Background
MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme (NAE) (IC50 = 4 nM) [1,2], 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) [2]. MLN4924 has a multifaceted mechanism of action that is characterized by the induction of DNA re-replication and DNA damage, increased oxidative stress, inhibition of NF-B activity, apoptotic cell death, and cellular senescence [3].
MLN4924 Treatment of HCT-116 cells for 24 h resulted in a dose-dependent decrease of Ubc12-NEDD8 thioester and NEDD8-cullin conjugates, with an IC50 < 0.1 μM [2], resulting in a reciprocal increase in the abundance of the known CRL substrates CDT1, p27 and NRF2 (also known as NFE2L2), c-Jun27, HIF1α, cyclin E29, CDC25A, EMI1 (also known as FBXO5) and phosphorylated IκBα, but not non-CRL substrates [2]. MLN4924 treatment of human-tumour-derived cell lines, including HCT-116(colon), Calu-6 (lung), SKOV-3 (ovarian), H460 (lung), DLD-1 (colon), CWR22 (prostate) and OCI-LY19 (lymphoma), resulted in S-phase-defective phenotypes [2]. Potent inhibition of MLN4924 of cell viability was observed across 17 lymphoma cell lines tested in an ATPlite viability assay, with EC50 values of 10 to 244nM [1].
In OCI-Ly10 xenografts, a dose- and time-dependent increase in pIκBα levels was observed after MLN4924 treatment (10, 30, or 60 mg/kg, subcutaneous), with peak elevation occurring 2 hours after dose and levels returning to baseline at 8 hours after dose. A consequence of inhibiting NAE and NF-κB signaling in OCI-Ly10 xenografts was the induction of apoptosis 8 to 12 hours after a single dose of 60 mg/kg MLN4924, as evidenced by detection of cleaved caspase-3 [1]. 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 MLN4924 (10, 30 or 60 mg/kg, subcutaneous), with maximal effect 1-2 h post-dose on HCT-116 tumour-bearing mice [2].
参考文献:
[1]. Milhollen M A, Traore T, Adams-Duffy J, et al. MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB-dependent lymphoma[J]. Blood, The Journal of the American Society of Hematology, 2010, 116(9): 1515-1523.
[2]. Soucy T A, Smith P G, Milhollen M A, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer[J]. Nature, 2009, 458(7239): 732-736.
[3]. Nawrocki S T, Griffin P, Kelly K R, et al. MLN4924: a novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy[J]. Expert opinion on investigational drugs, 2012, 21(10): 1563-1573.
MLN4924 是一种有效的选择性 NEDD8 激活酶 (NAE) 小分子抑制剂 (IC50 = 4 nM) [1,2]，并且相对于密切相关的酶 UAE、SAE、UBA6 和 ATG7 具有选择性（ IC50 = 1.5、8.2、1.8 和 >；分别为 10 μM）[2]。 MLN4924 具有多方面的作用机制，其特征在于诱导 DNA 再复制和 DNA 损伤、增加氧化应激、抑制 NF-B 活性、凋亡细胞死亡和细胞衰老[3] .
MLN4924 处理 HCT-116 细胞 24 小时导致 Ubc12-NEDD8 硫酯和 NEDD8-cullin 结合物呈剂量依赖性降低，IC50 为 <； 0.1 μM [2]，导致已知 CRL 底物 CDT1、p27 和 NRF2（也称为 NFE2L2）、c-Jun27、HIF1α、细胞周期蛋白 E29、CDC25A、EMI1 的丰度相互增加（也称为 FBXO5）和磷酸化 IκBα，但不是非 CRL 底物[2]。 MLN4924 治疗人肿瘤来源的细胞系，包括 HCT-116（结肠）、Calu-6（肺）、SKOV-3（卵巢）、H460（肺）、DLD-1（结肠）、CWR22（前列腺）和OCI-LY19（淋巴瘤）导致 S 期缺陷表型 [2]。在 ATPlite 活力测定中测试的 17 种淋巴瘤细胞系中观察到 MLN4924 对细胞活力的有效抑制，EC50 值为 10 至 244nM [1]。
在 OCI-Ly10 异种移植物中，在 MLN4924 治疗（10、30 或 60 毫克/千克，皮下注射）后观察到 pIκBα 水平呈剂量和时间依赖性升高，给药后 2 小时出现峰值升高，水平恢复给药后 8 小时达到基线。在 OCI-Ly10 异种移植物中抑制 NAE 和 NF-κB 信号传导的结果是在单剂量 60 mg/kg MLN4924 后 8 至 12 小时诱导细胞凋亡，检测到裂解的 caspase-3 [1] 。早在施用 MLN4924（10、30 或 60 mg/kg，皮下）后 30 分钟，单剂量 MLN4924 就会导致 NEDD8-cullin 水平的剂量和时间依赖性降低，在 1-2 小时后产生最大效果-对携带 HCT-116 肿瘤的小鼠的剂量[2]。
Protocol
| Cell experiment [1]: |
|
Cell lines
|
HCT-116 cells
|
|
Preparation Method
|
HCT-116 cells grown in 6-well cell-culture dishes were treated with 0.1% DMSO (control) or MLN4924 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.
|
|
Reaction Conditions
|
0-3μM for 24 hours
|
|
Applications
|
Treatment of HCT-116 cells with MLN4924 for 24 h resulted in a dose-dependent decrease of Ubc12-NEDD8 thioester and NEDD8-cullin conjugates, with an IC50 < 0.1 μM
|
| Animal experiment [2]: |
|
Animal models
|
Female athymic NCR mice
|
|
Preparation Method
|
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.
|
|
Dosage form
|
subcutaneous injection, once (QD) or twice (BID) daily,30 and 60 mg kg-1
|
|
Applications
|
MLN4924 administered on a BID schedule at 30 and 60 mg kg-1 inhibited tumour growth with T/C values of 0.36 and 0.15, respectively
|
|
参考文献:
[1]: Soucy T A, Smith P G, Milhollen M A, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer[J]. Nature, 2009, 458(7239): 732-736.
|
没有评价数据