Various ubiquitin and ubiquitin-like protein pathway enzymes have been shown by biochemical and genetic evidence to be involved in disease states and are thus regarded as molecular targets for drug discovery. Regarding neoplastic disease, for example, certain de-ubiquitinases (DUBs) are present in large amounts in malignant but not non-malignant cells or tissues and their functions have been linked to defined aspects of the transformed state. Progenra has developed an isopeptidase assay platform that can detect cleavage activity of one class of enzyme -- proteases of ubiquitin or ubiquitin-like protein fusions. The assay is based on the fusion of the amino terminus of a reporter enzyme requiring a free N-terminus for activity to the C-terminus of ubiquitin or a ubiquitin-like protein (UBL) such as NEDD8. Isopeptidase cleavage at the fusion junction liberates the active reporter. The assay has been validated for several DUBs and will be used to conduct a screening campaign to find inhibitors of DEN 1, an isopeptidase that cleaves the ubiquitin-like protein NEDD8 from its partner protein BCA3 (breast cancer associated protein 3), blocking the ability of BCA3 to prevent NFkB-induced activation of its pro-survival, anti-apoptotic transcription program. Inhibitors of DEN 1 are expected to permit BCA3 to block the pro-survival, anti-apoptotic program activated by NFkB in tumor cells treated with radiation or chemotherapeutic agents and thus to be candidates for single agent or adjuvant anticancer therapeutics. The introduction of such therapeutics to market is the commercial goal of this project. To find such inhibitors, the DEN1 assay will first be validated for high throughput screening. Next, two chemical libraries - a targeted protease inhibitor collection and a general diversity set - will be screened. Finally, selected positives from the screen will be evaluated for preclinical development (potency, selectivity, functional activity). Enzymes of the ubiquitin (and ubiquitin-like protein) pathway have become popular targets for drug discovery. The association of NEDD8, a ubiquitin-like protein, with another protein called BCA3 helps to reverse the malignant state by causing the cancer cells to die (apoptosis); this effect is magnified in the presence of many anticancer agents. A third protein called DEN 1 removes NEDD8 from association with BCA3 and thereby promotes the malignant state. An inhibitor of DEN 1 should prevent this interruption of NEDD8-BCA3 association and thereby should be useful in reversing cancers, particularly if given in concert with an established anticancer agent. Progenra proposes to find inhibitors of DEN 1 by screening selected chemical libraries using the assay it has developed. Compounds that are shown by the assay to inhibit DEN 1 will be evaluated further, and the best of these will be proposed for further development as antitumor agents. ? ? ?
| Lui, Tony T H; Lacroix, Celine; Ahmed, Syed M et al. (2011) The ubiquitin-specific protease USP34 regulates axin stability and Wnt/ýý-catenin signaling. Mol Cell Biol 31:2053-65 |
