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 #
5R01GM054072-16
Application #
8461167
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
1996-04-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
16
Fiscal Year
2013
Total Cost
$313,306
Indirect Cost
$103,129
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Dong, Jian; Zhuang, Zhihao; Song, Feng et al. (2012) A Thioester Substrate Binds to the Enzyme Arthrobacter Thioesterase in Two Ionization States; Evidence from Raman Difference Spectroscopy. J Raman Spectrosc 43:65-71
Papp-Wallace, Krisztina M; Bethel, Christopher R; Gootz, Thomas D et al. (2012) Inactivation of a class A and a class C ?-lactamase by 6?-(hydroxymethyl)penicillanic acid sulfone. Biochem Pharmacol 83:462-71
Nottingham, Micheal; Bethel, Christopher R; Pagadala, Sundar Ram Reddy et al. (2011) Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy. Bioorg Med Chem Lett 21:387-93
Carey, Paul R; Chen, Yuanyuan; Gong, Bo et al. (2011) Kinetic crystallography by Raman microscopy. Biochim Biophys Acta 1814:742-9
Gong, Bo; Chen, Jui-Hui; Yajima, Rieko et al. (2009) Raman crystallography of RNA. Methods 49:101-11
Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C et al. (2009) A catalytic metal ion interacts with the cleavage Site G.U wobble in the HDV ribozyme. Biochemistry 48:1498-507
Kalp, Matthew; Totir, Monica A; Buynak, John D et al. (2009) Different intermediate populations formed by tazobactam, sulbactam, and clavulanate reacting with SHV-1 beta-lactamases: Raman crystallographic evidence. J Am Chem Soc 131:2338-47
Kalp, Matthew; Bethel, Christopher R; Bonomo, Robert A et al. (2009) Why the extended-spectrum beta-lactamases SHV-2 and SHV-5 are "hypersusceptible" to mechanism-based inhibitors. Biochemistry 48:9912-20
Kalp, Matthew; Carey, Paul R (2008) Carbapenems and SHV-1 beta-lactamase form different acyl-enzyme populations in crystals and solution. Biochemistry 47:11830-7
Totir, Monica A; Cha, Jooyoung; Ishiwata, Akihiro et al. (2008) Why clinically used tazobactam and sulbactam are poor inhibitors of OXA-10 beta-lactamase: Raman crystallographic evidence. Biochemistry 47:4094-101

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