NEDD8-activating enzyme (NAE) (IC50 = 4.7 nM)

Pevonedistat (MLN4924) 选择性抑制密切相关的酶 UAE、SAE、UBA6 和 ATG7（IC50 分别为 1.5、8.2、1.8 和 >10 μM），同时是 NAE 的强效抑制剂（半数最大抑制剂量，IC50） =0.004μM）。使用 pevonedistat (MLN4924) 处理可降低生长的 HCT-116 细胞中的总蛋白周转率。 Pevonedistat (MLN4924) 对 HCT-116 细胞处理 24 小时，导致 NEDD8-cullin 缀合物和 Ubc12-NEDD8 硫酯呈剂量依赖性减少，IC50 < 0.1 μM。这伴随着已知 CRL 底物丰度的相互增加，例如 CDT1、p27 和 NRF2（也称为 NFE2L2），但非 CRL 底物则不然[1]。 Pevonedistat 通过诱导 CLL 细胞凋亡来克服基质介导的耐药性。 Pevonedistat 促进 CLL 细胞中 Noxa 和 Bim 的表达，从而导致 Bcl-2 家族成员重新平衡，有利于促凋亡 BH3-only 蛋白 [2]。 Pevonedistat (MLN4924) 通过转录激活 E-钙粘蛋白和抑制 MMP-9 来强烈抑制迁移。它还立即抑制 cullin 1 neddylation，并以剂量和时间依赖性方式显着降低胃癌细胞的增殖和存活以及迁移[3]。

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在追踪 E2-UBL 硫酯反应产物形成的纯化酶测定中，pevonedistat (MLN4924) 是 NAE 的有效抑制剂，并且对密切相关的酶 UAE、SAE、UBA6 和 ATG7 具有选择性（IC50=1.5、8.2、1.8，和 >10 μM，分别）。在纯酶和细胞测定中，与密切相关的泛素激活酶（UAE，也称为 UBA1）和 SUMO 激活酶（SAE；SAE1 和 UBA2 亚基的异二聚体）相比，pevonedistat (MLN4924) 优先抑制 NAE 活性。 MLN4924 对多种人类肿瘤来源的细胞系表现出强大的细胞毒活性[1]。

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MLN4924是NAE 的选择性抑制剂[1]
MLN4924是通过对n6 -苄基腺苷的反复药物化学研究发现的，n6 -苄基腺苷最初通过高通量筛选被鉴定为NAE抑制剂（见化学表征补充信息）。如图1a所示，MLN4924在结构上与腺苷5′-单磷酸腺苷（AMP）相关，AMP是NAE反应的紧密结合产物。AMP与MLN4924的主要区别在于：(1)MLN4924在N6处以氨基取代去氮嘌呤碱基取代腺嘌呤碱基；(2) MLN4924具有一个碳环代替核糖，并且缺少AMP的2′-羟基的等价物；(3) MLN4924用磺胺酸盐代替磷酸盐；(4)与AMP的立体化学结构相反，MLN4924的亚甲磺酸与去氮嘌呤呈非天然的反关系。x射线晶体学证实MLN4924结合在NAE的核苷酸结合位点。

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通过这种方法，研究人员确定了pevonedistat （MLN4924）和TNF-α之间的协同细胞毒性。Pevonedistat是nedd8活化酶（NAE）的抑制剂。NAE的抑制阻止cullin-RING连接酶的激活，而cullin-RING连接酶对于蛋白酶体介导的蛋白质降解至关重要。TNF-α是一种细胞因子，参与炎症反应和细胞死亡，以及其他生物功能。培酮远地与TNF-α联合（而非单独）治疗培养细胞可导致细胞快速死亡。确定这种细胞死亡是由caspase-8介导的。有趣的是，佩onedistat和TNF-α联合治疗也引起caspase-8的p10蛋白酶亚基的积累，这在细胞毒性剂量的TNF-α中没有观察到。在细胞凋亡被阻断的情况下，死亡机制转变为坏死下垂。［2］

