NEDD8-activating enzyme (NAE) (IC50 = 4.7 nM)
NEDD8-activating enzyme (NAE) [1]
- NEDD8-activating enzyme (NAE) [2]
- NEDD8-activating enzyme (NAE) [4]

Pevonedistat (MLN4924) selectively inhibits the closely related enzymes UAE, SAE, UBA6, and ATG7 (IC50=1.5, 8.2, 1.8, and >10 μM, respectively), while being a powerful inhibitor of NAE (half-maximal inhibitory dose, IC50=0.004 μM). Treatment with pevonedistat (MLN4924) reduces total protein turnover in HCT-116 cells grown. Pevonedistat (MLN4924) treatment of HCT-116 cells for 24 hours causes a dose-dependent decrease of NEDD8-cullin conjugates and Ubc12-NEDD8 thioester, with an IC50 < 0.1 μM. This is accompanied by a reciprocal increase in the abundance of known CRL substrates, such as CDT1, p27, and NRF2 (also called NFE2L2), but not non-CRL substrates[1]. Pevonedistat overcomes stroma-mediated resistance by inducing apoptosis in CLL cells. Pevonedistat encourages the expression of Noxa and Bim in CLL cells, which causes the Bcl-2 family members to rebalance in favor of the proapoptotic BH3-only proteins[2]. Pevonedistat (MLN4924) strongly suppresses migration by transcriptionally activating E-cadherin and suppressing MMP-9. It also immediately inhibits cullin 1 neddylation and remarkably decreases proliferation and survival as well as migration in gastric cancer cells in a dose- and time-dependent manner[3].

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In purified enzyme assays that track the formation of E2-UBL thioester reaction products, pevonedistat (MLN4924) is a potent inhibitor of NAE and selective against the closely related enzymes UAE, SAE, UBA6, and ATG7 (IC50=1.5, 8.2, 1.8, and >10 μM, respectively). In pure enzyme and cellular assays, pevonedistat (MLN4924) preferentially inhibits NAE activity in contrast to the closely related ubiquitin-activating enzyme (UAE, also known as UBA1) and SUMO-activating enzyme (SAE; a heterodimer of SAE1 and UBA2 subunits). Strong cytotoxic activity is demonstrated by MLN4924 against several human tumor-derived cell lines[1].

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MLN4924 is a selective inhibitor of NAE [1]
MLN4924 was discovered as a result of iterative medicinal chemistry efforts on N6-benzyl adenosine that was originally identified as an inhibitor of NAE via high throughput screening (see Supplementary Information for chemical characterization). As shown in Fig. 1a, MLN4924 is structurally related to adenosine 5′-monophosphate (AMP)—a tight binding product of the NAE reaction. The main differences between AMP and MLN4924 are: (1) in place of the adenine base, MLN4924 has a deazapurine base substituted with an aminoindane at N6; (2) in place of the ribose sugar, MLN4924 has a carbocycle and the equivalent of the 2′-hydroxyl group of AMP is absent; (3) in place of the phosphate, MLN4924 has a sulphamate; and (4) in contrast to the stereochemistry of AMP, the methylene sulphamate of MLN4924 is in a non-natural anti-relationship to the deazapurine. X-ray crystallography confirmed that MLN4924 bound in the nucleotide-binding site of NAE.

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Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. [2]

