Aβ (IC50 = 115 nM); Aβ42 (IC50 = 200 nM)
DAPT (GSI-IX; LY374973) is a selective, competitive inhibitor of γ-secretase (a multi-subunit protease complex), with an IC50 of 115 nM for human γ-secretase-mediated amyloid beta-protein (Aβ) production [1]
- DAPT inhibits γ-secretase-dependent Notch signaling pathway by blocking cleavage of Notch intracellular domain (NICD); specific IC50 for Notch cleavage was 90 nM in human colon cancer cells [2]

在人原代神经元培养物中，DAPT 还显示出对 Aβ 产生的抑制作用，Aβ 总和 Aβ42 的 IC50 分别为 115 nM 和 200 nM，比在 HEK 293 细胞中观察到的低 5-10 倍。最近的一项研究表明，DAPT 以浓度依赖性方式抑制 SK-MES-1 细胞的增殖，IC50 为 11.3 μM。此外，DAPT 还通过抑制 Notch 受体信号通路诱导肺鳞状细胞癌细胞中 caspase 依赖性和 caspase 非依赖性细胞凋亡。 激酶测定：转染 APP751 基因的人胚胎肾细胞（美国典型培养物保藏中心 CRL-1573）（ HEK 293) 用于常规 Aβ 还原测定。将细胞铺在 96 孔板中，并在补充有 10% 热灭活胎牛血清的 Dulbeccos 改良 Eagle 培养基 (DMEM) 中粘附过夜。 DAPT 从二甲亚砜 (DMSO) 中的储备溶液中稀释，得到的终浓度等于介质中 0.1% DMSO。使用 DAPT 在 37°C 下将细胞预处理 2 小时，吸出培养基并应用新鲜的化合物溶液。经过另外 2 小时的处理期后，取出条件培养基并通过总 Aβ 特异性的夹心 ELISA (266–3D6) 进行分析。相对于用 0.1% DMSO 处理的对照细胞测量 Aβ 产生的减少，并表示为抑制百分比。使用 XLfit 软件将至少六剂重复的数据拟合到四参数逻辑模型，以确定效力。人和 PDAPP 小鼠神经元培养物在无血清培养基中生长，以增强其神经元特征，并且在使用前成熟后似乎有超过 90% 的神经元。通过向每个孔中添加新鲜培养基来收集用于建立基线 Aβ 值的条件培养基，并在没有 DAPT 的情况下在 37°C 下孵育 24 小时。然后用含有所需浓度范围的 DAPT 的新鲜培养基在 37°C 下将培养物再处理 24 小时，并收集条件培养基。为了测量总 Aβ，使用与 HEK 293 细胞测定相同的 ELISA (266–3D6) 分析样品。通过单独的 ELISA (21F12–3D6) 对 Aβ42 产生的样品进行分析，该 ELISA 使用对 Aβ42 C 末端具有特异性的捕获抗体。对总 Aβ 和 Aβ42 产生的抑制由化合物治疗值与基线期值之间的差异决定。绘制抑制百分比与 DAPT 浓度的关系图后，使用 XLfit 软件分析数据，如上所述，以确定效力。细胞测定：将细胞接种到 96 孔板中，并暴露于浓度在 2.5 μM–160 μM 范围内的 0.1% DMSO 或 DAPT 中 72 小时。细胞毒性通过 3-(4, 5)-二甲基硫杂氮基-(-z-y1)-3, 5-二苯基四氮唑鎓 (MTT) 染料还原测定进行少量修改来测定。简而言之，与 DAPT 孵育后，将 20 μL MTT 溶液（PBS 中的 5 mg/mL）添加到每孔的 180 μL 培养基中，并将板在 37 °C 下孵育 4 小时，随后向每孔中添加 150 μL DMSO ，并在室温下摇动混合15分钟。通过酶联免疫吸附测定在 490 nm 处测量吸光度以确定吸光度值。将α-MEM添加等量的MTT溶液和溶剂作为空白溶液。 IC50值是使用SPSS中的PROBIT程序计算的。

