This proposal sets out to study the chemical reactions between compounds that are good inhibitor candidates and ?-lactamase enzymes. In the clinic, bacterial infections are becoming increasingly resistant to antibiotic-based treatments. A major source of resistance is the class of ? -lactamase enzymes produced by the bacteria. These enzymes hydrolyze, and thus inactivate, penicillin-like molecules before they can attack the bacteria. Here we set out to identify the intermediates formed by mechanism-based inhibitors that block the action of clinically problematic ? -lactamases. A focal point of the study will be the elucidation of the reactions of lead inhibitors with class A and class D ? -lactamases that function as carbapenemases (KPC-2 and OXA-24) and extended spectrum ? -lactamases (SHV-2, SHV-5 and OXA 10). A major thrust involves the use of Raman crystallography, where the reactions of the inhibitors within single crystals of the enzyme are followed in real time using a Raman microscope. This provides information on the identity and conformation of intermediates on the reaction pathway as well as kinetic data on their populations. The Raman data also provide an immediate link to X-ray crystallography with its detailed structural insights, and the two approaches are highly synergistic. In addition, the Raman analysis will be extended to solution studies, linking our knowledge of the reactions in crystals to the corresponding reaction in the aqueous phase. This is important because it is becoming clear that, for some inhibitors, the results from solution studies differs from those in crystals. Initially, our studies will use lead compounds that are 6-(pyridylmethylidene) penam sulfones possessing favorable IC50 values. Our results will identify reaction intermediates that block the active site under biologically relevant conditions and provide input into the design of better inhibitors, and thence potential therapeutic compounds.

Public Health Relevance

The threat to human health caused by drug resistance is a topic seldom out of the news'headlines. Bacteria are becoming increasingly resistant to elimination by "classical" antibiotic-based therapies. This project is part of a team-based effort to identify compounds that might serve as a basis for designing novel drugs effective against emerging resistant bacteria. Our contribution involves using newly-developed technology to characterize chemical reactions between lead compounds and the enzymes that are responsible for a large number of "drug resistance" outbreaks. By characterizing intermediates formed in these reactions we can propose compounds that can more effectively block the active sites of the enzymes and serve as a starting point for drug design. A focal point of the study will be the elucidation of the reactions of lead inhibitors with clinically problematic ? -lactamases that function as carbapenemases (KPC-2 and OXA-24) and extended spectrum ? -lactamases (SHV-2, SHV-5 and OXA 10). A major thrust involves the use of Raman crystallography, where the reactions of the inhibitors within single crystals of the enzyme are followed in real time using a Raman microscope. This provides information on the identity and conformation of intermediates on the reaction pathway as well as kinetic data on their populations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054072-15
Application #
8251222
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
1996-04-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
15
Fiscal Year
2012
Total Cost
$324,669
Indirect Cost
$106,869
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Antonopoulos, Ioanna H; Warner, Brittany A; Carey, Paul R (2015) Concerted Protein and Nucleic Acid Conformational Changes Observed Prior to Nucleotide Incorporation in a Bacterial RNA Polymerase: Raman Crystallographic Evidence. Biochemistry 54:5297-305
Heidari-Torkabadi, Hossein; Bethel, Christopher R; Ding, Zhe et al. (2015) "Mind the Gap": Raman Evidence for Rapid Inactivation of CTX-M-9 β-Lactamase Using Mechanism-Based Inhibitors that Bridge the Active Site. J Am Chem Soc 137:12760-3
Che, Tao; Rodkey, Elizabeth A; Bethel, Christopher R et al. (2015) Detecting a quasi-stable imine species on the reaction pathway of SHV-1 β-lactamase and 6β-(hydroxymethyl)penicillanic acid sulfone. Biochemistry 54:734-43
Heidari-Torkabadi, Hossein; Che, Tao; Lombardo, Michael N et al. (2015) Measuring propargyl-linked drug populations inside bacterial cells, and their interaction with a dihydrofolate reductase target, by Raman microscopy. Biochemistry 54:2719-26
Antonopoulos, Ioanna H; Murayama, Yuko; Warner, Brittany A et al. (2015) Time-resolved Raman and polyacrylamide gel electrophoresis observations of nucleotide incorporation and misincorporation in RNA within a bacterial RNA polymerase crystal. Biochemistry 54:652-65
Heidari Torkabadi, Hossein; Bethel, Christopher R; Papp-Wallace, Krisztina M et al. (2014) Following drug uptake and reactions inside Escherichia coli cells by Raman microspectroscopy. Biochemistry 53:4113-21
Che, Tao; Bethel, Christopher R; Pusztai-Carey, Marianne et al. (2014) The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine. J Biol Chem 289:6152-64
Sui, Xuewu; Kiser, Philip D; Che, Tao et al. (2014) Analysis of carotenoid isomerase activity in a prototypical carotenoid cleavage enzyme, apocarotenoid oxygenase (ACO). J Biol Chem 289:12286-99
Heidari Torkabadi, Hossein; Che, Tao; Shou, Jingjing et al. (2013) Raman spectra of interchanging ýý-lactamase inhibitor intermediates on the millisecond time scale. J Am Chem Soc 135:2895-8
Rodkey, Elizabeth A; McLeod, David C; Bethel, Christopher R et al. (2013) β-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme. J Am Chem Soc 135:18358-69

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