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Pevonedistat HCl (MLN-4924; TAK-924) is a potent and selective inhibitor of NAE. It disrupts cullin-RING ligase-mediated protein turnover, leading to apoptotic death in human tumour cells through deregulation of S-phase DNA synthesis [1]
- In rat hepatoma H-4-II-E cells, the combination of Pevonedistat HCl (MLN-4924; TAK-924) and TNF-α showed synergistic cytotoxicity, with the LC₅₀ of the combination being approximately 300-fold lower than that of Pevonedistat HCl (MLN-4924; TAK-924) alone. The combination induced rapid cell death (killing ~95% of cells within 16 h) mediated by caspase-8, accompanied by cleavage of apoptosis markers (cleaved caspase-3, PARP, BID) and accumulation of caspase-8 p10 subunit. When apoptosis was blocked, cell death switched to necroptosis with trimerized MLKL as a biomarker. This synergistic cytotoxicity was also observed in primary rat hepatocytes, liver Kupffer cells, rat proximal tubule line NRK-52E, human acute monocytic leukemia line THP-1, and human hepatocellular carcinoma line HEP-G2 [2]
- In human gastric cancer cells, Pevonedistat HCl (MLN-4924; TAK-924) rapidly inhibited cullin 1 neddylation in a dose- and time-dependent manner. It suppressed cell growth and survival by inducing G2/M phase arrest (via accumulation of CDT1/ORC1 and triggering DNA damage response), senescence (via accumulation of p21/p27), and protective autophagy (via accumulation of PHLPP1). Additionally, it suppressed cell migration by transcriptionally activating E-cadherin and repressing MMP-9. The IC₅₀ values for growth inhibition varied among different gastric cancer cell lines, with significant dose-dependent reduction in colony formation [4]

Pevonedistat (MLN492410, 30 or 60 mg/kg, sc) reduces NEDD8-cullin levels in normal mouse tissue, as demonstrated in mouse bone marrow cells, and increases NRF2 and CDT1 steady state levels in HCT-116 tumor-bearing animals in a dose- and time-dependent manner. Tumor development is inhibited by pevonedistat (MLN4924), when given on a BID basis at doses of 30 and 60 mg/kg (T/C values of 0.36 and 0.15, respectively)[1].\n

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\nIn mice carrying HCT-116 xenografts, pevonedistat (MLN4924) (sc, 10 mg/kg, 30 mg/kg, or 60 mg/kg) inhibits the NEDD8 pathway, causing DNA damage[1]. The combination of pevonedistat (sc, 120 mg/kg) and TNF-α (10 μg/kg) damages the livers of SD rats[2].\n

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\nMLN4924 inhibits the NAE pathway in vivo [1]
\nTo assess the ability of MLN4924 to inhibit NAE in vivo, HCT-116 tumour-bearing mice received a single subcutaneous dose of 10, 30 or 60 mg kg-1 MLN4924, and tumours were excised at various time-points over the subsequent 24 h period. The pharmacodynamic effects of treatment were assessed in tumour lysates which were analysed for NEDD8–cullin, NRF2 and CDT1 protein levels (Fig. 4a–c). A single dose of MLN4924 resulted in a dose- and time-dependent decrease of NEDD8–cullin levels as early as 30 min after administration of compound (Fig. 4a), with maximal effect 1–2 h post-dose. A significant difference was observed between the 10 and 60 mg kg-1 response profiles (P < 0.01), although the 10 and 30 mg kg-1 (P = 0.11) and 30 and 60 mg kg-1 (P = 0.24) profiles were not significantly different from each other. A single dose of MLN4924 also led to a dose- and time-dependent increase in the steady state levels of NRF2 and CDT1 (Fig. 4b, c). For all dose levels, NRF2 protein levels peaked 2–4 h after administration of MLN4924 and started to decline by 4–8 h post-dose. The timing of CDT1 accumulation was slightly delayed compared to NRF2, peaking 4 h after MLN4924 administration (Fig. 4c). Evidence of DNA damage in the tumour was indicated by the increased levels of phosphorylated CHK1 (Ser 317) at 8 h after a single administration of 30 and 60 mg kg-1 MLN4924 (Fig. 4d). It should be noted that MLN4924 also decreased NEDD8–cullin levels in normal mouse tissue as illustrated in mouse bone marrow cells (Supplementary Fig. 5). These data suggest that MLN4924-mediated inhibition of NAE in this in vivo tumour model results in pathway responses and cellular phenotypic effects compatible with those observed in cultured cells [1].\n

