A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase

Abstract

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor β-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC₅₀ = 24 nm) and IRAK-4 (IC₅₀ = 20 nm), with only minimal TAK1-inhibiting activity (IC₅₀ = 0.5 μm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Keywords: autoimmune disease; immunology; inhibitor; interleukin; tumor necrosis factor (TNF).

Figure 1.

Structural comparison of TAK1 against IRAK-1 and -4. a, overlay of the ribbon structure of IRAK-4 and TAK1. b, close-up view of the surface representation of takinib in the ATP binding pocket of TAK1 and modeled into IRAK-4. c, amino acid sequence alignment of TAK1, IRAK-4, and IRAK-1 highlighting key amino acid differences.

Figure 2.

Structure–activity studies of takinib analogs against IRAK-4. a, core aminobenzimidazole scaffold showing regions targeted for modification. b, outline of modifications made during the SAR campaign and corresponding IC₅₀ determined by mean IRAK-4 radioactive [³²P]ATP filter-binding assay inhibition from vehicle. c, titrations of takinib analogs against IRAK-4, presented as percentages of inhibition from vehicle control. The data points represent means ± S.E. (n = 2). d, comparison of percentages of inhibition of DMSO, takinib (1 μm), and HS-243 (1 μm) against IRAK-4. e, dose-dependent response of HS-243 compared with commercially available Sigma IRAK-1/4 compound. The data points represent means ± S.E. (n = 2).

Figure 3.

Structure and in vitro profiling of lead compound and other analogs against IRAK-4 and TAK1. a, structures of HS-243 IRAK-1/4 inhibitor, HS-242 dual TAK1/IRAK-4 inhibitor, HS-206 (takinib) TAK1 inhibitor, and HS-238 mixed TAK1/IRAK-4 inhibitor. b–e, data show kinase inhibition generated for each indicated analog against purified IRAK-4 and TAK1. The data points represent means ± S.E. (n = 2).

Figure 4.

a, docking pose of HS-243 in the ATP binding pocket of IRAK-4. π–π stacking interactions between HS-243 and Tyr-262 are in pink dots. Other interacting residues are labeled. Hydrogen bonds are shown with green dashes. b, comparison with TAK1 (PDB code 5V5N). c, kinase activity of IRAK-4 (WT), IRAK-4 (Y262A), and IRAK-4 (Y262T) tyrosine gatekeeper mutants. d, titrations of HS-243 against purified IRAK-4 Tyr-262 mutants (n = 2).

Figure 5.

Selectivity of HS-243 within the human kinome. a, kinome-wide screening of HS-243 against 468 human kinases. b, percentage of inhibition of top kinase hits from kinase screen (percentage of control from vehicle). c, dendrogram of human nononcogenic kinases show selectivity of HS-243 in the human kinome. The size of the dot is indicative of kinase potency. d, IC₅₀ values of top five kinases as determined by DiscoverX kinase assay. The data points represent means ± S.E. (n = 2). e, IC₅₀ values of HS-243 against IRAK-4, IRAK-1, TAK1, CLK4, and DYKR1B (n = 2).

Figure 6.

IRAK-1/4 inhibition has distinct cytokine profile from TAK1. Human RA-FLSs were stimulated with LPS and treated with either DMSO, HS-243, Sigma 1/4, takinib, or HS-242 at 10 μm for 24 h before cytokine profiling. a, representative cytokine proteome arrays with indicted cytokine positioning. b–e, quantification of CCL5 (b), CXCL12 (c), MIF (d), and IL-18 (e) expression. *, differences between DMSO and treatment; #, difference from HS-243. The data points represent means ± S.E. (n = 4). f, heat map of 30 cytokines/chemokines expression. g, schematic of IRAK-1/4 and TAK1 signaling. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7.

THP-1 human macrophages were differentiated with PMA for 72 h followed by a 24-h rest period in PMA-free medium. The cells were stimulated with LPS (10 ng/ml) immediately followed by 10 μm HS-243. 105 cytokines/chemokines were profiled from the supernatant of the media. a and b, significant changes seen in MIP-3a, Tim-3, uPAR, RANTES, Osteopontin, MMP-9, MIP-1a, MCP-1, I-TAC, IL-8, GDF-15, IP-10, and chitinase-3–like 1 protein expression (n = 3–4). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 8.

Percentage of survival of IRAK-1/4 inhibited cancer cells. Shown are the percentages of survival of seven cancer cell lines treated for 24 h in the presence of 10 μm HS-243 or HS-243 (10 μm) and IL-1β (30 ng/ml). The data points represent means (n = 2–4).