Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Chemical structures of representative HDAC8 PROTACs.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Design strategy of HDAC8 PROTACs. Compounds Z1 – Z7 and Z8 – Z13 were designed by linking compound 6 to CRBN ligand I and VHL ligand II via aliphatic linkers, respectively. Compounds Z14 and Z15 were designed by linking compound 6 to CRBN ligand I via rigid linkers. Connecting new CRBN ligand III and IV to compound 6 gave compounds Z16 and Z17 , respectively. Compound Z18 was obtained by linking compound 7 to CRBN ligand III .

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat cells were treated with indicated concentrations of compounds Z4 (A), Z12 (B), and 4 (C) for 6 h. HDAC8 levels were detected using Western blot. GAPDH was used as a loading control. Data were normalized to the DMSO-treated group and the dot plots were shown as mean ± SEM of at least two independent experiments. Nonlinear fitting was generated by the GraphPad Prism.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control, Generated

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat cells were treated with 100 nM of PROTACs Z1-Z7 and 4 (A), and Z8 – Z13 and 4 (B) for 6 h. HDAC8 level was detected using Western blot. GAPDH was used as a loading control.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat cells were treated with 100 nM of PROTACs Z14 – Z17 and 4 (A), and Z18 (B) for 6 h. HDAC8 level was detected using Western blot. GAPDH was used as a loading control.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat cells were treated with indicated concentrations of Z16 , and 10 μM pomalidomide (Poma) for 6 h. HDAC1/2/3/4/6/7/8/11 levels and IKZF1/IKZF3/GSPT1 levels were detected using Western blot. GAPDH was used as a loading control. HDAC8 levels were normalized to the DMSO-treated group and the dot plots were shown as mean ± SEM of at least two independent experiments. Nonlinear fitting was generated by the GraphPad Prism.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control, Generated

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: (A) Degradation kinetics of HDAC8 by PROTAC Z16 in Jurkat cells. Jurkat cells were treated with 100 nM of Z16 for the indicated time. HDAC8 levels were detected using Western blot and quantified using ImageJ. GAPDH was used as a loading control. Data are shown as mean ± SEM of two independent experiments. (B) Mechanistic investigation of HDAC8 degradation induced by PROTAC Z16 in Jurkat cells. Jurkat cells were pretreated with 5 μM of HDAC8 inhibitor 6 , CRBN ligand pomalidomide, and proteasome inhibitor bortezomib for 1 h, followed by treatment of Z16 at 100 nM for 6 h. Jurkat cells were treated with 100 nM of Z16 and NC-Z16 for 6 h. (C) Jurkat cells were treated with indicated concentrations of 32 for 6 h. HDAC8 levels were detected using Western blot. GAPDH was used as a loading control. Data are represented as mean ± SEM of two independent experiments. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs DMSO-treated group, one-way analysis of variance (ANOVA).

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: HCT116 (A), THP-1 (B), and A549 (C) cells were treated with the indicated concentrations of compound Z16 for 6 h. HDAC8 levels were detected using Western blot. GAPDH was used as a loading control. Data were normalized to the DMSO-treated group and the dot plots were shown as mean ± SEM of at least two independent experiments. Nonlinear fitting was generated by the GraphPad Prism.

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control, Generated

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat cells were treated with indicated concentrations of compound Z16 , 6 (A) and SAHA, NC-Z16 (B) for 6 h, respectively. (C) HCT116 cells were treated with indicated concentrations of compound Z16 , 6 and SAHA for 6 h. (D) Jurkat and HCT116 cells were treated with indicated concentrations of 32 for 6 h. (E) Jurkat cells were treated with indicated concentrations of compound Z16 for 24 h. (F) THP-1 cells were treated with indicated concentrations of Z16 and 32 for 24 h. The levels of Ac-SMC3, Ac-HH3, Ac-tubulin, and HDAC8 were detected using Western blot. GAPDH was used as a loading control. The levels of Ac-SMC3, Ac-HH3 and HDAC8 were quantified using ImageJ and normalized to the DMSO-treated group. Data are shown as mean ± SEM of two independent experiments. Nonlinear fitting was generated by the GraphPad Prism.ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs DMSO-treated group, one-way analysis of variance (ANOVA).

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control, Generated

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: Jurkat (A) and HCT116 (B) were either pretreated with 5 μM bortezomib (Bor) for 1 h, followed by treatment with 0.5 μM Z16 or treated with 0.5 μM Z16 for 6 h. The levels of Ac-HH3 and HDAC8 were detected using Western blot. GAPDH was used as a loading control. The levels of Ac-HH3 and HDAC8 were quantified using ImageJ and normalized to the DMSO-treated group. Data are shown as mean ± SEM of two independent experiments. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs DMSO-treated group, one-way analysis of variance (ANOVA).

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: Western Blot, Control

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: HDAC8 Degradation Efficiency for PROTACs Z1 – Z13

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: HDAC8 Degradation Efficiency for PROTACs Z14 – Z18

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors

doi: 10.1021/acs.jmedchem.4c00836

Figure Lengend Snippet: HDAC1/2/3/8 Inhibitory Activities of PROTAC Z16 a

Article Snippet: Then, 50 μL of the HDAC1/2/3 fluorogenic substrate Boc-Lys(Ac)-AMC (20 mM, Bachem, Germany) or the HDAC8 fluorogenic substrate Boc-Lys(TFA)-AMC (20 mM, Bachem, Germany) was added to the plates and incubated at 37 °C for 30 min. Then, 50 μL of the stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 6.0 mg/mL trypsin (porcine pancreas Type IX-S, Sigma-Aldrich) and 10 mM SAHA) was added to each well.

Techniques: