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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI076504-02
Application #
7883336
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Dempsey, Walla L
Project Start
2009-07-15
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
2
Fiscal Year
2010
Total Cost
$345,408
Indirect Cost
Name
University of California Riverside
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
627797426
City
Riverside
State
CA
Country
United States
Zip Code
92521
Malik-Chaudhry, Harbani Kaur; Gaieb, Zied; Saavedra, Amanda et al. (2018) Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways. Sci Rep 8:10108
Liu, Yan; Shen, Yali; Zheng, Shasha et al. (2015) A novel robust quantitative Förster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates. Mol Biosyst 11:3407-14
Jiang, Ling; Saavedra, Amanda N; Way, George et al. (2014) Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay. Mol Biosyst 10:778-86
Malik-Chaudhry, Harbani Kaur; Saavedra, Amanda; Liao, Jiayu (2014) A linker strategy for trans-FRET assay to determine activation intermediate of NEDDylation cascade. Biotechnol Bioeng 111:1288-95
Liu, Yan; Kieslich, Chris A; Morikis, Dimitrios et al. (2014) Engineering pre-SUMO4 as efficient substrate of SENP2. Protein Eng Des Sel 27:117-26
Liu, Yan; Liao, Jiayu (2013) Quantitative FRET (Förster Resonance Energy Transfer) analysis for SENP1 protease kinetics determination. J Vis Exp :e4430
Jiang, Ling; Liu, Yan; Song, Yang et al. (2013) Internal calibration Förster resonance energy transfer assay: a real-time approach for determining protease kinetics. Sensors (Basel) 13:4553-70
Song, Yang; Liao, Jiayu (2012) An in vitro Forster resonance energy transfer-based high-throughput screening assay for inhibitors of protein-protein interactions in SUMOylation pathway. Assay Drug Dev Technol 10:336-43
Song, Yang; Liao, Jiayu (2012) Systematic determinations of SUMOylation activation intermediates and dynamics by a sensitive and quantitative FRET assay. Mol Biosyst 8:1723-9
Zhao, Yongfeng; Pirrung, Michael C; Liao, Jiayu (2012) A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays. Mol Biosyst 8:879-87

Showing the most recent 10 out of 16 publications