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.