| dc.identifier.uri | http://hdl.handle.net/11401/76957 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | Slow-onset inhibitors are of particular interest in drug discovery programs as the slow dissociation of the inhibitor from the target-inhibitor complex prolongs target occupancy and improves in vivo efficacy. While slow-onset inhibition is observed in many enzymes and the kinetic equations used to describe slow-onset inhibition were derived more than 26 years ago, the structural basis for slow-onset inhibition is still not generally well understood, hindering prediction and control of slow-onset binding kinetics. An enzyme system with known experimental kinetics and structural data for multiple classic and slow-onset inhibition complexes would be an ideal model system for the study of structure basis of slow-onset inhibition. InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a validated target for the development of tuberculosis chemotherapeutics and an excellent model system for the study of slow-onset inhibition. Inhibition of InhA follows a two-step binding mechanism in which formation of the initial enzyme-inhibitor (EI) complex is followed by a slow conformational change that leads to the final enzyme-inhibitor complex (EI*). In this work, based on analyses of all available crystal structures, we found that the active-site conformation of InhA can be characterized as open or closed. Unrestrained molecular dynamics simulation started from the open conformation demonstrated that the active-site helix-6&7 region was moving toward to a semi-closed conformation in both rapid reversible and slow-onset inhibitors bound complexes. On the contrary, when simulation started from the closed conformation, the active-site helix-6&7 region maintained in a relative stable conformation in the slow-onset inhibitor bound complexes. We hypothesized that the open and closed conformations represent the initial EI and final EI* complexes, respectively, and conformational change from open to closed is the slow structural isomerization step. By using partial nudged elastic band (PNEB) and umbrella sampling (US) simulations, we were able to obtain a continued energy landscape along the open-closed conformational change path. For the rapid reversible inhibition complexes, the energy landscapes exhibit two types of energy profile, either preferring the open state, or having little preference and low energy barrier between the open and closed states. On the other hand, the slow-onset inhibition complexes have a relative stable closed state with a more significant energy barrier between the open and closed states. In order to modulate the life-time (residence time) of the enzyme-inhibitor complex, it is important to understand the interactions that modulate this induced-fit mechanism and, specifically, to determine the structure of the transition state that lies on the reaction coordinate between the open and closed states. Structural analyses identified several active-site residues that regulate the free energy barrier in the open-closed path. Replacement of these key residues with amino acids possessing smaller side chains results in a decrease of energy barrier. The energy barrier can be restored by rational reintroduction of a steric clash at the transition state. This was accomplished in two different ways: through mutation of a residue at a different position to a larger side chain, or through modification of the ligand with a bulky substituent. These loss and regain of function studies validate these key residues controlling conformational change, and will provide a platform for the design of inhibitors with longer residence time and better in vivo potency. Although the free energy profile derived from the PNEB/US approach provides a way to distinguish rapid reversible and slow-onset inhibitors, PNEB/US approach is computationally expensive and time-consuming. It is unlikely to use this approach to examine every inhibitor, thus, a rapid screening approach - using docking to rapidly examine inhibitors was designed. The dock score had same trend as the US results. The combined dock/PNEB/US protocol could provide an approach for slow-onset inhibitor screening. | |
| dcterms.available | 2017-09-20T16:51:32Z | |
| dcterms.contributor | Simmerling, Carlos L | en_US |
| dcterms.contributor | Green, David | en_US |
| dcterms.contributor | Tonge, Peter J | en_US |
| dcterms.contributor | Rizzo, Robert | en_US |
| dcterms.contributor | Drueckhammer, Dale. | en_US |
| dcterms.creator | Lai, Cheng-Tsung | |
| dcterms.dateAccepted | 2017-09-20T16:51:32Z | |
| dcterms.dateSubmitted | 2017-09-20T16:51:32Z | |
| dcterms.description | Department of Biochemistry and Structural Biology. | en_US |
| dcterms.extent | 211 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/76957 | |
| dcterms.issued | 2015-08-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:32Z (GMT). No. of bitstreams: 1
Lai_grad.sunysb_0771E_11744.pdf: 13864289 bytes, checksum: ae78b6e62725d3ca64f8c80ddc28d106 (MD5)
Previous issue date: 2014 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Biochemistry | |
| dcterms.subject | computer-aided drug design, enoyl-ACP reductase, molecular dynamics, residence time, Slow-onset inhibition, transition state | |
| dcterms.title | A Structural and Energetic Model for the Slow-onset Inhibition | |
| dcterms.type | Dissertation | |
