title: Novel approach for characterizing ubiquitin E3 ligase function.
abstract: The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.
authors.affiliation: Progenra, Inc., Malvern, Pennsylvania, USA. Marblestone@progenra.com
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Marblestone
authors.forename: K G
authors.initials: KG
authors.lastname: Suresh Kumar
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Eddins
authors.forename: Craig A
authors.initials: CA
authors.lastname: Leach
authors.forename: David E
authors.initials: DE
authors.lastname: Sterner
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mattern
authors.forename: Benjamin
authors.initials: B
authors.lastname: Nicholson
mesh_terms: D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D055808:Drug Discovery; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D049449:Luminescence; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C512817:CA11 protein, human; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C490846:RAD23 protein, S cerevisiae; C117909:SUMO2 protein, human; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D025841:Small Ubiquitin-Related Modifier Proteins; C470301:UBA2 protein, human; D025821:Polyubiquitin; C086985:RAD23A protein, human; D044767:Ubiquitin-Protein Ligases; D044764:Ubiquitin-Activating Enzymes; D045643:DNA Repair Enzymes