DNA-PK (IC50 = 0.28 μM); mTOR (IC50 = 5.3 μM)
Compound 401 is a potent inhibitor of DNA-PK (IC50=0.28 μM). Compound 401 reportedly performs poorly in vitro as an inhibitor of PI3K, ATM, and ATR but is effective against mTOR. mTOR is active against compound 401 (IC50=5.3 μM), but p110/p85 PI3K is not (IC50>100 μM). When cells are exposed to Compound 401, these sites—ribosomal protein S6 kinase 1 Thr389 and Akt Ser473—that have been altered by the mTOR-Rictor and mTOR-Raptor complexes are no longer phosphorylated. However, Akt Thr308 phosphorylation, which is PI3K-dependent, is not directly inhibited. Cells lacking DNA-PK show a similar pattern of behavior. Compound 401 inhibits endogenous or immunoprecipitated epitope-tagged mTOR in Raptor immunoprecipitates. At 5 μM or 10 μM, respectively, of compound 401, inhibition of 67% or 78% is obtained in both cases. Contrarily, dose response curves reveal that Compound 401 exhibits only modest inhibition of the p110α/p85α or p110β/p85α PI3K complexes at these concentrations. TSC1+/+ cells are resistant to Compound 401's proliferative inhibitory effects on TSC1-/- fibroblasts[1].

Compound 401 is a potent inhibitor of DNA-PK (IC50=0.28 μM). Compound 401 reportedly performs poorly in vitro as an inhibitor of PI3K, ATM, and ATR but is effective against mTOR. mTOR is active against compound 401 (IC50=5.3 μM), but p110/p85 PI3K is not (IC50>100 μM). When cells are exposed to Compound 401, these sites—ribosomal protein S6 kinase 1 Thr389 and Akt Ser473—that have been altered by the mTOR-Rictor and mTOR-Raptor complexes are no longer phosphorylated. However, Akt Thr308 phosphorylation, which is PI3K-dependent, is not directly inhibited. Cells lacking DNA-PK show a similar pattern of behavior. Compound 401 inhibits endogenous or immunoprecipitated epitope-tagged mTOR in Raptor immunoprecipitates. At 5 μM or 10 μM, respectively, of compound 401, inhibition of 67% or 78% is obtained in both cases. Contrarily, dose response curves reveal that Compound 401 exhibits only modest inhibition of the p110α/p85α or p110β/p85α PI3K complexes at these concentrations. TSC1+/+ cells are resistant to Compound 401's proliferative inhibitory effects on TSC1-/- fibroblasts[1].

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COMPOUND 401 inhibited immunoprecipitated epitope-tagged mTOR or endogenous mTOR in Raptor immunoprecipitates in vitro. Inhibition of 67% or 78% was obtained at 5 µM or 10 µM COMPOUND 401, respectively[1]
COMPOUND 401 showed poor inhibition of p110α/p85α or p110β/p85α PI3K complexes at concentrations up to 10 µM, unlike LY294002 which was a potent inhibitor[1]
COMPOUND 401 (25 µM) had no effect on the autophosphorylation of the p85α subunit of the p110α/p85α PI3K complex[1]
A selectivity screen against 40 protein kinases showed that most were inhibited less than 20% by 5 µM COMPOUND 401. Notable exceptions included MAPKAPK2 (40% inhibition), Pim1 (44%), and Aurora A (44%)[1]
In serum-starved Rat-1 fibroblasts stimulated with PDGF, pretreatment with COMPOUND 401 (1-10 µM) decreased phosphorylation of S6K1 Thr389 (mTORC1 site) and Akt Ser473 (mTORC2 site) in a dose-dependent manner. At 10 µM, inhibition was nearly complete. It did not inhibit PDGF-induced Erk phosphorylation[1]
COMPOUND 401 (10 µM) inhibited phenylephrine-induced S6K1 Thr389 phosphorylation in Rat-1 cells, a pathway independent of PI3K activity[1]
COMPOUND 401 inhibited insulin-induced phosphorylation of Akt Ser473 but not Thr308 in COS7 cells transfected with Akt phosphorylation site mutants, indicating it does not inhibit PDK1[1]
In M059J glioblastoma cells lacking DNA-PK, COMPOUND 401 (1-10 µM) decreased IGF-1-induced phosphorylation of Akt Ser473 and S6K1 Thr389, confirming its effects are not due to DNA-PK inhibition[1]
Long-term treatment (up to 24 h) of TSC1-/- mouse embryonic fibroblasts (MEFs) with 10 µM COMPOUND 401 blocked S6K1 Thr389 phosphorylation and maintained low levels of Akt Ser473 phosphorylation, unlike rapamycin which increased Akt Ser473 phosphorylation[1]
Proliferation of TSC1-/- MEFs was strongly inhibited by COMPOUND 401 in a dose-dependent manner (half-maximal effect at ~2 µM). A 54% decrease in cell number was observed after 1 day and 84% after 2 days at 10 µM. TSC1+/+ MEFs were resistant[1]
Growth inhibition by COMPOUND 401 was not due to general toxicity (TSC1+/+ MEFs were unaffected) or DNA-PK inhibition (TSC1-/- MEFs were resistant to the DNA-PK inhibitor AMA37)[1]
COMPOUND 401 (10 µM) induced a significant increase in apoptosis (measured by cytoplasmic nucleosome level) in TSC1-/- MEFs after 24 h, whereas rapamycin did not[1]

FreeStyle 293-F cells are transfected with cDNA for AU1-mTOR using 293fectin. The cells are lysed two days later, and mTOR immunoprecipitates are made using AU1 antibody. Raptor antibody can also be used to immunoprecipitate the mTORC1 complex from untransfected cells. As a substrate, glutathione S-transferase (GST)-4E-BP1 expressed by bacteria is used to measure the kinase activity in the immunoprecipitates in the presence of vehicle (DMSO) or Compound 401 (1, 5 and 10 M). Samples are subjected to SDS-PAGE after kinase reactions are stopped by boiling them in SDS sample buffer. Autoradiography can identify phosphorylated 4E-BP1. Scintillation counting is used to measure the radioactivity in the bands[1].

