The release of mitochondrial proapoptotic
proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of
caspases. Once in the cytosol,
cytochrome c triggers the formation of a
caspase-activating
protein complex called the
apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the
caspase inhibitory effect of
inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the
cytochrome c-induced formation of the active, approximately 700-kDa
apoptosome complex and
caspase activation. Using diarylureas to inhibit the formation of the
apoptosome complex, we demonstrated that
cytochrome c, rather than IAP antagonists, is the major mitochondrial
caspase activation factor in
tumor cells treated with
tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator
caspase 9 by directly inhibiting the formation of the
apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of
caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the
apoptosome in mitochondrion-dependent death paradigms.