A restricted role for TYK2 catalytic activity in human cytokine responses revealed by novel TYK2-selective inhibitors

Abstract

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including type I IFNs, IL-6, IL-10, IL-12, and IL-23. Extensive studies of mice that lack TYK2 expression indicate that the IFN-α, IL-12, and IL-23 pathways, but not the IL-6 or IL-10 pathways, are compromised. In contrast, there have been few studies of the role of TYK2 in primary human cells. A genetic mutation at the tyk2 locus that results in a lack of TYK2 protein in a single human patient has been linked to defects in the IFN-α, IL-6, IL-10, IL-12, and IL-23 pathways, suggesting a broad role for TYK2 protein in human cytokine responses. In this article, we have used a panel of novel potent TYK2 small-molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic activity in cytokine pathways in primary human cells. Our results indicate that the biological processes that require TYK2 catalytic function in humans are restricted to the IL-12 and IL-23 pathways, and suggest that inhibition of TYK2 catalytic activity may be an efficacious approach for the treatment of select autoimmune diseases without broad immunosuppression.

FIGURE 1.

Correlation of IC₅₀ values from JAK kinase reference cell assays with K_i values from JAK kinase JH1 domain biochemical assays for the panel of 51 reference small-molecule compounds. Log(K_i) of JAK1, JAK2, and TYK2 values are plotted against log(IC₅₀) values for inhibition of phosphorylation of STAT proteins in the JAK1, JAK2, and TYK2 reference cell line assays.

FIGURE 2.

Inhibition of primary human PBMC responses to IL-12 by TYK2- and JAK2-selective compounds. Log(IC₅₀) values for compound inhibition of (A) phospho-STAT4 and (B) IFN-γ production in IL-12–stimulated PBMCs are plotted against log(IC₅₀) values for inhibition of TYK2 or JAK2 reference cell assays. (C) Log(IC₅₀) values for compound inhibition of phospho-STAT4 are plotted against log(IC₅₀) values for compound inhibition IFN-γ production in IL-12–stimulated human PBMCs. (D) Western blot analysis of INCB018424, JAK1/2-selective compound 1, and TYK2-selective compound 26 inhibition of IL-12–induced phospho-STAT4 in primary human CD4 T cells.

FIGURE 3.

Inhibition of primary human Th17 memory cell responses to IL-23 by TYK2- and JAK2-selective compounds. Log(IC₅₀) values for compound inhibition of (A) phospho-STAT3 and (B) IL-17F production in IL-23–stimulated human Th17 memory cells are plotted against log(IC₅₀) values for inhibition of TYK2 or JAK2 reference cell assays. (C) Log(IC₅₀) values for compound inhibition of phospho-STAT3 in IL-23–stimulated human Th17 memory cells are plotted against the log(IC₅₀) values for inhibition of TYK2 or JAK2 reference cell assays for the 10 compounds with highest TYK2 selectivity. The unity line for equipotency between inhibition of phospho-STAT3 in human Th17 cells and inhibition of TYK2 or JAK2 reference cell assays is shown in red. (D) Log(IC₅₀) values for compound inhibition of phospho-STAT3 are plotted against log(IC₅₀) values for compound inhibition IL-17F production in IL-23–stimulated human Th17 memory cells.

FIGURE 4.

Inhibition of primary human fibroblast responses to IFN-α by TYK2- and JAK1-selective compounds. (A) Inhibition of phospho-STAT1 or the RQ of MxA mRNA by INCB018424. (B) Inhibition of phospho-STAT1 or the RQ of MxA mRNA by TYK2-selective compound 31 (purple), JAK1-selective compound 35 (green), and a combination of compounds 31 and 35 (black). (C) Inhibition of phospho-STAT1 or the RQ of MxA mRNA by TYK2-selective compound 32 (purple), JAK1-selective compound 34 (green), and a combination of compounds 32 and 34 (black). (D) Inhibition of phospho-STAT1 or the RQ of MxA mRNA by TYK2-selective compound 33 (purple), JAK1-selective compound 36 (green), and a combination of compounds 33 and 36 (black). Data are mean ± SEM of duplicate samples and are representative of four independent experiments, each using a different donor. (E) Western blot analysis of INCB018424, JAK1-selective compound 35, and TYK2-selective compound 31 inhibition of IFN-α–induced phospho-STAT1 in primary human fibroblasts.

