entrectinib t3678  (TargetMol)

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Entrectinib induces myocardial injury and left ventricular dysfunction. C57BL/6J mice were treated with vehicle or 200 mg/kg entrectinib for 6 weeks. n = 9. (A) The diagram of the experiment. (B–E) The cardiac function of mice was measured by echocardiography. (B) Representative M-mode echocardiographic images from each group in mice. (C) The statistics of ejection fraction. (D) The statistics of fractional shortening. (E) The statistics of heart rate. (F) Representative whole heart images. (G) Heart weight to tibia length ratio (HW:TL). (H) Representative images of cardiac sections stained by WGA. (I and J) Serum from the mice was analyzed for CK and CK-MB levels. (K) Representative images of cardiac sections stained by hematoxylin-eosin (H&E). (L–M) Representative images of cardiac longitudinal sections stained by Masson or sirius red. (N) the mRNA levels of cardiac remodeling gene. (O) CCC-HEH-2 cells were treated with entrectinib and the survival fraction was detected via SRB assay. (P) Representative images of cell morphology. (Q) Apoptosis rates were detected by PI and ANXA5 co-staining and flow cytometry. (R) CCC-HEH-2 cells were treated with entrectinib and Z-VAD-FMK. The protein levels of c-PARP were detected by western blot. GAPDH was used as the loading control. (S) The mitochondrial membrane potential was detected by JC-1 staining and flow cytometry. (T) Representative images of TUNEL staining of mouse heart tissue. (U) Representative images of transmission electron microscopy of mouse heart tissue. Results are presented as mean ± SD. All in vitro experiments were performed with three biological replicates. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. * p < 0.05, *** p < 0.001, **** p < 0.0001 (vs. The first group). #### p < 0.0001(vs. the second group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Staining, Sulforhodamine B Assay, Flow Cytometry, Western Blot, Control, Membrane, TUNEL Assay, Transmission Assay, Electron Microscopy, In Vitro

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Entrectinib-activated autophagy in cardiomyocytes mediates the occurrence of cardiotoxicity. (A-C) CCC-HEH-2 cells treated with entrectinib or vehicle were subjected to proteomic analysis. (A) Volcano plot of proteomics. (B) KEGG pathway terms of differentially expressed genes between entrectinib-treated and the control cells. (C) GSEA on autophagy pathway. (D and E) H9c2 and CCC-HEH-2 cells were infected with mCherry-GFP-LC3 virus and then treated with entrectinib or CQ after infection. Autophagic flux assays were performed with the confocal microscope. (F) Quantification of the number of LC3 puncta per cell for panel D. (G) Quantification of the number of LC3 puncta per cell for panel E. CCC-HEH-2 cells were transfected with siRNA targeting ATG7 , and then treated with or without entrectinib. (H) Representative images of cell morphology. (I) Apoptosis rates were detected by PI and ANXA5 co-staining and flow cytometry. (J) The protein levels of c-PARP and LC3 were detected by western blot. GAPDH was used as the loading control. (K) Construction and toxicity modeling diagram of mice with cardiac-specific heterozygous knockout of Atg7 . The number of mice in each of the four groups is 8, 7, 7, and 8 respectively. The cardiac function of mice was measured by echocardiography. (L) Representative M-mode echocardiographic images from each group in mice. (M) the statistics of ejection fraction. (N) the statistics of fractional shortening. (O) Representative images of cardiac sections stained by hematoxylin-eosin (H&E). (P) Representative images of TUNEL staining of mouse heart tissue. (Q) Representative images of transmission electron microscopy of mouse heart tissue. (R) Representative images of immunohistochemical image targeting LC3 protein of mouse heart tissue. Results are presented as mean ± SD. All in vitro experiments were performed with three biological replicates. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. **** p < 0.0001 (vs. The first group). ### p < 0.001, #### p < 0.0001(vs. the second group). p < 0.0001(vs. The fourth group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Control, Infection, Virus, Microscopy, Transfection, Staining, Flow Cytometry, Western Blot, Knock-Out, TUNEL Assay, Transmission Assay, Electron Microscopy, Immunohistochemical staining, In Vitro

