tyk2  (Cell Signaling Technology Inc)

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Effects of brepocitinib on MH7A cell viability and JAK-STAT pathway activation. A, cell viability of MH7A synoviocytes following treatment with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. MTT assay was performed, and absorbance at 570 nm was normalized to DMSO control (0.1%). Data represent mean ± SD from three independent experiments. * P < 0.05 vs. vehicle. Although small statistical differences were detected at 1 µM and 5 µM, all brepocitinib-treated groups maintained viability above the predefined non-cytotoxic threshold (≥ 85% of vehicle). Therefore, these changes are considered not biologically relevant and confirm that brepocitinib is non-cytotoxic within the tested range. doxorubicin (1 µM) was used as a positive control and showed a marked reduction (~20%) in viability, validating assay responsiveness. B, brepocitinib suppresses phosphorylation of JAK-STAT pathway proteins in MH7A synoviocytes. MH7A cells were treated with brepocitinib at concentrations of 0.5 µM, 1 µM, and 5 µM for 24 hours. Western blot analysis was performed to assess the expression of phosphorylated and unphosphorylated forms of TYK2, JAK1, STAT1, and STAT3. GAPDH was used as a loading control. Protein lysates (30 µg per lane) were resolved by SDS-PAGE and probed with specific antibodies as indicated. Blots are representative of three independent experiments. Regression analysis confirmed a significant concentration-dependent reduction in p-JAK1 and p-STAT3 (R² > 0.90, P < 0.01). C, validation in primary RA-FLS (HFLS-RA). Cells were treated with brepocitinib (0.5, 1, 5 µM, 24 h) or tofacitinib (1 µM) and analyzed by Western blot for p-TYK2, TYK2, p-JAK1, JAK1, p-STAT1, STAT1, p-STAT3, and STAT3. GAPDH served as loading control. Results represent mean ± SD from three independent experiments. Low, medium, and high doses correspond to 0.5, 1, and 5 µM brepocitinib, respectively. Regression analysis also confirmed dose-dependent suppression of p-TYK2, p-JAK1, p-STAT1, and p-STAT3 in RA-FLS (R² > 0.88, P < 0.01). D, primary RA-FLS (HFLS-RA) were treated with brepocitinib (1 µM) for 0, 30, 60, 240 minutes, and 24 hours. Western blot analysis was performed for p-STAT1, STAT1, p-STAT3, and STAT3, with GAPDH serving as the loading control. Values represent mean ± SD from at least three independent experiments. Significance compared with control: * P < 0.05, *** P < 0.001. Two-way ANOVA demonstrated significant effects of treatment, time, and their interaction (P < 0.05). In all experiments, low, medium, and high doses correspond to 0.5 µM, 1 µM, and 5 µM brepocitinib, respectively.

Article Snippet: Primary antibodies against p-TYK2, TYK2, p-JAK1, JAK1, p-STAT1, STAT1, p-STAT3, STAT3, BAX, BCL-2, cleaved caspase-3, total caspase-3, and GAPDH were purchased from cell signaling technology.

Techniques: Activation Assay, MTT Assay, Control, Positive Control, Phospho-proteomics, Western Blot, Expressing, SDS Page, Concentration Assay, Biomarker Discovery

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Dual TYK2/JAK1 Inhibition by Brepocitinib Reprograms Synoviocyte Pathobiology: Mechanistic Insights Into Targeted Therapy for Rheumatoid Arthritis

doi: 10.5812/ijpr-166019

Figure Lengend Snippet: Mechanistic summary of brepocitinib’s molecular and functional effects in MH7A synoviocytes. This diagram synthesizes findings from - into an integrated pathway model of brepocitinib action. The illustration highlights brepocitinib’s dual inhibition of TYK2 and JAK1, leading to downstream suppression of STAT1 and STAT3 phosphorylation. These upstream effects result in reduced transcription and secretion of key inflammatory cytokines (IL-6, TNF-α, and IFN-γ), which in turn drive reduced inflammation and impaired synoviocyte migration. Simultaneously, brepocitinib promotes mitochondrial-dependent apoptosis via increased BAX/BCL-2 ratio and activation of caspase-3. Notably, the inhibition of migration is independent of apoptosis, reflecting a cytokine-driven mechanism rather than loss of cell viability.

Article Snippet: Primary antibodies against p-TYK2, TYK2, p-JAK1, JAK1, p-STAT1, STAT1, p-STAT3, STAT3, BAX, BCL-2, cleaved caspase-3, total caspase-3, and GAPDH were purchased from cell signaling technology.

Techniques: Functional Assay, Inhibition, Phospho-proteomics, Migration, Activation Assay