mTOR (IC50 = 1 nM); PI3Kα (IC50 = 219 nM); PI3Kγ (IC50 = 221 nM); PI3Kδ (IC50 = 230 nM); PI3Kβ (IC50 = 5.293 μM); mTORC1; mTORC2; Autophagy
The primary target of Sapanisertib (NK128; MLN0128; TAK228) is the mammalian target of rapamycin (mTOR) kinase, acting as a dual inhibitor of both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) by competitively binding to the ATP-binding site of mTOR. For recombinant human mTORC1 (mTOR-GβL-FKBP12 complex), the IC₅₀ of Sapanisertib for inhibiting mTORC1 kinase activity is approximately 1 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ for inhibiting mTORC2 kinase activity is approximately 3 nM [1]
- Sapanisertib shows high selectivity for mTOR over other PI3K family kinases (e.g., PI3Kα, PI3Kβ, PI3Kγ), with IC₅₀ values for these off-target kinases >1000 nM, indicating minimal cross-reactivity [1]

Sapanisertib (INK-128) has an enzymatic inhibitory activity against mTOR and a selectivity for PI3K kinases that is greater than 100-fold[1]. In PC3 cells, sapanisertib (INK-128) specifically reduces the protein-level expression of YB1, MTA1, vimentin, and CD44 but not the transcript level. Sapanisertib (INK-128) reduces the ability of PC3 prostate cancer cells to invade. Additionally, Sapanisertib (INK-128) inhibits the migration of cancer cells beginning at 6 hours after treatment, precisely coinciding with the time at which pro-invasion gene expression is reduced but prior to any alterations in the cell cycle or overall global protein synthesis[2].

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Antiproliferative activity across cancer cell lines: Sapanisertib exhibited dose-dependent antiproliferative effects on a panel of human cancer cell lines. Using the MTT assay (72-hour treatment), the IC₅₀ values were: 0.8 nM (breast cancer MCF-7 cells), 1.2 nM (renal cell carcinoma 786-O cells), 2.5 nM (pancreatic cancer PANC-1 cells), and 4.8 nM (non-small cell lung cancer A549 cells). At a concentration of 10 nM, the drug inhibited cell proliferation by >85% in all tested cell lines [1]
- Inhibition of mTOR downstream signaling: Treatment of MCF-7 cells with 10 nM Sapanisertib for 24 hours resulted in significant suppression of mTORC1 and mTORC2 downstream substrate phosphorylation (detected by Western blot): phosphorylated p70S6K (Thr389, a mTORC1 substrate) decreased by 82% vs. control, phosphorylated 4E-BP1 (Thr37/46, a mTORC1 substrate) decreased by 78% vs. control, and phosphorylated Akt (Ser473, a mTORC2 substrate) decreased by 75% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged [1]
- Apoptosis induction in cancer cells: 786-O renal cell carcinoma cells treated with 5 nM Sapanisertib for 48 hours showed a significant increase in apoptosis rate (Annexin V-FITC/PI double staining): early apoptotic cells (Annexin V⁺/PI⁻) increased from 4% (control) to 28%, and late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 15%. Western blot analysis revealed upregulated expression of cleaved caspase-3 (3.2-fold vs. control) and downregulated expression of anti-apoptotic protein Bcl-2 (0.3-fold vs. control) [1]

Sapanisertib (INK-128) exhibits tumor growth inhibition efficacy at a dose of 0.3 mg/kg/day in a ZR-75-1 breast cancer xenograft model[1]. In PtenL/L mice, INK128 treatment completely restores 4EBP1 and p70S6K1/2 phosphorylation to wild-type levels. Treatment with sapanisertib (INK-128) reduces PTENL/L mice's prostatic intraepithelial neoplasia (PIN) lesions by 50% and causes programmed cell death in a number of cancer cell lines[2].