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Entrectinib activates autophagy through MTORC1 signaling. (A-C) CCC-HEH-2 cells treated with entrectinib or vehicle were subjected to phosphoproteomic analysis. (A) Volcano plot of phosphoproteomics. (B) KEGG pathway terms of differentially expressed genes between entrectinib-treated and the control cells. (C) Heatmap of the MTOR signaling pathway of phosphoproteomics. (D) Representative images of immunofluorescence image targeting p-RPS6 protein of CCC-HEH-2 cells treated with entrectinib. (E) CCC-HEH-2 cells were treated with entrectinib and the protein levels of c-PARP were detected by western blot. GAPDH was used as the loading control. (F) Representative images of immunohistochemical image targeting p-RPS6 protein of mouse heart tissue. (G) CCC-HEH-2 cells were infected with mCherry-GFP-LC3 virus and then transfected with siRNA targeting ULK1 . Autophagic flux assays were performed with the confocal microscope. (H) Quantification of the number of LC3 puncta per cell for panel G. (I) CCC-HEH-2 cells were transfected with siRNA targeting ULK1 , and then treated with or without entrectinib. Apoptosis rates were detected by PI and ANXA5 co-staining and flow cytometry. (J-K) The protein levels of c-PARP and p-RPS6 and LC3 were detected by western blot. ACTB was used as the loading control. (L) The protein levels of OTUD5 in proteomics. (M-O) CCC-HEH-2 cells were transfected with siRNA targeting OTUD5 . (M) Representative images of immunofluorescence image targeting p-RPS6 protein of CCC-HEH-2 cells. (N-O) the protein levels of p-RPS6 and LC3 and c-PARP were detected by western blot. ACTB was used as the loading control. Results are presented as mean ± SD. All in vitro experiments were performed with three biological replicates. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. ** p < 0.01 (vs. The first group). *** p < 0.001 (vs. The first group). **** p < 0.0001 (vs. The first group). # p < 0.05(vs. the second group). #### p < 0.0001(vs. the second group). p < 0.0001(vs. The second group). &&&& p < 0.0001(vs. the fourth group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Phospho-proteomics, Control, Immunofluorescence, Western Blot, Immunohistochemical staining, Infection, Virus, Transfection, Microscopy, Staining, Flow Cytometry, In Vitro

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Entrectinib inhibits OTUD5-MTORC1 by directly binding to HMGB1 and promoting its nuclear localization. (A and B) CCC-HEH-2 cells were treated with CHX (20 μg/mL) with or without entrectinib. (A) the protein levels of OTUD5 were detected by western blot. ACTB was used as the loading control. (B) relative quantification. (C) CCC-HEH-2 cells were treated with entrectinib and the mRNA levels of OTUD5 gene were detected by qPCR. (D) Dual-luciferase reporter assay for OTUD5 gene. Relative luciferase activity in CCC-HEH-2 cells treated with entrectinib. (E) Representative images of immunohistochemical image targeting HMGB1 protein of mouse heart tissue. (F) Representative images of immunofluorescence image targeting HMGB1 protein of CCC-HEH-2 cells. (G) CCC-HEH-2 cells were treated with 3 μM entrectinib for 24 h. Nuclear and cytoplasmic fractions were separated to detect the levels of HMGB1 by western blot. (H) relative luciferase activity in CCC-HEH-2 cells transfected with empty vector or HMGB1 plasmid. (I) CCC-HEH-2 cells were transfected with siRNA targeting HMGB1 and the mRNA levels of OTUD5 gene (left) and HMGB1 gene (right) were detected by qPCR. (J) Schematic showing the structure of HMGB1 protein. (K and L) CCC-HEH-2 cells were transfected with empty vector or HMGB1-Flag plasmid or HMGB1-Δ-Flag plasmid or HMGB1-mutant-Flag plasmid. (K) Representative images of immunofluorescence image targeting Flag. (L) Relative luciferase activity. (M) Molecular dynamics simulation of the interaction between HMGB1 protein and entrectinib. (N) Microscale thermophoresis (MST), illustrating the interaction between HMGB1 protein and entrectinib. (O) CCC-HEH-2 cells were transfected with plasmids as indicated. Representative images of immunofluorescence targeting Flag. Results are presented as mean ± SD. All in vitro experiments were performed with three biological replicates. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (vs. The first group). ### p < 0.001(vs. the second group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Binding Assay, Western Blot, Control, Quantitative Proteomics, Luciferase, Reporter Assay, Activity Assay, Immunohistochemical staining, Immunofluorescence, Transfection, Plasmid Preparation, Mutagenesis, Microscale Thermophoresis, In Vitro

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Inhibition of HMGB1 is protective against entrectinib-induced cardiotoxicity. (A–C) CCC-HEH-2 cells were transfected with siRNA targeting HMGB1 , and then treated with or without entrectinib. (A) Autophagic flux assays were performed with the confocal microscope. (B) Quantification of the number of LC3 puncta per cell for panel A. (B) Apoptosis rates were detected by PI and ANXA5 co-staining and flow cytometry. (D–F) CCC-HEH-2 cells were treated with tanshinone IIA with or without entrectinib. (D) Autophagic flux assays were performed with the confocal microscope. (E) Quantification of the number of LC3 puncta per cell for panel D. (F) Apoptosis rates were detected by PI and ANXA5 co-staining and flow cytometry. (G) construction and toxicity modeling diagram of mice with cardiac-specific knockout of Hmgb1 . The number of mice in each of the four groups is 8, 8, 6, and 6 respectively. (H) The statistics of ejection fraction. (I) the statistics of fractional shortening. (J) Representative images of cardiac sections stained by hematoxylin-eosin (H&E). (K–N) the mRNA levels of cardiac remodeling gene. (O) Representative images of TUNEL staining of mouse heart tissue. (P) Representative images of transmission electron microscopy of mouse heart tissue. Results are presented as mean ± SD. All in vitro experiments were performed with three biological replicates. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. *** p < 0.001, **** p < 0.0001 (vs. The first group). ## p < 0.01, ### p < 0.001, #### p < 0.0001 (vs. The second group). p < 0.0001(vs. The second group). &&&& p < 0.0001(vs. the fourth group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Inhibition, Transfection, Microscopy, Staining, Flow Cytometry, Knock-Out, TUNEL Assay, Transmission Assay, Electron Microscopy, In Vitro

