TBK1/IKKε Screening Cascade

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKε) have been validated as novel drug targets in inflammatory diseases such as psoriasis, rheumatoid arthritis and COPD. Kinase activity assays were established for both TBK1 and IKKε in 384 well format whereby reactions were performed at ATP Km and initial rate velocity. Structural and kinase activity data was generated to support medicinal chemistry with the result that low nanomolar compounds were identified (Figure 1). During further lead characterisation we demonstrated inhibition of the secreted cytokine IP10 in monocyte cell line THP1 following stimulation of the cells with lipopolysaccharide (LPS) (Figure 2). Mechanistic analysis demonstrated IP10 promoter IRF3 phosphorylation was inhibited by a TBK1 inhibitor in a concentration dependent manner (Figure 3). Whole blood taken from healthy human donors was stimulated with LPS and IP10 secretion measured using AlphaLISA. At an established fixed concentration of LPS, TBK1 IC50 values were resolved and demonstrated submicromolar potency (Figures 4a &b).

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Figure 1: Biochemical profiling of TBK1 inhibitors and assessment of ATP sensitivity. TBK1/IKKε inhibitors were titrated and incubated with TBK1 purified recombinant protein. Enzymatic reaction was initiated by the addition of peptide substrate at either Km or 30 times Km ATP levels. Assay was stopped after 2hrs and IC50 values generated using four parameter fit.


Figure 2: Inhibition profile of TBK1 inhibitors against LPS stimulated IP10 secretion. THP1 cells were pre-incubated for 1hr with a serial dilution of DMX compounds. Cells were subsequently stimulated with 0.1μg/ml LPS for 24hrs and the supernatant collected for IP10 assessment by AlphaLISA. Data was normalised to no stimulation (100%) and stimulation only (0%).


Figure 3: Effect of TBK1 inhibitor DMX on LPS stimulated IRF phosphorylation. THP1 monocytes were differentiated into macrophages by addition of PMA. IRF3 phosphorylation was stimulated after the addition of LPS. Levels of total and phosphorylated IRF3 were detected using Western blotting techniques. Figure shows total IRF3 is present in all samples and inhibition of phosphorylated IRF3 relative to total IRF3 with increasing concentrations of DMX following LPS stimulation.


LPS dose response effect on stimulation of IP10 in human whole blood from 3 human donors (D1, D2 and D3)

Figure 4a: LPS dose response effect on stimulation of IP10 in human whole blood from 3 human donors (D1, D2 and D3). Whole Blood was sourced from three healthy donors and treated with a serial dilution of LPS for 24hrs. IP10 levels were measured using AlphaLISA techniques and fold induction over unstimulated resolved. 1μg/ml LPS was selected for further experiments.


Figure 4b: Inhibition profile of DMX compound on LPS-stimulated IP10 secretion in human whole blood. Whole blood was pre-incubated for 1hr with a serial dilution of DMX compound. Subsequently stimulated with 1μg/ml LPS for 24hrs and the supernatant collected for AlphaLISA IP10 assessment. Data was normalised to no stimulation (100%) and stimulation only (0%).