Sumoylation (SUMO or small ubiquitin-like modifier) is a post-translational modification of many important proteins, such as p53, MDM2, STATs (signal transducer and activator of transcription), I:B, androgen and estrogen receptors. These proteins are involved in many critical physiological processes, including signal transduction, genome integrity, cell-cycle regulation, cell proliferation, and tumor progression. Studies of these processes would be greatly accelerated by identifying small-molecule inhibitors of sumoylation. To facilitate this work, we propose to develop assay protocols to identify and characterize inhibitors of the SUMO system. We will focus on the SUMO E2 and E3 ligases, Ubc9 and PIAS1, respectively. PIAS1 is the first SUMO E3 ligase identified in mammalian cells, and itmediates sumoylation of STAT1 in interferon 3-mediated antiviral activity and p53 activity in tumor suppression/genome integrity pathways. Ubc9 is the SUMO E2 ligase that catalyses SUMO peptide transfer to substrates with the help of E3 ligase, such as PIAS. Recent studies suggested that both PIAS1 and Ubc9 are intriguing targets for much-needed antiviral and anticancer drugs with new mechanisms of action. We propose to develop critical basic tools to screen for SUMO E2 and E3 ligase inhibitor(s) based our recently developed high efficiency Forster energy transfer (Fluorescence Energy Transfer or FRET) pair (CyPet and YPet) for cell-based and homogeneous high throughput screening (HTS) systems. Specifically, we will first develop Ubc9/SUMO1 and PIAS1/SUMO1 (FRET) assays that can be adapted to automatic high-throughput screening, corresponding to NIH program announcement (PA-07-320 for HTS assay development). We will optimize and validate these assays with pilot screens of compound collections available to us, including compound libraries from the Genomic Institute of UC Riverside and the Scripps Research Institute. Finally, we will then characterize the potential inhibitor(s) in biochemical and biological conformation assays. To our knowledge, these FRET based-HTS assays for SUMO E2/E3 ligases are not available in the research community. Potent, specific, and bioavailable Ubc9 and PIAS1 inhibitors will help the research community clarify sumoylation regulation in many physiological systems and to further validate these targets for the treatment of viral infections and tumorigenesis/metastasis. """"""""Proof of concept"""""""" studies with the PIAS1 and Ubc9 inhibitors may lead to improved therapeutic modalities for cancers and several infectious diseases. UC Riverside is a center of excellence for chemical genomics with a NSF-supported chemical genomics platform, and the applicant has a strong record in developing high-throughput screening assays for the identification and characterization of small-molecule pharmacological tools in th last few years.

Public Health Relevance

e major purpose of this Assay Development Proposal is to provide a new FRET screening platform and follow-up assays in a fully integrated effort towards the goal of finding small molecule pharmacological tools for the SUMO (Small Ubiquitin-like Modifier) E3 ligases, PIAS1, and E2 ligase, Ubc9, inhibitor(s) as chemical probes and potential anticancer reagents and antivirus. Sumoylation is a protein post-translational modification that results in changes to protein activities and subcellular localization in vivo. Ubc9 is the only SUMO E2 ligase that mediates all the sumoylation processes in mammalian cells so far. PIAS1 is a SUMO E3 ligase that mediates sumoylation and repression of p53 activity in the tumor suppression/genome integrity pathways and STAT1 in interferon 3-mediated antivirus activity. We propose to develop critical basic tools to screen SUMO E2 and E3 ligase inhibitor(s) and that can be applied to high- throughput biology. Potent, specific, and bioavailable PIAS1 inhibitors will help to further validate this target for the treatment of viral infections and tumorgenesis/metastasis. """"""""Proof of concept"""""""" studies with the Ubc9 and PIAS1 inhibitor(s) may lead to improved therapeutic modalities for cancers and several infectious diseases.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Instrumentation and Systems Development Study Section (ISD)
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Dempsey, Walla L
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University of California Riverside
Engineering (All Types)
Schools of Engineering
United States
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