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Tanshinone IIA can rescue entrectinib-induced cardiotoxicity. C57BL/6J mice were treated with vehicle, entrectinib (200 mg/kg), tanshinone IIA (20 mg/kg) or tanshinone IIA plus entrectinib for 6 weeks by means of intragastric administration. The number of mice in each of the four groups is 9, 9, 8, and 8 respectively. (A) Representative M-mode echocardiographic images from each group in mice. (B) The statistics of ejection fraction. (C) the statistics of fractional shortening. (D) Representative whole heart images. (E) Heart weight to tibia length ratio (HW:TL). (F) serum from the mice was analyzed for CK-MB levels. (G) Representative images of cardiac sections stained by hematoxylin-eosin (H&E). (H-I) Representative images of cardiac longitudinal sections stained by Masson or sirius red. (J) Representative images of TUNEL staining of mouse heart tissue. (K) Representative images of transmission electron microscopy of mouse heart tissue. (L) Representative images of immunohistochemical image of mouse heart tissue. Results are presented as mean ± SD. Unpaired t test was performed to detect the significance of difference between two groups. One-way ANOVA with Sidak’s multiple comparisons test was performed to detect the significance of difference among multiple groups. **** p < 0.0001 (vs. The first group). # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 (vs. The second group).

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques: Staining, TUNEL Assay, Transmission Assay, Electron Microscopy, Immunohistochemical staining

Journal: Autophagy

Article Title: Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity

doi: 10.1080/15548627.2025.2576619

Figure Lengend Snippet: Illustration of the proposed mechanism by which entrectinib induces cardiotoxicity. Entrectinib binds to HMGB1 and activates cardiomyocyte autophagy by inhibiting OTUD5-MTORC1 signaling to induce cardiotoxicity. Tanshinone IIA can rescue entrectinib-induced cardiotoxicity by inhibiting HMGB1.

Article Snippet: Entrectinib (TargetMol USA, T3678) and tanshinone IIA sulfonate sodium (TargetMol USA, T2946) were dissolved in cyclodextrin (Aladdin, H108813) to form a stock solution.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity

doi: 10.1016/j.jbc.2022.101731

Figure Lengend Snippet: ZAF is a novel inhibitor of TMEM16A channel. A , virtual screening flowchart. B , TMEM16A channel inhibitor binding pocket. C , whole-cell currents of HEK293T cells overexpressing (OE) TMEM16A channels were activated by 600 nM Ca 2+ in the pipette solution and inhibited by 10 μM of T16A inh -A01. WT HEK293T as vehicle control. The stimulation protocol: a holding potential of 0 mV for 100 ms, the membrane voltage was clamped in steps of 20 mV from −80 to +80 mV for 750 ms, then back down to −80 mV for 500 ms. D , statistical results of the TMEM16A whole-cell current in ( C ). Data are means ± SD (n = 3; ∗∗∗∗ p < 0.0001, OE TMEM16A + Ca 2+ versus control; ∗∗∗∗ p < 0.0001, OE TMEM16A + Ca 2+ + T16A inh -A01 versus OE TMEM16A + Ca 2+ ). E , the inhibition by Conivaptan, Entrectinib, Pimaricin, and Zafirlukast (100 μM) of TMEM16A current tested at +80 mV. Data are means ± SD (n = 5). F , representative current of TMEM16A inhibited by various concentrations of ZAF (0, 0.01, 0.1, 1, 10, and 100 μM). The stimulation protocol is consistent with ( C ). G , I-V curve of the TMEM16A currents inhibited with different concentrations of ZAF (n = 5). H , concentration response curves of ZAF inhibition of TMEM16A currents in HEK293T cells. The plot was fitted to the Hill equation (n = 5). HEK293T, human embryonic kidney 293T cells; I-V, current–voltage; ZAF, Zafirlukast.

Article Snippet: Entrectinib, Pimaricin, and T16A inh -A01 were purchased from TargetMol.

Techniques: Binding Assay, Transferring, Inhibition, Concentration Assay