Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 May 6;176(12):2095–2108. doi: 10.1111/bph.14653

Identification of the Raf kinase inhibitor TAK‐632 and its analogues as potent inhibitors of necroptosis by targeting RIPK1 and RIPK3

Xiaofei Chen 1,2,3,[Link], Chunlin Zhuang 3,4,[Link],✉, Yibin Ren 1,[Link], Hao Zhang 4, Xia Qin 1,2, Longmiao Hu 1,2, Jing Fu 1,2, Zhenyuan Miao 3, Yifeng Chai 3, Zheng‐gang Liu 5, Haibing Zhang 6, Zhenyu Cai 1,2,7,✉, Hong‐yang Wang 1,2,7,✉
PMCID: PMC6534794  PMID: 30825190

Abstract

Background and Purpose

Necroptosis is a form of programmed, caspase‐independent, cell death, mediated by receptor‐interacting protein kinases, RIPK1 and RIPK3, and the mixed lineage kinase domain‐like (MLKL). Necroptosis contributes to the pathophysiology of various inflammatory, infectious, and degenerative diseases. Thus, identification of low MW inhibitors for necroptosis has broad therapeutic relevance. Here, we identified that the pan‐Raf inhibitor TAK‐632 was also an inhibitor of necroptosis. We have further generated a more selective, highly potent analogue of TAK‐632 by targeting RIPK1 and RIPK3.

Experimental Approach

Cell viability was measured by MTT, propidium staining, or CellTiter‐Glo luminescent assays. Effects of TAK‐632 on necroptosis signalling pathways were investigated by western blotting, immunoprecipitation, and in vitro kinase assays. Downstream targets of TAK‐632 were identified by a drug affinity responsive target stability assay and a pull‐down assay with biotinylated TAK‐632. A mouse model of TNF‐α‐induced systemic inflammatory response syndrome (SIRS) was further used to explore the role of TAK‐632 in protecting against necroptosis‐associated inflammation in vivo.

Key Results

TAK‐632 protected against necroptosis in human and mouse cells but did not protect cells from apoptosis. TAK‐632 directly bound with RIPK1 and RIPK3 to inhibit kinase activities of both enzymes. In vivo, TAK‐632 alleviated TNF‐induced SIRS. Furthermore, we performed a structure–activity relationship analysis of TAK‐632 analogues and generated SZM594, a highly potent inhibitor of RIPK1/3.

Conclusions and Implications

TAK‐632 is an inhibitor of necroptosis and represents a new lead compound in the development of highly potent inhibitors of RIPK1 and RIPK3.

Abbreviations

DARTS

drug affinity responsive target stability assay

Nec‐1

necrostatin‐1

SAR

structure–activity relationship

SIRS

systemic inflammatory response syndrome

What is already known

  • Necroptosis is a form of programmed cell death with necrotic‐like morphology.

  • Two serine/threonine kinases, RIPK1 and RIPK3, are central components of the necroptotic machinery.

What this study adds

  • TAK‐632 and its analogues inhibit necroptosis by functioning as dual kinase inhibitors for RIPK1/RIPK3

What is the clinical significance

  • TAK‐632 and its analogues could be promising candidates for the treatment of necroptosis‐associated pathologies

1. INTRODUCTION

Necroptosis is a programmed necrosis characterized by cell swelling, plasma membrane rupture, and subsequent loss of intracellular contents to release damage‐associated molecular patterns, thereby triggering inflammatory responses in vivo (Galluzzi, Kepp, Chan, & Kroemer, 2017; Sarhan, Land, Tonnus, Hugo, & Linkermann, 2018). Recent studies suggest that necroptosis is involved in a variety of pathological processes including infectious diseases, ischaemia–reperfusion injury, atherosclerosis, hepatitis, inflammatory bowel diseases, and other inflammatory clinical disorders (Kaczmarek, Vandenabeele, & Krysko, 2013; Weinlich, Oberst, Beere, & Green, 2017). Necroptosis can be triggered by the engagement of death receptors, such as TNF receptor 1, CD95 (FAS), Toll‐like receptors (including TLR3 and TLR4), or IFN receptors (Grootjans, Vanden Berghe, & Vandenabeele, 2017). Downstream necroptotic signals induced by these receptors lead to formation of the necrosome. RIPK1 or other RIP homotypic interaction motif domain‐containing proteins interact with RIPK3 to initiate the formation of the necrosome and activate RIPK3 through phosphorylation (Cho et al., 2009; He et al., 2009). The activated RIPK3 subsequently recruits and phosphorylates another kinase, mixed lineage kinase domain‐like (MLKL; Sun et al., 2012; Zhao et al., 2012). The phosphorylated MLKL oligomerizes and translocates to the plasma membrane to trigger membrane rupture (Cai et al., 2014; Chen et al., 2014; Dondelinger et al., 2014; Wang et al., 2014). It is now known that these two serine/threonine kinases, RIPK1 and RIPK3, together with MLKL constitute the core of the necroptosis machinery. Thus, identification of these essential factors in necroptotic signalling pathway provides potential drug targets for therapeutic intervention in necroptosis‐associated diseases.

