Janus kinases (JAKs) are a class of cytoplasmic tyrosine kinases that are widely involved in signaling of cell surface receptors such as cytokine receptors. There are four JAK family members in mammals: JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). Binding of cytokines to their corresponding receptors activates JAK, which in turn initiates downstream signaling by phosphorylating the cytokine receptor and providing binding sites for signal transducers and activators of transcription (STAT).The JAK-STAT pathway is a central pathway for cytokine signaling and participates in a wide range of important cellular processes, playing a key role in normal physiology and pathology, especially in immune-mediated inflammatory diseases. It plays a key role in normal physiology and pathology, especially in immune-mediated inflammatory diseases.

TYK2 is widely expressed and is involved in the signaling of several key cytokines, such as type 1 interferon (IFN), interleukin (IL)-23, IL-12, IL-10 and IL-6. TYK2 is activated when cytokine binds to the receptor and then recruits STAT by phosphorylating the intracellular tail of the receptor and cooperating with other JAKs; the recruited STAT is phosphorylated and dimerized into the nucleus, regulating cytokine specific gene expression and cytokine secretion, which ultimately plays an important role in immune response and cellular function regulation.

TYK2 Structure and Mechanism of Inhibition

TYK2 (tyrosine kinase 2) is a non-receptor tyrosine kinase containing four functional domains: FERM, SH2, JH2 pseudokinase, and JH1 kinase.The JH2 pseudokinase structural domain, although it does not possess conventional catalytic activity, effectively inhibits the kinase activity of TYK2 by forming intermolecular autoinhibitory interactions with the JH1 kinase structural domain. Once the JH2 structural domain is stabilized, TYK2 cannot be activated by receptor-mediated activation, resulting in the blockage of its catalytic activity. Therefore, inhibitors targeting the structural domain of the JH2 pseudokinase can inhibit inflammatory and immune responses by maintaining the autoinhibitory state between JH2 and JH1, blocking TYK2 activation and downstream signaling pathways, such as IL-12, IL-23, and IFNα/β-mediated STAT1-5 phosphorylation. This strategy is highly selective and clinically tolerable, and represents an important direction for the development of novel TYK2 inhibitors.

1. ADP-Glo Assay

In the ADP-Glo Assay, we evaluated the effect of compounds on TYK2 kinase activity by measuring their inhibitory impact on ATP hydrolysis to ADP. At Km (Figure A) and 1 mM ATP concentrations (Figure B).

2. FP Assay

In the FP Assay, the inhibitory effect of compounds is evaluated by detecting their binding to pseudokinase domains, based on fluorescence shifts caused by kinase-substrate interactions. Binding analysis of BMS-986165 to different JAK proteins (Figure C) showed the strongest inhibition against TYK2(JH2).

Figure 1.  (A) The inhibitory potency of Tofacitinib and Filgotinib against in the TYK2 ADP-Glo Assay at Km ATP. (B) The IC50 for Tofacitinib and Filgotinib in the TYK2 ADP-Glo Assay at 1mM ATP. (C) FP Assay. BMS-98616 selectivity on the the JAK1(JH2), JAK2(JH2) and TYK2(JH2) FP assay.

Reporter Gene is a technique widely used in molecular biology, mainly for analyzing biological processes such as gene expression regulation and signal transduction. Under the stimulation of cytokines, the activation of relevant receptors triggers STAT phosphorylation through the TYK2 signaling pathway, which in turn regulates gene transcription and expression. Therefore, inhibition of the TYK2 signaling pathway can effectively block downstream signaling and provide an important basis for the evaluation of inhibitor activity.

Based on the reporter gene assay system for the TYK2 signaling pathway, we found that BMS 986165 had stronger inhibitory effects in the IL-23 and IL-12 pathways, Tofacitinib performed better in the IFN-γ and GM-CSF pathways, and both had similar inhibitory effects in the IL-6 pathway, BMS 986165 also showed significant inhibitory activity on the IFN-α/β and IL- 22 pathways showed significant inhibitory activity.

Figure 2. Reporter gene assays were used to evaluate the effects of BMS 986165 and Tofacitinib on TYK2-related pathways based on the IL-23 (A), IFN-γ (B), IL-6 (C), IFN-α/β (D), IL-12 (E), IL-22 (F), and GM-CSF (G) .

TYK2 inhibition in functional assays is used in primary cells to assess the effect of compounds on cytokine signaling pathways by detecting the phosphorylation level of STAT proteins and downstream cytokine secretion in cells. The technique uses primary cells such as peripheral blood mononuclear cells (PBMC) and isolated T cells. TYK2 plays a key role in the JAK/STAT signaling pathway, and inhibition of TYK2 reduces STAT phosphorylation induced by cytokine stimulation, thereby modulating the immune response.

Based on functional assays, we verified that tofacitinib and ruxolitinib effectively inhibited IFN-α, IL-6, and TPO-induced pSTAT (Figure A), whereas BMS 986165 demonstrated stronger effects in inhibiting IL-10 and IL-23-induced pSTAT3 (Figure A). In human whole blood, tofacitinib and BMS 986165 significantly inhibited IFN-α, IL-6, IL-10 and IL-12-induced pSTAT3 and pSTAT4 (Figure B). In addition, BMS 986165 and tofacitinib inhibited cytokine release in a concentration-dependent manner in different cell types and whole blood (Figure C). These functional assays effectively assess the specificity and potency of TYK2 inhibitors, highlighting their role in regulating STAT phosphorylation and cytokine secretion, and demonstrating their potential in modulating immune responses.

Figure 3. Assessment of the effects of compounds on cytokine-induced STAT phosphorylation in (A) primary cells, PBMCs, and T cells, and (B) human whole blood, measured using ELISA, AlphaLISA, and flow cytometry. (C) Evaluation of downstream cytokine secretion using a cytokine release assay in different cell types and whole blood.

ICE Bioscience provides multiple validated methods to evaluate the effects of TYK2 inhibitors. With the ADP-Glo Assay and FP Assay, we can accurately measure the inhibition of TYK2 kinase activity and molecular binding by our compounds, helping to assess their selectivity and potency. Reporter gene Assay is used to detect inhibition of the TYK2 signaling pathway, providing data on the inhibitory effects of different compounds on cytokine-mediated STAT phosphorylation. In addition, functional assays further validated the regulatory effects of inhibitors in the immune response by measuring STAT phosphorylation levels and cytokine secretion in primary cells. Overall, these technologies provide valuable data to support the screening, optimization, and clinical application of TYK2-targeted drugs. For more information, please feel free to contact us.