Smac mimetics are being developed as a novel class of anticancer drugs. three domains known as baculoviral IAP repeat (BIR) domains (5-7). Among these IAP proteins cellular IAP-1 (cIAP-1) and cIAP-2 play a critical role in regulation of tumor necrosis factor (TNF) receptor-mediated apoptosis (8-10) and X-linked IAP (XIAP) is a central regulator of both death receptor-mediated and mitochondria-mediated apoptosis pathways. XIAP inhibits apoptosis by suppressing EPZ004777 caspase activity (7 11 Whereas the third BIRdomain (BIR3) of XIAP selectively targets an initiator caspase-9 (7) the BIR2 domain together with the linker immediately preceding it inhibits effector caspase-3/caspase-7 (12-14). Consistent with their role in inhibition of apoptosis XIAP and cIAP-1 were found to be highly expressed in cancers of diverse tumor types (15-18) and are considered as attractive new cancer therapeutic targets (19 20 Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO) is an endogenous antagonist of IAP proteins (21 22 In its dimeric form Smac via its AVPI tetrapeptide binding motif interacts with both BIR2 and BIR3 domains in XIAP and abrogates the inhibition of caspase-3/caspase-7 and caspase-9 by XIAP (23 24 Smac also binds to cIAP-1/2 and can cause the degradation of cIAP-1 (25 26 Intense research efforts have been devoted to the design and development of small-molecule Smac mimetics as a new class of anticancer drugs (4 27 Two types of Smac mimetics have been reported (i.e. monovalent and bivalent Smac mimetics). The monovalent compounds are designed to mimic the binding of a single AVPI binding motif to IAP proteins (27-29) whereas the bivalent compounds contain two AVPI binding motif mimetics tethered together through a linker (4 30 Both types of Smac mimetics have been shown to induce apoptosis in solid tumor and leukemia cells EPZ004777 as single agents (4 27 28 30 33 Several recent independent studies have shown that Smac mimetics induce rapid degradation of cIAP-1/2 which leads to nuclear factor-κB activation production and secretion of TNFα and TNFα-dependent induction of apoptosis (31 32 34 35 These studies established that induction of cIAP-1/2 degradation is a key early EPZ004777 event Angpt1 in apoptosis induction by Smac mimetics and cIAP-1/2 are critical cellular targets for Smac mimetics. Interestingly although Smac mimetics were designed based on the interaction of XIAP and Smac (4 27 28 the role of XIAP in apoptosis induction EPZ004777 by Smac mimetics has not been well defined. In this study we investigated the mechanism of action and therapeutic potential of two different types of Smac mimetics: monovalent SM-122 and bivalent SM-164 (ref. 30; Fig. 1pharmacodynamic characterization For EPZ004777 pharmacodynamic studies the MDA-MB-231 xenograft tumor model was used. To develop EPZ004777 xenograft tumors 5 × 106 MDA-MB-231 cancer cells with Matrigel were injected s.c. on the dorsal side of severe combined immunodeficient (SCID) mice from Charles River one tumor per mouse. Mice bearing MDA-MB-231 xenograft tumors were treated with a single i.v. dose of SM-164 at 5 mg/kg Taxotere at 7.5 mg/kg or vehicle control. Tumor tissues and normal mouse tissues were harvested at indicated time points. Tumor tissues were..