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Structural analysis of beta-lactamase and resistant transpeptidase inhibition Gretes, Michael

Abstract

Beta-lactam antibiotics have achieved phenomenal success in the treatment of infections by inhibiting the transpeptidase enzymes that cross-link the bacterial cell wall. Beta-lactamase-producing pathogenic bacteria and multi-drug-resistant “superbugs” such as methicillin-resistant Staphylococcus aureus (MRSA) have emerged, however. Overcoming resistance factors is thus a research priority. BLIP (Beta-Lactamase Inhibitory Protein) from Streptomyces clavuligerus binds a variety of beta-lactamase enzymes with widely ranging specificity. Its interaction with Escherichia coli beta-lactamase TEM-1 is a well-established model system for protein-protein interaction studies. Presented in Chapter 2 are crystal structures of two BLIP relatives: BLIP-I (a highaffinity inhibitor, alone and in complex with TEM-1) and BLP (which appears not to inhibit beta-lactamases). Substantial variation appears possible in the sub-nanomolar binding of TEM-1 by two homologous proteinaceous inhibitors and such favorable interactions can be negated by a few, strongly unfavorable interactions. OXA-10 is a Pseudomonas aeruginosa beta-lactamase that is resistant to inhibitors in clinical use. Cyclobutanone beta-lactam mimics could be used instead. Chapter 3 reports the crystal structure of OXA-10 covalently modified at its catalytic serine nucleophile with a cyclobutanone inhibitor to form a hemiketal. Favorable and unfavorable contacts made at the active site are examined with a view to improved inhibitor design. PBP2a is the resistant transpeptidase that allows MRSA to maintain the bacterial cell wall in the presence of beta-lactam antibiotics. Ceftobiprole is the most clinically-advanced among a new generation of beta-lactams designed to treat MRSA by targeting PBP2a itself. Chapter 4 uses the crystal structure of a truncated, soluble form of PBP2a solved in complex iii with ceftobiprole to explain its inhibitory power and evaluate current anti-MRSA drug design hypotheses. Its efficacy appears to arise from improved binding affinity that overcomes the disfavored energetics of acylation. Ceftobiprole clinical trials reported no bacterial resistance, yet fully ceftobiproleresistant MRSA (MIC 128 !g/ml) were generated by passage through subinhibitory concentrations of ceftobiprole, discussed in Chapter 5. Resistance emerges in most cases via mutations to the gene encoding PBP2a. Computational modeling predicts that ceftobiprole resistance may be mediated in PBP2a by alteration of binding affinity, acylation efficiency, or by influencing interactions with other proteins.

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