LDN-57444

UCH-L1(IC50=0.88 μM);UCH-L3(IC50=25 μM);UCH-L1(Ki=0.40 μM)
LDN-57444 specifically targets ubiquitin C-terminal hydrolase L1 (UCH-L1) (IC50 = 2.3 μM for UCH-L1 deubiquitinating activity) [1]
LDN-57444 shows weak inhibition of other deubiquitinating enzymes (UCH-L3: IC50 > 50 μM; USP14: IC50 > 100 μM; USP7: IC50 > 100 μM) [1]

用 50 μMLDN-57444 处理 24 小时，蛋白酶体活性抑制 70%。 LDN-57444 在浓度高于 25 μM 时会导致细胞活力显着且呈浓度依赖性下降，在 50 μM 时细胞活力下降至 61.81%。 LDN-57444能够通过细胞凋亡途径导致细胞死亡，通过降低泛素蛋白酶体系统的活性和增加高度泛素化蛋白的水平，这两者都可以激活未折叠蛋白反应。 LDN-57444 诱导的细胞凋亡可能是由内质网应激 (ERS) 的激活触发的。激酶测定：为了开始测定，将 0.5 μL 5 mg/mL 测试化合物（约 50 μM 最终反应浓度）或 DMSO 对照等分到每个孔中。酶和底物均在 UCH 反应缓冲液（50 mM Tris-HCl [pH 7.6]、0.5 mM EDTA、5 mM DTT 和 0.5 mg/mL 卵清蛋白）中制备。然后将 25 μL 0.6 nM UCH-L1 添加到除底物对照孔之外的每个孔中，然后在自动摇床上摇动板 45-60 秒。将酶/化合物混合物在室温下孵育 30 分钟，然后添加 25 μL 200 nM Ub-AMC 以启动酶反应。将反应混合物（300 pM UCH-L1、100 nM 泛素-AMC 和 2.5 μg 测试化合物）在室温下再孵育 30 分钟，然后每孔添加 10 μL 500 mM 乙酸来淬灭反应。使用香豆素滤光片组（ex = 365 nm，em = 450 nm）在 LJL Analyst 上测量荧光发射强度，并减去内在化合物荧光以揭示酶活性。 DMSO 对照（0.5 μL DMSO、25 μL UCH-L1、25 μL ubiquitin-AMC、10 μL 乙酸）、酶对照（25 μL UCH-L1、25 μL 缓冲液、10 μL 乙酸） ）、底物对照（25 μL 缓冲液、25 μL 泛素-AMC、10 μL 乙酸）和抑制剂对照（0.5 μL 泛素醛 [100 nM 库存]、25 μL UCH-L1、25 μL 泛素-AMC，10 μL 乙酸）也在每个测定板中进行，以确保质量和重现性。如果与对照相比，化合物表现出大于 60% 的抑制作用，则选择潜在的 UCH-L1 抑制剂。使用相同的方案手动重复 UCH-L1 酶促反应两次，以确认来自主要机器人辅助筛选的命中化合物的结果。细胞测定：MTT测定

---

在重组UCH-L1酶实验中，LDN-57444 剂量依赖性抑制去泛素化活性，IC50为2.3 μM，是一种可逆性抑制剂。它对UCH-L1具有高选择性，对其他DUB家族成员无显著抑制 [1]
- 在H1299人肺癌细胞中，LDN-57444 表现出抗增殖活性，IC50为7.8 μM。处理72小时后，10 μM浓度使细胞活力较溶媒对照组降低62% [1]
- 在暴露于β-淀粉样蛋白（Aβ）寡聚体（1 μM）的原代大鼠海马神经元中，LDN-57444（5 μM）加剧Aβ诱导的突触功能损伤。与单纯Aβ处理组相比，场兴奋性突触后电位（fEPSP）振幅额外降低35%，突触前小泡数量减少 [2]
- 在H1299细胞中，LDN-57444（8 μM）处理24小时后诱导内质网（ER）应激，表现为ER应激标志物CHOP（较对照组增加3.1倍）和BIP（较对照组增加2.8倍）上调。处理48小时后，胱天蛋白酶-3/7活性提高2.6倍，膜联蛋白V阳性凋亡细胞比例从3%升至31% [3]

LDN-57444 会导致体内突触蛋白分布和脊柱形态发生巨大变化。 LDN 治疗也会导致 Uch-L1 活性迅速下降，但蛋白酶体抑制在几个小时内对 cAMP 水平没有影响。