Pevonedistat（MLN492410，30 或 60 mg/kg，皮下注射）可降低正常小鼠组织中的 NEDD8-cullin 水平，如小鼠骨髓细胞中所证明的那样，并增加 HCT-116 荷瘤动物中 NRF2 和 CDT1 的稳态水平- 和时间相关的方式。当每日两次给予 30 和 60 mg/kg 剂量（T/C 值分别为 0.36 和 0.15）时，pevonedistat (MLN4924) 可抑制肿瘤发展[1]。 在携带 HCT-116 异种移植物的小鼠中，pevonedistat (MLN4924)（皮下、10 mg/kg、30 mg/kg 或 60 mg/kg）抑制 NEDD8 通路，导致 DNA 损伤[1]。 pevonedistat（sc，120 mg/kg）和TNF-α（10 μg/kg）的组合会损害SD大鼠的肝脏[2]。

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MLN4924在体内抑制NAE通路[1]
为了评估MLN4924在体内抑制NAE的能力，HCT-116荷瘤小鼠接受单次皮下剂量10、30或60 mg kg-1 MLN4924，并在随后的24小时内的不同时间点切除肿瘤。在肿瘤裂解物中评估了治疗的药效学效果，分析了NEDD8-cullin、NRF2和CDT1蛋白水平（图4a-c）。单剂量MLN4924可导致NEDD8-cullin水平在给药后30分钟呈剂量依赖性和时间依赖性下降（图4a），在给药后1-2小时效果最大。10、30、30 mg kg-1 （P = 0.11）、30、60 mg kg-1 （P = 0.24）组间无显著差异，但10、60 mg kg-1组间差异显著（P < 0.01）。单剂量MLN4924也导致NRF2和CDT1的稳态水平呈剂量依赖性和时间依赖性增加（图4b， c）。对于所有剂量水平，NRF2蛋白水平在给药后2-4小时达到峰值，并在给药后4-8小时开始下降。与NRF2相比，CDT1积累的时间略有延迟，在MLN4924给药后4小时达到峰值（图4c）。在单次给药30和60 mg kg-1 MLN4924后8小时，磷酸化的CHK1 （Ser 317）水平升高表明肿瘤中DNA损伤的证据（图4d）。值得注意的是，MLN4924还降低了正常小鼠骨髓细胞中NEDD8-cullin的水平（补充图5）。这些数据表明，在这种体内肿瘤模型中，MLN4924介导的NAE抑制导致了与培养细胞[1]中观察到的通路反应和细胞表型效应相一致。

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Pevonedistat和TNF-α协同引起大鼠肝损伤[2]
在Sprague-Dawley大鼠的体内实验中，观察哌酮远地和TNF-α的作用。从先前的研究中得知，给予大鼠的佩维奈远的剂量耐受性良好，重组大鼠TNF-α的剂量激活了TNF信号，没有毒副作用每组（n=8）先给药或10 μg/kg TNF-α， 1 h后再给药或120 mg/kg哌酮。2只接受联合治疗的动物表现出垂死状态，并在10小时内被安乐死。单药与联合用药对大鼠肝损伤有明显差异。表1列出了各剂量组5只代表性动物肝脏显微镜检查结果的发生率和严重程度。给药pevondistat +TNF-α的动物肝脏出现轻微至轻度单细胞坏死和中性粒细胞浸润。图6a中具有代表性的组织学图像显示，单独接受佩伏奈远治疗的动物肝脏出现核肿大（白色箭头），联合治疗的动物肝脏出现坏死（黑色箭头）和中性粒细胞浸润（白色箭头）。与接受单药治疗的动物相比，接受联合治疗的动物血清标志物丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和山梨醇脱氢酶（SDH）显著升高约5倍（图6b）。肝提取物的Western blotting在所有动物中鉴定出未裂解的caspase-8（图6c，箭头），在接受pevonedistat±TNF-α（箭头）治疗的9/10只动物中观察到caspase-8的p32片段。p10和p18（数据未显示）均未检测到。在caspase-8切割的样品中，裂解的clip - l 43-kDa片段的染色最强。与对照组相比，pevonedistat±TNF-α组caspase-8活性升高4倍（图6d）。caspase-8激活是否是大鼠毒性的主要驱动因素尚不能确定。

