?-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. The production of ?-lactamases is singularly responsible for this phenotype. Added to this arsenal of resistance is the emerging importance of efflux pumps. The goal of this R01 renewal is to embark upon a multidisciplinary and integrative approach to gain insight into the molecular details that result in ?-lactam resistance by studying the AmpC cephalosporinase, ADC-7 (Acinetobacter derived cephalosporinase), and the efflux pumps of A. baumannii. Our work addresses the most important barriers to successful ?-lactam therapy. To address this significant problem, we will analyze how specific boronic acid inhibitors (BAIs) that contain different R1 and R2 side chains inactivate ADC-7. Our approach will entail kinetic and biophysical (isothermal calorimetry, protein thermal stability, and circular dichroism) measurements that will complement structural characterization. Together, we will gain unique insights into the mechanism of inactivation, the energetic requirements that define a potent inhibitor, and understand the evolution of ?-lactamase specificity. Once the BAIs are characterized and the structures of each BAI/ADC-7 complex are determined, we will also test our conclusions by performing site-directed mutagenesis of amino acid residues that interact with the BAI and test the impact of these changes on susceptibility, kinetics, protein stability, and structure. This comprehensive approach will reveal which amino acid positions are: (a) important for binding of BAI and ?-lactam compounds; and (b) critical for enzyme structure, stability, and/or function. Our efforts will provide a rational basis for the development and optimization of novel BAIs. In concert with these investigations, we will define the role of heretofore uncharacterized efflux pumps in ?-lactam/BAI resistance. Using knockouts of AdeABC and AdeIJK (adeABC/adeIJK), we will explore the role of secondary efflux pumps and transporters in ?-lactam resistance. Achieving these aims in two areas affecting ?-lactam resistance will help us design more potent ?-lactams against A. baumannii. Additionally, our work is a first step to understanding why the efflux systems of A. baumannii present a formidable barrier to antibiotics. Integrating this knowledge will provide the necessary insights regarding ?-lactam resistance that will assist medicinal chemists and physicians faced with this serious infectious disease threat.

Public Health Relevance

Resistance to ?-lactam antibiotics in Acinetobacter baumannii is one of the most serious problems facing physicians who treat patients in the hospital. This proposal seeks to understand the molecular details of how the ?-lactamase, Acinetobacter Derived Cephalosporinase, ADC -7 and efflux systems of Acinetobacter confer resistance to penicillins and cephalosporins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI072219-09
Application #
9273351
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Ernst, Nancy Lewis
Project Start
2006-12-01
Project End
2019-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
9
Fiscal Year
2017
Total Cost
$322,703
Indirect Cost
$36,207
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
Nukaga, Michiyoshi; Papp-Wallace, Krisztina M; Hoshino, Tyuji et al. (2018) Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam. Antimicrob Agents Chemother 62:
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Papp-Wallace, Krisztina M; Barnes, Melissa D; Alsop, Jim et al. (2018) Relebactam Is a Potent Inhibitor of the KPC-2 ?-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae. Antimicrob Agents Chemother 62:
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VanPelt, Jamie; Shurina, Ben A; Ramelot, Theresa A et al. (2018) 1H, 13C, and 15N backbone resonance assignments for KPC-2, a class A serine-?-lactamase. Biomol NMR Assign :
Papp-Wallace, Krisztina M; Nguyen, Nhu Q; Jacobs, Michael R et al. (2018) Strategic Approaches to Overcome Resistance against Gram-Negative Pathogens Using ?-Lactamase Inhibitors and ?-Lactam Enhancers: Activity of Three Novel Diazabicyclooctanes WCK 5153, Zidebactam (WCK 5107), and WCK 4234. J Med Chem 61:4067-4086
Ju, Lin-Cheng; Cheng, Zishuo; Fast, Walter et al. (2018) The Continuing Challenge of Metallo-?-Lactamase Inhibition: Mechanism Matters. Trends Pharmacol Sci 39:635-647
Bergstrom, Alexander; Katko, Andrew; Adkins, Zach et al. (2018) Probing the Interaction of Aspergillomarasmine A with Metallo-?-lactamases NDM-1, VIM-2, and IMP-7. ACS Infect Dis 4:135-145

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