Small Molecule Modulation of Caspase Enzymatic Activity

180 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.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.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD