MLN4924 is an effective and specific small molecule NEDD8-activating enzyme (NAE) inhibitor (IC50: 4.7 nM).
MLN4924 is a potent inhibitor of NAE, and is selective relative to the closely related enzymes UAE, SAE, UBA6 and ATG7 (IC50=1.5, 8.2, 1.8 and >10 μM, respectively) when evaluated in purified enzyme assays that monitor the formation of E2-UBL thioester reaction products. MLN4924 selectively inhibits NAE activity compared to the closely related ubiquitin-activating enzyme (UAE, also known as UBA1) and SUMO-activating enzyme (SAE; a heterodimer of SAE1 and UBA2 subunits), in purified enzyme and cellular assays. MLN4924 exhibits potent cytotoxic activity against a variety of human tumour-derived cell lines.
MLN4924 (sc, 10 mg/kg, 30 mg/kg, or 60 mg/kg) inhibits the NEDD8 pathway resulting in DNA damage in Mice bearing HCT-116 xenografts.Pevonedistat (sc, 120 mg/kg) and TNF-α (10 μg/kg) synergistically cause liver damage in SD rats.
Bcl-2 Binding affinity calculation: A predicted binding affinity for Obatoclax binding to BCL-2 is calculated using the SIE scoring function.  As a control in determining the reliability of the calculation, predicted binding affinities Ki) are calculated for a set of 12 small molecules with experimentally measured binding affinities to BCL-2.
MLN4924 is dissolved in DMSO and stored, and then diluted with appropriate medium before use. HCT-116 cells grown in 6-well cell-culture dishes are treated with 0.1% DMSO (control) or 0.3 μM MLN4924 for 24 h. Whole cell extracts are prepared and analysed by immunoblotting. For analysis of the E2-UBL thioester levels, lysates are fractionated by non-reducing SDS-PAGE and immunoblotted with polyclonal antibodies to Ubc12, Ubc9 and Ubc10. For analysis of other proteins, lysates are fractionated by reducing SDS-PAGE and probed with primary antibodies as follows: mouse monoclonal antibodies to CDT1, p27, geminin, ubiquitin, securin/PTTG and p53 or rabbit polyclonal antibodies to NRF2, Cyclin B1 and GADD34.