title: Quantifiable analysis of cellular pathway inhibition of a Nedd8-activating enzyme inhibitor, MLN4924, using AlphaScreen.
abstract: Cellular effects of a Nedd8-activating enzyme (NAE) inhibitor, MLN4924, using the AlphaScreen format were explored. MLN4924 acts as a substrate-assisted inhibitor of NAE by forming a tight binding Nedd8-MLN4924 adduct. The inhibited enzyme can no longer transfer Nedd8 downstream to modify and activate the E3 cullin-RING ligases. This results in the stabilization of proteins regulated by the proteasome, leading to cell death. These studies monitored the endogenous cellular changes to NAE∼Nedd8 thioester, the formation of the Nedd8-MLN4924 adduct, and the reduction in the Cul1-Nedd8. Lysates derived from MLN4924-treated HCT116 cells showed that whereas the β-subunit of NAE remained constant, reductions of both NAE∼Nedd8 thioester and Cul1-Nedd8 levels occurred with a concomitant rise of the adduct. Moreover, the formation of the Nedd8-MLN4924 adduct was approximately stoichiometric with the concentration of NAEβ. Higher density 384-well cell-based assays illustrated the kinetics of enzyme inactivation across a wider range of MLN4924 concentrations, showing a rapid loss of NAE∼Nedd8 thioester and Cul1-Nedd8. The reduction of NAE∼Nedd8 thioester precedes the loss of Cul1-Nedd8 at twice the rate. Finally, these results clearly demonstrate the utility of the homogeneous assay for quantitative assessment of these endogenous cellular components in a 384-well plate in response to inhibition of NAE by MLN4924.
authors.affiliation: Millennium Pharmaceuticals: The Takeda Oncology Company, Cambridge, MA 02139, USA.
authors.forename: Zhong-Hua
authors.initials: ZH
authors.lastname: Yan
authors.forename: Anne
authors.initials: A
authors.lastname: Burkhardt
authors.forename: Huay-Keng
authors.initials: HK
authors.lastname: Loke
authors.forename: Jesse
authors.initials: J
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Pam
authors.initials: P
authors.lastname: Brauer
authors.forename: Jingya
authors.initials: J
authors.lastname: Ma
authors.forename: Yafang
authors.initials: Y
authors.lastname: Lin
authors.forename: Khris
authors.initials: K
authors.lastname: Garcia
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bembenek
mesh_terms: D003517:Cyclopentanes / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D045325:HCT116 Cells; D006801:Humans; D011485:Protein Binding; D021122:Protein Subunits; D011743:Pyrimidines / Q000494:pharmacology*; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D003517:Cyclopentanes; D021122:Protein Subunits; D011743:Pyrimidines; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human; C539933:pevonedistat
keywords: AlphaScreen; Cell-based assay; MLN4924; Pathway inhibition