Home Ubiquitin E1 Activating inhibitor - NEDD8 inhibitor Pevonedistat (MLN4924)

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Pevonedistat (MLN4924) NAE inhibitor

Cat.No.S7109

Pevonedistat (MLN4924) is a small molecule inhibitor of Nedd8 activating enzyme (NAE) with IC50 of 4 nM.
Pevonedistat (MLN4924) E1 Activating inhibitor Chemical Structure

Chemical Structure

Molecular Weight: 443.52

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Quality Control

Cell Culture, Treatment & Working Concentration

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

Chemical Information, Storage & Stability

Molecular Weight 443.52 Formula

C21H25N5O4S

 Storage (From the date of receipt)
CAS No. 905579-51-3 Download SDF Storage of Stock Solutions

Solubility

In vitro
Batch:

DMSO : 89 mg/mL (200.66 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 22 mg/mL

Water : Insoluble

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Mass Concentration Volume Molecular Weight

In vivo
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Mechanism of Action

Features
A mechanism-based inhibitor of NAE, and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme.
Targets/IC50/Ki
NAE [1]
(Cell-free assay)
4 nM
In vitro
Pevonedistat (MLN4924) is structurally related to adenosine 59-monophosphate (AMP)—a tight binding product of the NAE reaction. It (3 μM) selectively inhibits NAE in HCT-116 cell lysates and inhibits overall protein turnover by <9% in HCT-116 cells. This compound 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. It (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] At the same concentration, it 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] At 1 μM, it triggers DNA replication and inhibits cell proliferation by stabilizing the DNA replication factor Cdt1, a substrate of cullins 1 and 4. This concentration, 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] Treatment with this compound 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. It 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.
In vivo
Pevonedistat (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. It also leads to a dose- and time-dependent increase in the steady state levels of NRF2 and CDT1 in HCT-116 tumour-bearing mice. This compound leads to DNA damage in the tumour indicated by the increased levels of phosphorylated CHK1 in HCT-116 tumour-bearing mice. When administered on a BID schedule at 30 mg/kg and 60 mg/kg, it inhibits tumour growth with T/C values of 0.36 and 0.15, respectively, in mice bearing HCT-116 xenografts. [1] It (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. This compound (60 mg/kg) results in NF-kappaB pathway inhibition accompanied by tumor regressions in primary human tumor mice models of ABC-DLBCL. [2]
References

Applications

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 S7109-WB1 28838998
Immunofluorescence RhoB / VE-cadherin / F-actin S7109-IF1 29358211
Growth inhibition assay Cell viability S7109-viability1 27333051

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

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

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