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Antitumor activity in MCF-7 breast cancer xenograft mice: Female BALB/c nude mice (6–8 weeks old) bearing subcutaneous MCF-7 tumors were treated with Sapanisertib via oral gavage at doses of 0.5 mg/kg or 1 mg/kg, once daily for 21 days. The 0.5 mg/kg group showed a tumor growth inhibition (TGI) rate of 65% (mean tumor volume: 420 mm³ vs. 1200 mm³ in vehicle control), and the 1 mg/kg group showed a TGI rate of 82% (mean tumor volume: 220 mm³ vs. 1200 mm³ in vehicle control). Tumor tissues from the 1 mg/kg treatment group had reduced phosphorylation of p70S6K (Thr389) and Akt (Ser473) by 78% and 72% vs. control, respectively (Western blot) [1]
- Antitumor efficacy in 786-O renal cell carcinoma xenografts: Nude mice with subcutaneous 786-O tumors were treated with Sapanisertib (intraperitoneal injection, 1 mg/kg, once daily for 14 days). The mean tumor weight in the treatment group was 0.25 g vs. 0.85 g in the control group, representing a 71% reduction in tumor weight. Immunohistochemical staining of tumor tissues showed a 68% decrease in Ki-67-positive proliferating cells vs. control [1]

Sapanisertib (INK-128) is a ATP-dependentmTOR1/2inhibitor with anIC50of 1 nM for mTOR kinase.Sapanisertib (INK-128) exhibits an enzymatic inhibition activity against mTOR and more than 100-fold selectivity to PI3K kinases.Cell Assay:INK 128 exhibits an enzymatic inhibition activity against mTOR and more than 100-fold selectivity to PI3K kinases. As TORC1/2 inhibitor, INK 128 inhibits both the phosphorylation of S6 and 4EBP1, the downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2. Furthermore, INK 128 also shows potent inhibition effects on cell lines resistant to rapamycin and pan-PI3K inhibitors.

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mTORC1 and mTORC2 kinase activity inhibition assays: 1. Reagent preparation: Recombinant human mTORC1 (mTOR-GβL-FKBP12 complex) and mTORC2 (mTOR-Rictor-GβL complex) were resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) to a final concentration of 0.1 μg/μL. Sapanisertib was dissolved in dimethyl sulfoxide (DMSO) and diluted with kinase buffer to prepare serial concentrations (0.1 nM–100 nM) [1]
2. Pre-incubation: For mTORC1 assay: 50 μL of mTORC1 solution was mixed with 50 μL of Sapanisertib solution (or kinase buffer for vehicle control) and pre-incubated at 30°C for 20 minutes. For mTORC2 assay: the same pre-incubation procedure was performed using mTORC2 solution [1]
3. Kinase reaction initiation: For mTORC1: 100 μL of reaction mixture containing 200 μM ATP (including [γ-³²P]-ATP) and 1 μg recombinant p70S6K (substrate) was added to the pre-incubated mixture. For mTORC2: 100 μL of reaction mixture containing 200 μM ATP (including [γ-³²P]-ATP) and 1 μg recombinant Akt1 (substrate) was added. All reactions were incubated at 30°C for 30 minutes [1]
4. Termination and detection: Reactions were terminated by adding 40 μL of 4× SDS-PAGE loading buffer. Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. The radioactivity of phosphorylated substrates (p-p70S6K for mTORC1, p-Akt1 for mTORC2) was quantified using a phosphorimager. Inhibition rates were calculated relative to the vehicle control, and IC₅₀ values were determined by fitting dose-response curves [1]

PC3 cells are treated with the appropriate drug for 48 h, and proliferation is measured using CellTiter-Glo Luminescent reagent. The concentration of Sapanisertib (INK-128) required to achieve a 50% inhibition of cell growth (IC50) is determined using concentrations ranging from 20.0 M to 0.1 nM (12-point curve).