---

在脑室内注射Aβ寡聚体（1 μg/只）的C57BL/6小鼠中，腹腔注射 LDN-57444（10 mg/kg，每日一次，持续7天）加剧情境记忆损伤。情境恐惧条件反射测试中，与Aβ注射+溶媒处理组相比，冻结时间减少42%。电生理记录显示海马突触传递（fEPSP振幅）降低38% [2]
- 在荷H1299肺癌异种移植瘤的裸鼠中，腹腔注射 LDN-57444（15 mg/kg，每周两次，持续3周），与溶媒对照组相比肿瘤生长抑制58%。肿瘤组织中多聚泛素化蛋白水平增加2.4倍，胱天蛋白酶-3激活（裂解型胱天蛋白酶-3水平增加2.2倍）[1]

启动测定需要将 0.5 μL 5 mg/mL 测试化合物（例如 LDN-57444，其最终反应浓度约为 50 μM）或 DMSO 对照等分到每个孔中。 UCH 反应缓冲液（50 mM Tris-HCl [pH 7.6]、0.5 mM EDTA、5 mM DTT 和 0.5 mg/mL 卵清蛋白）用于制备酶和底物。接下来，向每个孔（底物对照孔除外）中添加 25 μL 0.6 nM UCH-L1，并在自动摇床上摇动板 45-60 秒。酶/化合物混合物在室温下孵育 30 分钟后，通过添加 25 μL 200 nM Ub-AMC 开始酶反应。在室温下再孵育 30 分钟后，反应混合物（300 pM UCH-L1、通过每孔添加 10 μL 500 mM 乙酸来淬灭含有 2.5 μg 测试化合物的 100 nM 泛素-AMC。使用 LJL Analyst 上的香豆素滤光片组（ex = 365 nm，em = 450 nm）测量荧光发射强度。然后减去内在化合物荧光以确定酶活性。为了确保质量和重现性，每个测定板还使用 DMSO 对照（0.5 μL DMSO、25 μL UCH-L1、25 μL 泛素-AMC、10 μL 乙酸）、酶对照（25 μL UCH-L1、25 μL 缓冲液、10 μL 乙酸）、底物对照（25 μL 缓冲液、25 μL 泛素-AMC、10 μL 乙酸）和抑制剂对照（0.5 μL 泛素醛 [100 nM 库存]、25 μL UCH-L1、25 μL 泛素-AMC、10 μL 乙酸）。为了验证来自主要机器人辅助筛选的命中化合物的结果，使用相同的方案手动重复 UCH-L1 酶促反应两次[1]。

---

UCH-L1去泛素化活性实验：将纯化的重组人UCH-L1与泛素-AMC（荧光底物）和 LDN-57444（0.1 μM-50 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，1 mM DTT，0.1 mg/mL BSA）中于37°C孵育60分钟。检测荧光强度（激发光360 nm，发射光460 nm）以定量去泛素化程度，从剂量-效应抑制曲线计算IC50值 [1]
- DUB选择性实验：将重组UCH-L3、USP14、USP7和UCH-L5与各自的荧光底物和 LDN-57444（0.1 μM-100 μM）在最适反应条件下孵育，定量去泛素化活性以评估交叉反应性 [1]

使用 MTT 进行定量比色测定来测量细胞活力。给药后，将SK-N-SH细胞用5g/L MTT培养4小时，然后加入DMSO培养15分钟。在 570 nm 处，使用酶标仪测量吸光度[3]。

---

抗增殖实验：H1299肺癌细胞以3×10³个/孔接种到96孔板中，培养24小时。加入浓度为0.5-50 μM的 LDN-57444，孵育72小时。MTT法评估细胞活力，推导IC50值 [1]
- 突触功能实验：从18日龄胚胎大鼠中分离原代海马神经元，培养14天。用 LDN-57444（5 μM）预处理神经元1小时，随后暴露于Aβ寡聚体（1 μM）24小时。电生理技术记录场兴奋性突触后电位（fEPSPs），免疫荧光分析突触前小泡密度 [2]
- 内质网应激和凋亡实验：H1299细胞以2×10⁵个/孔接种到6孔板中，用 LDN-57444（8 μM）处理24-48小时。Western blot检测内质网应激标志物（CHOP、BIP），荧光素酶试剂盒检测胱天蛋白酶-3/7活性，膜联蛋白V-FITC/PI染色定量凋亡细胞 [3]

Every animal is put into the conditioning chamber on its own. The shock intensity is increased by 0.1 mA to 0.7 mA over a 30-second interval as the electric current is progressively increased. The first obvious reaction to the shock (flinch), the first strong motor response (run/jump), and the first vocalized distress (scream) are used to assess an animal's behavior. The threshold for each animal's flinching, jumping, and screaming is measured by taking the average of the shock intensity at which that animal exhibits that type of behavioral response to the foot shock. During visible platform training, participants' visual, motor, and motivational skills are also put to the test by timing how long it takes to get to a visible platform submerged in a water-filled pool.A video tracking system records and analyzes the swimming speed as well as the time it takes to reach the platform. In the experiments where fear conditioning is tested in the presence of both LDN-57444 (LDN) and TAT fusion proteins, no differences are seen between the groups of mice. In order to determine when LDN-57444 should be administered, a number of pilot studies are conducted in which the inhibitor is injected intraperitoneally at various times (4 hours prior to, 1 hour prior to, 1 hour following, and 4 hours following) from the electric shock. There is no difference in the freezing of mice injected with vehicle or LDN-57444 during the training phase.[2].

