β-lactamase inhibitors are inactive against these enzymes, and thus development of “second-generation” agents is a priority. We first studied boronic acid derivatives bearing side chains of the enzymes’ substrates as potential inhibitors. Insights about which recognition elements lead to low binding constants provide important leads for class C β-lactamase inhibitor design. We next examined how carbapenems, drugs of last resort for many resistant infections, may also behave as effective β-lactamase inhibitors through unique active site chemistry. Finally, mass spectrometry, kinetics, and susceptibility testing shed light on the possible reaction mechanisms of investigational inhibitors with diverse structures, revealing that inactivation of these enzymes is attainable, but significant barriers to in vivo activity remain.

Robert A. Bonomo, M.D. (Advisor)
Shu G. Chen, Ph.D. (Committee Chair)
Focco van den Akker, Ph.D. (Committee Member)
Michael Harris, Ph.D. (Committee Member)
Marion Skalweit, M.D., Ph.D. (Committee Member)
309 p.

Recommended Citations

Citations

  • Drawz, S. M. (2010). Inhibition of Class A and C β-Lactamases: Challenges and Promise [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1270163923

    APA Style (7th edition)

  • Drawz, Sarah. Inhibition of Class A and C β-Lactamases: Challenges and Promise. 2010. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1270163923.

    MLA Style (8th edition)

  • Drawz, Sarah. "Inhibition of Class A and C β-Lactamases: Challenges and Promise." Doctoral dissertation, Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1270163923

    Chicago Manual of Style (17th edition)