Ubiquitin E3 ligases and apoptosis in cancer drug discovery

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Abstract: CARP2 is an anti-apoptotic RING-domain protein that specifically binds to and negatively regulates death domain (DED) caspases 8 and 10 by acting as an E3 ubiquitin ligase, causing ubiquitination and ultimately proteasomal degradation of the target caspase. This ligase is over-expressed in many tumors, permitting them to escape apoptosis and survive. Inhibitors of CARP2 would thus be expected to have intrinsic antitumor activity in addition to facilitating antitumor activity of agents such as TRAIL, which act via the death receptor pathway. This proposal describes the development for high throughput screening of a yeast-based assay in which human CARP2, its p53-fused substrate, caspase 8, and a p53 reporter (p-galactosidase) are expressed in S. cerevisiae. Wnen all of these components are functional, CARP2, in collaboration with the endogenous yeast ubiquitin-proteasomal pathway elements (E1, E2, proteasomes), keeps the level pf Caspase 8-p53 at a minimum, resulting in no reporter activity. Inhibition of CARP2 permits Caspase-fused p53 to bind to its response element and activate the reporter, producing a positive signal (fluorescence). The assay will be configured, following cloning and construction of the necessary components, by transformation of CARP2, its substrate caspase 8 fused to p53, and a p53-responsive reporter, in yeast. After adjusting assay conditions to produce an acceptable signal:noise ratio and other parameters of high throughput screening, limited numbers of compounds and natural product extracts will be screened to evaluate the suitability of the assay for full scale high throughput screening for anticancer drug development. In addition, secondary screening assays, including a yeast based assay for CARP1, will be developed to filter primary hits for progression to preclinical development. The ultimate goal of the proposed work is to translate the assay to high throughput screening for a drug that is active as a single agent or a component of combination therapy against refractory cancers