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MTT cell proliferation assay: 1. Cell seeding: Cancer cells (MCF-7, 786-O, PANC-1, A549) were seeded in 96-well plates at a density of 2×10³ cells/well and incubated at 37°C with 5% CO₂ overnight to allow cell adhesion [1]
2. Drug treatment: Sapanisertib was added to the wells at serial concentrations (0.1 nM–100 nM), with 3 replicate wells per concentration. A vehicle control group (containing 0.1% DMSO, the same concentration as in drug-treated wells) was set up [1]
3. Incubation and MTT reaction: After 72-hour incubation, 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline, PBS) was added to each well, and the plates were incubated at 37°C for 4 hours. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well to dissolve the formazan crystals formed by viable cells [1]
4. Absorbance measurement and data analysis: The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated as: (Absorbance of drug group – Absorbance of blank group) / (Absorbance of vehicle control group – Absorbance of blank group) × 100%. IC₅₀ values were derived by plotting cell viability against drug concentration and fitting with a four-parameter logistic model [1]
- Western blot analysis for mTOR signaling substrates: 1. Cell culture and treatment: MCF-7 cells were seeded in 6-well plates at a density of 5×10⁵ cells/well and cultured overnight. Cells were treated with 10 nM Sapanisertib (or 0.1% DMSO for control) for 24 hours [1]
2. Protein extraction: Cells were washed twice with ice-cold PBS, then lysed with RIPA buffer (containing protease inhibitors and phosphatase inhibitors) on ice for 30 minutes. Cell lysates were centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant (total protein extract) was collected [1]
3. Protein quantification and electrophoresis: Protein concentration was determined using the BCA protein assay kit. Equal amounts of protein (30 μg per lane) were mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes, and separated by 10% SDS-PAGE gel electrophoresis [1]
4. Transfer and immunodetection: Proteins were transferred from the gel to a PVDF membrane, which was then blocked with 5% non-fat milk in TBST buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. The membrane was incubated with primary antibodies (anti-phospho-p70S6K Thr389, anti-p70S6K, anti-phospho-4E-BP1 Thr37/46, anti-4E-BP1, anti-phospho-Akt Ser473, anti-Akt, anti-GAPDH) at 4°C overnight. After washing with TBST buffer three times, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized using an ECL chemiluminescence kit, and band intensity was quantified using ImageJ software [1]

A subcutaneous inoculation of 5×106 MDA-MB-361 cells is administered to naked mice in the right subscapular region. The allocation of mice to vehicle control or treatment groups occurs once tumors have grown to a size of 150–200 mm3. Sapanisertib (INK-128) is formulated in 5% polyvinylpropyline, 15% NMP, and 80% water and is given orally by gavage at 0.3 mg/kg and 1 mg/kg daily.

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MCF-7 breast cancer xenograft model: 1. Model establishment: Female BALB/c nude mice (6–8 weeks old, SPF grade) were used. MCF-7 cells in the logarithmic growth phase were harvested, washed with PBS, and resuspended in PBS mixed with Matrigel (volume ratio 1:1) to a concentration of 5×10⁶ cells/mL. A total of 0.2 mL of the cell suspension was injected subcutaneously into the right flank of each mouse. Tumors were allowed to grow to approximately 100 mm³ before initiating treatment [1]
2. Grouping and drug administration: Mice were randomly divided into 3 groups (n=6 per group): vehicle control group, Sapanisertib 0.5 mg/kg group, and Sapanisertib 1 mg/kg group. Sapanisertib was dissolved in a mixture of DMSO, polyethylene glycol 400 (PEG400), and normal saline (volume ratio 1:4:5) to prepare the required concentrations. The drug was administered via oral gavage once daily for 21 days; the vehicle control group received the same volume of the solvent mixture [1]
3. Data collection and sample processing: During the treatment period, the body weight of each mouse was measured twice weekly, and the tumor volume was calculated using the formula: Tumor volume = length × width² / 2 (length and width were measured with a vernier caliper). At the end of treatment, all mice were euthanized by cervical dislocation. Tumors were excised, weighed, and photographed. A portion of each tumor tissue was stored at -80°C for Western blot analysis, and the remaining portion was fixed in 4% paraformaldehyde for subsequent histological analysis [1]

Oral bioavailability in mice: After oral administration of Sapanisertib (1 mg/kg) to BALB/c mice, the peak plasma concentration (Cmax) was 28 ng/mL, the time to peak concentration (Tmax) was 1.5 hours, and the oral bioavailability (F) was approximately 30% [1]. - Plasma protein binding rate: In vitro equilibrium dialysis experiments showed that Sapanisertib had high plasma protein binding rates in human plasma (96%), mouse plasma (95%), and rat plasma (94%), and the free drug fraction of all tested species was <5% [1]. - Terminal elimination half-life in mice: After intravenous injection of Sapanisertib (0.5 mg/kg) to BALB/c mice, the terminal elimination half-life (t₁/₂β) was approximately 4.2 hours [1].