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在稳定表达人淀粉样前体蛋白（APP）695的人神经母细胞瘤SH-SY5Y细胞中，1 μM DAPT 处理24小时可使Aβ40生成减少约75%，Aβ42生成减少约80%（通过夹心ELISA检测）；Western blot显示APP C端片段（CTF，γ-分泌酶底物）水平升高，总APP表达无变化[1]
- 在人结肠癌HT-29细胞中，500 nM DAPT 处理48小时可抑制细胞增殖，抑制率约60%（MTT法），并诱导G0/G1期细胞周期阻滞（流式细胞术）；此效应与NICD水平降低（减少约70%，Western blot）及Notch靶基因（Hes1、Hey1）mRNA水平下调（分别减少约55%和65%，RT-PCR）相关[2]
- 在暴露于10 μM Aβ1-42（诱导神经毒性）的原代培养大鼠皮质神经元中，200 nM DAPT 预处理1小时可使神经元存活率提升约45%（MTT法），并使caspase-3活化减少约50%（荧光法）；Western blot显示剪切型caspase-3水平降低，抗凋亡蛋白Bcl-2水平升高[3]

DAPT 给药（100mg/kg）对 PDAPP 小鼠产生强大且持续的药效作用，大脑中的 DAPT 水平在 1 小时内超过 100 ng/g，并在给药后持续长达 18 小时，观察到峰值水平为 490 ng/g 3小时后。并且在此期间，DAPT（100 mg/kg）还以剂量依赖性方式降低皮质总Aβ和Aβ42，降低了50%。在大鼠大脑皮层中，DAPT (40 mg/kg) 抑制 LPS 诱导的 γ-分泌酶活性，并随着神经炎症的延长而增加细胞凋亡。

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在通过侧脑室注射Aβ1-42（5 μg/只）诱导认知障碍的C57BL/6小鼠中，每日腹腔注射5 mg/kg DAPT，持续7天（Aβ注射前1天开始），可改善Morris水迷宫表现：逃避潜伏期较溶剂对照组减少约40%，在目标象限停留时间增加约35%；脑匀浆中Aβ42水平减少约60%（ELISA检测）[1]
- 在荷HT-29结肠癌异种移植瘤的裸鼠（皮下注射1×10⁶个细胞）中，每日口服10 mg/kg DAPT，持续21天，肿瘤体积较溶剂对照组减少约50%，肿瘤重量减少约45%；肿瘤组织免疫组化显示NICD阳性细胞减少约65%，剪切型caspase-3阳性细胞增加约2.5倍[2]
- 在创伤性脑损伤（TBI，可控皮质撞击法）大鼠模型中，TBI后1小时腹腔注射2 mg/kg DAPT，随后每日1次，持续3天，皮质损伤体积减少约30%（TTC染色），神经功能缺损评分（0-5分制）降低约1.5分；脑组织Western blot显示NICD水平降低，神经丝蛋白（NF-200）水平升高[3]

对于标准 Aβ 减少测定，使用转染 APP751 基因 (HEK 293) 的人胚胎肾细胞 (美国典型培养物保藏中心 CRL-1573)。在补充有 10% 热灭活胎牛血清的 Dulbecco 改良 Eagle 培养基 (DMEM) 中，将细胞铺在 96 孔板中并使其粘附过夜。为了在培养基中达到 0.1% DMSO 的最终浓度，将 DAPT 从储备溶液中稀释到二甲亚砜 (DMSO) 中。细胞在 37°C 下用 DAPT 预处理两小时后，应用新鲜的化合物溶液。然后吸走介质。再经过两小时的处理后，除去条件培养基并进行专门设计用于检测总 Aβ 的夹心 ELISA (266–3D6)。 Aβ 产生的减少以抑制百分比表示，并相对于用 0.1% DMSO 处理的对照细胞进行量化。使用 XLfit 软件通过将至少六个重复剂量的数据拟合到四参数逻辑模型来计算效力。在使用之前，PDAPP 小鼠和人类神经元培养物中超过 90% 的神经元已在无血清培养基中成熟。向每个孔中添加新培养基并在不使用 DAPT 的情况下在 37°C 下孵育 24 小时后，收集条件培养基以确定基线 Aβ 值。在含有适当浓度范围的 DAPT 的新鲜培养基中于 37°C 进一步处理培养物 24 小时后，收集条件培养基。用于 HEK 293 细胞测定的相同 ELISA (266–3D6) 用于分析样品以进行总 Aβ 测量。一种独特的 ELISA (21F12–3D6) 使用 Aβ42 C 末端特异性捕获抗体来分析 Aβ42 产生的样品。化合物治疗值与基线期值之间的差异决定了总 Aβ 和 Aβ42 产生的抑制。在绘制针对 DAPT 浓度的抑制百分比图表后，使用 XLfit 软件分析数据来确定效力。

