DAPT (GSI-IX; LY374973)

Alias: LY-374973; DAPT; LY 374973; LY374973; GSI-IX; DAPT peptide; GSI-IX; GSI IX; N-(2FPhAc)Ala-phenyl-Gly t-butyl ester
Cat No.:V0714 Purity: ≥98%
DAPT (also known as GSI-IX; LY-374973) is a novel, potent and selective γ-secretase inhibitor used in the study of the Notch signaling pathway and is reported to be able to reduce the levels of beta-amyloid in a mouse model of Alzheimer's disease.
DAPT (GSI-IX; LY374973) Chemical Structure CAS No.: 208255-80-5
Product category: Gamma-secretase
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

DAPT (also known as GSI-IX; LY-374973) is a novel, potent and selective γ-secretase inhibitor used in the study of the Notch signaling pathway and is reported to be able to reduce the levels of beta-amyloid in a mouse model of Alzheimer's disease. Through its indirect inhibition of Notch, a substrate of γ-secretase, DAPT inhibits the production of Aβ in HEK 293 cells, with an IC50 of 20 nM.

Biological Activity I Assay Protocols (From Reference)
Targets
Aβ (IC50 = 115 nM); Aβ42 (IC50 = 200 nM)
ln Vitro
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]
ln Vivo
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]
Enzyme Assay
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.
Cell Assay
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.
Animal Protocol
Mice: The APPV717F 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.
References

[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.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C23H26F2N2O4
Molecular Weight
432.46
Exact Mass
432.19
Elemental Analysis
C, 63.88; H, 6.06; F, 8.79; N, 6.48; O, 14.80
CAS #
208255-80-5
Appearance
Solid powder
SMILES
C[C@@H](C(=O)N[C@@H](C1=CC=CC=C1)C(=O)OC(C)(C)C)NC(=O)CC2=CC(=CC(=C2)F)F
InChi Key
DWJXYEABWRJFSP-XOBRGWDASA-N
InChi Code
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
Chemical Name
tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate
Synonyms
LY-374973; DAPT; LY 374973; LY374973; GSI-IX; DAPT peptide; GSI-IX; GSI IX; N-(2FPhAc)Ala-phenyl-Gly t-butyl ester
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO: ~86 mg/mL (~198.9 mM)
Water: <1 mg/mL
Ethanol: ~50 mg/mL (~115.6 mM)
Solubility (In Vivo)
4% DMSO+corn oil: 10 mg/mL
 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.3124 mL 11.5618 mL 23.1235 mL
5 mM 0.4625 mL 2.3124 mL 4.6247 mL
10 mM 0.2312 mL 1.1562 mL 2.3124 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Clinical Trial Information
NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT03606642 Active
Recruiting
Drug: Dual Antiplatelet
(DAPT) Therapy
Device: The Synergy® stent
Atherosclerosis
Stent Placement
HonorHealth Research Institute November 19, 2018 Phase 2
NCT04135677 Recruiting Drug: DAPT
Drug: Rivaroxaban
Thrombi
Stroke
Shanghai Zhongshan Hospital November 11, 2022 Phase 4
NCT03462498 Active
Recruiting
Drug: 1-months DAPT
Drug: 12-month DAPT
Coronary Artery Disease
Acute Coronary Syndrome
Kyoto University, Graduate
School of Medicine
April 2, 2018 Phase 4
NCT06013020 Recruiting Drug: NOAC+DAPT
Drug: DAPT
STEMI - ST Elevation
Myocardial Infarction
Zunyi Medical College August 28, 2023 Phase 4
NCT02619760 Active
Recruiting
Drug: 1-month DAPT
Drug: 12-month DAPT
Coronary Artery Disease Kyoto University, Graduate
School of Medicine
December 2015 Phase 4
Biological Data
  • DAPT (GSI-IX)

  • DAPT (GSI-IX)

  • DAPT (GSI-IX)

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