Home Ubiquitin E1 Activating inhibitor Pevonedistat (MLN4924) For research use only.

Pevonedistat (MLN4924)

MLN4924 is a small molecule inhibitor of Nedd8 activating enzyme (NAE) with IC50 of 4 nM.

Pevonedistat (MLN4924) Chemical Structure

Pevonedistat (MLN4924) Chemical Structure

CAS No. 905579-51-3

Purity & Quality Control

Pevonedistat (MLN4924) Related Products

Signaling Pathway

E1 Activating Signaling Pathways

E1 Activating Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
K562 Antiproliferative assay 72 hrs Antiproliferative activity against human K562 cells after 72 hrs by CellTiter-Glo assay, EC50 = 0.108 μM. 24900352
U2OS Antitumor assay 72 hrs Antitumor activity against human U2OS cells after 72 hrs by MTT assay, IC50 = 0.16 μM. 28388520
HCT116 Antitumor assay 72 hrs Antitumor activity against human HCT116 cells after 72 hrs by MTT assay, IC50 = 0.19 μM. 28388520
Caco2 Function assay 16 hrs Inhibition of NAE-mediated Ubcl2-NEDD8 conjugation in human Caco2 cells after 16 hrs by Western blot analysis, EC50 = 3.4 μM. 29232579
Caco2 Function assay 16 hrs Inhibition of NAE-mediated Ubcl2-NEDD8 conjugation in human Caco2 cells after 16 hrs by Western blot analysis, EC50 = 3.4 μM. 29232579
Caco2 Cytotoxicity assay 72 hrs Cytotoxicity against human Caco2 cells after 72 hrs by MTT assay, IC50 = 4.4 μM. 29232579
Click to View More Cell Line Experimental Data

