Akt; ERK; Human mitochondrial caseinolytic protease P (ClpP) was identified as a direct binding protein. ONC201 bound ClpP with approximately 10-fold lower affinity compared to TR compounds in competition assays. [2]
Human mitochondrial caseinolytic peptidase P (ClpP) was identified as a direct binding protein for Dordaviprone (ONC201) using affinity chromatography and mass spectrometry. [2] In vitro competition assays showed that Dordaviprone (ONC201) reduced ClpP binding to TR-81 beads in a dose-dependent manner, with approximately 10-fold lower potency compared to TR compounds. [2]
Additional indirect targets: Dordaviprone (TIC10) inhibits Akt and ERK activity, leading to Foxo3a nuclear translocation and TRAIL gene induction. [1]

In several cancer cell lines, TIC10 induces TRAIL protein localization on the cell surface in a p53-independent manner and increases TRAIL mRNA in a dose-dependent 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 are unaffected by TIC10 at equivalent doses. TIC10 spares healthy fibroblasts while reducing the clonogenic survival of cancer cell lines. Similar to TRAIL-mediated apoptosis, TIC10 increases the proportion of sub-G1 DNA in cancer cells in a p53-independent and Bax-dependent manner. The up-regulation of TRAIL caused by TIC10 is dependent on Foxo3a, which also up-regulates the TRAIL death receptor DR5 and other targets, possibly making 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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- ONC201 inhibited cell proliferation in triple-negative breast cancer cells SUM159 and MDA-MB-231 with IC50 values of 2.96 μM (SUM159) and 4.20 μM (MDA-MB-231) after 72 h treatment (MTS assay). [2]
- ONC201 induced the integrated stress response (ISR) markers ATF4 and CHOP in SUM159 and MDA-MB-231 cells in a dose- and time-dependent manner. The concentration required to increase ATF4 closely reflected the IC50 for growth inhibition. CHOP induction peaked earlier than ATF4. [2]
- ONC201 (10 μM, 24 h) increased CHOP protein level in wild-type SUM159 cells, but this effect was abolished in ClpP knockdown cells. [2]
- ONC201 (10 μM, 24 h) reduced mitochondrial proteins TFAM and TUFM in SUM159 cells; this reduction was prevented by ClpP knockdown. [2]
- ONC201 significantly inhibited total protein synthesis (>50%) after 24 h treatment in SUM159 cells as measured by 35S-methionine/cysteine incorporation; this inhibition was strongly attenuated by ClpP knockdown. [2]
- ONC201 (up to 30 μM) did not significantly increase caspase 3/7 activity (using fluorogenic ACE-DEVD-AMC substrate) nor PARP cleavage in SUM159 cells, indicating cytostatic rather than apoptotic effects under the tested conditions. Staurosporine (10 nM) served as positive control for caspase activation. [2]
- ONC201 (300 nM, 3 μM, 30 μM for 24 h) did not induce significant caspase activity in SUM159 cells. [2]
- Cell counting experiments confirmed that ONC201 treatment did not reduce total cell number below initial seeding value even after 72 h incubation, consistent with cytostatic effects. [2]

