mouse p ripk1  (Cell Signaling Technology Inc)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: Identification of RIPK1/3 dual inhibitors. (A) Bar graph showing the protective effects of RIPK3i and derivatives in L929 cells. Cells were treated with TNF α at 40 ng/mL in the presence of DMSO or compounds for 15 h before the cell viability test. (B) The chemical structure of the derivatives of 10 . (C) Dose–response curves showing the protective effects of each compound compared to DMSO in HT-29 cells stimulated by TNF α (10 ng/mL), SM164 (25 nmol/L) and zVAD (25 μmol/L) for 24 h. (D) HT-29 cells stimulated by TNF α , SM164, and zVAD in the presence of DMSO or compounds at 5 μmol/L for 4 h, then analyzed by Western blotting. (E) Bar graph showing the anti-necroptotic effects of each compound at 10 μmol/L in FKBP-RIPK3-expressing NIH-3T3 cells. Cells were treated with the FKBP-dimerizing agent, AP20187 (100 nmol/L), in the presence of DMSO or compounds for 15 h before the viability test. All data represent mean ± SD ( n = 3); ∗∗ P < 0.01, ∗∗∗ P < 0.001, unpaired Student's t -test.

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: Western Blot, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: Determination of the co-crystal structure of RIPK1‒ 20 complex. (A) Superimposition of the co-crystal structures of RIPK1 (green ribbons) in complex with 1 (purple sticks, PDB 4ITH ) or 2 (grey sticks, PDB 5TX5 ). (B) The co-crystal structure of RIPK1 (green ribbons) in complex with 20 (gold sticks, PDB 8I2N ). (C) Superimposition of the co-crystal structures of RIPK1‒ 20 complex (green ribbons and gold sticks) and RIPK1– 5 complex (gray ribbons and sticks, PDB 4NEU ). (D) Cartoon showing interactions between 20 and the ATP-binding pocket of RIPK1, key residues were shown as sticks, and H-bonds were indicated as red dashes. (E) The residue side chains of RIPK1 potentially interacting with 20 through van der Waals forces.

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: Binding Assay, Residue

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: The predicted binding mode of 39 (azure sticks) based on the RIPK1- 20 (yellow sticks) co-crystal structure. The docking study predicted an additional H-bond (red dash) for 39 with the hinge region of RIPK1 (A), and similar but distinct interactions for the tail subunit of 39 compared to that of 20 (B). In panel B, the structure of mRIPK1 (gray ribbons and sticks) was aligned with that of hRIPK1 (green ribbons and sticks).

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: The predicted binding mode of 49 (orange sticks) and 62 (magenta sticks) based on the RIPK1- 20 co-crystal structure, which indicates an intramolecular H-bond (red dash) in 49 (A) and a highly similar conformation for 62 (B).

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: Interrogating RIPK1 in cellular models with 62 . (A) Human FADD −/− Jurkat or mouse L929 cells were treated with TNF α (40 ng/mL) and SM164 (25 nmol/L) for 15 h in the presence of 1 , 2 , or 62 at indicated concentrations, then lysed and followed by Western blotting analysis using species-specific antibodies. (B) Bar graph showing the anti-necroptotic effects of each compound at 10 μmol/L in mouse 3T3 cells engineered with an FKBP-RIPK3-fusion. Cells were treated with the FKBP-dimerizing agent, AP20187 (100 nmol/L), in the presence of DMSO or compounds for 15 h before the viability test. Data represent means ± SD ( n = 3); ∗∗ P < 0.01, ∗∗∗ P < 0.001, unpaired Student's t -test. (C) Human lung organoids were infected with SARS-CoV-2 (0.3 MOI) for 1 h, then washed out with PBS and cultured with normal medium and indicated compounds (5 μmol/L) for an additional 48 h, then lysed and followed by Western blotting analysis. The viral loads of SARS-CoV-2 were indicated by its N protein levels.

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: Western Blot, Infection, Cell Culture

Journal: Acta Pharmaceutica Sinica. B

Article Title: Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

doi: 10.1016/j.apsb.2023.10.021

Figure Lengend Snippet: The anti-inflammatory effects of 62 in vivo . (A) Evaluation of 62 ( po ) in TNF α (10 μg) induced SIRS mouse model measuring reduction in body temperature and survival rates ( n = 7). (B) Quantification of the bodyweight for each group of Ripk1 K612R/K612R mice treated with vehicle, 1 or 62 ( po ) ( n = 4). (C) Quantification of the RA symptoms induced by collagen antibodies and LPS for each group of mice treated with vehicle or 62 ( po ) ( n = 3). Data represent means ± SD; ∗∗ P < 0.01, ∗∗∗ P < 0.001, paired Student's t -test.

Article Snippet: The following antibodies were used in this study: p-S345-MLKL (ab196436, mouse-specific), p-S358-MLKL (ab187091, human-specific), MLKL (ab172868, mouse-specific), MLKL (ab189612, human-specific) and RIPK3 (ab72106, human-specific) were purchased from Abcam (Cambridge, UK); RIPK1 (3493), p-S166-RIPK1 (31122, human-specific), p-T231/S232-RIPK3 (57220, mouse-specific) and p-S227-RIPK3 (93654, human-specific) were purchased from Cell Signaling Technology (Danvers, MA, USA); RIPK3 (AHP1797, mouse-specific) was purchased from BIO-RAD (Hercules, CA, USA); β -actin (I10813) and tubulin (I11107) were purchased from TransGen (Beijing, China); SARS-CoV-2 N protein (40588-T62) was purchased from Sino Biological (Beijing, China); p-S166-RIPK1 (mouse specific) was purchased from Biolynx (Hangzhou, China).

Techniques: In Vivo