The suppression of caspase activity occurs in the presence o

The suppression of caspase activity occurs in the presence of specific members of the IAP family. In particular cIAP1 and cIAP2 are indirect inhibitors of caspases activity, whereas XIAP is able to directly inhibit both initiator and effector caspases. All IAPs host one to three BIR domains that are critical for their anti-apoptotic activity. In particular, it has been shown that the XIAP-BIR2 domain is responsible for the inhibition of effector caspases, whereas XIAPBIR3 directly binds to and inhibits initiator caspase-9, which can also be recognized by cIAP1-BIR3. The caspase inhibitory activity of XIAP is endogenously antagonized by Smac which is released from mitochondria together with cytochrome in response to death stimuli. The N-terminal tetrapeptides of Smac/DIABLO and caspases competitively bind to the same XIAP active pocket, resulting in activation or inhibition of apoptosis, respectively. Since the structural details of IBM interactions with XIAP and cIAPs have been previously described, the IBM peptides provide a natural basis for the design of Smac-mimetics. These compounds have been shown to displace caspases from XIAP-BIR2 and BIR3 inhibitory pockets, and to induce 3PO (inhibitor of glucose metabolism) biological activity auto-ubiquitination and degradation of cIAPs by perturbing BIR3/RING domain interaction. Therefore, the Smac-mimetics can restore the apoptotic cascade operating in a variety of signaling pathways. Over the last few years several Smac-mimetics have been designed, with the aim of exploiting their pro-apoptotic properties, alone or in combination with other pro-apoptotic compounds such as TRAIL ; these initiatives led to the progressive development of new and potent compounds, some of which are currently in phase I clinical trials. One of the most promising Smacmimetics is SM164, a divalent molecule composed of two moieties, connected by a flexible linker, aimed to target simultaneously two BIR domains. Taking advantage of the experience gathered with monovalent Smac-mimetics design, we generated a library of twenty divalent compounds, belonging to three structural sub-classes, each characterized by distinct linkers or central scaffold-substitutions, to explore different molecular rigidity patterns and to test related metabolic assumptions. All divalent compounds were fully profiled in vitro, and GSK1016790A biological activity compared in terms of overall druglike properties. In particular, 9a displayed in vitro low nM affinity values for the BIR3 domains of XIAP, cIAP1 and cIAP2, but also for XIAP-BIR2BIR3; it also showed good cytotoxicity properties against a selected breast cancer cell line. Notably, due to its ionisable secondary amino groups, 9a is soluble in physiological buffer and could be administered in vivo;