With the aim to identify new potential drugs for the treatment of diseases characterized by excessive apoptosis, a nonpeptide small-molecule library was screened employing an apoptosome-dependent caspase activation assay. This assay is based on the finding that the in vivo induction of the intrinsic cell death pathway can be mimicked in vitro by adding cytochrome c
and dATP to cytosolic extracts (16
). Such an assay should allow not only the identification of direct caspase inhibitors but also putative inhibitors of the formation of the apoptosome complex. Screening 392 selected compounds representing a nonpeptide molecular library of 5,000 compounds resulted in 11 hits that conferred more than 50% inhibition of cytochrome c
- and dATP-induced DEVDase activity. Most of the 11 compounds were highly toxic to cells; therefore, the further studies were performed with three of the compounds best tolerated by cells (i.e., NS3694, NS1784, and NS1764). All three compounds belonged to the chemical class of diarylureas and fulfilled the Lipinski rules for bioavailability, i.e., molecular weight under 500, less than five hydrogen bond donors, less than 10 hydrogen bond acceptors, and calculated log P
value under 5 (17
). This may explain the similar levels of inhibition and toxicities for the three compounds.
The in vitro studies on the mechanism of action of NS3694 revealed that it inhibited the formation of the active apoptosome complex rather than the activity of caspases. In the cytosolic extracts of HeLa cells, NS3694 inhibited the cytochrome c
- and dATP-induced coimmunoprecipitation of caspase 9 and Apaf-1 and the processing of caspase 9 and all its downstream targets tested. In the whole-cell lysates of THP.1 cells, NS3694 inhibited the dATP-induced formation of the active 700-kDa apoptosome complex and the processing and activation of caspase 9 and caspase 3. However, NS3694 failed to inhibit the dATP-induced formation of the 1.4-MDa apoptosome complex in THP.1 lysates. Although this complex contains processed caspase 9, it does not cleave and activate the effector caspases (3
), and in the presence of NS3694, all of the caspase 9 that eluted in this fraction was present as the inactive proform. Thus, the results obtained from dATP-stimulated THP.1 lysates suggest that NS3694 inhibits only the formation of the active apoptosome complex without interfering with the formation of the inactive complex of Apaf-1 and procaspase 9 (Fig. ). Interestingly, in the coimmunoprecipitation experiments with HeLa cell lysates (Fig. ), the anti-caspase 9 antibody did not precipitate all of the Apaf-1. Therefore, it may be that the interaction between the ~1.4-MDa complex and caspase 9 is fairly weak due to the inappropriate oligomerization of Apaf-1 (2
), and as a result, the Apaf-1 in this complex cannot be immunoprecipitated. Alternatively, it could be possible that the 1.4-MDa complex is not readily formed in HeLa cytosol. However, we have obtained essentially similar results with coimmunoprecipitation studies in dATP-activated THP.1 cell lysates (K. Cain and D. Brown, unpublished results). This indicates that the anti-caspase 9 antibody immunoprecipitates Apaf-1 only when caspase 9 is correctly incorporated in an active apoptosome holoenzyme complex, whose formation is inhibited by NS3694.
Supporting the data obtained from the in vitro model systems, NS3694-mediated protection from cell death in living cells occurred downstream of cytochrome c release but upstream of caspase activation. Furthermore, NS3694 inhibited death receptor-induced caspase activation in type II cells that require mitochondrial involvement for the effective effector caspase activation but not in type I cells, where caspase 8 can directly activate effector caspases. The precise mechanism by which diarylureas inhibit the formation of the active apoptosome remains to be studied. Since the formation of the apoptosome is triggered by cytochrome c and dATP, it is possible that NS3694 prevents the association of either cytochrome c or dATP to their respective domains on Apaf-1. Another possibility is that NS3694 interferes with the caspase recruitment domain (CARD)-CARD interaction between caspase 9 and Apaf-1. Diarylurea compounds are a new class of apoptosis inhibitors acting at a different step of the apoptosis signaling process than other available pharmacological inhibitors.
In contrast to most intrinsic death pathways that depend on the formation of the apoptosome complex, the role of the apoptosome in the postmitochondrial death signaling induced by death receptors is still controversial (5
). Recently, Deng and coworkers suggested that the release of Smac/Diablo, but not the release of cytochrome c
, from the mitochondria of TNF-related apoptosis-inducing ligand (TRAIL)-treated cells is required and sufficient to induce effector caspase activation and apoptosis (6
). The ability of NS3694 to specifically inhibit the formation of the apoptosome complex allowed us to study the contribution of the apoptosome in caspase activation and death of TNF-treated tumor cells. In all three cell types studied, NS3694 almost completely inhibited the effector caspase activation, indicating that in these cells, Smac/Diablo-mediated inhibition of IAPs is not sufficient for the effective activation of downstream caspases. This conclusion is further supported by our preliminary data showing that the small interference RNA-mediated depletion of Apaf-1 in MCF-7 cells confers a level of protection against TNF similar to that of NS3694. Our data also demonstrate that even though the apoptosome is clearly required for caspase activation in the models used here, it is not necessarily required for cell death. For example, in TNF-treated WEHI-S cells and staurosporine-treated ME-180as cells, NS3694 almost completely inhibited caspase activation without any effect on cell viability. Thus, the death of these cells is likely to be mediated by caspase-independent signaling pathways (14
As demonstrated above, NS3694 offers a novel tool to study the role of the apoptosome in various apoptosis paradigms. Meticulous in vivo studies on different disease models will, however, be required to evaluate the potential of NS3694 or related molecules as drug candidates for the treatment of apoptosis-associated diseases.