Biological Activity

Description MLN4924 is a small molecule inhibitor of Nedd8 activating enzyme (NAE) with IC50 of 4 nM.
Features A mechanism-based inhibitor of NAE, and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme.
Targets
NAE [1]
(Cell-free assay)
4 nM
In vitro
In vitro MLN4924 is structurally related to adenosine 59-monophosphate (AMP)—a tight binding product of the NAE reaction. MLN4924 (3 μM) selectively inhibits NAE in HCT-116 cell lysates. MLN4924 (3 μM) inhibits overall protein turnover by <9% in HCT-116 cells. MLN4924 results in a dose-dependent decrease of Ubc12–NEDD8 thioester and NEDD8–cullin conjugates with an IC50 < 0.1 μM in HCT-116 cells, resulting in a reciprocal increase in the abundance of the known CRL substrates CDT1, p27 and NRF2, but not non-CRL substrates. MLN4924 (3 μM) leads cells to accumulate in S-phase as early as 8 hours and results in a significant fraction of cells contained 4N DNA content by 24 hours in HCT-116 cells. [1] MLN4924 (3 μM) results in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition in ABC DLBCL cells. [2] MLN4924 (1 μM) triggers DNA replication and inhibit cell proliferation by stabilizing the DNA replication factor Cdt1, a substrate of cullins 1 and 4. MLN4924 (1 μM) , which is sufficient to elevate Cdt1 for 4-5 hours, is found to be sufficient to induce DNA replication and to activate apoptosis and senescence pathways. [3] MLN4924 treatment induces the characteristics of senescence phenotypes as evidenced by enlarged and flattened cellular morphology and positive staining of senescence-associated β-Gal. MLN4924-induced senescence is associated with cellular response to DNA damage, triggered by accumulation of DNA-licensing proteins CDT1 and ORC1, as a result of inactivation of CRL/SCF E3s. MLN4924-induced senescence is irreversible and coupled with persistent accumulation of p21 and sustained activation of DNA damage response. [4]
Kinase Assay In vitro E1-activating enzyme assays
A time-resolved fluorescence energy transfer assay format is 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, is 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 PHYCOLINK allophycocyanin (XL-APC)-labelled GST-specific antibody. After incubation for 2 hours at 24 ℃, the plate is read on the LJL Analyst HT Multi-Mode instrument using a time-resolved fluorescence method. A similar assay protocol is used to measure other E1 enzymes.
Cell Research Cell lines HCT-116 cells
Concentrations 3 μM
Incubation Time 72 hours
Method Cell suspensions are seeded at 3,000–8,000 cells per well in 96-well culture plates and incubated overnight at 37 ℃. MLN4924 are added to the cells in complete growth media and incubated for 72  hours at 37 ℃. Cell number is quantified using the ATPlite assay.
Experimental Result Images Methods Biomarkers Images PMID
Western blot Culin 3 / CDT2 / CDT1 / SET8 / p21 / p-p53 / p-CHK1 / p-CHK2 / CHK2 / γH2AX / H2AX / PARP / c-PARP Culin 1 / WEE1 / p27 / p-H3 / cyclin B1 Cleaved caspase-3 / Cleaved PARP pro-apoptotic and anti-apoptotic proteins p-c-Jun / c-Jun p-H2A / p-CHK2 p-AKT / p-mTOR / p-70S6K 28838998
Immunofluorescence RhoB / VE-cadherin / F-actin 29358211
Growth inhibition assay Cell viability 27333051
In Vivo
In vivo MLN4924 (60 mg/kg) results in a dose- and time-dependent decrease of NEDD8–cullin levels as early as 30 min after administration in HCT-116 tumour-bearing mice, with maximal effect 1–2 hours post-dose. MLN4924 (60 mg/kg) also leads to a dose- and time-dependent increase in the steady state levels of NRF2 and CDT1 in HCT-116 tumour-bearing mice. MLN4924 (60 mg/kg) leads to DNA damage in the tumour indicated by the increased levels of phosphorylated CHK1 in HCT-116 tumour-bearing mice. MLN4924 administered on a BID schedule at 30 mg/kg and 60 mg/kg inhibits tumour growth with T/C values of 0.36 and 0.15, respectively, in mice bearing HCT-116 xenografts. [1] MLN4924 (60 mg/kg) blocks NAE pathway biomarkers and results in complete tumor growth inhibition in mice bearing human xenograft tumors of ABC- and GCB-DLBCL. MLN4924 (60 mg/kg) results in NF-kappaB pathway inhibition accompanied by tumor regressions in primary human tumor mice models of ABC-DLBCL. [2]
Animal Research Animal Models mice bearing HCT-116 xenografts
Dosages 60 mg/kg
Administration Subcutaneously injection
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT04985656 Withdrawn
Myelodysplastic Syndromes (MDS)
Takeda
 October 1 2021 Phase 2
NCT04800627 Terminated
Locally Advanced Malignant Solid Neoplasm|Metastatic Malignant Solid Neoplasm|Unresectable Malignant Solid Neoplasm
M.D. Anderson Cancer Center
 March 29 2021 Phase 1|Phase 2
NCT04266795 Active not recruiting
Acute Myeloid Leukemia (AML)
Takeda
 October 13 2020 Phase 2
NCT03770260 Completed
Recurrent Multiple Myeloma|Refractory Multiple Myeloma
National Cancer Institute (NCI)
 February 10 2020 Phase 1
NCT04172844 Active not recruiting
Acute Myelogenous Leukemia
Medical College of Wisconsin
 January 13 2020 Phase 1

Chemical Information & Solubility

Molecular Weight 443.52 Formula

C21H25N5O4S
CAS No. 905579-51-3 SDF Download Pevonedistat (MLN4924) SDF
Smiles C1CC2=CC=CC=C2C1NC3=C4C=CN(C4=NC=N3)C5CC(C(C5)O)COS(=O)(=O)N
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 89 mg/mL ( (200.66 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 22 mg/mL

Water : Insoluble


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In vivo
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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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