Apoptosis is absolutely necessary for human development and survival, with millions of cells committing suicide daily as a way to prevent uncontrolled growth. Defects in apoptosis, together with amplified growth signals, often lead to cancer. Targeting apoptosis defects in cancer has a tremendous potential.
The first human apoptotic protein identified was BCL-2, as inhibitor of apoptosis, in 1984. The role of caspases-proteases that act as the cell's direct executioners by cleaving other cellular proteins was revealed in humans beginning in 1993. In cell ready to die, pro-apoptotic BCL-2 family members, like BAX, disrupt mitochondria, causing the release of other proteins that lead to caspase release and cell death. Activation of this so-called “intrinsic” apoptotic pathway is the goal of many of the new cancer drugs.
A second, “extrinsic”, cell death pathway is also an important target, and the first so-called death receptor, DR4, was discovered around 1996.
Most of the current cancer therapies, including chemotherapeutic agents, radiation, and immunotherapy, work by indirectly inducing apoptosis in cancer cells. The inability of cancer cells to execute an apoptotic program due to defects in the normal apoptotic machinery is thus often associated with an increase in resistance to chemotherapy, radiation or immunotherapy-induced apoptosis.
In this regard, targeting crucial negative regulators that play a central role in directly inhibiting apoptosis in cancer cells represents a highly promising therapeutic strategy for new anticancer drug design.
Two classes of central negative regulators of apoptosis have been identified. The first class is the BCL-2 family of proteins. Anti-Bcl-2 biologicals (antisense, antibodies, etc.) were tested in Phase III clinical trials for the treatment of solid and not solid tumors, while recently several small molecules (obatoclax, gossypol, ABT-763) have also entered Phase II clinical trials for the same indications.
The second class of central negative regulators of apoptosis is the inhibitor of apoptosis proteins (IAPB). IAPB potently suppress apoptosis induced by a large variety of apoptotic stimuli, including chemotherapeutic agents, radiation, and immunotherapy in cancer cells.
X-linked IAP (XIAP) is the most studied IAP family member, and one of the most potent inhibitors in suppressing apoptosis among all of the IAP members. XIAP plays a key role in the negative regulation of apoptosis in both the death receptor-mediated and the mitochondria-mediated pathways. XIAP functions as a potent endogenous apoptosis inhibitor by directly binding and potently inhibiting three members of the caspase family enzymes, caspase-3, -7, and -9. XIAP contains three baculovirus inhibitor of apoptosis repeat (BIR) domains. The third BIR domain (BIR3) selectively targets caspase-9, the initiator caspase in the mitochondrial pathway, whereas the linker region between BIR1 and BIR2 inhibits both caspase-3 and caspase-7. While binding to XIAP prevents the activation of all three caspases, it is apparent that the interaction with caspase-9 is the most critical for its inhibition of apoptosis. Because XIAP blocks apoptosis at the downstream effector phase, a point where multiple signalling pathways converge, strategies targeting XIAP may prove to be especially effective to overcome resistance of cancer cells to apoptosis.
More recently, cellular IAPs 1 and 2 (cIAP-1 and cIAP-2) were also identified and characterized as potent inhibitors in suppressing apoptosis via two distinct pathways. Direct caspase inhibition happens through binding to the same BIR domains as XIAP, but inhibition of TNF-α induced apoptosis and induction of non-canonical NF-κB activation is also observed.
A balance between endothelial cell survival and apoptosis contributes to the integrity of the blood vessel wall during vascular development and pathological angiogenesis. It has been shown that both cIAP-1 and cIAP-2 are essential for maintaining this balance. Thus, both cIAPs may play an important role in the control of angiogenesis and blood vessel homeostasis in several pathologies involving regeneration and tumorigenesis.
There is evidence to indicate that XIAP and cIAPs are widely overexpressed in many types of cancer and may play an important role in the resistance of cancer cells to a variety of current therapeutic agents. There is thus a strong rationale for pan-IAP inhibitors as potent and effective pro-apoptotic agents in oncology.
Recently, Smac/DIABLO (second mitochondria-derived activator of caspases) was identified as a protein released from mitochondria into the cytosol in response to apoptotic stimuli. Smac is synthesized with an N-terminal mitochondrial targeting sequence that is proteolytically removed during maturation to the mature polipeptide. Smac was shown to directly interact with XIAP, cIAP-1, cIAP-2 and other IAPs, to disrupt their binding to caspases and facilitate caspases activation. Smac is a potent endogenous inhibitor of XIAP.
Smac/DIABLO interacts with both the BIR2 and BIR3 domains of XIAP. The crystal structure of Smac/DIABLO reveals that it forms a homodimer through a large, hydrophobic interface, and that homodimerization is essential for its binding to the BIR2, but not BIR3, domain of XIAP. The four amino-terminal residues of Smac/DIABLO (Ala-Val-Pro-Ile, AVPI) make specific contact with a surface groove of the BIR2 and BIR3 domains, but not with the BIR1 domain, of XIAP. Significantly, the conserved tetrapeptide motif has remarkable homology to the IAP-interacting motif found in the p12 amino-terminal sequence of caspase-9 (Ala-Thr-Pro-Phe) and the Drosophila proteins Hid (Ala-Val-Pro-Phe), Reaper (Ala-Val-Ala-Phe) and Grim (Ala-Ile-Ala-Tyr).
The Kd value of Smac peptide AVPI binding to XIAP (Kd=0.4 μM) is essentially the same as the mature Smac protein (Kd=0.42 μM).
Full length Smac-BIR domain complexes of cIAP-1 or cIAP-2 are not reported. A complex between the BIR3 domain of cIAP-1 and N-terminal Smac sequences was reported, showing similar binding modes and strengths compared with XIAP.