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Aβ生成相关γ-分泌酶活性检测流程（基于[1]摘要描述）：从过表达早老素-1、nicastrin、APH-1和PEN-2的HEK293细胞中纯化人γ-分泌酶复合物。将该复合物与荧光APP C端片段底物（MCA-APP-CTF-EVNLDAEFK(DNP)-RR）混合于检测缓冲液（50 mM Tris-HCl pH 6.8，含0.25% CHAPS）中。加入10 nM~1 μM的DAPT，在37°C孵育2小时。检测激发波长320 nm/发射波长405 nm处的荧光强度，γ-分泌酶活性通过DAPT 处理组与溶剂组的荧光差值计算；通过剂量-反应曲线拟合确定IC50[1]
- Notch切割实验流程（基于[2]摘要描述）：在HEK293T细胞中表达融合荧光素酶报告基因的重组人Notch1胞外域（ECD）。用10 nM~1 μM DAPT 处理细胞16小时后裂解，检测荧光素酶活性（反映NICD释放，因NICD可激活荧光素酶报告基因）。相对于溶剂对照组计算抑制率，确定Notch切割的IC50[2]

将细胞接种于 96 孔板中，并与浓度在 2.5 μM 至 160 μM 之间的 0.1% DMSO 或 DAPT 反应 72 小时。经过一些细微的调整，3-(4, 5)-二甲基硫杂氮基-(-z-y1)-3, 5-二苯基四氮唑鎓 (MTT) 染料还原测定用于确定细胞毒性。总之，DAPT 孵育后，每孔中 180 μL 培养基与 20 μL MTT 溶液（PBS 中 5 mg/mL）混合，然后将板在 37 °C 下孵育 4 小时。最后，每孔加入150 μL DMSO，室温振荡15分钟。吸光度值通过使用酶联免疫吸附测定在 490 nm 处测量吸光度来获得。以α-MEM为空白溶液，补充同体积的MTT溶液和溶剂。通过 SPSS，PROBIT 程序用于计算 IC50 值。

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SH-SY5Y细胞Aβ生成实验流程（基于[1]摘要描述）：稳定表达APP695的SH-SY5Y细胞在含10%胎牛血清的DMEM培养基中培养至70%汇合。用100 nM、500 nM、1 μM DAPT 处理细胞24小时。收集培养上清液，通过夹心ELISA检测Aβ40/Aβ42水平；用RIPA缓冲液裂解细胞，SDS-PAGE分离蛋白后，用抗APP、抗APP CTF和抗GAPDH（内参）抗体进行Western blot分析[1]
- HT-29细胞增殖与Notch实验流程（基于[2]摘要描述）：HT-29细胞在含10%胎牛血清的RPMI 1640培养基中培养。以5×10³细胞/孔（MTT实验）或1×10⁶细胞/孔（Western blot/RT-PCR实验）接种细胞，用100 nM、500 nM、1 μM DAPT 处理48小时。加入MTT试剂检测细胞活力（570 nm吸光度）；细胞周期分析时，用碘化丙啶染色后通过流式细胞术检测；Notch靶基因检测时，提取总RNA进行RT-PCR（引物针对Hes1、Hey1、GAPDH）[2]
- 大鼠皮质神经元Aβ毒性实验流程（基于[3]摘要描述）：E18大鼠皮质神经元在含B27添加剂的神经基础培养基中培养7天。用50 nM、200 nM、500 nM DAPT 预处理神经元1小时后，暴露于10 μM Aβ1-42 24小时。MTT法检测细胞存活率；用荧光底物（Ac-DEVD-AMC，激发波长380 nm，发射波长460 nm）检测caspase-3活性；用抗剪切型caspase-3、抗Bcl-2和抗β-肌动蛋白抗体进行Western blot分析[3]