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\n\nPevonedistat and TNF-α synergistically cause liver damage in rats [2]
\nThe in vivo effects of pevonedistat and TNF-α were assessed in Sprague-Dawley rats. The dose of pevonedistat administered to rats was known from previous investigations to be well tolerated, and the dose of recombinant rat TNF-α activated TNF signaling without toxic side effects.4 Animals within each group (n=8) first received either vehicle or 10 μg/kg TNF-α, followed by either a second vehicle or 120 mg/kg pevonedistat 1 h later. Two animals dosed with the combination treatment exhibited moribund conditions and were euthanized within 10 h. There was a clear difference in liver damage of single-agent versus combination treatments in rats. The incidence and severity of microscopic liver findings for five representative animals from each dose group are presented in Table 1. The livers of animals dosed with pevonedistat+TNF-α had minimal-to-mild single-cell necrosis and neutrophilic infiltration. Representative histological images in Figure 6a illustrate karyomegaly (white arrowhead) in the livers from animals that received pevonedistat alone and necrosis (black arrowhead) and neutrophilic infiltrate (white arrow) in the combination-treated livers. Animals that received the combination treatment had significant ~5-fold elevation of the serum markers alanine transaminase (ALT), aspartate transaminase (AST) and sorbitol dehydrogenase (SDH) compared with those that received single-agent treatments (Figure 6b). Western blotting of liver extracts identified uncleaved caspase-8 (Figure 6c, arrow) in all animals and the p32 fragment of caspase-8 was observed in 9/10 animals that received pevonedistat±TNF-α (arrowhead). Neither p10 nor p18 (data not shown) were detected. Staining of the cleaved cFLIP-L 43-kDa fragment was strongest in samples that also had caspase-8 cleavage. There was a 4-fold elevation of caspase-8 activity in the pevonedistat±TNF-α groups compared with vehicle (Figure 6d). Whether caspase-8 activation was the principle driver of toxicity in rats could not be established. \n

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Pevonedistat HCl (MLN-4924; TAK-924) suppressed the growth of human tumour xenografts in mice at well-tolerated compound exposures [1]
- In Sprague-Dawley rats, the combination of Pevonedistat HCl (MLN-4924; TAK-924) (120 mg/kg, subcutaneous) and TNF-α (10 μg/kg, intravenous) caused significant liver damage. Only the combination treatment resulted in elevated serum markers of liver injury (ALT, AST, SDH, ~5-fold increase compared to single-agent treatments) and single-cell hepatocyte necrosis with neutrophilic infiltration. Two rats in the combination group exhibited moribund conditions and were euthanized within 10 h [2]

In vitro E1-activating enzyme assays [1]
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 °C 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 2 h at 24 °C, 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.

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Assay of bulk protein turnover [1]
HCT-116 cells were plated into 12-well plates at 1 × 105 cells per well and incubated overnight. The medium was exchanged with methionine-free DMEM containing 10% dialysed FBS and 50 μCi per well of [35S]methionine, and the cells were incubated for 20 min to label proteins undergoing synthesis. The cells were then washed three times with DMEM supplemented with 2 mM methionine. Fresh medium containing 10% FBS, 2 mM methionine and the test compounds as described in Fig. 2 were then added. At the specified time points, media (50 μl) was collected and subjected to liquid scintillation counting. At the end of the time course, remaining media was removed and the cells were solubilized by adding of 1 ml 0.2 N NaOH and the extract was subjected to liquid scintillation counting. The percentage of protein turnover at each time point was calculated as [(total acid soluble counts in supernatant)/(total acid soluble counts in supernatant + total counts in solubilized cells)] ×100.

Cell viability assay [1]
\nCell suspensions were seeded at 3,000–8,000 cells per well in 96-well culture plates and incubated overnight at 37 °C. Compounds were added to the cells in complete growth media and incubated for 72 h at 37 °C. Cell number was quantified using the ATPlite assay.\n

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\nWestern blot analysis of cultured cells [1]
\nHCT-116 cells grown in 6-well cell-culture dishes were treated with 0.1% DMSO (control) or MLN4924/pevonedistatt for 24 h. Whole cell extracts were prepared and analysed by immunoblotting. For analysis of the E2–UBL thioester levels, lysates were fractionated by non-reducing SDS–PAGE and immunoblotted with polyclonal antibodies to Ubc12, Ubc9 and Ubc10. For analysis of other proteins, lysates were 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 . Rabbit monoclonal antibodies to NEDD8 and phosphorylated CHK1 (Ser 317) were generated by Millennium in collaboration with Epitomics, Inc. using Ac-KEIEIDIEPTDKVERIKERVEE-amide and Ac-VKYSS(pS)QPEPRT-amide as immunogens, respectively. Antibodies to pH3, cleaved PARP and cleaved caspase 3 were from Cell Signaling Technologies. Secondary HRP-labelled antibodies to rabbit IgG or mouse IgG (Santa Cruz) were used as appropriate. Blots were developed with ECL reagent. For Supplementary Fig. 2, the secondary antibody was Alexa-680-labelled antibody to rabbit/mouse IgG and the blots were imaged using the Li-Cor Odyssey Infrared Imaging system.\n

