Despite progress in the discovery and design of b-lactams, b-lactamases continue to pose the most significant threat to our antibiotic armamentarium. Among the serine b-lactamases, the most widespread and problematic are the class A extended-spectrum (ES) ?-lactamases, the AmpC (class C) cephalosporinases, and the ES AmpCs. These ?-lactamases are found in ceftazidime resistant Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., Acinetobacter baumannii, and Pseudomonas aeruginosa. In this proposal we will continue our work with boronic acid transition state inhibitors (BATSIs) as molecular probes to target the ES SHV of K. pneumoniae and the AmpC of P. aeruginosa, PDC-3. Our central hypothesis is that a better understanding of the molecular details of catalysis and inhibition of ?-lactamase enzymes can lead to the design of more effective inhibitors. With this, we formulate the following specific aims: 1. Complete our studies of ES SHV ?-lactamases by testing novel BATSIs possessing unique R1 side chains. We will use information obtained from susceptibility tests, kinetics, and X-ray crystallography to inform the design of novel functionalities that improve inhibitor affinity and specificity. 2. Define the sequence requirements, molecular, and kinetic interactions that characterize the catalysis and inhibition of PDC-3 ?-lactamase;a) To understand the interactions of PDC-3 with cephalosporins, carbapenems, and BATSIs on a deeper level, we will test the role of Thr105Ala, Thr289, Asn343, Asn346 and Arg349 in PDC-3 using site- saturation and Ala replacement mutagenesis. b) To investigate the properties of PDC-3 that permit the evolution of the ES and carbapenemase profile, we will introduce specific deletion mutations into the R2-loop of this PDC (D 303 - 306);3. Determine the apo crystal structure of PDC-3 and the structure of PDC-3 complexed with BATSIs possessing ceftazidime (LP06) and cefotaxime R1 side chains. This work defines important structure-function relationships in class A and C ?-lactamases and brings us closer to understanding the evolution of the ES cephalosporinases in the clinic as well as discovering effective inhibitors for this challenging drug target. We chose these ?-lactamases as they are among the most important class A and class C enzymes in Gram-negative bacteria.
This proposal is a continuation of our investigations that use boronic acid transition state inhibitors (BATSIs) to understand why Gram-negative bacteria become resistant to extended-spectrum cephalosporins and carbapenems. We chose the beta-lactamases of Klebsiella pneumoniae and Pseudomonas aeruginosa as our target enzymes;these are among the most problematic pathogens found in the hospital setting. We will engineer mutations in these beta-lactamases to understand their catalytic properties, study the interactions of new inhibitors with these enzymes, and determine their structures by X-ray crystallography.
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