Smac/DIABLO was recently discovered as a potent pro-apoptotic protein released from mitochondria. Smac functions as an endogenous antagonist for the Inhibitors of Apoptosis Proteins (lAPs) by binding to the BIR3 domain of X-linked lAP (XIAP) and other lAP proteins and displace caspase-9 from this site. High resolution 3D structures of Smac in complex with the BIR3 domain of XIAP showed that the interaction between them is mediated by only four Smac residues at its N-terminus and a small but well-defined binding groove in the XIAP BIR3 domain, which is suitable for designing small molecule inhibitors. Several recent studies have shown that short Smac peptides (4-8 residues) tethered to a carrier peptide for intra-cellular delivery are effective to overcome apoptosis-resistance of cancer cells in vitro and in vivo by directing targeting lAP proteins, while having no toxicity to normal tissues or to animals. Hence, Smac mimetics that bind to the BIR3 domain in XlAP where Smac and caspase-9 bind may have great therapeutic potential to be developed as an entirely new class of anticancer drugs through overcoming apoptosis-resistance of cancer cells as mediated by lAP protein overexpression. In this project, we propose to design, synthesize and characterize highly potent Smac peptido-mimetics and nonpeptidic mimetics with much improved cell permeability over Smac peptides through the following Specific Aims:
Aim 1. (a). Structure-based design of Smac peptido-mimetics and nonpeptidic mimetics based upon the published high-resolution experimental structures of Smac in complex with XlAP BIR3. (b). Determination of high-resolution structures of several most potent Smac mimetics through X-ray crystallography.
Aim 2. Chemical synthesis of these Smac mimetics designed in Aim 1.
Aim 3. (a). Determination of the binding affinities of these Smac mimetics to the BIR3 domain of XIAP by the fluorescence polarization-based method; (b). Conclusive confirmation of the binding of the most potent Smac mimetics to the XlAP BIR3 protein by NMR methods.
Aim 4. Investigation of the activity, specificity and molecular mechanisms of action of the most potent Smac mimetics. Designing highly potent and cell-permeable Smac mimetics is a new and exciting area of research. Our current proposed research represents the first but an essential step toward our long-term goal of developing a novel anticancer drug through targeting apoptosis-resistance in cancer cells mediated by lAP proteins.
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