体外e1活化酶测定[1]
采用时间分辨荧光能量转移法测定NAE的体外活性。酶促反应含50 μl 50 mM HEPES， pH 7.5, 0.05% BSA, 5 mM MgCl2, 20 μM ATP， 250 μM谷胱甘肽，10 nM Ubc12-GST， 75 nM NEDD8-Flag和0.3 nM重组人NAE酶，在284孔板上24°C孵育90 min，终止前用25 μl停止/检测缓冲液(0.1 M HEPES, pH 7.5, 0.05% Tween20, 20 mM EDTA, 410 mM KF，0.53 nM euroium - crypate标记的单克隆flag - m2特异性抗体和8.125 μg ml-1 PHYCOLINK allophycocyanin （XL-APC）标记的gst特异性抗体)。24°C孵育2小时后，在LJL Analyst HT Multi-Mode仪器上使用时间分辨荧光法读取板。其他E1酶也采用了类似的测定方法。

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体蛋白周转测定[1]
将HCT-116细胞以每孔1 × 105个细胞的速度镀于12孔板中，孵育过夜。培养基中加入不含蛋氨酸的DMEM，其中含有10%的透析FBS和50 μCi /孔的[35S]蛋氨酸，细胞孵育20分钟，标记合成蛋白。然后用添加2 mM蛋氨酸的DMEM洗涤细胞三次。然后加入含有10%牛血清、2 mM蛋氨酸和图2所示试验化合物的新鲜培养基。在规定的时间点，收集50 μl的介质，进行液体闪烁计数。时间过程结束时，除去剩余培养基，加入1 ml 0.2 N NaOH溶解细胞，提取液进行液体闪烁计数。每个时间点的蛋白质周转百分比计算为[（上清液中总酸溶性计数）/（上清液中总酸溶性计数+溶解细胞中总酸溶性计数）]×100。

细胞活力测定[1]
细胞悬液以每孔3000 - 8000个细胞的速度接种于96孔培养板中，在37℃下孵育过夜。将化合物添加到完全生长培养基中的细胞中，在37℃下孵育72 h。使用ATPlite法定量细胞数量。

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Western blot分析培养细胞[1]
在6孔细胞培养皿中培养的HCT-116细胞用0.1% DMSO（对照）或MLN4924/pevonedistat处理24 h。制备全细胞提取物并进行免疫印迹分析。为了分析E2-UBL的硫酯水平，裂解物用非还原SDS-PAGE分离，并用Ubc12、Ubc9和Ubc10多克隆抗体进行免疫印迹。对于其他蛋白的分析，裂解物通过还原SDS-PAGE进行分离，并用以下一抗进行探针检测：小鼠CDT1、p27、geminin、ubiquitin、securin/PTTG和p53单克隆抗体或兔NRF2、Cyclin B1和GADD34多克隆抗体。兔NEDD8单克隆抗体和磷酸化CHK1 （Ser 317）单克隆抗体由Millennium与Epitomics， Inc.合作，分别以Ac-KEIEIDIEPTDKVERIKERVEE-amide和Ac-VKYSS(pS)QPEPRT-amide作为免疫原制备。pH3、cleaved PARP和cleaved caspase 3抗体来自Cell Signaling Technologies。根据需要使用兔IgG或小鼠IgG 二级酶标抗体。用ECL试剂进行印迹。对于补充图2，二抗为alexa -680标记的兔/小鼠IgG抗体，印迹使用Li-Cor Odyssey红外成像系统成像。