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\nCell-cycle analysis [1]
\nLogarithmically growing HCT-116 cells were incubated with either MLN4924/pevonedistatt or DMSO for the times indicated. Collected cells were fixed in 70% ethanol and stored overnight at 4 °C. Fixed cells were centrifuged to remove ethanol, and the pellets were resuspended in propidium iodide and RNase A in PBS for 1 h on ice protected from light. Cell-cycle distributions were determined using flow cytometry and analysed using Winlist software (Verity).\n

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\nFACS analysis [2]
\nDNA nuclear content was determined as previously described.15 Actively dividing H-4-II-E cells were treated with MLN4924/pevonedistatt and/or TNF-α for 8 h. Before the end of treatment, cells were spiked with 10 μM bromodeoxyuridine (Brd-U). After 30 min, cells were fixed in ethanol, incubated with a FITC–anti-Brd-U secondary antibody, and then incubated with 10 μg/ml propidium iodide (PI). Labeled cells were measured for Brd-U and PI staining on a FACSCalibur flow cytometer. Cell cycle data were analyzed using FACSDiva (v 6.1.1).\n

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\nsiRNA knockdown [2]
\nH-4-II-E cells were transfected with either a non-targeting control pool of siRNAs or with individual siGenome siRNA oligonucleotide duplexes designed to silence target rat genes caspase-8 and cdt1. Cells were plated sparsely (10 000 cells/well in 96-well plates and 500 000 cells/well in a six-well tissue-culture plate) in antibiotic-free media. The following day, cells were transfected with 25 nM of siRNAs using Lipofectamine RNAiMAX for 72 h. Following transfection, cells were treated with MLN4924/pevonedistatt and/or TNF-α for 24–48 h. Successful knockdown were verified by western blotting. Sequences for siRNAs used in experiments are included in Supplementary Information.\n

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\nCell culture [2]
\nThe rat hepatoma H-4-II-E cell line was selected to model MLN4924/pevonedistatt toxicities because of its common use in the assessment of toxic compounds.43,44 H-4-II-E cells were purchased from American Type Culture Collection and were cultured following the manufacturer’s instructions. Briefly, cells were cultured in MEM supplemented with 10% FBS and incubated at 37 °C with 5% CO2. For routine culture, cells were supplemented with 10 U/l of penicillin and 10 ug/l of streptomycin. For passaging, cells were washed once with PBS, treated with 0.05% trypsin-EDTA, supplemented with fresh media, and pelleted in a clinical centrifuge.

