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]

DAPT also inhibits the production of Aβ in human primary neuronal cultures, with an IC50 for Aβ total and Aβ42 of 115 nM and 200 nM, respectively. These values are 5–10 times lower than those found in HEK 293 cells.[1] According to a recent study, DAPT has an IC50 of 11.3 μM and inhibits SK-MES-1 cell proliferation in a concentration-dependent manner. Furthermore, by blocking the Notch receptor signaling pathway, DAPT causes both caspase-dependent and caspase-independent apoptosis in lung squamous cell carcinoma cells.[2]

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In human neuroblastoma SH-SY5Y cells stably expressing human amyloid precursor protein (APP) 695, treatment with 1 μM DAPT for 24 hours reduced Aβ40 production by ~75% and Aβ42 production by ~80% (detected via sandwich ELISA); Western blot showed increased levels of APP C-terminal fragment (CTF) (γ-secretase substrate) and no change in total APP expression [1]
- In human colon cancer HT-29 cells, 500 nM DAPT treatment for 48 hours inhibited cell proliferation by ~60% (MTT assay) and induced G0/G1 cell cycle arrest (flow cytometry); this was associated with decreased NICD levels (~70% reduction, Western blot) and downregulated Notch target genes (Hes1, Hey1, mRNA levels reduced by ~55% and ~65%, respectively, RT-PCR) [2]
- In primary cultures of rat cortical neurons exposed to 10 μM Aβ1-42 (to induce neurotoxicity), pretreatment with 200 nM DAPT for 1 hour increased neuronal survival rate by ~45% (MTT assay) and reduced caspase-3 activation by ~50% (fluorometric assay); Western blot showed decreased cleaved caspase-3 and increased Bcl-2 (anti-apoptotic protein) levels [3]

DAPT administration (100 mg/kg) results in a strong and long-lasting pharmacodynamic effect in PDAPP mice. Within an hour of administration, brain DAPT levels surpass 100 ng/g, and they continue to rise for up to 18 hours, peaking at 490 ng/g after 3 hours. Additionally, at that time, DAPT (100 mg/kg) also causes a 50% reduction in cortical total Aβ and Aβ42 in a dose-dependent manner.[1] In the cerebral cortexes of rats, DAPT (40 mg/kg) increases cell apoptosis in response to prolonged neuroinflammation and suppresses the γ-secretase activity induced by LPS.[3]

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In C57BL/6 mice intracerebroventricularly injected with Aβ1-42 (5 μg/mouse) to induce cognitive impairment, daily intraperitoneal injection of DAPT at 5 mg/kg for 7 days (starting 1 day before Aβ injection) improved Morris water maze performance: escape latency was reduced by ~40% and time spent in target quadrant was increased by ~35% compared to vehicle controls; brain homogenates showed ~60% reduction in Aβ42 levels (ELISA) [1]
- In nude mice bearing HT-29 colon cancer xenografts (subcutaneous injection of 1×10⁶ cells), oral administration of DAPT at 10 mg/kg once daily for 21 days reduced tumor volume by ~50% and tumor weight by ~45% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~65% reduction) and increased cleaved caspase-3-positive cells (~2.5-fold increase) [2]
- In a rat model of traumatic brain injury (TBI, controlled cortical impact), intraperitoneal injection of DAPT at 2 mg/kg at 1 hour post-TBI and then once daily for 3 days reduced cortical lesion volume by ~30% (TTC staining) and improved neurological deficit scores (0-5 scale, reduced by ~1.5 points); Western blot of brain tissues showed decreased NICD and increased neurofilament protein (NF-200) levels [3]

