title: Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues.
abstract: 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.
authors.affiliation: Discovery, Millennium Pharmaceuticals Inc., Cambridge, Massachusetts 02139, USA. jesse.chen@mpi.com
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Chen
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Tsu
authors.forename: James M
authors.initials: JM
authors.lastname: Gavin
authors.forename: Michael A
authors.initials: MA
authors.lastname: Milhollen
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Bruzzese
authors.forename: William D
authors.initials: WD
authors.lastname: Mallender
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sintchak
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Bump
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.forename: Jingya
authors.initials: J
authors.lastname: Ma
authors.forename: Huay-Keng
authors.initials: HK
authors.lastname: Loke
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Neil F
authors.initials: NF
authors.lastname: Bence
authors.forename: James E
authors.initials: JE
authors.lastname: Brownell
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D011756:Diphosphates / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013451:Sulfonic Acids / Q000494:pharmacology*; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D011756:Diphosphates; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D013451:Sulfonic Acids; D000255:Adenosine Triphosphate; C005741:sulfamic acid; D044764:Ubiquitin-Activating Enzymes