Akt; ERK
TIC10 Analogue (ONC-201 isomer) is a small molecule that dual-inhibits Akt and ERK signaling pathways, which are critical for cell proliferation and survival. In recombinant enzyme assays, it has IC50 values of 3.5 μM for Akt1 and 4.8 μM for ERK2 [1]
- TIC10 is also a selective DRD2 (dopamine D2 receptor) antagonist with a Ki value of 1.2 μM, as determined by radioligand binding assays [24]
- The compound transcriptionally induces TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in a p53-independent manner through Foxo3a nuclear translocation, which is a key mechanism for its antitumor activity [1][4]

In several cancer cell lines, TIC10 increases TRAIL mRNA in a dose-dependent manner and induces TRAIL protein localization on the cell surface in a p53-independent manner. While TIC10 exhibits broad-spectrum anti-tumor activity in vitro and causes TRAIL-sensitive HCT116 p53/p53 cells to exhibit an increase in sub-G1 DNA content indicative of cell death, normal fibroblasts do not experience similar changes in their cell cycle profiles. Cancer cell lines' ability to reproduce clonally is reduced by TIC10 while healthy fibroblasts are unaffected. Similar to TRAIL-mediated apoptosis, TIC10 raises the proportion of sub-G1 DNA in cancer cells in a p53-independent and Bax-dependent manner. Foxo3a is required for TIC10-induced TRAIL up-regulation, and it also up-regulates the TRAIL death receptor DR5, among other targets, which may make some TRAIL-resistant tumor cells susceptible. ERK and Akt kinases are inactivated by TIC10, which causes Foxo3a to move into the nucleus and bind to the TRAIL promoter to activate gene transcription. Foxo3a is then transported into the nucleus. The effective antitumor drug TIC10 works by increasing the levels of the naturally occurring tumor suppressor TRAIL in tumor cells and their surrounding tissue. [1]

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In human colon cancer HCT116 p53^-/- cells, TIC10 (0.5-20 μM) dose-dependently inhibited proliferation with an IC50 of 4.8 μM after 72 hours. At 10 μM, it reduced viable cells by 80% compared to control (MTT assay) [1]
- In glioblastoma (GBM) cell lines (T98G, SF767), TIC10 (5 μM) induced TRAIL expression on the cell surface after 72 hours, with 2.5- to 4-fold increases compared to untreated cells (flow cytometry) [1]
- In HCT116 p53^-/- cells, TIC10 (5-10 μM) increased the sub-G1 population (apoptotic cells) to 35% after 72 hours, compared to 4% in control cells. This effect was blocked by the pan-caspase inhibitor zVAD-fmk (10 μM), confirming caspase-dependent apoptosis [1]
- In freshly resected glioblastoma tissue, TIC10 (10 μM) was more effective than temozolomide (10 μM) in reducing cell viability after 72 hours, with a 55% reduction compared to 30% for temozolomide (viability assay) [1]
- In normal human foreskin fibroblasts (HFF), TIC10 (up to 10 μM) had minimal cytotoxicity, with <15% reduction in viability after 72 hours, demonstrating its selective toxicity toward cancer cells [1]

TIC10 and TRAIL treatment causes tumor regression in the HCT116 p53−/− xenograft to a comparable extent when both are administered as multiple doses. Additionally, TIC10 causes the MDA-MB-231 human triple-negative breast cancer xenografts to regress, whereas the tumors advanced when TRAIL was administered. TIC10 induces tumor stasis in DLD-1 colon cancer xenografts one week after treatment, whereas TRAIL-treated tumors advance after a single dose. The fact that TIC10 is equally effective when administered orally or intraperitoneally and causes a sustained regression of the SW480 xenograft suggests that TIC10 has a good oral bioavailability. Through direct and indirect effects of TRAIL, TIC10 kills tumor-specific cells. Glioblastoma multiforme tumors in humans that are orthotopic can be effectively treated with TIC10. [1]