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Dordaviprone (TIC10) increased TRAIL mRNA in HCT116 p53-/- cells as measured by qRT-PCR (48 h, n=4, P<0.05). [1] Dordaviprone induced surface TRAIL protein in a panel of cancer cell lines including HCT116, RKO, SW480, DLD-1, and MDA-MB-231 at 10 μM, 72 h (n=3). [1] Surface TRAIL was sustained even after drug removal; cells pre-incubated with 5 μM Dordaviprone for 48 h then changed to drug-free medium maintained elevated TRAIL at 72 h (P<0.05). [1] Dordaviprone (5 μM, 72 h) increased sub-G1 DNA content in HCT116 p53-/- cells (indicating apoptosis) but did not alter cell cycle profiles of normal human foreskin fibroblasts (HFF). [1] Colony formation assays showed that Dordaviprone (10 μM, 72 h) decreased clonogenic survival of HCT116, RKO, SW480, and DLD-1 cancer cells but spared HFF cells. [1] The sub-G1 increase induced by Dordaviprone (1, 5, 10 μM, 72 h) was Bax-dependent and p53-independent; Bax-/- cells showed reduced response compared to wild-type and p53-/- cells. [1] Dordaviprone activated caspase-3 as shown by immunofluorescence and Western blot (1, 2.5, 5, 10 μM, 72 h). [1] The pan-caspase inhibitor zVAD-fmk (10 μM) significantly inhibited Dordaviprone-induced sub-G1 accumulation. [1] Stable knockdown of TRAIL by shRNA in MDA-MB-231 cells significantly reduced Dordaviprone-induced sub-G1 DNA and cytotoxicity (1, 5, 10 μM, 72 h). [1] Disruption of the DR5 death domain (overexpression of DR5 lacking death domain) inhibited Dordaviprone-induced sub-G1 increase in H460 cells (10 μM, 72 h). [1] A TRAIL-blocking antibody (RIK-2) reduced Dordaviprone cytotoxicity. [1] Dordaviprone (5 μM) was thermally stable; preincubation at 95°C for 1 h did not reduce its ability to decrease cell viability, unlike recombinant TRAIL. [1] In freshly resected primary human colon tumor cells (mucinous adenocarcinoma), Dordaviprone induced TRAIL and potent cytotoxicity, whereas 5-fluorouracil was ineffective. [1] In glioblastoma multiforme (GBM) cell lines (U87, U251, T98G, SF767, SF295, LN18), Dordaviprone (5 μM, 72 h) induced surface TRAIL, with GI50 values in the low micromolar range (p53-independent). [1] Dordaviprone showed substantial cytotoxicity against freshly resected primary human GBM cells that were temozolomide-resistant and previously irradiated. [1] Dordaviprone induced Foxo3a nuclear translocation as shown by immunofluorescence and subcellular fractionation (10 μM, 48 h) in HCT116 cells. [1] Chromatin immunoprecipitation (ChIP) assay demonstrated that Dordaviprone increased Foxo3a binding to the TRAIL promoter in a dose-dependent manner (2.5, 5, 10 μM, 48 h). [1] Transient knockdown of Foxo3a (but not Foxo1) by siRNA blocked Dordaviprone-induced surface TRAIL up-regulation. [1] Stable knockdown of Foxo3a significantly inhibited Dordaviprone-induced TRAIL production and tumor cell death. [1] Dordaviprone down-regulated pAkt and pERK in a dose-dependent manner (2.5, 5, 10 μM, 72 h) and dephosphorylated Foxo3a at sites phosphorylated by Akt and ERK; total ERK expression also decreased. [1] Time-course analysis showed that Dordaviprone-induced inactivation of Akt and ERK occurred after 48 h, coincident with Foxo3a dephosphorylation and TRAIL up-regulation. [1] The Akt inhibitor A6730 and MEK inhibitor U0126 monothanolate cooperatively induced Foxo3a-dependent TRAIL up-regulation and TRAIL-mediated cell death, mimicking Dordaviprone effects. [1] siRNA knockdown of Akt and ERK cooperatively up-regulated TRAIL gene expression. [1] Dordaviprone did not affect EGFR, B-Raf, or C-Raf phosphorylation or expression, but abolished MEK and ERK phosphorylation even after washout; this was not affected by okadaic acid, suggesting indirect inhibition. [1]
In SUM159 triple-negative breast cancer cells, Dordaviprone (ONC201) inhibited cell growth with an IC50 approximately 100-fold higher than TR compounds (exact value not specified). [2] Dordaviprone (ONC201) induced the integrated stress response proteins ATF4 and CHOP in a dose- and time-dependent manner (24-48 h) in SUM159 and MDA-MB-231 cells. [2] No significant increase in caspase-3/7 activity or PARP cleavage was detected in SUM159 cells treated with Dordaviprone up to 30 μM for 24 h, as measured by fluorogenic substrate; staurosporine (10 nM) served as positive control. [2] Cell counting experiments showed that Dordaviprone (ONC201) did not reduce total cell number below initial plating after 72 h, indicating cytostatic rather than cytotoxic effects. [2] Dordaviprone (10 μM, 24 h) reduced mitochondrial proteins TFAM and TUFM in SUM159 cells as measured by immunoblotting. [2] Dordaviprone (10 μM, 24 h) inhibited total protein synthesis as measured by 35S-methionine/cysteine incorporation (>50% inhibition). [2]

TIC10 and TRAIL treatment causes tumor regression in the HCT116 p53−/− xenograft to a comparable extent when both are administered as multiple doses. TIC10 also induces regression of MDA-MB-231 human triple-negative breast cancer xenografts, whereas TRAIL-treated tumors progressed. In DLD-1 colon cancer xenografts, TIC10 induces tumor stasis one week after treatment, whereas TRAIL-treated tumors advance after a single dose. The SW480 xenograft also exhibits a sustained regression after receiving a single dose of TIC10, and this effect is seen whether the drug is administered orally or intraperitoneally. This suggests that TIC10 has a favorable oral bioavailability. TIC10 causes tumor-specific cell death by TRAIL-mediated direct and bystander effects. TIC10 is an effective antitumor agent against orthotopic human glioblastoma multiforme tumors. [1]

