Discovery of inhibitors against ubiquitin specific protease in human DNA damage r

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DESCRIPTION (provided by applicant): The overall goal of this project is to identify potent and specific inhibitors for human deubiquitylating enzyme through a quantitative high-throughput screening (qHTS). Besides the well-known roles in protein degradation, protein ubiquitylation also plays essential roles in many other cellular processes, including membrane trafficking, innate immunity, kinase signaling, chromatin dynamics and DNA damage response. Deregulation of protein ubiquitylation often leads to devastating human diseases, including cancer and neurodegenerative disease. Given the large number of deubiquitylating enzymes (DUBs) existing in the human proteome and the implication of many DUBs in human diseases, it is imperative to identify potent and specific inhibitors for the individual deubiquitylating enzymes. Human ubiquitin specific protease 1, a prototypical USP, forms a stable complex with a WD40 repeat-containing protein. It plays important roles in the human DNA damage response pathways, i.e. the Fanconi anemia pathway and the translesion DNA synthesis pathway. Inhibitors of ubiquitin specific protease 1 will serve as useful chemical probes for investigating the role of deubiquitylation in both pathways. The inhibitors will also have therapeutic potential for the recalcitrant diseases cancer. Together with Anton Simeonov's group at the NIH Chemical Genomics Center (NCGC), we have developed a fluorescence- based primary assay at a low 4 ¿L volume in 1536-well format. Secondary assay orthogonal to the fluorescence assay was developed to eliminate the false positive hits obtained in the primary screening. The specificity and selectivity of the small molecules' effect on USP1 will be determined by screening the compound library on other human and yeast USPs. The optimized lead compounds will be used to probe the in vivo deubiquitylation in DNA damage response. Moreover, we will test the compounds as sensitizers for cancer cells to DNA-damaging drugs in a cellular model. The inhibitors of deubiquitinating enzymes hold promise as the next-generation therapeutics that can overcome the narrow therapeutic index and toxic side effects facing the proteasome inhibitors. PUBLIC HEALTH RELEVANCE: Ubiquitin specific proteases (USPs) are emerging as promising targets for pharmacological intervention because of their connection to many human diseases, including prostate, colon and breast cancer, pediatric acute lymphoblastic leukemia, and familial cylindromatosis. So far few small molecule inhibitors have been developed for inhibiting the human USPs. The goal of this project is to develop lead compounds that can specifically inhibit a USP in DNA damage response and thus sensitize cancer cells to DNA crosslinking drugs.)