Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for~60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (ΔΨ m ). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-x L overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-x L blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the deathinducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of ΔΨ m . Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-x L , converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-x L . In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartatespecific cysteine proteases (ASCPs) Apoptosis is a fundamental biochemical cell-death pathway essential for normal tissue homeostasis, cellular differentiation, and development within a multicellular organism (for review, see refs. 1-3). Members of the growing family of aspartate-specific cysteine proteases (ASCPs) that include mammalian interleukin 13 converting enzyme (ICE) (4,5), Nedd2 (ICH-1) (6,7), CPP32 (8), Mch2 (9), Mch3 (10), TX (ICH-2, ICErel-II) (11)(12)(13), and ICErel-Ill (13) have been implicated as mediators of all apoptotic cell death (for review, see ref. 14).Cytotoxic T lymphocytes (CTL) induce apoptosis in their target cells possibly by activating members of the ASCP family (15). This hypothesis was supported by the observation that granzyme B, the CTL granule aspartate-specific serine protease, can cleave CPP32 (16). Recent observations suggested that the Fas-interacting protein FADD/MORT1 (17,18)

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.