A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data

Abstract

The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.

Figure 1. Activation of p38 MAPK in response to certain stimuli.

Dashed lines refer to one or more stimuli. Bold arrows refer to translocation of the kinase in response to activation by upstream kinases.

Figure 2. Cellular MK2-EGFP translocation assay to screen for p38 inhibitors.

(A) Schematical outline of MK2-EGFP translocation assay. Nuclear DAPI segmentation facilitates determination of the nucleocytoplasmic MK-2-EGFP fluorescence intensity ratio (grey ring). In the ground state U2OS cells stably expressing MK2-EGFP shows a slightly higher fluorescent intensity in the nucleus compared to the cytoplasm. For activation of the Rac/p38 pathway cells were subjected to hyperosmotic stress (175 mM NaCl/350 mOsm) leading to a rapid relocation of MK2-EGFP from the nucleus to the cytoplasm. Co-treatment with p38 inhibitors abolishes this redistribution, which is visualized in high content analysis. Determination of fluorescent intensities provides a cellular read-out with appropriate z’ values above 0.5. (B) Representative pictures of activated U2OS MK2-EGFP cells either non-treated (DMSO) or treated with p38 inhibitors Skepinone-L or SB203580, scale bar 20 µM. (C) Workflow of the p38 inhibitor screen, validation procedures and number of identified compounds (D) Sorted results of the entire primary p38 inhibitor screen represented as average cellular nucleocytoplasmic fluorescence intensity ratios reflecting the distribution of MK2-EGFP (E) Rank comparison of the first and second biological replica of the primary screen at 15 µM showing the reproducibility of the results. R² of >0.85 indicate reliability for further cherry-pick listing.

Figure 3. Validation of the screening results using the MK2-EGFP translocation assay at decreasing compound concentrations.

Confirmation of p38 inhibition of selected 378 cherry-picks at (A) 1.5 µM, (B) 150 nM, and (C) 15 nM. Green stripe: Skepinone-L top level bench mark of inhibition of nuclear export of MK2-EGFP. Yellow strip: nuclear intensity in activated cells treated with DMSO. Top 40 inhibitors are shown, including Skepinone-L and negative control (DMSO). (D) Comparison of lead inhibitor activity at 1.5 µM, 150 nM and 15 nM. Nucleocytoplasmic fluorescent ratio after treatment with Skepinone-L is set to 1. For direct comparison nucleocytoplasmic fluorescent ratios induced by all validated compounds are normalized to Skepinone-L.

Figure 4. Structures of identified top 10 inhibitors.

Figure 5. Comparison of imidazole derived inhibitor SB203580 with the dibenzosuberone derived inhibitor Skepinone-L in the MK2-EGFP translocation assay.

(A) Nucleocytoplasmic intensity ratios of activated U2OS MK2-EGFP cells after incubation with increasing concentrations (0 µM –100 µM) of SB203580 (blue) or Skepinone-L (red) Incubation with Skepinone-L induces an increased nucleocytoplasmic ratio of <1 up to 10 nM of compound concentration indicating a strong inhibitory effect on p38 MAPK. (B) Representative cellular images of the selected data points (scale bar: 20 µm).

Figure 6. Phospho-Ser63-c-Jun/JNK kinase assay in living cells.

(Left) representative pictures of phospho-Ser63-c-Jun antibody signal in the nucleus upon pathway activation. (Right) Top ten lead inhibitors identified from the MK2-EGFP translocation screen have negligible effects on JNK/phosphor-c-jun activity at relevant concentrations (<15 µM). Significant effects (* p<0.05) for several p38 inhibitors are detected at very high concentrations (> = 150 µM). As positive control a specific JNK inhibitor SP600125 is shown. SD derived from 3 independent experiments.

Figure 7. Determination of cellular toxicity of top ten lead inhibitors.

Analysis of metabolic viabilities using the AlamarBlue assay after incubation of the cells with the top ten p38 inhibitors for 24 µM. Incubation with DMSO serves as negative control. Shown is standard deviation derived from 3 independent experiments.