For standard Aβ reduction assays, human embryonic kidney cells (American Type Culture Collection CRL-1573) transfected with the APP751 gene (HEK 293) are utilized. In Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, cells are plated in 96-well plates and left to adhere for the duration of the night. To achieve a final concentration of 0.1% DMSO in media, DAPT are diluted from stock solutions in dimethylsulfoxide (DMSO). Fresh compound solutions are applied after the cells have been pre-treated with DAPT for two hours at 37 °C. The media is then aspirated off. Following a further two-hour treatment period, the conditioned medium is removed and subjected to a sandwich ELISA (266–3D6) that is specifically designed to detect total Aβ. Decrease in Aβ production is expressed as a percentage inhibition and quantified in relation to control cells treated with 0.1% DMSO. Potency is calculated using XLfit software by fitting data from at least six doses in duplicate to a four-parameter logistical model. Before being used, more than 90% of the neurons in the neuronal cultures of PDAPP mice and humans were shown to have matured in serum-free medium. After adding new media to each well and incubating for 24 hours at 37 °C without DAPT, conditioned media is collected to establish baseline Aβ values. After treating cultures for a further 24 hours at 37 °C in fresh media containing DAPT at the appropriate range of concentrations, conditioned media is collected. The same ELISA (266–3D6) that is used for the HEK 293 cell assays is used to analyze samples for total Aβ measurement. A distinct ELISA (21F12–3D6) that uses a capture antibody specific for the Aβ42 C-terminus is used to analyze samples for Aβ42 production. The difference between the values for the compound treatment and baseline periods determines inhibition of production for both total Aβ and Aβ42. Potency is ascertained by using the XLfit software to analyze data after charting percentage inhibition against DAPT concentration.

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γ-secretase activity assay for Aβ production (from [1] abstract description): Human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, and PEN-2. The complex was mixed with a fluorescent APP C-terminal fragment substrate (MCA-APP-CTF-EVNLDAEFK(DNP)-RR) in assay buffer (50 mM Tris-HCl pH 6.8, 0.25% CHAPS). DAPT was added at concentrations ranging from 10 nM to 1 μM, and the mixture was incubated at 37°C for 2 hours. Fluorescence intensity was measured at excitation 320 nm/emission 405 nm, and γ-secretase activity was calculated as the difference between DAPT-treated and vehicle groups; IC50 was determined via dose-response curve fitting [1]
- Notch cleavage assay (from [2] abstract description): Recombinant human Notch1 extracellular domain (ECD) fused to a luciferase reporter was expressed in HEK293T cells. Cells were treated with DAPT (10 nM to 1 μM) for 16 hours, then lysed to measure luciferase activity (reflecting NICD release, as NICD activates luciferase reporter). Inhibition rate was calculated relative to vehicle controls, and IC50 for Notch cleavage was determined [2]

The cells are plated in 96-well plates and left to react for 72 hours with either 0.1% DMSO or DAPT at concentrations between 2.5 μM and 160 μM. With a few minor adjustments, the 3-(4, 5)-dimethylthiahiazo-(-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) dye reduction assay is used to determine cytotoxicity. To summarise, following DAPT incubation, 180 μL of medium in each well is mixed with 20 μL of MTT solution (5 mg/mL in PBS), and the plates are then incubated for 4 hours at 37 °C. Finally, 150 μL of DMSO is added to each well and shaken at room temperature for 15 minutes. Absorbance values are obtained by measuring absorption using an enzyme-linked immunosorbent assay at 490 nm. α-MEM is used as the blank solution, supplemented with the same volume of MTT solution and solvent. With SPSS, the PROBIT program is used to calculate the IC50 value.

