Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate 
intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). 
MLN4924 is an adenosine sulfamate analogue that was identified as a selective, 
mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, 
by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, 
a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., 
Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. 
A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, 
T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., 
Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. 
F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In 
the present study, substrate-assisted inhibition of human UAE (Ube1) by another 
adenosine sulfamate analogue, 
5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a 
nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent 
ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 
transthiolation in a dose-dependent manner. Mechanistic studies on Compound I 
and its purified ubiquitin adduct demonstrate that the proposed 
substrate-assisted inhibition via covalent adduct formation is entirely 
consistent with the three-step ubiquitin activation process and that the adduct 
is formed via nucleophilic attack of UAE thioester by the sulfamate group of 
Compound I after completion of step 2. Kinetic and affinity analysis of Compound 
I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of 
adduct formation and the affinity between the adduct and E1 contribute to the 
overall potency. Because all E1s are thought to use a similar mechanism to 
activate their cognate ubiquitin-like proteins, the substrate-assisted 
inhibition by adenosine sulfamate analogues represents a promising strategy to 
develop potent and selective E1 inhibitors that can modulate diverse biological 
pathways.