Bacterial infections pose serious threats to human health. Furthermore, viral infections such as the flu are frequently accompanied by bacterial infections which is often a deadly combination. Due to the development of resistance, the options for treating infections have dwindled substantially. This resistance is in large part due to the bacterial expression of beta-lactamases that degrade beta-lactam antibiotics including penicillins, cephalosporins, and the """"""""last resort"""""""" carbapenem antibiotics. An alternative approach for treating p-lactam resistant gram-negative infections is co-administering a p-lactamase inhibitor in addition to penicillin-like antibiotics. Regrettably, p-lactamases have also evolved an inhibitor-resistant phenotype able to overcome this treatment option. The p-lactamase variants that hydrolyze these inhibitors are called inhibitor resistant [3-lactamases, those that hydrolyze cephalosporins are called extended-spectrum p-lactamases (ESBLs), and those that hydrolyze carbapenems are known as carbapenemases. The overarching goals of this proposal are to understand the structural basis of the phenotypes of ESBL-, carbapenemase-, and inhibitor resistant p-lactamases, and to develop novel inhibition strategies. Our structure-function studies involve a novel synergy between X-ray and Raman crystallography and this innovative inter-disciplinary approach allows us to identify and track reaction intermediates inside crystals prior to X-ray analysis and provides a unique advantage to accomplish our Aims.
Aim 1 : To further improve our novel designed beta-lactamase inhibitor SA2-13 by modifying the overall charge and carboxyl linker to improve uptake and trans-enamine stabilization.
Aim 2 : To test the hypothesis that the changes in or near D179 which are present in ESBL's SHV-6, -8, and -24 have evolved to hydrolyze ceftazidime by shifting the omega loop thereby extending the active site to accommodate ceftazidime.
Aim 3 : To test the hypothesis that class A carbapenemases such as KPC-2 have adopted a shallower active site and flexible catalytic 870 side chain to efficiently hydrolyze carbapenems.
Aim 4 : To test the hypothesis that inhibitors that either can carry out bi-cyclization (such as LN1-255) or fra/is-enamine inhibitors (SA2-13) are capable of forming stable inhibitory complexes with inhibitor-resistant class A and inhibitor-insensitive class D p-lactamases. The impact of beta-lactamase mediated antibiotic resistance on human health is enormous, costing billions of dollars in health care costs. Detailed understanding is needed and our targeted structural knowledge will provide for molecular insights into cases such as the recent outbreaks of KPC mediated carbapenem-resistant K. pneumoniae in New York. These resistance insights will lead to new therapeutic approaches and our goal is to study and develop new broad-spectrum beta-lactamase inhibitors. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI062968-02
Application #
7463633
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Xu, Zuoyu
Project Start
2007-07-15
Project End
2011-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
2
Fiscal Year
2008
Total Cost
$265,238
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Rodkey, Elizabeth A; Winkler, Marisa L; Bethel, Christopher R et al. (2014) Penam sulfones and ?-lactamase inhibition: SA2-13 and the importance of the C2 side chain length and composition. PLoS One 9:e85892
Rodkey, Elizabeth A; McLeod, David C; Bethel, Christopher R et al. (2013) ?-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme. J Am Chem Soc 135:18358-69
Ke, Wei; Pattanaik, Priyaranjan; Bethel, Christopher R et al. (2012) Structures of SHV-1 ?-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation. PLoS One 7:e49035
Ke, Wei; Bethel, Christopher R; Papp-Wallace, Krisztina M et al. (2012) Crystal structures of KPC-2 ?-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226. Antimicrob Agents Chemother 56:2713-8
Rodkey, Elizabeth A; Drawz, Sarah M; Sampson, Jared M et al. (2012) Crystal structure of a preacylation complex of the ?-lactamase inhibitor sulbactam bound to a sulfenamide bond-containing thiol-?-lactamase. J Am Chem Soc 134:16798-804
Ke, Wei; Rodkey, Elizabeth A; Sampson, Jared M et al. (2012) The importance of the trans-enamine intermediate as a ?-lactamase inhibition strategy probed in inhibitor-resistant SHV ?-lactamase variants. ChemMedChem 7:1002-8
Ke, Wei; Laurent, Abigail H; Armstrong, Morgan D et al. (2012) Structure of an engineered ?-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation. PLoS One 7:e39168
Ke, Wei; Sampson, Jared M; Ori, Claudia et al. (2011) Novel insights into the mode of inhibition of class A SHV-1 beta-lactamases revealed by boronic acid transition state inhibitors. Antimicrob Agents Chemother 55:174-83
Sampson, Jared M; Ke, Wei; Bethel, Christopher R et al. (2011) Ligand-dependent disorder of the Omega loop observed in extended-spectrum SHV-type beta-lactamase. Antimicrob Agents Chemother 55:2303-9
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

Showing the most recent 10 out of 13 publications