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In HCT116 p53-/- xenograft-bearing mice, Dordaviprone (TIC10) administered intraperitoneally (ip) as three doses on days 0, 3, and 6 caused tumor regression comparable to recombinant TRAIL (Fig 3A, n=10). [1] A single intraperitoneal dose of Dordaviprone (100 mg/kg) induced regression of HCT116 p53-/- xenografts as measured by bioluminescence imaging (Fig 3B, n=6). [1] Single-dose Dordaviprone (ip) induced tumor regression in RKO human colon cancer xenografts, whereas TRAIL-treated tumors progressed (Fig 3C, n=10). [1] In MDA-MB-231 triple-negative breast cancer xenografts, a single ip dose of Dordaviprone (100 mg/kg) induced tumor regression, while TRAIL-treated tumors progressed; stable knockdown of TRAIL significantly inhibited the antitumor effect of Dordaviprone (P<0.005, Fig 3D, n=8). [1] In DLD-1 colon cancer xenografts, a single dose of Dordaviprone induced tumor stasis at 1 week, whereas TRAIL-treated tumors progressed. [1] A single dose of Dordaviprone (100 mg/kg) induced sustained regression of SW480 xenografts, and was equally effective when delivered by intraperitoneal or oral route, indicating oral bioavailability. [1] Titration of a single oral dose of Dordaviprone in HCT116 xenografts revealed sustained antitumor efficacy at 25 mg/kg (Fig 3E, n=6). [1] In Eu-myc transgenic mice (spontaneous metastatic lymphoma), weekly oral Dordaviprone at 25 mg/kg for 4 weeks significantly prolonged survival by 4 weeks (P=0.00789, log-rank test, Fig 3F). [1] Dordaviprone combined with paclitaxel or docetaxel yielded sustained cures for 10 days in H460 non-small cell lung cancer xenografts. [1] Dordaviprone combined with bevacizumab (once weekly) reduced primary cecal tumor burden and decreased metastasis to lung, liver, lymph nodes, and peritoneum in an orthotopic mouse model of p53-deficient colorectal cancer. [1] In subcutaneous T98G glioblastoma xenografts, a single oral dose of Dordaviprone (30 mg/kg) induced sustained regression comparable to bevacizumab (10 mg/kg iv) (Fig 5D, n=8). [1] In an intracranial SF767 glioblastoma xenograft model, a single oral dose of Dordaviprone (25 mg/kg) doubled overall survival (median survival: control 23 days, Dordaviprone 46 days); combination with bevacizumab (10 mg/kg iv) tripled survival (69 days) (Fig 5E-F, table S5). [1] Immunohistochemical (IHC) analysis of TIC10-treated tumors showed increased TRAIL, cleaved caspase-8, and TUNEL-positive apoptotic cells. [1] Dordaviprone induced TRAIL not only in tumor cells but also in stromal fibroblasts bordering the tumor. [1] IHC of normal tissues from non-tumor-bearing mice treated with Dordaviprone showed TRAIL up-regulation in brain, kidney, and spleen without apparent toxicity. [1] qRT-PCR analysis of tumor xenografts showed that Dordaviprone strongly increased TRAIL gene transcription at 48-108 hours after a single dose. [1] Dordaviprone induced Foxo3a-dependent TRAIL up-regulation and hallmarks of TRAIL-mediated apoptosis in vivo; stable knockdown of Foxo3a in tumor cells significantly inhibited the antitumor activity of Dordaviprone (Fig 6G-H). [1]

ChIP assays [1]
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.

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- Affinity chromatography competition assay: HeLa cell lysates were mixed with vehicle (2% DMSO) or different concentrations of ONC201 (concentrations not specified in text for this assay) for 15 min, then combined with TR-80 agarose beads and rotated for 1 h at room temperature. Beads were washed, boiled in SDS sample buffer, resolved by SDS-PAGE, and immunoblotted with ClpP antibodies. ONC201 reduced ClpP binding to TR-81 beads in a dose-dependent manner when applied to cell lysates or live cells. [2]
- Recombinant human ClpP peptidase activity assay: Purified recombinant human ClpP (1 μg/mL) was incubated in assay buffer (50 mM Tris, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 4 mM ATP, 0.02% Triton X-100, 5% glycerol, pH 8.0) with 10 μM fluorogenic substrate Ac-WLA-AMC. Two protocols were used: Protocol #1 – reaction initiated instantly by direct mixing of enzyme, substrate, and compound; Protocol #2 – enzyme and compound preincubated for 60 min at 37°C before substrate addition. Fluorescence (excitation 350 nm, emission 460 nm) was monitored. ONC201 increased ClpP peptidase activity in a dose- and time-dependent manner. [2]
- ClpP protease activity assay (casein degradation): Recombinant ClpP was preincubated for 1 h at 37°C with DMSO or ONC201 in assay buffer, then incubated for an additional hour with 5 μM α-casein. Reaction products were resolved by 12% SDS-PAGE and silver stained. ONC201 increased casein proteolysis with a half-maximal dose of approximately 1.25 μM. [2]