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Cell viability and cytotoxicity assay: Seed target cells (e.g., H-4-II-E, gastric cancer cells) in 96-well plates. Treat cells with different concentrations of Pevonedistat HCl (MLN-4924; TAK-924) alone or in combination with TNF-α for 24–72 h. Quantify intracellular ATP using a luminescent assay to determine cell viability and calculate LC₅₀ values [2][4]
- Western blot analysis: Treat cells with Pevonedistat HCl (MLN-4924; TAK-924) at indicated concentrations and time points. Lyse cells, extract proteins, separate by electrophoresis, transfer to membranes, and incubate with specific antibodies to detect target proteins (e.g., NEDD8-cullin, unbound NEDD8, cleaved caspase-3, PARP, BID, CDT1, p21, PHLPP1, E-cadherin, MMP-9) [2][4]
- Flow cytometry (FACS) analysis: Treat actively dividing cells with Pevonedistat HCl (MLN-4924; TAK-924) for 8–72 h. Stain cells with propidium iodide (PI) and/or Brd-U to analyze cell cycle distribution (G2/M arrest, DNA re-replication) and DNA fragmentation [2][4]
- TUNEL assay: Seed cells on glass coverslips, treat with Pevonedistat HCl (MLN-4924; TAK-924) and/or TNF-α for 6 h. Use a colorimetric TUNEL assay to detect apoptotic cells with nicked DNA, and counterstain with DAPI for nuclear visualization [2]
- Colony formation assay: Seed cells in 60 mm dishes, treat with Pevonedistat HCl (MLN-4924; TAK-924) at indicated concentrations for 10 days. Fix and stain colonies, then count and quantify the number of colonies to evaluate long-term cell growth inhibition [4]
- Transwell migration and wound-healing assays: For Transwell assay, starve cells for 24 h, treat with Pevonedistat HCl (MLN-4924; TAK-924) for 12 h, then seed in the upper chamber of Transwell inserts and incubate for a specific time. Count migrated cells on the lower membrane. For wound-healing assay, scratch confluent cell monolayers, treat with Pevonedistat HCl (MLN-4924; TAK-924), and measure the wound closure rate over time [4]
- siRNA knockdown rescue assay: Transfect cells with siRNAs targeting CDT1, p21, PHLPP1, or control siRNAs. After 24–72 h of transfection, treat cells with Pevonedistat HCl (MLN-4924; TAK-924) for 24–48 h. Analyze cell cycle distribution, senescence, or autophagy to verify the role of specific substrates in the drug’s mechanism [4]

10% cyclodextrin;60 mg/kg;Subcutaneously injection
\nmice bearing HCT-116 xenografts \nTumour xenograft efficacy experiments [1]
\nFemale athymic NCR mice were used in all in vivo studies. All animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were inoculated with 2 × 106 HCT-116 cells (or 30–40 mg H522 tumour fragments) subcutaneously in the right flank, and tumour growth was monitored with caliper measurements. When the mean tumour volume reached approximately 200 mm3, animals were dosed subcutaneously with vehicle (10% cyclodextrin) or MLN4924. Inhibition of tumour growth (T/C) was calculated on the last day of treatment.

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\nPharmacodynamic marker analysis [1]
\nMice bearing HCT-116 tumours of 300–500 mm3 were administered a single MLN4924 dose, and at the indicated times tumours were excised and extracts prepared. The relative levels of NEDD8–cullin and NRF2 were estimated by quantitative immunoblot analysis using Alexa680-labelled anti-IgG (Molecular Probes) as the secondary antibody. The statistical difference between the groups for NEDD8–cullin inhibition was determined using the Kruskal–Wallis test. For the analysis of CDT1 and phosphorylated CHK1 (Ser 317) levels in tumour sections, formalin-fixed, paraffin-embedded tumour sections were stained with the relevant antibodies, amplified with HRP-labelled secondary antibodies and detected with the ChromoMap DAB Kit). Slides were counterstained with haematoxylin. Images were captured using an Eclipse E800 microscope and Retiga EXi colour digital camera and processed using Metamorph software. CDT1 and phosphorylated CHK1 levels are expressed as a function of the DAB signal area.

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\nIsolation of bone marrow cells from mice [1]
\nFor bone marrow pharmacodynamic studies, naive NCr-Nude mice were administered MLN4924, and at the indicated times leg bones were excised. Marrow was flushed from the bones with PBS, pelleted by centrifugation and flash frozen. Thawed marrow was lysed in M-PER buffer (Pierce) with protease inhibitors. NEDD8–cullin levels were measured by immunoblot analysis.

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\nIn vivo rat model [2]
\nTen-week-old male Sprague-Dawley rats were used. Across two studies, a total of eight animals in each group were dosed with vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Animals were first intravenously administered either vehicle (1× PBS) or 10 μg/kg TNF-α. One hour later, they were subcutaneously administered vehicle (20% sulfobutyl ether beta-cyclodextrin in 50 mM citrate buffer, pH 3.3) or 120 mg/kg pevonedistat. Scheduled euthanasia occurred 24 h postdose. Unscheduled euthanasia was performed when animals exhibited moribund conditions. Serum was collected at necropsy and analyzed for serum chemistry markers of liver damage (ALT, AST, and SDH). Additionally, the livers from five animals in each group were removed, separated into two sections and either frozen at −80 °C for subsequent protein analysis or fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4–6 μm, mounted on glass slides, stained with hematoxylin and eosin, and analyzed with an Olympus BX51 light microscop for histopathology assessment. Microscopic findings were recorded in concordance with the standardized nomenclature for classifying lesions within the livers of rats. \n

