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BioAssay: AID 651722

Summary assay for small molecule inhibitors of cullin neddylation

Cullin-RING ubiquitin ligases (CRLs) are responsible for ~20% of all intracellular protein degradation by the proteasome. This critical role in maintaining protein homeostasis is emphasized by links between CRLs and diseases such as cancer and neurodegenerative disorders, inflammation, host-pathogen interactions, and the immune response. The activation of CRLs by covalent modification with the more ..
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AID: 651722
Data Source: Burnham Center for Chemical Genomics (SBCCG-A934-UBC12-Summary-Assay)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-10-28
Modify Date: 2013-04-24
Targets
Related Experiments
AIDNameTypeComment
651699uHTS identification of inhibitors of cullin neddylation in a TR-FRET assayScreeningdepositor-specified cross reference
652217Single concentration confirmation of inhibitors of cullin neddylation in a TR-FRET assayScreeningdepositor-specified cross reference
652247Dose-response confirmation of inhibitors of cullin neddylation in a TR-FRET assayConfirmatorydepositor-specified cross reference
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: R03 DA034599-01
Assay Provider: Matthew D. Petroski, Ph.D., Sanford-Burnham Medical Research Institute

Cullin-RING ubiquitin ligases (CRLs) are responsible for ~20% of all intracellular protein degradation by the proteasome. This critical role in maintaining protein homeostasis is emphasized by links between CRLs and diseases such as cancer and neurodegenerative disorders, inflammation, host-pathogen interactions, and the immune response. The activation of CRLs by covalent modification with the ubiquitin-like protein NEDD8 functions as a major regulatory mechanism to control these enzymes. NEDD8 modifications are essential for cancer cell survival as a small molecule known as MLN4924 that generally inhibits all neddylation pathways selectively induces cancer cell death by triggering S-phase DNA re-replication and inhibits tumor growth in xenograft studies.

UBC12, a member of the 50+ ubiquitin and ubiquitin-like protein conjugating enzyme (E2) family, is responsible for the direct covalent modification of a subset of cullins with NEDD8 and represents a novel target for small molecule probes. Although E2s have been considered unfavorable targets for probe development due to the absence of obvious catalytic pockets or small molecule binding sites, recent work on the ubiquitin E2 Cdc34 identified a cryptic binding pocket that when occupied by the small molecule CC0651 inhibits the covalent transfer of ubiquitin by preventing a variety of subtle allosteric changes throughout the enzyme. Intriguingly, similar pockets can now be discerned in other E2 structures including UBC12. However, the critical residues necessary for CC0651 binding to Cdc34 are not conserved and it remains unknown if this allosteric activation is a general mechanism used by all E2s or is specific to Cdc34.

The overall objective of this proposal is to identify small molecule probes that inhibit both ATP dependent Nedd8 activation of NAE (E1) and UBC12(E2)-mediated transfer of NEDD8 from UBC12 onto its cullin substrates.

References
1. Watson IR, Irwin MS, Ohh M. NEDD8 pathways in cancer, Sine Quibus Non. Cancer Cell. 2011;19(2):168- 76.
2. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol. 2009;10(5):319-31. PMCID: PMC2712597.
3. Pan ZQ, Kentsis A, Dias DC, Yamoah K, Wu K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene. 2004;23(11):1985-97.
4. Petroski MD, Deshaies RJ. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005;6(1):9-20.
5. Duda DM, Borg LA, Scott DC, Hunt HW, Hammel M, Schulman BA. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell. 2008;134(6):995-1006. PMCID: 2628631.
6. Furukawa M, Zhang Y, McCarville J, Ohta T, Xiong Y. The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol Cell Biol. 2000;20(21):8185-97. PMCID: 86428.
Protocol
Please see pertinent AIDs: 651699
Comment
Probe molecules are defined as the positives of this assay and assigned a score of 100. Testing has not progressed to the point where a probe molecule has been identified.
Additional Information
Grant Number: R03 DA034599-01

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