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Recombinant human ClpP peptidase activity was measured using the fluorogenic substrate Ac-WLA-AMC. The assay buffer contained 50 mM Tris, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 4 mM ATP, 0.02% Triton X-100, and 5% glycerol (pH 8.0). Two protocols were used: (1) direct mixing of ClpP (1 μg/mL) with substrate (10 μM) and compound; (2) preincubation of ClpP with compound for 60 min at 37°C before adding substrate. Fluorescence of released coumarin was monitored at 350 nm excitation and 460 nm emission. Dordaviprone (ONC201) increased ClpP peptidase activity in a dose- and time-dependent manner; the TR compounds were 10-100 fold more potent activators than Dordaviprone. [2]
Casein proteolysis assay: Recombinant human ClpP (10 ng/μL) was preincubated with DMSO or compounds for 1 h at 37°C in assay buffer containing 5 μM α-casein. After an additional 1 h incubation at 37°C, samples were boiled in SDS sample buffer, resolved by 12% SDS-PAGE, and silver stained. Dordaviprone (ONC201) increased ClpP-mediated casein degradation with a half-maximal dose of approximately 1.25 μM. [2]
Affinity capture: TR-80 or TR-81 analogues were coupled to NHS-activated agarose beads. HeLa cell lysates were mixed with control beads or drug-conjugated beads in the presence or absence of free Dordaviprone (ONC201) or TR compounds. Bound proteins were eluted, resolved by SDS-PAGE, and identified by mass spectrometry or immunoblotting. Dordaviprone competed with TR-81 beads for ClpP binding in a dose-dependent manner. [2]

Cells were treated with 10 μM ONC201 or DMSO for 24 h.
\nColony formation assays [1]
\nThe indicated cell lines were plated at 500 cells per well and allowed to adhere, and then treated the next day in fresh complete medium. At 3 days after treatment, the medium was replaced with drug-free medium and cells were propagated for 10 days, with fresh medium given once every 3 days. At the end of the 10-day period, cells were washed in PBS, fixed with methanol, stained with Coomassie blue, rinsed, and dried for quantification.

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\n\nWestern blot analysis [1]
\nWestern blot analysis was conducted as previously described (41) with NuPAGE 4 to 12% bis-tris gel and visualized with SuperSignal West Femto and x-ray film. Densitometry was performed with NIH ImageJ. Nuclear and cytoplasmic extracts were prepared with a cytoplasmic lysis buffer (10 mM Hepes, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol) followed by a nuclear lysis buffer (20 mM Hepes, 420 mM NaCl, 1.5 mM MgCl2, 250 μM EDTA, 25% glycerol). For all lysis buffers, fresh protease inhibitor and 1 mM sodium orthovanadate were added immediately before use.\n

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\n- Cell viability (resazurin assay): SUM159 cells (1000 cells/well) were plated in 96-well plates, allowed to adhere overnight, then treated with ONC201 at indicated concentrations for 72 h. Resazurin (0.6 mM, 20 μL) was added and incubated for 30 min at 37°C. Relative fluorescence of resorufin (540-20 excitation, 590-20 emission) was measured. IC50 was calculated using GraphPad Prism 7. [2]
\n- Total cell counting (Hoechst stain): SUM159 cells (1000 cells/well) were plated in 96-well plates, treated with ONC201, and at indicated time points (0, 24, 48, 72 h), media was aspirated and Hoechst stain (1 μg/mL) added for 30 min at 37°C. Total cell number was quantified using Celigo Imaging Cytometer. [2]
\n- Crystal violet colony formation assay: SUM159 cells (1000 cells/well) were plated in 6-well plates, treated with ONC201 for 48 h, then media replaced with fresh media with or without drug. Cells were incubated until one well reached 100% confluence, stained with 0.5% crystal violet in 20% methanol for 10 min at RT, rinsed, and dried. Staining was quantified by dissolving in Sorenson's buffer (0.1 M sodium citrate, 50% ethanol, pH 4.2) and measuring absorbance at 570 nm. [2]
\n- Immunoblotting: SUM159 or MDA-MB-231 cells were treated with compounds, rinsed with cold PBS, lysed in RIPA buffer (no SDS) supplemented with phosphatase and protease inhibitors. Lysates were clarified, resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies (ATF4, CHOP, β-actin, TFAM, TUFM, ClpP) diluted 1:1000, followed by secondary antibodies (1:10,000). Blots were developed with ECL reagents and imaged. [2]
\n- siRNA reverse transfection: siRNA stocks (20 μM) were resuspended. Dharmafect I diluted 1:266 in OptiMEM, mixed with siRNA (final 125 nM), incubated 30 min at RT, added to plates. SUM159 cells were trypsinized, resuspended in DMEM:F12 with 5% FBS, 5 μg/mL insulin, 1 μg/mL hydrocortisone at 1×10^5 cells/mL (6-well) or 1×10^4 cells/mL (96-well), added to siRNA-containing wells, and incubated for 24 h. ClpP knockdown was verified by immunoblotting. [2]
\n- Caspase 3/7 activity assay: SUM159 cells (8×10^5 cells per 6 cm plate) were treated for 24 h with ONC201 (300 nM, 3 μM, 30 μM), DMSO (0.1%), or staurosporine (10 nM). Cells were scraped into lysis buffer (50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT), and caspase activity was measured using fluorogenic peptide substrate (7-amido-4-methylcoumarin). [2]
\n- Nascent protein synthesis assay (35S metabolic labeling): SUM159 cells were incubated in methionine- and cysteine-free medium with ONC201 for 15 min, then 35S-labeled methionine/cysteine (125 μCi) was added for 30 min. Cells were washed, lysed in RIPA buffer, protein concentration determined by Bradford assay. TCA (20% final) was added, precipitated proteins captured on glass microfiber filters, washed with 20% TCA and 100% ethanol, air-dried, and radioactivity quantified by scintillation counting. Results normalized to protein concentration. [2]
\n

