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. 2011 Nov 25;286(47):40867-77.
doi: 10.1074/jbc.M111.279984. Epub 2011 Oct 3.

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Jesse J Chen  1 , Christopher A Tsu, James M Gavin, Michael A Milhollen, Frank J Bruzzese, William D Mallender, Michael D Sintchak, Nancy J Bump, Xiaofeng Yang, Jingya Ma, Huay-Keng Loke, Qing Xu, Ping Li, Neil F Bence, James E Brownell, Lawrence R Dick
Affiliations

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Jesse J Chen et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Proposed mechanisms of Ubl activation and E1 inhibition. A, three-step Ubl activation leading to a ternary complex via a Ubl-adenylate intermediate. B, structures of AMP and two adenosine sulfamate analogues, MLN4924 and Compound I. C, proposed mechanism of E1 inhibition by adenosine sulfamates.
FIGURE 2.
FIGURE 2.
Inhibition of UAE by Compound I or Ub-I using the ATP-PPi exchange assay. A, wild-type UAE. B, C632A UAE. The assay mixtures contained 1 mm ATP. Results are summarized in Table 1.
FIGURE 3.
FIGURE 3.
Inhibition of UAE-S∼ubiquitin thioester by Compound I. A, Western blots showing dose-dependent loss of UAE-S∼ubiquitin thioester with 0.1 mm ATP (top panel) or 1 mm ATP (bottom panel) using N-FLAG-ubiquitin as the substrate. Blots were probed with mouse anti-FLAG antibody. B, quantitation and data analysis of Western blots shown in A. The protein bands were visualized and quantitated using a fluorescently labeled secondary antibody (A.U., arbitrary fluorescence unit). IC50 values of 2.8 ± 0.2 and 12.9 ± 2.4 μm were obtained with 0.1 mm ATP (filled circles) and 1 mm ATP (open circles), respectively. The concentration range of Compound I is from 0.15 to 100 μm in a 3-fold dilution series.
FIGURE 4.
FIGURE 4.
Ub-I adduct formation during the course of Compound I-mediated inhibition of UAE-S∼ubiquitin thioester. A, Western blots showing dose-dependent formation of Ub-I with 0.1 mm ATP (top panel) or 1 mm ATP (bottom panel) using N-FLAG-ubiquitin as the substrate. Blots were probed with rabbit anti-Compound I antibody. B, quantitation and data analysis of Western blots shown in A. The protein bands were visualized and quantitated using a fluorescently labeled secondary antibody (A.U., arbitrary fluorescence unit). K1/2 values of 4.4 ± 1.3 and 16.8 ± 1.1 μm were obtained with 0.1 mm ATP (filled circles) and 1 mm ATP (open circles), respectively. The concentration range of Compound I is from 0.15 to 100 μm in a 3-fold dilution series.
FIGURE 5.
FIGURE 5.
Time-dependent UAE inhibition demonstrated by progress curve analysis. A, time course of radioactive ATP production in ATP-PPi exchange assays at various [Compound I]. B, time course of AMP production during UAE activation/UAE-UbcH10 transthiolation monitored using an ADT-ATP cycling system at various [Compound I]. For curves in A and B, the observed rate of inactivation (kobs) was estimated by fitting the curves using a slow, tight-binding inhibition model: Y = vbkg × t + A0 × (1 − exp(−kobs t)). C, estimation of kinact/Ki by linear fitting kobs versus [Compound I]. Similar kinact/Ki values were obtained in these two assays (2.6 ± 0.2 × 104 m−1 s−1 from ATP-PPi exchange assays and 2.5 ± 0.2 × 104 m−1 s−1 from transthiolation). The concentration of ATP in both assays is 0.1 mm.
FIGURE 6.
FIGURE 6.
Determination of the rate of Ub-I formation under single-turnover conditions. The observed rates (kobs) were plotted versus [Compound I]. Ki (27 ± 6 μm) and kinact (8.3 ± 0.7 s−1) were obtained by fitting the data to a hyperbolic equation: kobs = kinact × [Compound I]/([Compound I] + Ki).
FIGURE 7.
FIGURE 7.
Binding interaction between UAE and Ub-I studied by surface plasmon resonance. A, sensorgrams of Ub-I (0.1, 0.2, and 0.4 μm, black, red, and green traces, respectively) binding to UAE immobilized on the sensor chip surface. The binding phase of the data were fit to a single-exponential rise to derive apparent association rates (kobs). No measurable dissociation was observed during the course of experiments. B, kobs was plotted against [Ub-I] to obtain the association rate (ka) of 1.9 × 106 m−1 s−1.
FIGURE 8.
FIGURE 8.
Steady-state formation of the UAE-catalyzed ubiquitin-MLN4924 (Ub-4924) adduct analyzed by reverse-phase HPLC. Peaks corresponding to Ub-4924 are highlighted in red. The area under the Ub-4924 peaks were integrated and converted to the amount of Ub-4924 using a purified sample as a standard, which yielded a steady-state rate of 1.4 × 10−3 s−1 or ∼5 h−1.
FIGURE 9.
FIGURE 9.
Western blot analysis demonstrating the ubiquitination pathway inhibition in Compound I-treated cells. HCT116 cells were treated with DMSO or 10 μm Compound I for 1 h. The cells were then harvested and whole cell extracts were subjected to SDS-PAGE and Western blot analysis under nonreducing conditions. Upper panel, anti-UbcH10, middle panel, anti-ubiquitin; bottom panel, anti-tubulin.
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