Inhibition of class A and C beta-lactamases: Challenges and promise

Since the introduction of penicillin, beta-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 beta-lactamase enzymes. To overcome beta-lactamase-mediated resistance, beta-lactamase inhibitors were introduced (clavulanate, sulbactam, and tazobactam). These inhibitors greatly enhance the efficacy of their partner beta-lactams in the treatment of Gram-negative infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to beta-lactam/-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant beta-lactamases that are intrinsically resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of beta-lactams.;Here, we demonstrate that the Asn276Asp substitution confers resistance to clavulanate in the class A SHV beta-lactamase. Unlike the Asn276Asp substitution in the related TEM enzyme, and inhibitor-resistant beta-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 beta-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 beta-lactamases from Acinetobacter spp. and Pseudomonas aeruginosa, pathogens of increasing clinical concern for which few effective therapeutic options remain. The currently available beta-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 beta-lactamase inhibitor design. We next examined how carbapenems, drugs of last resort for many resistant infections, may also behave as effective beta-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.