The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway 
essential for cancer cell growth and survival. MLN4924 is a selective inhibitor 
of NAE currently in clinical trials for the treatment of cancer. Here, we show 
that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent 
NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles 
NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be 
further utilized in subsequent intraenzyme reactions. The stability of the 
NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby 
accounting for the potent inhibition of the NEDD8 pathway by MLN4924. 
Importantly, we have determined that compounds resembling MLN4924 demonstrate 
the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) 
catalyzed by their cognate-activating enzymes. These findings reveal insights 
into the mechanism of E1s and suggest a general strategy for selective 
inhibition of UBL conjugation pathways.