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For mTOR kinase assay, AU1-mTOR was immunoprecipitated from transfected FreeStyle 293-F cells or the mTORC1 complex was immunoprecipitated from untransfected cells using Raptor antibody. Kinase activity in the immunoprecipitates was assayed in the presence of vehicle or compound using bacterially expressed GST-4E-BP1 as a substrate in a reaction containing ATP. Phosphorylated 4E-BP1 was detected by autoradiography and quantified by scintillation counting[1]
For PI3K lipid kinase assay, vehicle or compound was dried in tubes, redissolved in PI3K assay buffer, and mixed with enzyme (p110α/p85α or p110β/p85α complex). The kinase reaction was started by adding sonicated L-α-phosphatidylinositol and a reaction mix containing ATP and [γ-32P]ATP. After incubation, lipids were extracted, separated by thin-layer chromatography, and the radioactive PI 3-phosphate spot was visualized by autoradiography and quantified by scintillation counting[1]
For PI3K autophosphorylation assay, vehicle or compound was dried, redissolved in autophosphorylation buffer, and mixed with p110α/p85α PI3K. The reaction was started by adding ATP and [γ-32P]ATP. After incubation, the reaction was stopped and samples were analyzed by SDS-PAGE and autoradiography to detect phosphorylated p85α[1]

For signaling pathway analysis, cells (Rat-1, COS7, M059J, MEFs) were serum-starved (typically overnight or 20-22 hours) and then preincubated with COMPOUND 401 (dissolved in DMSO) or vehicle (DMSO) for 20 minutes at the indicated concentrations. Cells were then stimulated with the appropriate agonist (PDGF, phenylephrine, insulin, IGF-1) for specified times (5-20 minutes). Cells were rinsed with cold PBS and lysed. Equal amounts of protein from cleared lysates were analyzed by SDS-PAGE and Western blotting using phospho-specific antibodies (e.g., anti-phospho-S6K1 Thr389, anti-phospho-Akt Ser473/Thr308) and total protein antibodies. Signals were detected using HRP-linked secondary antibodies and chemiluminescence[1]
For proliferation assays, TSC1-/- or TSC1+/+ MEFs were plated at a density of 0.5 x 10^5 cells per 6-cm dish. The next day, drugs or vehicle (DMSO) were added. Live cells were counted using trypan blue exclusion 1 or 2 days later[1]
For apoptosis assays, TSC1-/- MEFs were seeded in a 96-well plate (2000 cells/well). Drugs or vehicle were added the next day. After 24 hours, cells were lysed and nucleosomes in the cytoplasmic fraction were quantified using a commercial enzyme-linked immunosorbent assay kit[1]

[1]. Inhibition of mammalian target of rapamycin signaling by 2-(morpholin-1-yl)pyrimido[2,1-alpha]isoquinolin-4-one. J Biol Chem. 2007 Aug 17;282(33):24463-70.

Compound 401 is a synthetic small molecule derived from LY294002, originally designed as a DNA-PK inhibitor, but it has also been found to inhibit mTOR[1]. It is a tool compound used to study mTOR kinase inhibition, avoiding the side effects of PI3K inhibition[1]. Unlike rapamycin, which only inhibits mTORC1, compound 401 can simultaneously inhibit the mTORC1 (responsible for S6K1 phosphorylation) and mTORC2 (responsible for Akt Ser473 phosphorylation) complex[1]. In cells with overactivated mTOR signaling (e.g., TSC1-deficient cells), compound 401 is more effective than rapamycin in inhibiting cell proliferation and inducing apoptosis, possibly because compound 401 does not upregulate survival-promoting Akt activity as rapamycin does. Studies have shown that mTOR kinase inhibitors (such as compound 401) may be more effective than rapamycin analogs in treating proliferative diseases that depend on elevated mTOR activity (e.g., tumors associated with tuberous sclerosis).[1]

C16H15N3O2

281.32

281.116

C, 68.31; H, 5.37; N, 14.94; O, 11.37

168425-64-7

10039361

White to off-white solid powder

1.4±0.1 g/cm3

456.9±55.0 °C at 760 mmHg

214 °C

230.1±31.5 °C

0.0±1.1 mmHg at 25°C

1.695

2.36

0

4

1

21

532

0

O1C([H])([H])C([H])([H])N(C2=C([H])C(N3C([H])=C([H])C4=C([H])C([H])=C([H])C([H])=C4C3=N2)=O)C([H])([H])C1([H])[H]

BVRDQVRQVGRNHG-UHFFFAOYSA-N

InChI=1S/C16H15N3O2/c20-15-11-14(18-7-9-21-10-8-18)17-16-13-4-2-1-3-12(13)5-6-19(15)16/h1-6,11H,7-10H2

2-morpholin-4-ylpyrimido[2,1-a]isoquinolin-4-one

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: 1 mg/mL (3.55 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO 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 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: 1 mg/mL (3.55 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 10.0 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: 1 mg/mL (3.55 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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