Small Molecule Modulation of Caspase Enzymatic Activity
Goode, David Ryan
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/84292
Description
Title
Small Molecule Modulation of Caspase Enzymatic Activity
Author(s)
Goode, David Ryan
Issue Date
2008
Doctoral Committee Chair(s)
Hergenrother, Paul J.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Pharmaceutical
Language
eng
Abstract
Programmed cell death, also known as apoptosis, is a critical process in the survival of multi-cellular organisms, as it allows for development and maintenance. The misregulation of apoptosis is the underlying cause of various disease states. While apoptosis is a complex signaling cascade, the key mediators of the apoptotic signal are caspase enzymes. Caspases, a family of cysteine proteases, specifically cleave substrates C-terminal to an aspartic acid residue. Small molecule modulation of caspase activity can play an important role in the study of the apoptotic cascade, the role caspases have within apoptosis, and the various diseases that arise from misregulation of apoptosis. These small molecules could also be therapeutically relevant. Thus, the underlying theme in the following work is the identification and characterization of small molecule modulators of caspase activity. Studies of both small molecule caspase inhibition and activation were conducted. In order to increase the selectivity in the design of small molecule inhibitors of caspases, a systematic probing of the S1' site in caspase-3 and caspase-7 was conducted using designed peptidic ketone inhibitors. Synthesis and evaluation of a series of these ketonic peptide inhibitors showed selectivity difference due solely to the P1' group (which binds the S1' pocket). The incorporation of the discovered biases should guide future production of selective non-peptidic caspase inhibitors. On the activator front, the characterization of the in vitro mechanism of a previously identified procaspase-3 activator, PAC-1, was conducted. Through enzymatic assays under various buffer conditions, the in vitro mechanism of PAC-1 activation of procaspase-3 was conclusively found to be through relief of enzyme inhibition by metal ions. Zinc, the most physiologically relevant metal inhibitor, was further focused upon. PAC-1 was found to bind Zn with a KD of ∼45 nM. PAC-1 and other metal binders were shown to reactivate procaspase-3 and caspase-3 in reconstituted system. Preliminary studies were performed to link this in vitro mechanism to the cell culture effects of PAC-1. Also presented in this work was the serendipitous discovery of promiscuous enzyme activators while high-throughput screening (HTS) for procaspase-2 activators. The mechanism of these promiscuous activators was linked to detergent-like effects. Characterization of these promiscuous activators should save others time and effort in future HTS campaigns.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
