Type
Text
Type
Dissertation
Advisor
Boon, Elizabeth | Tonge, Peter J. | Wang, Jin | Walker, Stephen.
Date
2015-12-01
Keywords
Chemistry
Department
Department of Chemistry.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/77172
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Tuberculosis (TB) infection is the single most lethal bacterial infections in the world. Isoniazid (INH), one of the most effective anti-TB drugs is usually administered to treat latent TB infection and is used in combination therapy to treat active TB. However resistance to INH is common and the emergence of multi-drug-resistant, extensively drug-resistant and now totally drug-resistant strains of TB, has resulted in an urgent need to develop novel anti-TB drugs. INH targets InhA, the enoyl-ACP reductase (FabI) from the Type II fatty acid biosynthesis pathway, and resistance to INH usually results from mutations in KatG, the enzyme that activates INH. Thus compounds that target InhA directly should be active against drug-resistant TB. Drug-target residence time has recently emerged as a crucial parameter for drug discovery since drugs with long residence times on their targets will have sustained target occupancy leading to improved pharmacodynamics and safety. However, the molecular basis of drug-target residence time is poorly understood. In addition, methods to quantitate the lifetime of drug-target complexes are also lacking. This gap in knowledge hinders the rational design of inhibitors with improved residence times. Utilizing InhA as our model system, we have developed a robust assay to accurately quantitate drug-target residence time. Using a combination of enzyme kinetics, computational modeling and X-ray crystallography, we have rigorously characterized the structure-kinetic relationships for 24 diphenyl ether-based InhA inhibitors, and have investigated the mechanism of time-dependent enzyme inhibition at the molecular level. This has led to the development of a 2-step slow-onset inhibition model in which the InhA substrate-binding loop (SBL) undergoes an “open-to-closed†conformational change on the binding reaction coordinate. Site-directed mutagenesis suggests that the energy barrier associated with slow-onset inhibition results from a large- scale refolding of the SBL. Based on this finding, we have successfully designed a novel InhA inhibitor (PT504) with a 10-fold longer residence time compared to INH. Cellular studies suggest that PT504 is active against Mycobacterium tuberculosis and that the long residence time can be translated into a prolonged post-antibiotic effect. Further studies using a fragment-based approach together with phenotypic screening, have led to the identification of 3 different classes of InhA inhibitors with unique inhibition mechanisms, thus diversifying the current InhA drug candidate portfolio. | 292 pages
Recommended Citation
Yu, Weixuan, "Targeting the Enoyl-ACP Reductase in Mycobacterium tuberculosis: Mechanism of Time Dependent Inhibition and Cellular Consequence of Target Engagement" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 3007.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/3007