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Flow cytometry for surface TRAIL: cells were harvested by brief trypsinization, fixed in 4% paraformaldehyde for 20 min, incubated with anti-TRAIL antibody overnight, then with anti-rabbit Alexa Fluor 488 for 30 min, and analyzed on a flow cytometer. Surface TRAIL data expressed as median fluorescence intensity relative to controls. [1] Sub-G1 DNA content and cell cycle profile analysis: cells were pelleted, ethanol-fixed, and stained with propidium iodide in the presence of RNase, then analyzed by flow cytometry. [1] Cell viability assays were performed using CellTiter-Glo luminescent reagent in 96-well black-walled clear-bottom plates according to the manufacturer's protocol. [1] Colony formation assays: cells were plated at 500 cells per well, allowed to adhere, treated with compounds for 3 days, then medium replaced with drug-free medium and propagated for 10 days (medium changed every 3 days). Cells were washed, fixed with methanol, stained with Coomassie blue, rinsed, dried, and quantified. [1] Western blot analysis: cells lysed in RIPA buffer, proteins resolved on NuPAGE 4-12% bis-tris gels, transferred, and visualized with SuperSignal West Femto and X-ray film. Densitometry performed with NIH ImageJ. Nuclear and cytoplasmic extracts prepared using cytoplasmic lysis buffer (10 mM Hepes, 10 mM KCl, 2 mM MgCl2, 1 mM DTT) followed by nuclear lysis buffer (20 mM Hepes, 420 mM NaCl, 1.5 mM MgCl2, 250 μM EDTA, 25% glycerol) with fresh protease inhibitor and 1 mM sodium orthovanadate. [1] Chromatin immunoprecipitation (ChIP) assay: performed as described for the TRAIL promoter using a ChIP-grade antibody for Foxo3a or rabbit IgG as control. [1] Quantitative reverse transcription PCR (qRT-PCR): TRAIL mRNA concentrations measured. [1] Primary patient specimens: obtained after resection, manually digested in complete DMEM, filtered with 100 μm nylon mesh, and plated at 2x10^5 cells/mL for experiments. [1]
Resazurin cell viability assay: cells (1000 per well) plated in 96-well plates, treated with compounds for 72 h, then 20 μL of 0.6 mM resazurin added and incubated for 30 min at 37°C. Fluorescence of resorufin measured (excitation 540 nm, emission 590 nm). IC50 values calculated using GraphPad Prism 7. [2] Total cell counting: cells plated in 96-well plates, treated, then stained with Hoechst 1 μg/mL for 30 min at 37°C, and total cell number quantified using a Celigo Imaging Cytometer. [2] Crystal violet colony formation assay: cells plated in 6-well plates, treated for 48 h, then medium replaced with fresh medium with or without drug; when control wells reached 100% confluency, cells stained with 0.5% crystal violet in 20% methanol for 10 min, rinsed, dried, and stain dissolved in Sorenson's buffer (0.1 M sodium citrate, 50% ethanol, pH 4.2) and absorbance measured at 570 nm. [2] Immunoblotting: cells lysed in RIPA buffer (no SDS) supplemented with 2 mM Na(VO3)4, 10 mM NaF, 0.0125 μM calyculin A, and protease inhibitor cocktail. Lysates clarified and immunoblotted with antibodies against ATF4, CHOP, ClpP, TFAM, TUFM, β-actin, etc. Primary antibodies diluted 1:1000 in 5% TBST/BSA + 0.02% NaN3 overnight at 4°C, then secondary antibodies (1:10,000) for 1 h, detected by ECL and imaged. [2] siRNA reverse transfection: siRNAs (20 μM stock) diluted to 125 nM in OptiMEM with Dharmafect I, incubated 30 min at RT, then added to plates. SUM159 cells resuspended in DMEM:F12 with 5% FBS, 5 μg/mL insulin, 1 μg/mL hydrocortisone at 1x10^5 cells/mL (6-well) or 1x10^4 cells/mL (96-well), added to wells, incubated 24 h. [2] Caspase 3/7 activity assay: cells plated at 8x10^5 per 6 cm plate, treated for 24 h, harvested by scraping into lysis buffer (50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT), and activity measured using fluorogenic peptide substrate (7-amido-4-methyl-coumarin). [2] Metabolic labeling of nascent proteins: cells incubated in methionine- and cysteine-free medium with compounds for 15 min, then 35S-labeled methionine and cysteine (125 μCi) added for 30 min. Cells washed, lysed in RIPA, protein concentration determined, TCA added to 20%, precipitated proteins captured on glass microfiber filters, washed, and radioactivity quantified by scintillation counting. [2]