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In HCT116 p53^-/- xenograft models, a single intraperitoneal dose of TIC10 (100 mg/kg) resulted in a 40% reduction in tumor volume after 7 days, compared to vehicle-treated controls. The antitumor effect was sustained for at least 14 days [1]
- In RKO colon cancer xenografts, a single intraperitoneal dose of TIC10 (100 mg/kg) reduced tumor volume by 50% after 13 days. Near-infrared imaging with AngioSense 680 showed decreased tumor perfusion in treated mice [1]
- In orthotopic SF767 glioblastoma models, a single oral dose of TIC10 (25 mg/kg) significantly prolonged median survival to 45 days, compared to 30 days in vehicle-treated mice (p < 0.05). Combined treatment with TIC10 (25 mg/kg) and bevacizumab (10 mg/kg) further increased median survival to 55 days [1]
- In Eμ-myc transgenic mice (lymphoma model), weekly oral administration of TIC10 (25 mg/kg) for 4 weeks significantly prolonged overall survival by 30% compared to untreated controls. Histopathological analysis showed reduced lymphoma burden in lymph nodes [1]
- In a pediatric diffuse midline glioma model, TIC10 (ONC201) administered at 625 mg every 3 weeks orally achieved a 71% 6-month overall survival rate and 53% 9-month overall survival rate in patients with recurrent glioblastoma [22]

ChIP assays were carried out as previously described for the TRAIL promoter with a ChIP-grade antibody for Foxo3a or an equivalent concentration of rabbit immunoglobulin G as a nonspecific control [1].

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Akt Kinase Assay: Recombinant human Akt1 (0.1 μg/reaction) was incubated with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), 20 μM substrate peptide (RXRXXS/T), and serial dilutions of TIC10 (0.1-10 μM) in a 50 μL reaction volume. After 30 minutes at 30°C, reactions were terminated with 25 μL of 30% trichloroacetic acid. Phosphorylated peptides were captured on P81 phosphocellulose paper, washed with 1% phosphoric acid, and radioactivity was measured by liquid scintillation counting. IC50 values were calculated using four-parameter logistic regression [1]
- ERK2 Kinase Assay: Recombinant human ERK2 (0.2 μg/reaction) was incubated with 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), 5 μM myelin basic protein (substrate), and TIC10 (0.1-10 μM) in a 50 μL reaction volume. After 45 minutes at 30°C, reactions were terminated with SDS sample buffer. Phosphorylated proteins were separated by 12% SDS-PAGE, and radioactivity was detected by autoradiography. IC50 values were determined by dose-response curves [1]
- DRD2 Binding Assay: Membranes from HEK293 cells stably expressing human DRD2 were incubated with [3H]spiperone (0.5 nM), 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and TIC10 (0.01-10 μM) in a 200 μL reaction volume. After 60 minutes at 25°C, bound ligand was separated from free ligand by filtration through GF/B filters. Radioactivity was measured by liquid scintillation counting. Non-specific binding was determined in the presence of 10 μM haloperidol. Ki values were calculated using the Cheng-Prusoff equation [24]

TIC10 causes a dose-dependent increase in TRAIL mRNA and induces TRAIL protein localization on the cell surface of several cancer cell lines in a p53-independent manner. TIC10 has broad-spectrum activity against multiple malignancies in vitro and induces an increase in sub-G1 DNA content suggestive of cell death in TRAIL-sensitive HCT116 p53−/− cells, but does not alter the cell cycle profiles of normal fibroblasts at equivalent doses. TIC10 decreases the clonogenic survival of cancer cell lines and spares normal fibroblasts. TIC10 increases the percentage of sub-G1 DNA in cancer cells in a p53-independent and Bax-dependent manner, as previously reported for TRAIL-mediated apoptosis. TIC10-induced TRAIL up-regulation is Foxo3a-dependent, which also up-regulates TRAIL death receptor DR5 among other targets, potentially allowing for sensitization of some TRAIL-resistant tumor cells. TIC10 inactivates kinases Akt and extracellular signal–regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.

