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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063517-09
Application #
8471045
Study Section
Special Emphasis Panel (ZRG1-IDM-S (04))
Program Officer
Korpela, Jukka K
Project Start
2005-02-15
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2013
Total Cost
$293,139
Indirect Cost
$60,489
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Ruggiero, Melina; Papp-Wallace, Krisztina M; Taracila, Magdalena A et al. (2017) Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 ?-Lactamase. Antimicrob Agents Chemother 61:
Marshall, Steven; Hujer, Andrea M; Rojas, Laura J et al. (2017) Can Ceftazidime-Avibactam and Aztreonam Overcome ?-Lactam Resistance Conferred by Metallo-?-Lactamases in Enterobacteriaceae? Antimicrob Agents Chemother 61:
Rojas, Laura J; Hujer, Andrea M; Rudin, Susan D et al. (2017) NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas. Antimicrob Agents Chemother 61:
Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C et al. (2017) Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-?-lactamase-1. J Med Chem 60:7267-7283
Hujer, Andrea M; Higgins, Paul G; Rudin, Susan D et al. (2017) Nosocomial Outbreak of Extensively Drug-Resistant Acinetobacter baumannii Isolates Containing blaOXA-237 Carried on a Plasmid. Antimicrob Agents Chemother 61:
Imai, Waka; Sasaki, Masakazu; Aoki, Kotaro et al. (2017) Simple Screening for Carbapenemase-Producing Enterobacteriaceae by Moxalactam Susceptibility Testing. J Clin Microbiol 55:2276-2279
Palacios-Baena, Zaira Raquel; Gutiérrez-Gutiérrez, Belén; De Cueto, Marina et al. (2017) Development and validation of the INCREMENT-ESBL predictive score for mortality in patients with bloodstream infections due to extended-spectrum-?-lactamase-producing Enterobacteriaceae. J Antimicrob Chemother 72:906-913
Moya, Bartolome; Barcelo, Isabel M; Bhagwat, Sachin et al. (2017) Potent ?-Lactam Enhancer Activity of Zidebactam and WCK 5153 against Acinetobacter baumannii, Including Carbapenemase-Producing Clinical Isolates. Antimicrob Agents Chemother 61:
Vázquez-Ucha, Juan Carlos; Maneiro, María; Martínez-Guitián, Marta et al. (2017) Activity of the ?-Lactamase Inhibitor LN-1-255 against Carbapenem-Hydrolyzing Class D ?-Lactamases from Acinetobacter baumannii. Antimicrob Agents Chemother 61:
Mojica, Maria F; Papp-Wallace, Krisztina M; Taracila, Magdalena A et al. (2017) Avibactam Restores the Susceptibility of Clinical Isolates of Stenotrophomonas maltophilia to Aztreonam. Antimicrob Agents Chemother 61:

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