Smac-based antagonists of the inhibitors of apoptosis

Abstract

Apoptosis signal pathways converge on the Caspases, a family of proteases that form a self-amplifying cascade, the activation of which generates the characteristic morphological and biochemical features of apoptotic cell death. The Inhibitors of Apoptosis (IAPs) are a family of proteins that bind and inactivate Caspases at both the initiation and terminal effector stages of this cascade. Over-expression of IAPs, as observed in cancer cell lines and tumour biopsy samples, results in a cellular phenotype of resistance to a variety of apoptotic stresses including chemotherapeutic drugs and irradiation. One negative regulator of IAP function, Smac (second mitochondrial activator of Caspases a.k.a.Diablo) has been identified as a mitochondrial protein that is processed and released concomitantly with cytochrome c following an apoptotic stress. Proteolytic processing of the mitochondrial signal peptide sequence generates a novel amino terminus that interacts with XIAP and disrupts XIAP-meditated Caspase-9 inhibition. By disrupting the interaction of XIAP with Caspase-9, Smac is believed to promote apoptotis. Furthermore, the structure determinations of XIAP, in conjunction with either Caspases or Smac, have led to the identification of a critical pocket and groove on the surface of each BIR domain ( Baculovirus IAP Repeat). Within XIAP BIR3, the binding pocket interacts with Caspase-9 and can be disrupted by the presence of Smac, which competes for the same site. In this thesis work, the utility of Smac as a chemo-sensitizing agent for the treatment of cancer was enhanced by three different strategies. The first approach involved the development and characterization of an ubiquitin-Smac fusion system that circumvents the targeting of Smac to the mitochondria and results in the expression of a fully mature, biologically active Smac protein. The second approach entailed developing a strategy for increasing the affinity of Smac binding to the IAPs. A random peptide phage display screen was used to identify novel, high affinity peptide ligands that bind to the IAP-BIR3 motifs. These peptide sequences were similar in structure to the amino terminus of Smac and Caspase-9. The data generated from these two approaches showed that both over-expressed, mature Smac and Smac-like peptide sequences bind to the IAPs in vitro and in vivo and sensitize cells to chemotherapeutic drugs. The third and final approach involved developing a Smac system that was a more potent apoptosis-inducer than wild-type Smac, which would be predicted to have greater therapeutic potential. It has been shown that Smac interactions with the IAP BIR domains may accelerate IAP auto-ubiquitination and destruction via the IAP carboxy terminal RING (Really Interesting New Gene) domain. Adding an IAP-RING domain to the carboxy terminus of Smac was predicted to enhance IAP degradation while maintaining the IAPs in a non-functional protein complex. Indeed, it was observed that generating a Smac protein with RING-conferred E3 ligase activity significantly decreased IAP levels, with a concomitant increase in the sensitivity to apoptosis in the presence of chemotherapeutic agents.