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RAMAN SPECTROSCOPIC STUDIES OF INHIBITOR REACTIONS IN CLASS A, B AND D beta-LACTAMASES
- Publication Year :
- 2015
-
Abstract
- The driving force behind my research is the need to develop improved antibiotic therapies to combat the drug resistance. The production of ß-lactamase is the major cause of failure of ß-lactam-based therapy against Gram-negative bacteria, such as Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. However, currently there are only three ß-lactamase inhibitors in clinical use and they narrowly target the class A enzymes. A clinically useful inhibitor needs to have a significantly broader inhibitory spectrum, ideally targeting all serine ß-lactamases (class A, C and D).My work is focused on studying the catalytic mechanisms of serine ß-lactamases, and thence aiding in the design new mechanism-based inhibitors. A major thrust involves use of Raman spectroscopy which is a laser light scattering technique that provides structural information of the bound ligand in the active site of the enzymes in real time. With the combination of Raman spectroscopy and other structural or microbiological methodologies, we aim to address two fundamental questions in drug discovery: 1) why is a compound a good inhibitor for a target enzyme in vitro? 2) how effectively is a drug taken up by cells?We have designed and implemented a new method by combining rapid mix-rapid freeze of aqueous solution with Raman spectroscopy. This enables us to trap intermediates early on the reaction pathway- within milliseconds. By studying the reaction of OXA-24 and OXA-1 and a penicillin derivative inhibitor in crystallo and in solution, we have shown that carboxylation of the lysine is critical for class D enzymes' catalysis, while decarboxylation can be induced by inhibitors and therefore, leading to enzyme inactivation.Penetration barriers and efflux pumps are two major mechanisms bacteria adopt to exclude drug molecules. The difficulty in directly measuring compound penetration is a major reason for the lack of progress in antibiotic discovery. Using a new protocol, the intermediates or products formed inside the bacterial cells have been captured, which allows us to investigate reactions inside bacterial cells.
Details
- Language :
- English
- Database :
- OpenDissertations
- Publication Type :
- Dissertation/ Thesis
- Accession number :
- ddu.oai.etd.ohiolink.edu.case1421514785