Dysregulated apoptosis, a hallmark of cancer, significantly limits the effectiveness of
chemotherapeutic agents, which mainly work through the induction of apoptosis. Inhibitors of apoptosis
(IAPs) are a family of conserved proteins of the intrinsic apoptotic pathway whose deregulated expression
in tumours confers chemoresistance. Human IAPs comprise eight members, including XIAP, cIAP1, cIAP2
and Survivin, each characterized by one or more structural Baculovirus IAP repeat (BIR) domains. Five
IAPs, additionally, host a RING domain endowed with E3 ubiquitin ligase activity. Through the BIR and
RING domains, IAPs regulate caspase activity and survival pathways. XIAP, the most studied of IAPs,
contains three BIR domains, of which BIR2 binds to and inhibits effector caspases-3 and -7, while BIR3
binds and blocks the initiator caspase-9.
XIAP is naturally antagonized by SMAC/DIABLO, a mitochondrial protein which, upon release to the
cytosol in response to apoptogenic stimuli, interacts through its N-teminus AVPI sequence with the BIR
domains of XIAP, relieving caspase-3 and caspase-7 inhibition and allowing cleavage of effector caspase
substrates and consequent cell death. These IAP antagonists of BIR domains are therefore considered
attractive tools as anticancer therapeutics. Small molecules mimicking the N-terminal AVPI tetrapeptide
of activated SMAC/DIABLO and interacting with XIAP-BIR3, have been shown to suppress inhibition of
caspase-9 and to sensitize cells to apoptosis, especially in response to death ligands. Interestingly, while
competing with the caspases, SMAC-mimetics concurrently stimulate the E3 ubiquitin ligase activity of
cIAP1 and cIAP2, triggering their auto-ubiquitination and proteasomal degradation. This phenomenon
further potentiates the pro-apoptotic activity of SMAC-mimetics.
Based on crystal and molecular structure determinations of XIAP-BIR3/ligand complexes, docking
simulations, synthetic chemistry and cytotoxicity assays, we have recently designed and chemically
synthesized a library of small monomeric and dimeric SMAC-mimetics with nanomolar affinities for XIAP
that variably kill cancer cells, particularly in combination with TRAIL (tumor necrosis factor related
apoptosis-inducing ligand). Upon binding the TRAIL-R1/R2 receptor, TRAIL triggers the activation of the
extrinsic apoptotic pathway and owing to its limited side effects is currently undergoing clinical trials as
anticancer agent. TRAIL-induced extrinsic apoptosis is potentiated by the activation of the intrinsic
apoptotic pathway as that elicited by SMAC-mimetics.
How effective SMAC-mimetic molecules are as antitumor drugs in in vivo preclinical models
remains to be fully investigated. To this end, we propose the following specific aims:
1) Design novel monomeric, homodimeric and heterodimeric SMAC-mimetics targeting the BIR2/BIR3
domains of XIAP, cIAP1 and cIAP2, aided by crystal structure determination of these proteins in complex
with SMAC-mimetics, in silico modeling, and structure activity relations (SAR) studies. Hit molecules will
be chemically synthesized in large scale, optimized and experimentally tested in cell-free systems and in
preclinical in vitro and in vivo models.
2) Elucidate the biochemical mechanisms by which SMAC-mimetics activate the IAP-dependent apoptotic
pathway alone or in combination with TRAIL ligand, focusing on the interaction of IAPs with caspases,
ubiquitination/degradation events involving cIAP1 and cIAP2, and TRAIL receptor complex status and
downstream signalling.
3) Identify anticancer agents that potentiate the cytotoxic activity of bioactive SMAC-mimetics.
4) Assess the pharmacokinetics and anticancer therapeutic activity of lead monomeric and dimeric
SMAC-mimetics using mouse models xenografted with human tumors.
Overall, this research will shed light on a series of pro-apoptotic peptide-mimetic compounds and their
intracellular mechanism of action. Coupled to 3D structure analyses, this study will lead to the
development of drug leads active in cancer therapy.