Identification of low molecular weight compounds mediating apoptosis by directly inducing cleavage of procaspase 3

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Apoptosis or programmed cell death is a mechanism in normal cells that leads to the complete elimination of the target cells. Apoptosis is required during a variety of physiological processes like development, immune response or normal cell turnover. Defects in the ability to activate apoptosis can lead to several diseases. Tumor cells commonly fail to activate apoptosis a fact that is usually not due to alterations in the basic apoptotic machinery since an appropriate stimulus induces apoptosis in nearly all tumor cells. Caspases, a family of cysteine proteases, are major players in the apoptotic pathway. They are synthesized as inactive proenzymes and are acti vated by proteolytic processing. A proteolytic cascade of different caspases is required to transmit and enhance the apoptotic signal. Caspase 3, for example, one of the major downstream caspases, is physiologically activated via cleavage into two polypeptides by upstream caspase 8 or 9. Due to an upregulation of procaspase 3 in certain tumor cell types, elevation of the amount of active caspase 3 might primarily induce apoptosis in these cells. Recent reports demonstrated that procaspase 3 can be activated directly by peptides containing the amino acid RGD motif suggesting the possibility that even low molecular weight compounds might be able to functionally mimic these peptides. The aim of this study was to identify compounds that are able to directly activate procaspase 3. We have developed an HTS compatible in vitro procaspase 3 activation assay based on purified recombinant procaspase 3 and using cleavage of a fluoremetic substrate as indicator for the conversion into active caspase 3. Using this assay, we have screened a library comprising about 800 different compounds and identified several substances that induce activation of procaspase 3. We will present in vitro and in vivo data showing that these compounds can induce apoptosis and cleavage of procaspase 3. To confirm that the apoptotic response observed in tumor cell lines was dependent on the presence of procaspase 3 we used human MCF7 cells that do not express functional procaspase 3 and transfected them either with a plasmid encoding for procaspase 3 or the vector alone. Treatment with one of the identified compound results in enhanced apoptosis in those cells that stably express procaspase 3 compared to control cells. Western Blot analyses demonstrated that the observed apoptosis is accompanied by an increase in the amount of cleaved caspase 3. In summary, the results show that we have developed an HTS assay that allows the identification of small molecules that can directly activate procaspase 3. The compounds identified will be used in future studies to validate the novel therapeutic anti-cancer approach that based on the activation of procaspase 3.