In vitro E1-activating enzyme assays

A time-resolved fluorescence energy transfer assay format was used to measure the in vitro activity of NAE. The enzymatic reaction, containing 50 μl 50 mM HEPES, pH 7.5, 0.05% BSA, 5 mM MgCl2, 20 μM ATP, 250 μM glutathione, 10 nM Ubc12–GST, 75 nM NEDD8–Flag and 0.3 nM recombinant human NAE enzyme, was incubated at 24℃ for 90 min in a 384-well plate, before termination with 25 μl of stop/detection buffer (0.1 M HEPES, pH 7.5, 0.05% Tween20, 20 mM EDTA, 410 mM KF, 0.53 nM Europium-Cryptate-labelled monoclonal Flag-M2-specific antibody and 8.125 μg ml-1 PHYCOLINK allophycocyanin (XL-APC)-labelled GST-specific antibody). After incubation for 2h at 24℃, the plate was read on the LJL Analyst HT Multi-Mode instrument using a time-resolved fluorescence method. A similar assay protocol was used to measure other E1 enzymes.

Cell lines

HCT-116 cells

Preparation method

Limited solubility. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

24 h

Applications

Treatment of HCT-116 cells with MLN4924 for 24h results in a dose-dependent decrease of Ubc12–NEDD8 thioester and NEDD8–cullin conjugates, with an IC50<0.1 μM, causing a reciprocal increase in the abundance of the known CRL substrates CDT1, p27 and NRF2. In HCT-116 cells, the most prominent phenotype observed was the disruption of S-phase regulation leading to cellular death. By 24h, a significant fraction of cells contained≥4N DNA content.

Animal models

HCT-116 tumour-bearing mice

Dosage form

Subcutaneous injection once (QD) or twice (BID) daily

Applications

30 mgkg-1 and 60 mgkg-1 MLN4924 significantly inhibits tumor growth on a once daily. Moreover, MLN4924 administered once daily for three cycles of two-day treatment followed by five treatment-free days, results in modest but significant tumor growth inhibition. All doses and schedules were well tolerated, with an average weight loss for all dose groups at the end of treatment of less than 10%.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

MLN4924 is a potent and selective inhibitor of NEDD8-activating enzyme (NAE) with IC50 value of 4nM [1].

MLN4924 binds NAE within the nucleotide-binding site competing with AMP. It is a potent inhibitor of NAE and is selective relative to the closely related enzymes UAE, SAE, UBA6 and ATG7 with IC50 values of 1.5, 8.2, 1.8 and 10μM, respectively. MLN4924 shows no effect to other ATP-using enzymes. In HCT-116 cells, MLN4924 treatment results in a decrease of Ubc12–NEDD8 thioester and NEDD8–cullin conjugates, thus resulting in an inhibition of protein degradation mediated by CRL-ubiquitinylation. CDT1 is one of these proteins. The accumulation of CDT1 can cause cell-cycle defects. In mice bearing HCT-116 xenografts, administrations of MLN4924 at 30mg/kg and 60mg/kg significantly inhibit the tumor growth and these doses are well tolerated. The anti-tumour activity of MLN4924 is also found both in mice bearing H522 lung tumour xenografts and in mice bearing Calu-6 lung carcinoma xenografts [1].

References:
[1] Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, Brownell JE, Burke KE, Cardin DP, Critchley S, Cullis CA, Doucette A, Garnsey JJ, Gaulin JL, Gershman RE, Lublinsky AR, McDonald A, Mizutani H, Narayanan U, Olhava EJ, Peluso S, Rezaei M, Sintchak MD, Talreja T, Thomas MP, Traore T, Vyskocil S, Weatherhead GS, Yu J, Zhang J, Dick LR, Claiborne CF, Rolfe M, Bolen JB, Langston SP. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009 Apr 9;458(7239):732-6.