Alterations in apoptotic pathways have been implicated in many debilitating diseases such as cancer and neurodegenerative disorders. 1, 2 Thus, targeting cell death pathways has always been therapeutically attractive. In particular, as it is conceptually easier to kill than to sustain cells, abundant attention has been focused on anti-cancer therapies using pro-apoptotic agents such as conventional radiation and chemo-therapy. These treatments are generally believed to trigger activation of the mitochondria-mediated apoptotic pathways. However, these therapies lack molecular specificity. Over the last year or so, the discovery and structural characterization of an IAP-binding peptide motif have generated much enthusiasm in screening for an anti-cancer drug tailored for the caspase pathways. 3 Apoptosis is primarily executed by activated caspases, a family of cysteine proteases with aspartate specificity in their substrates. Caspases are produced in cells as catalytically inactive zymogens and must be proteolytically processed to become active proteases during apoptosis. In normal surviving cells that have not received an apoptotic stimulus, most caspases remain inactive. Even if some caspases are aberrantly activated, their proteolytic activity can be fully inhibited by a family of evolutionarily conserved proteins called IAPs (inhibitors of apoptosis proteins). 4 Each of the IAPs contains 1±3 copies of the so-called BIR (baculoviral IAP repeat) domains and directly interacts with and inhibits the enzymatic activity of mature caspases. Several distinct mammalian IAPs including XIAP, survivin and Livin/MLIAP, 5±7 have been identified, and they all exhibit antiapoptotic activity in cell culture. 4 As IAPs are expressed in most cancer cells, 6, 8, 9 they may directly contribute to tumor progression and subsequent resistance to drug treatment. In normal cells signaled to undergo apoptosis, however, the IAP-mediated inhibitory effect must be removed, a process at least in part performed by a mitochondrial protein named Smac10(second mitochondria-derived activator of caspases) or DIABLO11 (direct IAP binding protein with low pI). Smac, synthesized in the cytoplasm, is targeted to the inter-membrane space of mitochondria. Upon apoptotic stimuli, Smac is released from mitochondria back into the cytosol, together with cytochrome c. 10 Whereas cytochrome c directly activates Apaf-1 (apoptotic protease activating factor 1) to facilitate the formation of an apoptosome holoenzyme involving caspase-9, Smac interacts with multiple IAPs and relieves their inhibitory effect on both the initiator caspases, such as caspase-9, and the effector caspases, such as caspase-3 and-7. The full-length Smac protein contains 239 amino acids, with the N-terminal 55 residues encoding the mitochondriatargeting sequence that is removed upon import. 10 Thus the mature Smac has a fresh N-terminus Ala1-Val2-Pro3-Ile4 (AVPI). These N-terminal residues were found to play an indispensable role in binding IAPs and relieving IAP-mediated inhibition of caspases. 12, 13 A 7-residue peptide beginning with AVPI can efficiently remove XIAP-mediated inhibition of caspase-9. 12 The specificity of this interaction was demonstrated by a complete loss of interactions with XIAP and concomitant loss of function associated with a single missense mutation of the N-terminal residue Ala (to Met) in the peptide. 12 These findings suggest potential therapeutic applications using these peptides or peptidomimetics as prototypical anti-cancer drugs. The potential of using Smac N-terminal peptide for drug design was significantly elevated by timely structural analyses, which reveal that the Smac N-terminal …