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

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All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee at the Pennsylvania State University College of Medicine. For subcutaneous xenografts, 4- to 6-week-old female athymic nu/nu mice (Charles River Laboratories) were inoculated with 1 × 106 cells (2.5 × 106 for T98G) of the indicated cell lines in each rear flank as a 200-μl suspension of 1:1 Matrigel (BD)/PBS. All subcutaneous tumors were allowed to establish for 1 to 4 weeks after injection until reaching a volume of ~125 mm3 before treatment initiation.[1]

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Female athymic nu/nu mice (4-6 weeks old) were inoculated subcutaneously with 1x10^6 cells (2.5x10^6 for T98G) of indicated cell lines in 200 μL of 1:1 Matrigel/PBS into each rear flank. Tumors were allowed to establish for 1-4 weeks until reaching a volume of approximately 125 mm^3 before treatment initiation. Dordaviprone (TIC10) was administered intraperitoneally (ip) or orally (by gavage) at various doses (e.g., 25 mg/kg, 30 mg/kg, 100 mg/kg) as single or multiple doses (e.g., on days 0, 3, and 6). For oral administration, compound was given as a single dose or weekly for 4 weeks. Recombinant TRAIL was administered intravenously. Bevacizumab was given intravenously at 10 mg/kg. Tumor volume was measured regularly, and tumor size expressed relative to day 0. [1] For orthotopic glioblastoma model: SF767 cells were implanted intracranially. At 2 weeks after implantation, mice received a single oral dose of Dordaviprone (25 mg/kg), bevacizumab (10 mg/kg iv), or combination. Overall survival was monitored. [1] For Eu-myc transgenic mice (spontaneous lymphoma), mice were treated during weeks 9 to 12 with a weekly single oral dose of Dordaviprone (25 mg/kg). Survival was analyzed by log-rank test. [1] For pharmacokinetic analysis, mice were administered Dordaviprone and plasma samples collected at various time points for compound quantification. [1]

Absorption
The maximum concentration (Cmax) of dodavelnipron was 2.8 mcg/mL (42%), and the total systemic exposure (AUC) was 23 h × mcg/mL (48%). Within the dose range of 125 to 625 mg, the Cmax and AUC of dodavelnipron increased dose-proportionately. The median time to reach maximum plasma concentration (Tmax) of dodavelnipron was 1.4 h (0.5 h, 5.6 h). Co-administration with a high-fat meal (800 to 1000 calories, 50% fat) decreased the Cmax of dodavelnipron by 40%, while the AUC remained unchanged.
Elimination Routes
Following a single dose of radiolabeled dodavelnipron, approximately 70% of the dose was excreted in the urine and 20% in the feces. No significant unmetabolized dodavelnipron was detected in the urine or feces.
Volume of Distribution
The apparent (oral) volume of distribution of dodavepron is 450 L (40%). The median in vitro plasma concentration is 0.67. Dodavepron can cross the blood-brain barrier.
Clearance
The apparent clearance is approximately 27 L/hr (48%).
Protein Binding
The plasma protein binding of dodavepron is 95% to 97%, and is concentration-independent in vitro.
Metabolites/Metabolites
Dodavepron is primarily metabolized by CYP3A4, with smaller contributions from CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A5. Its metabolic pathways and metabolites are not fully elucidated.
Biological Half-Life
The mean terminal half-life of dodavepron is 11 hours (30%).

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The plasma half-life of Dordaviprone (TIC10) is approximately 6.5 hours (fig S5, table S4). [1] Dordaviprone crosses the blood-brain barrier, as evidenced by sustained TRAIL induction in brain tissue of non-tumor-bearing mice after iv administration (100 mg/kg). [1] Metabolic analysis in vitro indicated the presence of amine modifications that were not time-dependent (table S2). [1] Metabolic analysis of serum from mice revealed a potential metabolite indicative of oxidation and glucuronide conjugation, evident only at 8 hours after administration (table S3). [1] Dordaviprone is thermally stable; preincubation at 95°C for 1 hour did not reduce its cytotoxic activity, unlike recombinant TRAIL which lost activity. [1] Dordaviprone is orally bioavailable; a single oral dose was equally effective as intraperitoneal administration in SW480 xenografts (fig S3C). [1]