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细胞周期分析[1]
对数生长的HCT-116细胞用MLN4924/pevonedistatt 或DMSO孵育指定时间。收集的细胞在70%乙醇中固定，在4°C下保存过夜。将固定细胞离心去除乙醇，将微球在PBS中碘化丙啶和RNase A中重悬1小时，避光。流式细胞术测定细胞周期分布，用Winlist软件（Verity）分析。

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FACS分析[2]
DNA核含量的测定如前所述积极分裂的h -4- ii - e细胞用MLN4924/pevonedistat 和/或TNF-α处理8小时。在处理结束前，给细胞加10 μM溴脱氧尿苷（Brd-U）。30min后，将细胞固定在乙醇中，用fitc抗brd - u二抗孵育，再用10 μg/ml碘化丙啶（PI）孵育。在FACSCalibur流式细胞仪上检测标记细胞的Brd-U和PI染色。使用FACSDiva （v 6.1.1）分析细胞周期数据。

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siRNA敲低[2]
H-4-II-E细胞转染了非靶向siRNA对照库或单个siGenome siRNA寡核苷酸双链，旨在沉默靶大鼠基因caspase-8和cdt1。细胞稀疏地镀在无抗生素培养基中（96孔板10000个细胞/孔，6孔组织培养板500000个细胞/孔）。第二天，用Lipofectamine RNAiMAX转染细胞25 nM sirna 72 h。转染后，用MLN4924/pevonedistat 和/或TNF-α处理细胞24-48小时。western blotting证实敲除成功。实验中使用的sirna序列包含在补充信息中。

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细胞培养[2]
选择大鼠肝癌H-4-II-E细胞系模拟MLN4924/pevonedistat毒性，因为它常用于毒性化合物的评估。43,44个H-4-II-E细胞从美国型培养收集公司购买，并按照制造商的说明进行培养。简单地说，细胞在添加10%胎牛血清的MEM中培养，在37°C和5% CO2中孵育。常规培养时，细胞中分别添加10 U/l青霉素和10 ug/l链霉素。传代时，细胞用PBS洗涤一次，用0.05%胰蛋白酶- edta处理，补充新鲜培养基，在临床离心机中成粒。

Tumour xenograft efficacy experiments [1]
Female 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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Pharmacodynamic marker analysis [1]
Mice 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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Isolation of bone marrow cells from mice [1]
For 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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In vivo rat model [2]
Ten-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.

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10% cyclodextrin;60 mg/kg;Subcutaneously injection

mice bearing HCT-116 xenografts

[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 that is 7H-pyrrolo[2,3-d]pyrimidine which is substituted by a (1S)-2,3-dihydro-1H-inden-1-ylnitrilo group at position 4 and by a (1S,3S,4S)-3-hydroxy-4-[(sulfamoyloxy)methyl]cyclopentyl group at position 7. It is a potent and selective NEDD8-activating enzyme inhibitor with an IC50 of 4.7 nM, and currently under clinical investigation for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes. It has a role as an apoptosis inducer and an antineoplastic agent. It is a pyrrolopyrimidine, a secondary amino compound, a member of cyclopentanols, a sulfamidate and a member of indanes.
Pevonedistat has been used in trials studying the treatment of Lymphoma, Solid Tumors, Multiple Myeloma, Hodgkin Lymphoma, and Metastatic Melanoma, among others.
Pevonedistat is a small molecule inhibitor of Nedd8 activating enzyme (NAE) with potential antineoplastic activity. Pevonedistat binds to and inhibits NAE, which may result in the inhibition of tumor cell proliferation and survival. NAE activates Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8), an ubiquitin-like (UBL) protein that modifies cellular targets in a pathway that is parallel to but distinct from the ubiquitin-proteasome pathway (UPP). Functioning in diverse regulatory activities, proteins conjugated to UBLs like Nedd8 typically are not targeted for proteasomal degradation.

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Drug Indication
Treatment of acute myeloid leukaemia, Treatment of myelodysplastic syndromes.

