Class A carbapenemases in combination with other resistance mechanisms are capable of producing resistance to virtually all available antibiotics and pose a very serious challenge for the successful treatment of life-threatening infections. The KPC- and GES-type carbapenemases are of a special clinical concern as they are widely distributed in various Gram-negative pathogens, isolated from different parts of the world. Lack of a detailed pre-steady-state kinetic studies and structural information regarding interaction of these enzymes with carbapenem antibiotics constitutes the major impediment for elucidation of mechanism(s) of these deleterious bacterial enzymes. Our studies would provide structural and mechanistic basis for rational design of the future generations of ?-lactam antibiotics and inhibitors of class A carbapenemases. The long-term goal of our studies is to delineate detailed kinetics of class A carbapenemases and elucidate structures of these enzymes and their interactions with carbapenem antibiotics. The objectives in this application is to conduct detailed studies of class A carbapenemases by pursuing three Specific Aims: 1) Perform structural and kinetic studies of clinically important class A carbapenemases;2) Perform studies of novel class A carbapenemases;and 3) Elucidate pathways for evolution of carbapenemase activity in class A ?-lactamases. To elucidate the mechanistic aspects of these clinically important antibiotic-resistance enzymes, we will utilize a multidisciplinary approach that includes molecular biology, protein chemistry, detailed enzymology, mass spectrometry, and X-ray crystallography of the native enzymes and their complexes with four clinically used carbapenem antibiotics. Elucidation of structural and kinetic mechanisms that ultimately resulted in acquisition of carbapenemase activity by class A ?-lactamases would greatly contribute to our understanding of the evolutionary potential of these clinically important enzymes and would provide an important guidance for the future utility of carbapenems, antibiotics of the last resort for treatment of life-threatening infections.

Public Health Relevance

The proposed research is relevant to public health because it elucidates the biomedical process that leads to obsolescence of antibiotics, life-saving drugs that are needed by humanity. Elucidation of the catalytic mechanisms and structures of class A carbapenemases has the potential to result in development of the next generations of ?-lactam antibiotics and inhibitors of these enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI089726-03
Application #
8418729
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Korpela, Jukka K
Project Start
2011-02-07
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
3
Fiscal Year
2013
Total Cost
$352,500
Indirect Cost
$117,500
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Stewart, Nichole K; Smith, Clyde A; Frase, Hilary et al. (2015) Kinetic and structural requirements for carbapenemase activity in GES-type ?-lactamases. Biochemistry 54:588-97
Lamoureaux, Toni L; Vakulenko, Viktoria; Toth, Marta et al. (2013) A novel extended-spectrum *-lactamase, SGM-1, from an environmental isolate of Sphingobium sp. Antimicrob Agents Chemother 57:3783-8
Toth, Marta; Vakulenko, Viktoria; Antunes, Nuno T et al. (2012) Class A carbapenemase FPH-1 from Francisella philomiragia. Antimicrob Agents Chemother 56:2852-7