Abstract:

This study focuses on two proteins in the mitochondrial pathway of programmed cell death (apoptosis), Smac (second mitochondrial activator of caspases) and XIAP (X-linked inhibitor of apoptosis protein). Briefly, at the onset of apoptosis, Smac is released from the mitochondria. In the cytosol, Smac antagonizes XIAP, which inhibits apoptosis by sequestering the caspases (cysteine-aspartate proteases). The caspases are responsible for the cascade of events which produces the morphological changes associated with cell death. Smac displaces the caspases from XIAP and allows the apoptotic process to proceed.

The overexpression of XIAP has been associated with the resistance of cancer cells to cytotoxic drugs, presumably due to its anti-apoptotic activity. Therefore, it is of interest to design compounds that can mimic Smac and counter an abundance of XIAP in cancer cells. The crystal structure of Smac bound to XIAP provides a basis for the design of these "Smac mimics." The binding interaction between these two proteins primarily consists of the N-terminal tetrapeptide of Smac (AVPI) interacting with the BIR3 domain of XIAP.

In order to probe the effects of the Smac mimics in cancer cell lines where XIAP is overexpressed, the peptide character would need to be minimized in order to increase stability and cell-permeability. In the study described herein, a series of compounds was designed to mimic the Smac N-terminal tetrapeptide while replacing the A-V peptide bond by an oxazole or thiazole ring. The heterocycle-containing Smac mimics were synthesized and assayed for binding to the BIR3 domain of XIAP. The binding affinity varied with heterocycle identity, heterocycle modification, and modification to the peptidic half of the molecule (P-F). Crystallographic data revealed a different mode of binding for the oxazole-containing Smac mimics relative to the N-terminal tetrapeptide of Smac. Comparison of the crystal structures of AoxSPW, AVPF and AVPI bound to BIR3-XIAP demonstrated that the protein is displaced more in the AoxSPW structure relative to the peptide-bound structures. Biological data demonstrated the ability of one Smac mimic to reduce cell viability in a breast cancer cell line where the Smac peptide had no effect.