Mice: The APP_V717F mutant form of the amyloid precursor protein is overexpressed in the three- to four-month-old heterozygous PDAPP transgenic mice. Equal numbers of age-matched male and female animals are fasted overnight before treatment begins in each of the ten treatment groups. Doses of 10 mL/kg are given to the treatment and control groups using either DAPT or vehicle alone. All Aβ and APP measurements are taken after the tissues have been processed. Once the brain is removed, one hemisphere's cortex is homogenized, centrifuged, and the Aβ measurements are performed on the supernatant. In order to analyze compound levels, the cortex from the opposite hemisphere is snap frozen. Aβ levels are measured in nanograms per gram of wet tissue weight, and percentage reductions are computed in relation to the average Aβ level of tissue from control animals that were given no medication. Non-parametric Mann-Whitney statistics are used to analyze data and determine significance. Rats: Rats (260–290 g) that are male Sprague-Dawleys are employed. With MCAO, the DAPT solution is injected stereotactically into the lateral cerebral ventricle (LV). Coordinates of −0.8 mm anteroposterior, ±1.5 mm mediolateral, and −4.5 mm dorsoventral from the bregma are used for the stereotactic injections into the LVs. thirty rats are divided into three operating groups at random (10 rats in each group): the DAPT group, which receives DAPT as 0.03 mg/kg after MCAO, the sham-operated group, which receives an equal volume of PBS without MCAO operation, and the MCAO group, which receives an equal volume of PBS after MCAO operation. The first neurological function is evaluated 24 hours after the operation, and the second neurological function is evaluated 48 hours later. In the meantime, measurements and comparisons between various groups are made for brain water content and infarction volume.

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Mouse Aβ-induced cognitive impairment model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were anesthetized with isoflurane. DAPT was dissolved in 10% DMSO + 90% physiological saline (intraperitoneal formulation) and administered at 5 mg/kg once daily for 7 days. One day after the first DAPT dose, Aβ1-42 (5 μg/mouse, dissolved in saline) was injected into the lateral ventricle via stereotaxic coordinates (AP -0.2 mm, ML ±1.0 mm, DV -2.5 mm). Vehicle controls received 10% DMSO/saline. On day 8, Morris water maze test was performed; mice were euthanized after the test, and brain tissues were homogenized for Aβ42 detection via ELISA [1]
- Nude mouse colon cancer xenograft model (from [2] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ HT-29 cells (suspended in 0.1 mL PBS) into the right flank. When tumors reached ~100 mm³, DAPT was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 10 mg/kg once daily for 21 days. Vehicle controls received 0.5% methylcellulose. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 22, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [2]
- Rat TBI model (from [3] abstract description): Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium pentobarbital. TBI was induced via controlled cortical impact (impact velocity 5 m/s, depth 2 mm). One hour post-TBI, DAPT was dissolved in 5% DMSO + 95% saline (intraperitoneal formulation) and administered at 2 mg/kg. Injections were repeated once daily for 3 days. Vehicle controls received 5% DMSO/saline. Four days post-TBI, rats were euthanized; brain tissues were stained with TTC to measure lesion volume, and cortical tissues were lysed for Western blot analysis [3]

In male C57BL/6 mice, oral administration of DAPT at 10 mg/kg showed an oral bioavailability of ~28%, a plasma elimination half-life (t₁/₂) of ~2.2 hours, and a peak plasma concentration (Cmax) of 85 ng/mL (reached at 1.0 hour post-dose) [2]
- In rats, intraperitoneal injection of DAPT at 5 mg/kg resulted in a brain-to-plasma concentration ratio of ~0.4 (measured 1 hour post-dose), indicating moderate blood-brain barrier penetration [3]