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Cell Proliferation Assay: Cancer cells (5×103 cells/well) were seeded in 96-well plates and treated with TIC10 (0.5-20 μM). After 72 hours, 20 μL of MTT (5 mg/mL) was added, and plates were incubated for 4 hours. Formazan crystals were dissolved in 150 μL of DMSO, and absorbance was measured at 570 nm. IC50 values were calculated as the concentration that reduced absorbance by 50% compared to control wells [1]
- TRAIL Expression Assay: Cells (1×106 cells/mL) were treated with TIC10 (5 μM) for 72 hours. Cells were harvested, washed with PBS, and stained with phycoerythrin-conjugated anti-TRAIL antibody (1:100) for 30 minutes at 4°C. After washing, cells were analyzed by flow cytometry. TRAIL expression was quantified as mean fluorescence intensity relative to untreated control cells [1]
- Apoptosis Assay: Cells (1×106 cells/mL) were treated with TIC10 (5-10 μM) for 72 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's protocol. Apoptotic cells were quantified as the percentage of Annexin V-positive/PI-negative cells by flow cytometry. For caspase inhibition studies, cells were pre-treated with zVAD-fmk (10 μM) for 1 hour before TIC10 treatment [1]
- Glioblastoma Tissue Culture Assay: Freshly resected glioblastoma tissue was minced into 1-2 mm3 pieces and cultured in 24-well plates with RPMI 1640 medium containing 10% FBS. TIC10 (10 μM) or temozolomide (10 μM) was added, and tissue pieces were incubated for 72 hours. Viability was assessed by measuring ATP content using the CellTiter-Glo assay. Results were expressed as percentage of viability relative to vehicle-treated controls [1]

Female athymic nu/nu mice
25, 50, 100 mg/kg
Intraperitoneal/oral

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HCT116 p53^-/- Xenograft Model: Female athymic nude mice (6-8 weeks old, n=10/group) were subcutaneously injected with 2×106 HCT116 p53^-/- cells in 100 μL of PBS into the right flank. When tumors reached 100 mm3, mice were randomized to receive a single intraperitoneal injection of TIC10 (100 mg/kg) or vehicle (5% DMSO in PBS). Tumor volume was measured every 3 days (volume = length × width2 / 2) for 14 days. At day 7, mice were sacrificed, and tumors were harvested for immunohistochemical analysis of TRAIL expression and cleaved caspase-3 [1]
- SF767 Glioblastoma Orthotopic Model: Male athymic nude mice (6-8 weeks old, n=7-8/group) were stereotactically implanted with 5×105 SF767 glioblastoma cells expressing luciferase into the right striatum. Two weeks after implantation, mice received a single oral dose of TIC10 (25 mg/kg), bevacizumab (10 mg/kg, intravenously), or combination treatment. Mice were monitored daily for survival. Bioluminescence imaging was performed weekly to assess tumor burden [1]
- Eμ-myc Lymphoma Model: Eμ-myc transgenic mice (8-10 weeks old, n=5/group) received weekly oral doses of TIC10 (25 mg/kg) for 4 weeks, starting at 9 weeks of age. Mice were monitored twice weekly for survival and weight. At study end, lymph nodes were harvested for histopathological analysis [1]
- Pediatric Diffuse Midline Glioma Clinical Trial: Patients with H3K27M-mutant diffuse midline glioma received TIC10 (ONC201) at 625 mg orally every 3 weeks. Median overall survival was 41.6 weeks, with 71% of patients alive at 6 months and 53% at 9 months. Patients were monitored for adverse events and radiographic responses every 6-8 weeks [22][31]

In patients with advanced solid tumors, a single oral dose of 625 mg of TIC10 (ONC201) achieved a Cmax of 1.5–7.5 μg/mL (3.9–19.4 μmol/L), a mean half-life of 11.3 hours, and a mean AUC of 37.7 h·μg/mL [15]. In pediatric patients with diffuse midline gliomas, oral administration of 625 mg of TIC10 (ONC201) every 3 weeks resulted in a T1/2 of 8.4 hours, a Tmax of 2.1 hours, a Cmax of 2.3 μg/mL, and an AUC0-tlast of 16.4 h·μg/mL [13][19]. According to measurements, TIC10 (ONC201) has a Cmax of 1.5–7.5 μg/mL (3.9–19.4 μmol/L), a mean half-life of 11.3 hours, and a mean AUC0-tlast of 37.7 h·μg/mL [15]. It has a high plasma protein binding rate (>90%) in the human body and is balanced by dialysis [15]
- This compound has good oral bioavailability, reaching peak plasma concentration within 2-3 hours after administration. It can cross the blood-brain barrier and reach therapeutic concentrations in the central nervous system [4][1]
- In mouse tissue distribution studies, TIC10 was found to accumulate in the liver (highest concentration), followed by the spleen, lungs, and brain. The brain concentration was sufficient to inhibit the growth of glioblastoma in an orthotopic model [1]

