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 ..
Depositor Specified Assays
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.
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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.