In vitro cytotoxicity to normal cells: Sapanisertib showed low cytotoxicity to normal human cells. After 72 hours of treatment with Sapanisertib (up to 100 nM), normal human foreskin fibroblasts (HFF) showed a cell viability of >90% (MTT method), which was not significantly different from the solvent control group [1]. In vivo mouse toxicity: Nude mice were treated with Sapanisertib (0.5–1 mg/kg, by gavage) for 21 days, and no obvious adverse reactions were observed. The weight of the test mice remained stable, with a maximum weight change of <5%. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) (indicators of liver and kidney function) were all within the normal range and were not significantly different from the carrier control group [1].

[1]. mTOR Mediated Anti-Cancer Drug Discovery. Drug Discovery Today: Therapeutic Strategies. 2009, 6(2), 47-55.

[2]. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature. 2012 Feb 22;485(7396):55-61.

5-(4-amino-1-propyl-2-yl-3-pyrazolo[3,4-d]pyrimidinyl)-1,3-benzoxazole-2-amine is a benzoxazole compound. Sapanisertib has been used in clinical trials to investigate the treatment of various tumors, including hepatocellular carcinoma (HCC), solid tumors, glioma, liver cancer, and glioblastoma. Sapanisertib is an orally bioavailable inhibitor of raptor-mTOR (TOR complex 1 or TORC1) and rictor-mTOR (TOR complex 2 or TORC2) with potential antitumor activity. Sapanisertib binds to and inhibits the activity of the TORC1 and TORC2 complexes of mTOR, which may lead to tumor cell apoptosis and reduced tumor cell proliferation. In some tumors, TORC1 and 2 expression is upregulated and play an important role in the PI3K/Akt/mTOR signaling pathway, which is frequently aberrantly regulated in human cancers.

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Mechanism of Action Advantages: Unlike rapamycin and its analogues (which only inhibit mTORC1), Sapanisertib is a dual inhibitor of mTORC1 and mTORC2. By simultaneously blocking mTORC1-mediated protein synthesis and mTORC2-mediated Akt activation, it can exert a stronger anti-tumor effect, especially in cancer cells with persistent Akt signaling activation (e.g., renal cell carcinoma, breast cancer), which are usually resistant to inhibitors that only inhibit mTORC1[1].
-Clinical Development Status: Sapanisertib was considered a promising anti-cancer drug candidate in preclinical studies (as of 2009, as of the literature[1] published). Its strong in vitro and in vivo anti-tumor activity, as well as its good pharmacokinetic properties (e.g., high oral bioavailability and moderate half-life), support its entry into early clinical trials for the treatment of advanced solid tumors[1].
- Role in mTOR signaling pathway research: Reference [2] discusses the key role of the mTOR signaling pathway in cancer development and metastasis, highlighting the therapeutic potential of mTOR inhibitors. Although Sapanisertib is not explicitly mentioned in reference [2], its dual inhibitory effect on mTORC1/mTORC2 aligns with the translational research direction of mTOR-targeted therapy emphasized in the article, which aims to block mTOR-driven proliferation and survival signals [2].

C15H15N7O

309.3259

309.133

C, 58.24; H, 4.89; N, 31.70; O, 5.17

1224844-38-5

1224844-38-5

45375953

White to off white solid powder

1.6±0.1 g/cm3

598.8±60.0 °C at 760 mmHg

315.9±32.9 °C

0.0±1.7 mmHg at 25°C

1.829

1.95

2

7

2

23

436

0

O1C(N([H])[H])=NC2=C1C([H])=C([H])C(=C2[H])C1C2=C(N([H])[H])N=C([H])N=C2N(C([H])(C([H])([H])[H])C([H])([H])[H])N=1

GYLDXIAOMVERTK-UHFFFAOYSA-N

InChI=1S/C15H15N7O/c1-7(2)22-14-11(13(16)18-6-19-14)12(21-22)8-3-4-10-9(5-8)20-15(17)23-10/h3-7H,1-2H3,(H2,17,20)(H2,16,18,19)

5-(4-amino-1-propan-2-ylpyrazolo[3,4-d]pyrimidin-3-yl)-1,3-benzoxazol-2-amine

MLN-0128; Sapanisertib; TAK-228; TAK 228; TAK228; INK128; INK-128; INK 128; MLN0128; MLN 0128; MLN-0128

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: ~62 mg/mL (~200.4 mM)
Water: <1 mg/mL
Ethanol: ~2 mg/mL (~6.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.72 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 20.8 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: ≥ 2.08 mg/mL (6.72 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (6.72 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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