FIGURE 5.

Inhibition of primary human hepatocyte responses to IL-6 by TYK2- and JAK1-selective compounds. (A) Inhibition of phospho-STAT3 or CRP production by INCB018424. (B) Inhibition of phospho-STAT3 or CRP production by TYK2-selective compound 31 (purple), JAK1-selective compound 35 (green), and a combination of compounds 31 and 35 (black). (C) Inhibition of phospho-STAT3 or CRP production by TYK2-selective compound 32 (purple), JAK1-selective compound 34 (green), and a combination of compounds 32 and 34 (black). (D) Inhibition of phospho-STAT3 or CRP production by TYK2-selective compound 33 (purple), JAK1-selective compound 36 (green), and a combination of compounds 33 and 36 (black). Data are mean ± SEM of duplicate samples and are representative of four independent experiments, each using a different donor. (E) Western blot analysis of INCB018424, JAK1-selective compound 35, and TYK2-selective compound 31 inhibition of IL-6–induced phospho-STAT3 in primary human hepatocytes.

FIGURE 6.

Inhibition of primary human MDDC responses to IL-10 by TYK2- and JAK1-selective compounds. (A) Inhibition of phospho-STAT3 or the suppression of LPS-induced TNF-α production by INCB018424. (B) Inhibition of phospho-STAT3 or the suppression of LPS-induced TNF-α production by TYK2-selective compound 31 (purple), JAK1-selective compound 35 (green), and a combination of compounds 31 and 35 (black). (C) Inhibition of phospho-STAT3 or the suppression of LPS-induced TNF-α production by TYK2-selective compound 32 (purple), JAK1-selective compound 34 (green), and a combination of compounds 34 and 32 (black). (D) Inhibition of phospho-STAT3 or the suppression of LPS-induced TNF-α production by TYK2-selective compound 33 (purple), JAK1-selective compound 36 (green), and a combination of compounds 33 and 36 (black). (E) Western blot analysis of INCB018424, JAK1-selective compound 35, and TYK2-selective compound 31 inhibition of IL-10–induced phospho-STAT3 in human MDDCs. (F) Inhibition of PDL1 upregulation by 0.3 (light colored bars) or 1 μM (dark colored bars) INCB018424 control compound (blue) or TYK2- and JAK1-selective compounds (purple and green) or 0.3 μM of both control or TYK2- and JAK1-selective compounds (black). (G) Flow cytometry profiles of PD-L1 staining intensity on MDDCs with (dark blue) and without (orange) IL-10 stimulation, and with IL-10 stimulation plus 0.3 μM INCB018424 (light blue, left graph), TYK2-selective compound 31 (purple, right graph), JAK1-selective compound 35 (green, right graph), or a combination of both compounds 31 and 35 (black, right graph). (A–D) Data are mean ± SEM of two samples and are representative of six independent experiments, each using a different donor. (F and G) Data are representative of six independent experiments, each using a different donor.

FIGURE 7.

Inhibition of primary human keratinocyte responses to IL-22 by TYK2- and JAK1-selective compounds. (A) Inhibition of phospho-STAT3 by INCB018424. (B) Inhibition of phospho-STAT3 by TYK2-selective compound 31 (purple), JAK1-selective compound 35 (green), and a combination of compounds 31 and 35 (black). (C) Inhibition of phospho-STAT3 by TYK2-selective compound 34 (purple), JAK1-selective compound 32 (green), and a combination of compounds 34 and 32 (black). (D) Inhibition of phospho-STAT3 by TYK2-selective compound 33 (purple), JAK1-selective compound 36 (green), and a combination of compounds 33 and 36 (black). Data are mean ± SEM of two samples and are representative of two independent experiments, each using a different donor.

FIGURE 8.

Inhibition of IL-6– and IL-10–mediated STAT3 phosphorylation by pan-JAK control compound INCB018424, JAK1-selective compound 35, and TYK2-selective compound 31 in bone marrow–derived macrophages from TYK2 wild-type mice. The cytokine-dependent response of cells from TYK2-deficient mice is also shown for reference in each panel. (A) IL-6–induced phospho-STAT3. (B) IL-10–induced phospho-STAT3.