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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-IDM-P (02))
Program Officer
Korpela, Jukka K
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Meini, María-Rocío; Llarrull, Leticia I; Vila, Alejandro J (2014) Evolution of Metallo-?-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution. Antibiotics (Basel) 3:285-316
Vazquez Melendez, Elsa L; Farrell, John J; Hujer, Andrea M et al. (2014) Culture negative empyema in a critically ill child: an opportunity for rapid molecular diagnostics. BMC Anesthesiol 14:107
Papp-Wallace, Krisztina M; Mallo, Susana; Bethel, Christopher R et al. (2014) A kinetic analysis of the inhibition of FOX-4 *-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic *-lactams and carbapenems. J Antimicrob Chemother 69:682-90
Marchiaro, Patricia M; Brambilla, Luciano; Morán-Barrio, Jorgelina et al. (2014) The complete nucleotide sequence of the carbapenem resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain reveals a chimeric design formed by modules derived from both environmental and clinical bacteria. Antimicrob Agents Chemother 58:1816-21
González, Lisandro J; Moreno, Diego M; Bonomo, Robert A et al. (2014) Host-specific enzyme-substrate interactions in SPM-1 metallo-?-lactamase are modulated by second sphere residues. PLoS Pathog 10:e1003817
Aitha, Mahesh; Marts, Amy R; Bergstrom, Alex et al. (2014) Biochemical, mechanistic, and spectroscopic characterization of metallo-?-lactamase VIM-2. Biochemistry 53:7321-31
Bogan, Christopher; Kaye, Keith S; Chopra, Teena et al. (2014) Outcomes of carbapenem-resistant Enterobacteriaceae isolation: matched analysis. Am J Infect Control 42:612-20
Perez, Federico; Hujer, Andrea M; Marshall, Steven H et al. (2014) Extensively drug-resistant pseudomonas aeruginosa isolates containing blaVIM-2 and elements of Salmonella genomic island 2: a new genetic resistance determinant in Northeast Ohio. Antimicrob Agents Chemother 58:5929-35
Kiedrowski, Lee M; Guerrero, Dubert M; Perez, Federico et al. (2014) Carbapenem-resistant Enterobacter cloacae isolates producing KPC-3, North Dakota, USA. Emerg Infect Dis 20:1583-5
Perez, Federico; Bonomo, Robert A (2014) Vaccines for Acinetobacter baumannii: thinking "out of the box". Vaccine 32:2537-9

Showing the most recent 10 out of 27 publications