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β-lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β-lactams is the expression of β-lactamase enzymes. To overcome β-lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β-lactamases also contain the characteristic β-lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non-β-lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem-hydrolyzing class D β-lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β-lactamase inhibitors, nor are they effectively inhibited by the newest, non-β-lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β-lactamases, and are not extensively characterized as inhibitors of class D β-lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β-lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA-24/40. Several compounds were identified as effective inhibitors of OXA-24/40, with Ki values as low as 5 μM. The X-ray crystal structures of OXA-24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β-lactamases.

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C3/C4 carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C3/C4 carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C3/C4 β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C3/C4 β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.

The molecular basis of resistance to β-lactams and β-lactam-β-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β-lactam therapy. Recent crystal structures of KPC-2 and other class A β-lactamases suggest that Ambler position Trp105 may be of importance in binding β-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β-lactamase displayed improved catalytic efficiencies, k(cat)/K(m), toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k(cat)/K(m) toward cefotaxime and imipenem when compared to WT β-lactamase. The Trp105Asn variant exhibited increased K(m)s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k(cat)/K(m)) for all substrates. With respect to clavulanic acid, the K(i)s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the β-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β-lactam and β-lactamase inhibitor discrimination.