Abstract B44: Probing the effects of linker flexibility in second mitochondria-derived activator of caspases mimetic dimers and assessing their ability to induce apoptosis in a MDA-MB-231 cell line

Abstract

Abstract Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (lAPs). X-linked inhibitor of apoptosis protein (XIAP) is one of the best characterized lAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), in particular caspase −3, −7, and −9 through one of its baculovirus IAP repeat (BIR) domains. Second mitochondria-derived activator of caspases (SMAC) is an antagonist protein released from the mitochondria and into the cytosol, to disrupt the complexes produced between XIAP and caspase. SMAC binds to the BIR domains in XIAP through its tetra-peptide motif, AVPI. Studies have demonstrated that the first and third amino acid positions of this tetra-peptide motif are important for binding, while positions two and four can be modified to increase binding affinity (AXPX). It has also been shown that dimers of SMAC mimetics show greater potency than their monomer counter parts. Previously, our laboratory has synthesized and tested SMAC dimers using three glycine amino acids to link AXPX sequences. These compounds showed very little activity. We also synthesized and tested SMAC dimers comprised of a lysine amino acid, linking two AXPX sequences; these mimetics did display significant activity. In particular an (AKPF)2K sequence showed 50% cell death at 10 uM when combined with death ligand TRAIL. Synthesis of these compounds was conducted using solid phase peptide synthesis (SPPS) on a polystyrene resin. We hypothesized that flexibility in the linker joining the two AXPX sequences was necessary to increase binding affinity. Consequently several SMAC mimetic dimers built on two different linkers (gamma-amino butyric acid and ornithine), both of which have a four carbon spacer between each terminus, have been prepared and evaluated for activity using a MDA-MB-231 breast cancer cell line. The first series of SMAC mimetic compounds consists of MeAXPX-GABA-MeAXPX amino acid sequence while the second series of SMAC mimetics are comprised of a MeAXPX-Ornithine-XPXMeA amino acid sequence. All data and results, including synthesis, biological assays, and the testing of these compounds will be the major focus of this presentation. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B44.