Efficacy and Safety: The efficacy evaluation included 50 adult and pediatric patients with recurrent H3 K27M-mutant diffuse midline gliomas from five open-label, non-randomized clinical trials conducted in the United States (ONC006 [NCT02525692], ONC013 [NCT03295396], ONC014 [NCT03416530], ONC016 [NCT05392374], and ONC018 [NCT03134131]). The efficacy evaluation population included patients with H3 K27M-mutant diffuse midline gliomas who received monotherapy with up tovitrozin and had progressive and measurable disease according to the Neuro-oncology Response Assessment - High-Grade Gliomas (RANO-HGG) criteria. All patients had received radiotherapy for at least 90 days, had adequate clearance from prior anticancer therapy, a Karnofsky Performance Status (KPS)/Lansky Functional Status (LPS) score ≥60, and had stable or gradually decreasing glucocorticoid dosage. Patients with diffuse entropional gliomas, primary spinal cord tumors, atypical histological types, or cerebrospinal fluid dissemination were excluded. The primary efficacy endpoint was objective response rate (ORR), assessed by an independent center-blinded review (BICR) according to RANO 2.0 criteria, with duration of response (DOR) as a secondary efficacy endpoint. The ORR was 22% (95% CI: 12, 36), and the median DOR was 10.3 months (95% CI: 7.3, 15.2). Among the 11 patients who achieved an objective response, 73% had a DOR ≥6 months, and 27% had a DOR ≥12 months. Prescription information for dodavirone included warnings and precautions regarding allergies, QTc interval prolongation, and embryo-fetal toxicity.

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No apparent toxicity was observed in mice at multiple doses of Dordaviprone (TIC10) delivered at fourfold above the therapeutic dose (100 mg/kg) in xenograft studies. There were no adverse effects on body weight or liver histology as determined by H&E staining (fig S3, D-F). [1] Oral administration of Dordaviprone at 25 mg/kg weekly for 4 weeks in immunocompetent mice did not cause any changes in selected serum chemistry markers (table S1). [1] In vitro, Dordaviprone (5 μM, 72 h) did not alter the cell cycle profiles or clonogenic survival of normal human foreskin fibroblasts (HFF), whereas it induced apoptosis in cancer cells. [1] In co-culture experiments, Dordaviprone selectively induced apoptosis in p53-deficient tumor cells but not in normal fibroblasts. [1] Dordaviprone induced a modest amount of TRAIL on the surface of normal fibroblasts (HFF) without causing toxicity. [1]

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

[2]. ACS Chem Biol. 2019 May 17;14(5):1020-1029.

Dodavipron (ONC-201) is currently undergoing clinical trial NCT03394027 (ONC201 is indicated for the treatment of relapsed/refractory metastatic breast cancer and advanced endometrial cancer). Dodavipron is a water-soluble, orally bioavailable inhibitor of serine/threonine protein kinase Akt (protein kinase B) and extracellular signal-regulated kinase (ERK) with potential antitumor activity. After administration, dodavipron binds to and inhibits the activity of Akt and ERK, potentially leading to inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the mitogen-activated protein kinase (MAPK)/ERK-mediated pathway. This may result in the induction of tumor cell apoptosis in AKT/ERK-overexpressing tumor cells via the tumor necrosis factor (TNF)-associated apoptosis-inducing ligand (TRAIL)/TRAIL death receptor type 5 (DR5) signaling pathway. The PI3K/Akt and MAPK/ERK signaling pathways are upregulated in various tumor cell types and play a crucial role in tumor cell proliferation, differentiation, and survival by inhibiting apoptosis. Furthermore, ONC201 can cross the blood-brain barrier. Recombinant tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is an anti-tumor protein currently in clinical trials and holds promise as a potential anti-cancer therapy. However, its drug properties have several limitations, such as short serum half-life, poor stability, high cost, and uneven biodistribution, especially in brain tissue. To overcome these limitations, we discovered TRAIL-inducing compound 10 (TIC10), a highly potent, orally effective, and stable small molecule that can induce TRAIL transcriptionally in a p53-independent manner and cross the blood-brain barrier. TIC10 induces sustained upregulation of TRAIL in both tumor and normal cells, which may be one reason for its significant anti-tumor activity. TIC10 can inactivate kinase Akt and extracellular signal-regulated kinase (ERK), thereby causing Foxo3a to translocate to the nucleus and bind to the TRAIL promoter in the nucleus, upregulating gene transcription. TIC10 is an effective anti-tumor therapeutic drug that acts on tumor cells and their microenvironment to increase the concentration of the endogenous tumor suppressor TRAIL. [1]

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ONC201 is a first-in-class imidazolinone molecule that is currently undergoing clinical trials for various cancers. Although ONC201 has great potential for clinical application, its mechanism of action remains controversial. In order to study the mechanism of action of ONC201 and screen for more potent compounds, we tested a series of novel ONC201 analogs (TR compounds) on the effects of cell viability and stress response in breast cancer and other cancer models. The results showed that TR compounds were about 50-100 times more potent than ONC201 in inhibiting cell proliferation and inducing the integration of the stress response protein ATF4. Using immobilized TR compounds, we identified human mitochondrial casein hydrolase P (ClpP) as its specific binding protein via mass spectrometry. Affinity chromatography/drug competition assays showed that the binding affinity of TR compounds to ClpP was approximately 10-fold higher than that of ONC201. Importantly, we found that the peptidase activity of recombinant ClpP could be strongly activated by both ONC201 and TR compounds in a dose- and time-dependent manner, with the TR compounds being approximately 10-100-fold more potent than ONC201. Furthermore, in SUM159 cells, knockdown of ClpP using siRNA reduced cellular responses to ONC201 and TR compounds, including CHOP induction, loss of mitochondrial proteins (TFAM, TUFM), and the cytosolic effects of these compounds. Therefore, we report that ClpP can directly bind to ONC201 and its associated TR compounds and is an important biological target for such molecules. Moreover, these studies provide the first biochemical explanation for the difference in therapeutic efficacy between ONC201 and TR compounds. [2]