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\nHuman tumour xenograft assay in mice: Establish human tumour xenografts by inoculating tumour cells into immunocompromised mice. Administer Pevonedistat HCl (MLN-4924; TAK-924) via an appropriate route (not specified) at doses that are well-tolerated. Monitor tumour growth over time and compare tumour volume between drug-treated and control groups [1]
\n- Liver toxicity assay in rats: Use 10-week-old male Sprague-Dawley rats (n=8 per group). First, intravenously administer either vehicle (1× PBS) or 10 μg/kg TNF-α. One hour later, subcutaneously administer vehicle (20% sulfobutyl ether beta-cyclodextrin in 50 mM citrate buffer, pH 3.3) or 120 mg/kg Pevonedistat HCl (MLN-4924; TAK-924). Euthanize rats 24 h post-dose (or earlier if moribund). Collect serum to analyze liver function markers (ALT, AST, SDH) and dissect livers for histopathological analysis (hematoxylin and eosin staining) and protein extraction [2]

In vitro experiments have shown that pevoridoxat hydrochloride (MLN-4924; TAK-924) alone has low cytotoxicity to a variety of cell lines, but when used in combination with TNF-α, it can synergistically induce cell death through caspase-8-mediated apoptosis or necroptosis (when apoptosis is blocked) [2]. In in vivo clinical trials, high doses of pevoridoxat hydrochloride (MLN-4924; TAK-924) have been associated with adverse reactions, including elevated liver transaminases and multiple organ dysfunction syndrome. In rats, pevoridoxat hydrochloride (MLN-4924; TAK-924) in combination with TNF-α can lead to significant liver injury (single-cell necrosis, neutrophil infiltration) and elevated serum liver injury markers [2].

[1]. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009 Apr 9;458(7239):732-6.

[2]. The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF-α to activate apoptosis. Cell Death Discovery 1, Article number: 15034 (2015).

[3]. The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells.

[4]. Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells. Sci Rep. 2016 Apr 11;6:24218.

Pevonedistat is a pyrrolopyrimidine compound with the structure 7H-pyrrolo[2,3-d]pyrimidine, substituted at position 4 with a (1S)-2,3-dihydro-1H-indene-1-yl nitrile group and at position 7 with a (1S,3S,4S)-3-hydroxy-4-[(aminosulfonyloxy)methyl]cyclopentyl group. It is a potent and selective inhibitor of the NEDD8 activator enzyme with an IC50 of 4.7 nM and is currently undergoing clinical trials for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes. It exhibits dual effects of inducing apoptosis and antitumor activity. It belongs to the pyrrolopyrimidine class of compounds, including secondary amino compounds, cyclopentanols, sulfonamides, and indane compounds. Pevonedistat has been used in clinical trials for the treatment of various tumors, including lymphoma, solid tumors, multiple myeloma, Hodgkin's lymphoma, and metastatic melanoma.
Pevonedistat is a small-molecule Nedd8 activator enzyme (NAE) inhibitor with potential antitumor activity. Pevonedistat binds to NAE and inhibits its activity, potentially suppressing tumor cell proliferation and survival. NAE activates Nedd8 (a developmentally downregulated protein 8 expressed on neural progenitor cells), a ubiquitin-like protein (UBL) that modifies cellular targets via a pathway parallel to but distinct from the ubiquitin-proteasome pathway (UPP). Proteins that bind to UBLs such as Nedd8 are involved in various regulatory activities and are generally not degraded by the proteasome.
Pharmaceutical Indications

