Currently, very few cancer therapies result in long-term tumor stabilization or regression. This may be because most current therapies target a relatively small number of proteins and pathways. For this reason, we have developed evidence validating antizyme inhibitor (AZI) as a novel therapeutic target. AZI gene amplification and overexpression are common in a variety of tumor types, including cancers of the prostate, breast, ovary, testis, liver, lung, and skin. In addition, we have found that AZI silencing suppresses cell proliferation and experimental cancer in animal models. AZI promotes tumor growth by several pathways. These include binding and sequestering the ornithine decarboxylase (ODC) antagonist antizyme (AZ), inhibiting polyamine uptake, and inactivating ODC, the rate-limiting enzyme in polyamine synthesis. AZ sequestration also derepresses cyclin D1, SMAD1, and aurora A kinase. Inhibition of only one of these (ODC) with DFMO has resulted in anticancer activity in clinical trials, but its success has been modest, likely because of a compensatory increase in uptake of extracellular polyamines that is blocked by AZ. Because of the increased range of oncogenic activities regulated by AZ, molecules that increase available intracellular AZ by blocking AZI are expected to be more active than DFMO. Such molecules have not yet been discovered; however, they are likely to exist because AZI retains a pocket analogous to the known substrate-binding pocket in ODC to which it is homologous. Given the overlap between this pocket and the AZ binding site, small molecule inhibitors represent an opportunity to block AZ binding and sequestration and increase available AZ in the cell. We propose to develop a drug discovery pipeline designed to identify molecules that inhibit the AZ-AZI interaction. The first stage of this pipeline will be high throughput FRET assays to identify inhibitors of AZ-AZI interaction, using AZ-ODC and AZ-AZIN2 FRET interaction assays as counterscreens. These assays will be developed in the Rogers Lab, which has developed two different protein-protein FRET assays, has screened >250,000 compounds with each, and has successfully identified compounds with antiangiogenic activity in vivo. Secondary assays to assess the ability of hit compounds to disrupt AZ-AZI complexes in vivo will be developed in the lab of Dr. Bruce Zetter, who has substantial experience in cancer research and is an expert on the role of the AZ-AZI pathway in cancer. Assays to be developed include BLI (BioLayer Interferometry) and in vivo FRET assays to assess AZ-AZI interaction in cultured cells. AZI antagonism will be confirmed with a polyamine production assay and BLI assays for SMAD1, cyclin D1, and Aurora A kinase. Finally, the labs will collaborate to test the whole pipeline on a library of 10,000 compounds at the Institute of Chemistry and Cell Biology (ICCB) facility at Harvard Medical School in Boston. Small molecule AZI antagonists identified in the proposed screening pipeline will be used as lead compounds for the design of anticancer therapeutics and as probes of the molecular recognition and cellular effects of AZ release.
We have recently validated antizyme inhibitor (AZI) as a target for therapy in prostate cancer. Here we propose to develop a drug screening pipeline that will be used to identify leads that can be developed into drugs that target AZI. Given the widespread overexpression of AZI among cancers, therapeutics developed by this pipeline are likely to be effective in treating a wide variety of tumor types including: prostate, breast, ovary, testis, liver, lung, stomach, esophagus, pancreas, kidney, brain, and skin.