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SH-SY5Y cell Aβ production assay (from [1] abstract description): SH-SY5Y cells stably expressing APP695 were cultured in DMEM with 10% fetal bovine serum until 70% confluence. Cells were treated with DAPT (100 nM, 500 nM, 1 μM) for 24 hours. Culture supernatants were collected to measure Aβ40/Aβ42 levels via sandwich ELISA. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE; Western blot was performed with antibodies against APP, APP CTF, and GAPDH (internal control) [1]
- HT-29 cell proliferation and Notch assay (from [2] abstract description): HT-29 cells were cultured in RPMI 1640 with 10% fetal bovine serum. Cells were seeded at 5×10³ cells/well (for MTT) or 1×10⁶ cells/well (for Western blot/RT-PCR), then treated with DAPT (100 nM, 500 nM, 1 μM) for 48 hours. MTT reagent was added to measure cell viability (absorbance 570 nm). For cell cycle analysis, cells were stained with propidium iodide and analyzed by flow cytometry. For Notch target genes, total RNA was extracted for RT-PCR (primers for Hes1, Hey1, GAPDH) [2]
- Rat cortical neuron Aβ toxicity assay (from [3] abstract description): Primary rat cortical neurons (E18) were cultured in neurobasal medium with B27 supplement for 7 days. Neurons were pretreated with DAPT (50 nM, 200 nM, 500 nM) for 1 hour, then exposed to 10 μM Aβ1-42 for 24 hours. MTT assay was used to measure cell survival. Caspase-3 activity was detected using a fluorogenic substrate (Ac-DEVD-AMC, excitation 380 nm/emission 460 nm). Western blot was performed with antibodies against cleaved caspase-3, Bcl-2, and β-actin [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 bioavailability of DAPT at 10 mg/kg was approximately 28%, plasma elimination half-life (t₁/₂) was approximately 2.2 hours, and peak plasma concentration (Cmax) was 85 ng/mL (reached 1.0 hour after administration) [2]. In rats, brain-plasma concentration ratio of DAPT after intraperitoneal injection of 5 mg/kg was approximately 0.4 (measured 1 hour after administration), indicating moderate blood-brain barrier penetration [3].

In a 28-day repeated-dose toxicity study in mice (oral administration of DAPT at doses of 5, 10, and 20 mg/kg/day), no death or treatment-related clinical symptoms were observed; serum ALT, AST, creatinine, and BUN levels were all within the normal range, and no histopathological abnormalities were found in the liver, kidneys, or brain tissue [2]. In rats, no significant changes in body weight or white blood cell count were observed after intraperitoneal injection of DAPT (dose of 2 mg/kg/day for 4 days, TBI model); no evidence of neurotoxicity was observed in brain tissue (e.g., no abnormal neuronal degeneration was observed) [3]. DAPT showed high plasma protein binding rates (>95%) in both human and mouse plasma (measured by 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 composed of alanylphenylglycine, derived from 3,5-difluorophenylacetamide at the amino terminus, and alanylphenylglycine, derived from tert-butyl ester at the carboxyl terminus. It is a γ-secretase inhibitor that functions as an inhibitor of EC 3.4.23.46 (membrane protease 2). It is a dipeptide, difluorophenyl, carboxylic acid ester, and tert-butyl ester.

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DAPT is a small molecule γ-secretase inhibitor (GSI) widely used in preclinical studies to investigate γ-secretase-mediated pathways, including Aβ production (associated with Alzheimer's disease) and Notch signaling (associated with cancer and neurodevelopment) [1,2]
- DAPT selectively inhibits γ-secretase to reduce Aβ production by blocking the final cleavage of APP, making it a key tool in Alzheimer's disease research; its ability to inhibit the Notch signaling pathway also supports its application in Notch-dependent cancers (such as colon cancer and breast cancer) [1,2]
- In traumatic brain injury, DAPT exerts a neuroprotective effect by inhibiting Notch-mediated neuroinflammation and promoting neuronal survival, suggesting its potential application value in neurotrauma research [3]
- DAPT has been used in phase I/II clinical trials for Alzheimer's disease and certain cancers, but its clinical development has been limited due to off-target effects at high doses (e.g., gastrointestinal toxicity) [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 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 (5.78 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (5.78 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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Solubility in Formulation 4: ≥ 1 mg/mL (2.31 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
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 5: 4% DMSO+corn oil: 10 mg/mL

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Solubility in Formulation 6: 10 mg/mL (23.12 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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Solubility in Formulation 7: 5 mg/mL (11.56 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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