The global dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of morbidity and mortality. Carbapenemases, or carbapenem-hydrolyzing b-lactamases, are severely limiting the treatment of infections caused by bacteria possessing these enzymes. Among the carbapenemases, metallo- b-lactamases (MBLs) are rapidly emerging and are the most concerning. Although reports of the epidemiology of MBLs are increasing, little knowledge is available regarding the important reaction mechanisms and structure-function properties of MBLs. Guided by the collective experience with serine carbapenemases and MBLs, our unique partnership is initiating in-depth studies to address this important knowledge gap and to acquire mechanistic insights that will ultimately support novel drug design efforts. We propose that a common anionic intermediate is formed in the hydrolysis of carbapenems by MBLs. Recognizing this common anionic intermediate is an important "first step" in the mechanistic understanding of the reaction pathway of MBLs. Therefore, our investigations will test the following hypotheses: 1) structurally divergent MBLs hydrolyze b-lactams, especially carbapenems, by proceeding through common reaction intermediates;2) the mobile loops flanking the active sites of MBLs are important in recognizing a broad repertoire of substrates;and 3) compounds that mimic the common reaction intermediates and interact with the mobile loops will serve as inhibitors of the reaction mechanism. To test these hypotheses we will endeavor to accomplish the following specific aims: 1) characterizing the populated reaction intermediates in clinically relevant MBLs (i.e., NDM-1, VIM-2, IMP-1, and SPM-1);2) identify biochemical features of the mobile loops flanking the active sites of common MBLs that assist in recognizing a broad repertoire of substrates and evaluate these loops as potential pharmacophores for inhibitor development;and 3) design compounds that will mimic the common anionic intermediates to give insight into the mechanism of inhibition. In this proposal, we will provide strong evidence that a common intermediate exists along the reaction pathway of MBLs from all subclasses and that this species will lead us to a deeper understanding of the reaction mechanism. Our consortium is experienced in structural studies of many clinically important b- lactamases, as well as in clinical microbiology, crystallography, NMR, steady-state and pre-steady state kinetics, mass spectrometry and b-lactamase inhibitor testing. The joining together of diverse talents and personnel will ensure the findings obtained from these studies will advance the translational nature of this work.

Public Health Relevance

Carbapenemases, or carbapenem-hydrolyzing b-lactamases that potentially inactivate all b- lactams including carbapenems, can severely limit the treatment of infections caused by Gram- negative bacteria. Among the carbapenemases, metallo- b-lactamases (MBLs such as NDM-1, VIM-2, IMP-1, and SPM-1) are rapidly emerging world-wide and pose one of the most serious public health threats. We seek to study these clinically relevant MBLs to provide evidence that a common reaction intermediate exists and can be exploited to make an effective inhibitor.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI100560-03
Application #
8604370
Study Section
Special Emphasis Panel (ZRG1-IDM-P (02))
Program Officer
Palker, Thomas J
Project Start
2011-02-01
Project End
2017-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
3
Fiscal Year
2014
Total Cost
$409,487
Indirect Cost
$31,943
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
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