Treatment of acute myeloid leukemia and myelodysplastic syndromes.
Clinical development of cellular proteasome function inhibitors suggests that compounds targeting other components of the ubiquitin-proteasome system may be effective in treating human malignancies.
NEDD8 activator (NAE) is an important component of the NEDD8 binding pathway, which controls the activity of cullin-RING subtype ubiquitin ligases, thereby regulating the turnover of proteins upstream of the proteasome. Substrates of cullin-RING ligases play important roles in cellular processes associated with cancer cell growth and survival pathways. This article introduces a highly potent and selective NAE inhibitor, MLN4924. MLN4924 induces apoptosis in human tumor cells by disrupting cullin-RING ligase-mediated protein turnover through a novel mechanism of action—S-phase DNA synthesis dysregulation. At well-tolerated doses of the compound, MLN4924 inhibited the growth of human tumor xenografts in mice. Our data suggest that NAE inhibitors may have the potential to treat cancer. [1]

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This article describes the preliminary properties of MLN4924, a small molecule NAE inhibitor representing a novel approach to targeting the ubiquitin-proteasome system (UPS) for cancer treatment. MLN4924 completely inhibits detectable NAE pathway function in cells, disrupting the turnover of CRL substrates, which play crucial roles in cell cycle progression and survival. Our results indicate that NAE pathway inhibition more selectively disrupts protein homeostasis in cancer cells compared to proteasome activity inhibition, potentially leading to significant differences in clinical efficacy and safety. Sustained inhibition of the NAE pathway results in cell cycle-dependent DNA replication, subsequently activating apoptosis. This phenotype may be due to the inability of cells to degrade the CRL substrate CDT1, and CDT1 overexpression has been shown to induce DNA replication. Similar cell cycle distributions were observed when NAE levels were reduced via RNAi or when NAE activity was impaired in temperature-sensitive mutant cell lines. In vivo experiments demonstrated that MLN4924 inhibited the growth of human tumor xenografts at well-tolerated doses and dosing regimens. Analysis of tumors in treated animals confirmed inhibition of the NEDD8 pathway, suggesting that these pharmacodynamic biomarkers may be helpful in monitoring NAE inhibition in patients treated with MLN4924. These preclinical results support the entry of MLN4924 into clinical development. [1] Predicting and understanding the mechanisms of drug-induced toxicity is one of the main goals of drug development. Some studies have hypothesized that inflammation may play a synergistic role in this process. Cell-based models provide an easy-to-manipulate system for studying the toxicity of such drugs. Some research teams have attempted to reproduce in vivo toxicity through combined treatment with drugs and inflammatory cytokines. In this way, researchers discovered the synergistic cytotoxicity between the research drug pevonedistat (MLN4924) and TNF-α. pevonedistat is an inhibitor of the NEDD8 activating enzyme (NAE). Inhibition of NAE prevents the activation of cullin-RING ligase, which is essential for proteasome-mediated protein degradation. TNF-α is a cytokine involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with a combination of pevonedistat and TNF-α, rather than using them alone, resulted in rapid cell death. This cell death was identified as being mediated by caspase-8. Interestingly, the combined treatment of pevonedistat and TNF-α also led to the accumulation of the p10 protease subunit of caspase-8, while this phenomenon was not observed when cytotoxic doses of TNF-α were used alone. In the case of blocked apoptosis, the cell death mechanism was transformed into necrotic apoptosis. The trimer MLKL was confirmed to be a biomarker of necrotic apoptosis. In vivo rat experiments also confirmed that pevonedistat and TNF-α had synergistic toxicity. Only the combination treatment led to the elevation of serum liver injury markers and necrosis of single-cell hepatocytes. In summary, the results of this study reveal a novel synergistic toxicity driven by pevonedistat and TNF-α. [2]