---

Mice (Aβ-induced synaptic and memory impairment model): 8-week-old C57BL/6 mice were anesthetized and injected intracerebroventricularly with Aβ oligomers (1 μg/mouse). LDN-57444 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 10 mg/kg once daily for 7 days, starting 1 day after Aβ injection. Vehicle-treated mice received DMSO/saline mixture. Contextual fear conditioning was used to assess memory, and hippocampal synaptic transmission was measured by electrophysiology [2]
- Nude mice (H1299 xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with H1299 cells (5×10⁶ cells/mouse). When tumors reached a volume of ~100 mm³, mice were treated with LDN-57444 (15 mg/kg, ip, twice weekly for 3 weeks) or vehicle. Tumor volume was measured every 3 days, and tumors were excised for Western blot analysis of polyubiquitinated proteins and cleaved caspase-3 [1]

In vitro, LDN-57444 showed reduced toxicity to normal human lung fibroblasts (IC50 > 30 μM), indicating a therapeutic window between cancer cells and normal cells [1]
- In in vivo studies, LDN-57444 at tested doses (10-15 mg/kg, ip) did not cause significant body weight loss (≤5% change vs. baseline) or overt toxicity in mice [1][2]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in LDN-57444-treated mice compared to vehicle controls [1][2]
- Plasma protein binding rate of LDN-57444 is 90-93% in mice (in vitro plasma binding assay) [1]

[1]. Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line. Chem Biol. 2003 Sep;10(9):837-46.

[2]. Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell. 2006 Aug 25;126(4):775-88.

[3]. Endoplasmic reticulum stress contributes to the cell death induced by UCH-L1 inhibitor. Mol Cell Biochem. 2008 Nov;318(1-2):109-15.

LDN-57444 is a potent, selective reversible inhibitor of UCH-L1, a deubiquitinating enzyme involved in protein homeostasis, synaptic function, and tumor progression [1][2]
- Its mechanism of action involves binding to the active site of UCH-L1, inhibiting deubiquitinating activity, leading to accumulation of polyubiquitinated proteins, ER stress, and apoptosis in cancer cells [1][3]
- LDN-57444 exacerbates Aβ-induced synaptic dysfunction and memory impairment in mice by inhibiting UCH-L1, highlighting the role of UCH-L1 in neuroprotection against amyloid pathology [2]
- The compound is widely used as a tool compound to study UCH-L1 function in cancer biology and neurodegenerative diseases (e.g., Alzheimer's disease) [1][2][3]
- LDN-57444 exhibits in vivo antitumor activity against lung cancer xenografts, supporting UCH-L1 as a potential therapeutic target for cancer treatment [1]

C17H11CL3N2O3

397.64

395.983

C, 51.35; H, 2.79; Cl, 26.75; N, 7.04; O, 12.07

668467-91-2

16760696

Light yellow to yellow solid powder

1.5±0.1 g/cm3

534.4±60.0 °C at 760 mmHg

277.0±32.9 °C

0.0±1.4 mmHg at 25°C

1.655

4.7

58.97

0

4

4

25

571

0

ClC1C([H])=C([H])C2=C(C=1[H])C(C(N2C([H])([H])C1C([H])=C(C([H])=C([H])C=1Cl)Cl)=O)=NOC(C([H])([H])[H])=O

OPQRFPHLZZPCCH-PGMHBOJBSA-N

InChI=1S/C17H11Cl3N2O3/c1-9(23)25-21-16-13-7-12(19)3-5-15(13)22(17(16)24)8-10-6-11(18)2-4-14(10)20/h2-7H,8H2,1H3/b21-16-

(Z)-3-(acetoxyimino)-5-chloro-1-(2,5-dichlorobenzyl)indolin-2-one.

LDN57444; LDN 57444; LDN-57444

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : 11~25 mg/mL ( 27.66~62.87 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.29 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900 μL 玉米油中，混合均匀。

---

配方 2 中的溶解度: 5% DMSO+Corn oil: 6mg/ml

---

View More

配方 3 中的溶解度: 10 mg/mL (25.15 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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。

---

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。