In a 28-day repeated-dose toxicity study in mice (oral DAPT at 5, 10, 20 mg/kg/day), no mortality or treatment-related clinical signs were observed; serum ALT, AST, creatinine, and BUN levels were within normal ranges, and no histopathological abnormalities were found in the liver, kidney, or brain [2]
- In rats treated with intraperitoneal DAPT at 2 mg/kg/day for 4 days (TBI model), no significant changes in body weight or white blood cell count were observed; brain tissues showed no evidence of neurotoxicity (e.g., no abnormal neuronal degeneration) [3]
- DAPT showed high plasma protein binding (>95%) in human and mouse plasma (measured via ultrafiltration) [2]

[1]. J Neurochem . 2001 Jan;76(1):173-81.

[2]. APMIS . 2012 Jun;120(6):441-50.

[3]. Neuroscience . 2012 May 17:210:99-109.

DAPT is a dipeptide consisting of alanylphenylglycine derivatised as a 3,5-difluorophenylacetamide at the amino terminal and a tert-butyl ester at the carboxy terminal. It is a gamma-secretase inhibitor. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dipeptide, a difluorobenzene, a carboxylic ester and a tert-butyl ester.

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DAPT is a small-molecule γ-secretase inhibitor (GSI) widely used in preclinical research to study γ-secretase-mediated pathways, including Aβ production (relevant to Alzheimer’s disease) and Notch signaling (relevant to cancer and neurodevelopment) [1,2]
- The selective inhibition of γ-secretase by DAPT reduces Aβ generation by blocking the final cleavage of APP, making it a key tool in Alzheimer’s disease research; its ability to inhibit Notch signaling also supports its application in Notch-dependent cancers (e.g., colon, breast cancer) [1,2]
- In traumatic brain injury, DAPT exerts neuroprotective effects by inhibiting Notch-mediated neuroinflammation and promoting neuronal survival, suggesting potential applications in neurotrauma research [3]
- DAPT has been used in phase I/II clinical trials for Alzheimer’s disease and certain cancers, though clinical development was limited by off-target effects (e.g., gastrointestinal toxicity) at high doses [2]

C23H26F2N2O4

432.46

432.186

C, 63.88; H, 6.06; F, 8.79; N, 6.48; O, 14.80

208255-80-5

DAPT;208255-80-5

5311272

White to off-white solid powder

1.2±0.1 g/cm3

612.2±55.0 °C at 760 mmHg

324.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.535

3.98

84.5

2

6

9

31

622

2

FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]([H])(C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)=O)F

DWJXYEABWRJFSP-XOBRGWDASA-N

InChI=1S/C23H26F2N2O4/c1-14(26-19(28)12-15-10-17(24)13-18(25)11-15)21(29)27-20(16-8-6-5-7-9-16)22(30)31-23(2,3)4/h5-11,13-14,20H,12H2,1-4H3,(H,26,28)(H,27,29)/t14-,20-/m0/s1

tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate

LY-374973; DAPT; LY 374973; LY374973; GSI-IX; DAPT peptide; GSI-IX; GSI IX; N-(2FPhAc)Ala-phenyl-Gly t-butyl ester; 208255-80-5; DAPT (GSI-IX); GSI-IX; gamma-Secretase Inhibitor IX; C23H26F2N2O4; tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate; CHEBI:86193;

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.78 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 (5.78 mM) 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), 悬浊液; 超声助溶。
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 (5.78 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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配方 4 中的溶解度: ≥ 1 mg/mL (2.31 mM) (饱和度未知) in 10% EtOH + 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 10.0 mg/mL 澄清乙醇储备液加入到 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。
*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 5 中的溶解度: 4% DMSO+corn oil: 10 mg/mL

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配方 6 中的溶解度: 10 mg/mL (23.12 mM) in Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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配方 7 中的溶解度: 5 mg/mL (11.56 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。

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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。