Chemical Antagonists of IAP-Family Anti-Apoptotic Proteins
Principal Investigator: J C Reed
Abstract: [unreadable] DESCRIPTION (provided by applicant): Apoptosis plays essential roles in many aspects of normal development and physiology, becoming dysregulated in myriad diseases characterized by insufficient or excessive cell death. Caspases are the executioners of apoptosis. These intracellular proteases are suppressed by Inhibitor of Apoptosis Proteins (IAPs), a family of evolutionarily conserved anti-apoptotic proteins. Proteins released from mitochondria (SMAC and HtrA2) can competitively displace IAPs from the Caspases, thus helping to drive apoptosis. It has been shown that only a few residues at the N-terminus of activated SMAC protein (4mer) are sufficient to affect the release of IAPs from Caspases. Thus, it is plausible to identify chemical compounds that mimic the effect of SMAC in antagonizing IAPs by causing them to release Caspases. Non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of cell permeability, stability, and in vivo pharmacology. To this end we have developed a binding assay based upon fluorescence polarization, using a short peptide representing residues from the N-terminus of activated SMAC with an attached fluorochrome. This fluorescence polarization assay (FPA) forms the basis for a high-throughput competitive displacement assay that we have optimized for chemical library screening. We propose to screen the NIH compound library using this FPA and thus identify chemical compounds that compete with SMAC peptide for binding to IAPs. Then, using 3 types of secondary assays we have already devised, the hits will be independently confirmed. Structure Activity Relations (SAR) studies of analogs will be performed for a prototypical member of the IAP-family, XIAP. Finally, to define the selectivity of the compounds, SAR studies will be performed using assays configured for additional members of the IAP family (cIAP1, cIAP2, ML-IAP, ILP2). Altogether, these efforts will result in validated chemical probes for studying the biology of IAPs in a variety of cellular and organismal contexts. Cell death is a normal facet of physiology. The average human produces and in parallel eradicates 50-70 billion cells in his or her body, with most of this cell death occurring via a process known as "apoptosis." Defects in the normal regulation of apoptosis are at the core of many diseases, including cancer where insufficient cell death permits abnormal cell accumulation, and degenerative diseases where excessive cell death leads to tissue loss and organ dysfunction. Apoptosis is accomplished by intracellular proteases, called Caspases. Like all proteolytic systems, the Caspases are under fine control by networks of proteins that either promoter their activation or suppress their activity. The chief endogenous antagonists of Caspases are IAPs (Inhibitor of Apoptosis Proteins), an evolutionarily conserved family of proteins that bind to and inhibit the activity of Caspases or that induce Caspase degradation by ubiquitin-dependent mechanisms. Our objective is to identify chemical compounds that bind sites on IAPs, competitively displacing Caspases. The resulting compounds will be useful as research tools for understanding the biology of IAPs and for ascertaining their roles in diseases such as cancer, where IAP over-expression is commonly observed. [unreadable] [unreadable] [unreadable]
Funding Period: 2007-06-01 - 2008-05-31
more information: NIH RePORT
- Expedient synthesis of highly potent antagonists of inhibitor of apoptosis proteins (IAPs) with unique selectivity for ML-IAPMitchell Vamos
Program in Apoptosis and Cell Death and NCI Designated Cancer Center, Sanford Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
ACS Chem Biol 8:725-32. 2013..Furthermore, computational modeling was performed, revealing key contacts between the IAP proteins and antagonists, suggesting a structural basis for the observed potency...