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The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.[1]

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Here we have described the initial characterization of MLN4924, a small molecule inhibitor of NAE that represents a new approach to targeting the UPS for the treatment of cancer. MLN4924 completely inhibits detectable NAE pathway function in cells, disrupting the turnover of CRL substrates, with important roles in cell-cycle progression and survival. Our results indicate that inhibition of the NAE pathway disrupts cancer cell protein homeostasis more selectively than the inhibition of proteasome activity, which may contribute to useful differences in clinical efficacy and safety profiles. Sustained NAE pathway inhibition was found to result in the activation of apoptosis as a consequence of cell-cycle-dependent DNA re-replication. This phenotype was presumably a result of the inability of the cell to degrade the CRL substrate CDT1, which has been shown to induce re-replication when overexpressed. Similar cell-cycle profiles were obtained when NAE levels were reduced by RNAi or when NAE activity was compromised in a temperature-sensitive mutant cell line.
In vivo, we demonstrated that MLN4924 suppressed the growth of human tumour xenografts at doses and schedules that were well tolerated. Analysis of tumours from treated animals confirmed inhibition of the NEDD8 pathway, suggesting that these pharmacodynamic markers may have use in monitoring NAE inhibition in patients treated with MLN4924. These preclinical findings have supported the transition of MLN4924 into clinical development. [1]

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Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. 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. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.[2]

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The results of this study demonstrate that the combination of the NAE inhibitor pevonedistat and the pro-inflammatory cytokine TNF-α is toxic. The driver of in vitro toxicity appears to be enhanced cleavage/activation of the caspase-8 p10 protease, which in turn activated apoptosis. However, the molecular mechanism that links pevonedistat to caspase-8 remains unclear in the pevonedistat and TNF-α cytotoxicity model. As cullin-3 can ubiquitinate caspase-8 (Jin et al.36) and is also inhibited by pevonedistat, it was an obvious candidate for investigation, but cullin-3 knockdown did not increase sensitivity to single-agent TNF-α (Supplementary Figure S4). Ultimately, a role for cullin-3 in mediating the synergistic toxicity was not established. Single-agent pevonedistat is known to stabilize the expression of ≥120 different proteins,42 none of which are known to interact with caspase-8. A higher-throughput approach is needed to determine if any unrecognized proteins become stabilized in response to pevonedistat+TNF stimulation. Further investigations using pevonedistat as a tool compound will lead to a better understanding of the molecular mechanisms that underlie programmed cell death.[2]

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MLN4924 is a recently discovered small molecule inhibitor of NEDD8-Activating Enzyme (NAE). Because cullin RING ligase (CRL), the largest family of E3 ubiquitin ligase, requires cullin neddylation for its activity, MLN4924, therefore, acts as an indirect inhibitor of CRL by blocking cullin neddylation. Given that CRLs components are up-regulated, whereas neddylation modification is over-activated in a number of human cancers, MLN4924 was found to be effective in growth suppression of cancer cells. Whether MLN4924 is effective against gastric cancer cells, however, remains elusive. Here we showed that in gastric cancer cells, MLN4924 rapidly inhibited cullin 1 neddylation and remarkably suppressed growth and survival as well as migration in a dose-and time-dependent manner. Mechanistic studies in combination with siRNA knockdown-based rescue experiments revealed that MLN4924 induced the accumulation of a number of CRL substrates, including CDT1/ORC1, p21/p27, and PHLPP1 to trigger DNA damage response and induce growth arrest at the G2/M phase, to induce senescence, as well as autophagy, respectively. MLN4924 also significantly suppressed migration by transcriptionally activating E-cadherin and repressing MMP-9. Taken together, our study suggest that neddylation modification and CRL E3 ligase are attractive gastric cancer targets, and MLN4924 might be further developed as a potent therapeutic agent for the treatment of gastric cancer. [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

140.74

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.21 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.21 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.21 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。