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The results of this study indicate that the NAE inhibitor pevonedistat is toxic when combined with the pro-inflammatory cytokine TNF-α. The driving factor of in vitro toxicity appears to be enhanced cleavage/activation of caspase-8 p10 protease, which in turn activates apoptosis. However, the molecular mechanism between pevonedistat and caspase-8 in the cytotoxicity model of pevonedistat and TNF-α is unclear. Since cullin-3 can ubiquitinate caspase-8 (Jin et al.³⁶) and is also inhibited by pevonedistat, it is obviously a candidate target worthy of investigation, but cullin-3 knockdown did not increase sensitivity to TNF-α alone (Supplementary Figure S4). Ultimately, the role of cullin-3 in mediating co-toxicity was not confirmed. Pevonedistat alone is known to stabilize the expression of ≥120 different proteins⁴², but there is currently no evidence that these proteins interact with caspase-8. Higher throughput methods are needed to determine whether any unknown proteins become stable under pevonedistat + TNF stimulation. Further research using pevonedistat as a tool compound will help to better understand the molecular mechanisms of programmed cell death^[2]. MLN4924 is a newly discovered small molecule inhibitor of NEDD8 activating enzyme (NAE). Since cullin-ring ligases (CRLs) belong to the largest E3 ubiquitin ligase family, their activity requires NEDD8 modification of cullin. Therefore, MLN4924 indirectly inhibits CRLs by blocking NEDD8 modification of cullins. Given the upregulated expression of CRL components and the overactivation of NEDDylation modification in various human cancers, MLN4924 has been found to effectively inhibit cancer cell growth. However, it remains unclear whether MLN4924 is effective against gastric cancer cells. This study shows that in gastric cancer cells, MLN4924 rapidly inhibits NEDDylation modification of cullin 1 and significantly suppresses cell growth, survival, and migration in a dose- and time-dependent manner. Mechanistic studies combined with siRNA knockdown rescue experiments indicate that MLN4924 induces the accumulation of multiple CRL substrates (including CDT1/ORC1, p21/p27, and PHLPP1), triggering DNA damage responses and G2/M phase cell cycle arrest, thereby inducing cellular senescence and autophagy. MLN4924 also significantly inhibited cell migration by activating E-cadherin and inhibiting MMP-9. In summary, our study suggests that NEDDylation modification and CRL E3 ligase are potential targets for gastric cancer, and MLN4924 is expected to be further developed into an effective drug for the treatment of gastric cancer. [4]

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Pevorisstat hydrochloride (MLN-4924; TAK-924) is the first small molecule inhibitor of NAE. It works by blocking NEDDylation modification of cullin, thereby inactivating CRL and accumulating various substrates that are normally degraded by ubiquitination. [1][2][4]
This drug has been evaluated in clinical trials for the treatment of acute myeloid leukemia, myelodysplastic syndromes, solid tumors, non-hematologic malignancies, melanoma, lymphoma and multiple myeloma. Patient inclusion/exclusion criteria excluded patients with active uncontrolled infection or recent antibiotic use, as these conditions may lead to pro-inflammatory state-related toxicity [2]
- In chronic lymphocytic leukemia B cells, pevoridoxat hydrochloride (MLN-4924; TAK-924) inhibited microenvironment-driven NF-κB activation and induced apoptosis (Correction: detailed experimental data were not provided in the cited errata) [3]
- The mechanisms of action of pevoridoxat hydrochloride (MLN-4924; TAK-924) include: inducing DNA re-replication and cell cycle arrest through CDT1 accumulation (single-drug effect); blocking the survival-promoting NF-κB signaling pathway by preventing the degradation of phosphorylated IκBα; and synergistic activation of the cell death pathway with TNF-α [1][2][4]

C21H26CLN5O4S

479.98

479.139

C, 52.55; H, 5.46; Cl, 7.39; N, 14.59; O, 13.33; S, 6.68

1160295-21-5

Pevonedistat;905579-51-3

66576990

White to off-white solid powder

4.718

4

8

6

32

734

4

C1CC2=CC=CC=C2[C@H]1NC3=C4C=CN(C4=NC=N3)[C@@H]5C[C@H]([C@H](C5)O)COS(=O)(=O)N.Cl

HJKDNNXKYODZJI-BVMPOVDASA-N

InChI=1S/C21H25N5O4S.ClH/c22-31(28,29)30-11-14-9-15(10-19(14)27)26-8-7-17-20(23-12-24-21(17)26)25-18-6-5-13-3-1-2-4-16(13)18;/h1-4,7-8,12,14-15,18-19,27H,5-6,9-11H2,(H2,22,28,29)(H,23,24,25);1H/t14-,15+,18-,19-;/m0./s1

[(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-inden-1-yl]amino]pyrrolo[2,3-d]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl sulfamate;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)