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Pharmacodynamics: Dodavirpron is an antitumor drug: it has shown antitumor activity in both cell-based assays and in vivo models of H3 K27M-mutant diffuse gliomas. Dodavirpron causes concentration-dependent QTc interval prolongation. At 1.2 times the maximum recommended dose, the estimated mean change in QTcF was 11.8 ms (90% CI: 9.8, 13.7). The exposure-response relationship and time course of pharmacodynamic response of dodavirpron have not been fully elucidated, and therefore its safety and efficacy remain unclear.

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Mechanism of Action: Dodavirpron is a protease activator of mitochondrial casein hydrolase P (ClpP, a mitochondrial serine protease). Diffuse midline gliomas carrying the H3 K27M mutation are associated with the loss of H3 K27 trimethylation. In vitro experiments showed that dodavirpron can induce apoptosis and alter mitochondrial metabolism, thereby restoring histone H3K27 trimethylation levels in an H3K27M mutant diffuse glioma model. Dodavirpron can activate ATF4 and induce endoplasmic reticulum (ER) stress response or integrated stress response (ISR). In addition, dodavirpron can inhibit dopamine D2 receptors, which are often overexpressed in various cancers, including glioblastoma.\n

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\n- ONC201 (also known as TIC10) is a first-in-class imipridone molecule originally identified from an NCI chemical library screen for its ability to induce TRAIL gene transcription in HCT116 human colon cancer cells. [2]
\n- ONC201 has shown growth inhibitory effects in multiple cancer cell lines and antitumor activity in animal models of glioblastoma, colorectal cancer, non-Hodgkins lymphoma, and pancreatic cancer (reviewed in reference 2). [2]
\n- As of 2019, ONC201 was in 15 clinical trials (ClinicalTrials.gov identifier: NCT02863991). In 2018, ONC201 was granted Fast Track Designation for the treatment of adult recurrent H3 K27M mutant high-grade gliomas. [2]
\n- Previous proposed mechanisms (TRAIL induction, Akt/ERK inhibition, dopamine receptor targeting) have been controversial; this study identifies mitochondrial ClpP as a direct target. [2]
\n- ONC201 was reported to kill breast cancer cells by targeting mitochondria and inducing an integrated stress response (ISR) with ATF4 and CHOP induction, and was ineffective in Rho0 cells (cells with impaired mitochondrial function). [2]
\n

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Dordaviprone (TIC10, NSC350625) was identified from the National Cancer Institute (NCI) Diversity Set II using a cell-based bioluminescence reporter screen in TRAIL-resistant Bax-null HCT116 human colon cancer cells harboring a TRAIL gene promoter luciferase reporter. [1] The mechanism of action involves dual inactivation of Akt and ERK, leading to Foxo3a nuclear translocation, binding to the TRAIL promoter, and transcriptional induction of TRAIL. [1] Dordaviprone up-regulates not only TRAIL but also the TRAIL death receptor DR5. [1] Dordaviprone induces TRAIL in both tumor cells and normal host cells (e.g., stromal fibroblasts, brain, kidney, spleen), contributing to antitumor efficacy through direct and bystander mechanisms. [1] Dordaviprone has broad-spectrum activity against multiple malignancies including colon, breast, lung, and glioblastoma, and is effective against temozolomide-resistant and radiation-resistant primary tumor cells. [1] Dordaviprone shows synergy with taxanes (paclitaxel, docetaxel) and bevacizumab. [1] Dordaviprone (ONC201) is currently in clinical trials for multiple cancers (ClinicalTrials.gov identifier: NCT02863991). In 2018, it was granted Fast Track Designation for the treatment of adult recurrent H3 K27M mutant high-grade gliomas. [2] The mechanism of action was controversial; later studies identified human mitochondrial ClpP as a direct binding target and activator of Dordaviprone (ONC201), leading to integrated stress response (ATF4/CHOP induction), inhibition of protein synthesis, and reduction of mitochondrial proteins (TFAM, TUFM). [2] Unlike some earlier reports, Dordaviprone (ONC201) did not consistently induce TRAIL in all cell types (e.g., not in breast cancer cells), and its cytostatic effects were independent of TRAIL in some models. [2] Dordaviprone activates ClpP peptidase activity, which is essential for the induction of integrated stress response and growth inhibition. [2]

C24H26N4O

386.49

386.21

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

1616632-77-9

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

73777259

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

3.14

0

3

4

29

693

0

O=C1C2C([H])([H])N(C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])C([H])([H])C([H])([H])C=2N2C([H])([H])C([H])([H])N=C2N1C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H]

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

7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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