In normal human cells (PBMC, HFF), TIC10 (at concentrations up to 20 μM) exhibits extremely low cytotoxicity with a CC50 value >15 μM, indicating selective toxicity to cancer cells [1][16]. In animal toxicology studies, the No Adverse Effect Level (NOAEL) was ≥42 mg/kg in dogs and ≥125 mg/kg in rats, which, assuming a standard allometric growth rate, is equivalent to approximately 1.25 g in humans [17]. In a Phase I clinical trial, TIC10 (ONC201) was administered every 3 weeks at doses up to 625 mg and was well tolerated with no Grade 1 or higher adverse events. The most common side effects were mild fatigue, nausea, and transient loss of appetite[15][24]
- In a rat chronic toxicity study, oral administration of TIC10 for 28 days at doses up to 200 mg/kg did not cause significant changes in hematology, clinical chemistry, or major organ histopathology. Mild and reversible decreases in activity and food intake were observed at the highest dose[16]
- In patients with H3K27M mutant glioblastoma, TIC10 (ONC201) treatment was associated with very low neurotoxicity, and no serious adverse events were reported in compassionate use trials[21]

[1].Sci Transl Med. 2013 Feb 6;5(171):171ra17.

The TIC10 analogue (ONC-201 isomer) was the first imipyrone compound initially developed as a TRAIL inducer, but was later found to be a selective DRD2 antagonist [24][4]. The chemical structure of TIC10 was later confirmed to be a angular [3,4-e] isomer of the initially disclosed linear [4,3-d] structure, explaining its unique pharmacological properties [7]. TIC10 induces TRAIL expression through a novel mechanism involving dual inhibition of Akt and ERK, thereby preventing phosphorylation of Foxo3a and causing its nuclear translocation to bind to the TRAIL promoter [1]. This dual inhibition of pro-survival kinases (Akt and ERK) produces a synergistic effect, making TIC10 more effective than a single inhibitor. Two pathways [1] - TIC10 (ONC201) is currently in multiple Phase II clinical trials for various cancers, including glioblastoma, triple-negative breast cancer, non-small cell lung cancer, and colorectal cancer [20][28] - The compound has shown particular efficacy in H3K27M mutant gliomas, which are highly aggressive childhood brain tumors with limited treatment options [25][31] - Unlike traditional chemotherapy drugs, TIC10 does not cause significant myelosuppression or neurotoxicity, making it suitable for long-term administration [16][17]

C24H26N4O

386.49

386.21

C, 74.58; H, 6.78; N, 14.50; O, 4.14

41276-02-2

41276-02-2 (isomer);1616632-77-9;1638178-82-1 (HCl);1777785-71-3 (HBr);2007141-57-1 (2HBr);

336423

White to off-white solid powder

1.2±0.1 g/cm3

559.7±60.0 °C at 760 mmHg

292.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.672

3.19

0

3

4

29

693

0

O=C(C(C1)=C(N2CC3=C(C)C=CC=C3)CCN1CC4=CC=CC=C4)N5C2=NCC5

RSAQARAFWMUYLL-UHFFFAOYSA-N

InChI=1S/C24H26N4O/c1-18-7-5-6-10-20(18)16-28-22-11-13-26(15-19-8-3-2-4-9-19)17-21(22)23(29)27-14-12-25-24(27)28/h2-10H,11-17H2,1H3

TIC10 isomer; TIC 10 isomer; TIC10 isomer; ONC201 isomer; ONC 201 isomer; ONC201 isomer

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: ~11 mg/mL (28.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.29 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 5.0 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: ≥ 0.5 mg/mL (1.29 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 5.0 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: ≥ 0.5 mg/mL (1.29 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 5.0 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.)