2.3. In vitro kinase assays

Compounds were assayed on Latha screen Assay (Invitrogen, PV4813) against B‐RAF kinase with ATP concentration at K _M according to the manufacturer's instructions. The compounds were tested from 10 μM, fourfold dilution, 10 points. B‐RAF kinase was prepared in 1 × kinase buffer (50‐mM HEPES, pH 7.5, 0.01% BRIJ‐35, 10‐mM MgCl₂, 1‐mM EGTA). The substrate solution of Fluorescein‐MAP 2K1 and ATP in 1 × kinase reaction buffer was prepared at fourfold of the final concentration of each reagent desired in the assay. This substrate solution was added to each well of the assay plate to start the reaction. the assay plate was covered and incubated at room temperature (25^oC) for 1 hr. The detection solution was prepared at double the final concentration in antibody dilution buffer and added (10 μl) to each well, mixed briefly, and incubated at 25^oC, for at least 30 min in the dark before reading the fluorescence on a plate reader.

The binding affinities of compounds for RIPK1 and RIPK3 kinase were measured by KINOMEscan™ assay. Briefly, the RIPK1 and RIPK3 kinase‐tagged T7 phage strains lysate was tagged with DNA for qPCR detection. Binding reaction mixtures were assembled by combining kinases, liganded affinity beads and test compounds in 1× binding buffer (20% SeaBlock, 0.17 × PBS, 0.05% Tween 20, 6‐mM DTT). Compounds were prepared as 10 mM in 100% DMSO. Equilibrium K _Ds were determined using a 10‐point threefold compound dilution series with three DMSO control points. All reactions were performed in polypropylene 384‐well plate. The assay plates were incubated at room temperature with shaking for 1 hr, and the affinity beads were washed with wash buffer (1× PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1× PBS, 0.05% Tween 20, 0.5‐μM non‐biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by qPCR.

The enzymic RIPK1 kinase assays were established by using the Kinase‐Glo Luminescent Kinase Assays Kit (Promega, Madison, WI). Briefly, the purified recombinant His‐hRIPK1 1–479 (150 nM, produced in our laboratory) was incubated with its substrate MBP (Invitrogen) in the kinase reaction buffer (50‐μM ATP, 100‐mM HEPES, pH 7.3, 5‐mM MgCl2, 5‐mM MnCl2, 750‐mM NaCl, 0.5% BSA) in the presence of increasing concentrations of TAK‐632. for 4 hr at room temperature. Luminescence was recorded with a BioTek 312e microplate reader (Winooski, VT) with integration time of 1 s.

The enzymic RIPK3 kinase assays were measured by Reaction Biology Corp (Malvern, PA) with a radiometric kinase method ([γ‐³²P]ATP) according to their online protocol (http://www.reactionbiology.com/webapps/site/).

KINOMEscan™ Profiling for TAK‐632 and SZM594 in a panel of 97 kinases distributed across the kinome was performed by DiscoverX (Fremont, CA).

Drug affinity responsive target stability assay (DARTS) experiments for identifying the targets of TAK‐632 were performed as previously reported (Lomenick et al., 2009). In brief, cells were lysed and treated with TAK‐632 (50 μM) followed by digestion with 0.01% Pronase for 30 min at room temperature. The digestion was stopped by directly adding SDS‐PAGE loading buffer and Pronase inactivated by boiling. Protein samples were separated with 4–20% SDS‐PAGE and analysed by immunoblotting.

The antibody‐based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology. Cells were collected and lysed in M2 buffer (20‐mM Tris, pH 7.0, 0.5% NP40, 250‐mM NaCl, 3‐mM EDTA, 3‐mM EGTA, 2‐mM DTT, 0.5‐mM PMSF, 20‐mM‐glycerol phosphate, 1‐mM sodium vanadate, 1 μg·ml⁻¹ of leupeptin). Cell lysates were separated by SDS‐PAGE and analysed by immunoblotting. The dilution of the antibodies used for western blotting is 1:1000. The proteins were visualized by enhanced chemiluminescence, according to the manufacturer's instruction (Amersham).

For immunoprecipitation analysis, cells were washed three times with ice‐cold PBS solution and were lysed in M2 buffer. The samples were precipitated with the indicated antibodies (1 μg) and protein A/G‐agarose beads (Santa Cruz) by incubating at 4°C overnight. Beads were washed four times with 1‐ml M2 buffer, and the bound proteins were removed by boiling in SDS buffer and resolved in 4–20% SDS‐PAGE for western blotting analysis.