The first identified inhibitor of necroptosis was necrostatin‐1 (Nec‐1; Degterev et al., 2005). By targeting RIPK1, Nec‐1 provides a valuable tool to empirically dissect the necroptosis pathway (Degterev et al., 2008). However, its poor metabolic stability (t 1/2 < 5 min) limits its further application in drug discovery (Teng et al., 2005). In addition, Nec‐1 and other highly potent RIPK1 inhibitors cannot block RIPK3‐independent necroptosis (Cai & Liu, 2014). With the discovery of two other kinases, RIPK3 and MLKL, downstream of RIPK1, compounds targeting these two essential components in necroptosis pathway were developed (Degterev & Linkermann, 2016; Li, Qian, & Yuan, 2017). However, few of them have been applied to potential medical uses to date. Thus, identification of novel inhibitors of necroptosis is critical for the development of new drugs associated with this pathway. In this study, we found that the pan‐Raf inhibitor TAK‐632 was a novel inhibitor of necroptosis, and we further used the TAK‐632 scaffold to develop new, highly potent and selective inhibitors of RIPK1 and RIPK3.

2. METHODS

2.1. Cell culture and transfection

HT‐29 (NCI‐DTP Cat# HT‐29, RRID:CVCL_0320), L929 (ECACC Cat# 14112101, RRID:CVCL_AR58), J774A.1 (ICLC Cat# ATL98011, RRID:CVCL_0358), and HEK293T (ICLC Cat# HTL04001, RRID:CVCL_0063) cells were cultured in DMEM. THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) and U937 (CLS Cat# 300368/p474_U‐937, RRID:CVCL_0007) cells were cultured in RPMI 1640; these cell lines were obtained from the ATCC (Manassasa,VA). Wild‐type (WT) and RIPK1 or RIPK3‐deficient mouse embryo fibroblast (MEF) cells were kindly provided by Haibing Zhang (Chinese Academy of Sciences, China) and cultured in DMEM. All media were supplemented with 10% FBS (v/v), 2 mM of L‐glutamine and 100 U·ml−1 of penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere with 5% CO2 and were harvested in all experiments from exponentially growing cultures.

The plasmids were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol in HEK293T cells. After 24 hr, the cell lysates were analysed by immunoblotting or immunoprecipitation.

2.2. Necroptosis induction and cell viability assays

Necroptosis was induced by pretreatment with z‐VAD‐fmk (20 μM) and Smac mimetic (10 nM) for 30 min and followed by TNF‐α (20 ng·ml−1) for 16 hr. For MEF cells, necroptosis was induced by Poly (I:C) 100 ng·ml−1 and z‐VAD‐fmk (40 μM) for 48 hr. Apoptosis was induced by TNF‐α (20 ng·ml−1) and Smac mimetic (10 nM) for 16 hr. The compounds were incubated with the cells exposed to one of the above combinations at the indicated concentrations for 16 hr. Cell viability was then examined by using the CellTiter‐Glo Luminescent Cell Viability Assay kit (Promega) or MTT assay. Luminescence or absorbance was recorded with a BioTek 312e microplate reader (BioTek Instruments, Winooski, VT). For FACS, cells were trypsinized, collected by centrifugation, washed once with PBS buffer and then resuspended in PBS containing 5 μg·ml−1 of PI. The proportions of PI‐negative cells were quantified with BD FACSCalibur (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401).

2.3. In vitro kinase assays

2.3.1. B‐RAF kinase assay

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 (25oC) 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 25oC, for at least 30 min in the dark before reading the fluorescence on a plate reader.

2.3.2. RIPK1 and RIPK3 kinase assay

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).

2.3.4. Drug affinity responsive target stability assay

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.

2.3.5. Immunoblotting and immunoprecipitation

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.

2.6. Pull‐down analysis with biotinylated TAK‐632

Pull down of biotinylated TAK‐632 was performed by adding DMSO or biotinylated TAK‐632 (100 and 500 μM) to HT‐29 cell lysates and incubating with Streptavidin Agarose Resins (Pierce, Catalogue Number: 20347). After incubation with cell lysates, streptavidin resins were washed with ice‐cold M2 lysis buffer for one time and ice‐cold PBS (0.1‐M phosphate and 0.15‐M NaCl) for three times. The resins were eluted by directly adding 2× SDS‐PAGE loading buffer and were analysed by immunoblotting.

2.6.7. Animal experiments

All animal care and experimental procedures complied with the National Institutes of Health guidelines and were approved by the animal care and use committee of the Second Military Medical University. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. For TNF‐induced systemic inflammatory response syndrome (SIRS), female C57BL/6 J mice (6–8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and raised in a pathogen‐free environment (23 ± 2°C and 55 ± 5% humidity). Compounds were suspended in distilled water containing 0.5% carboxymethyl cellulose sodium. Overnight‐fasted mice were randomly divided into vehicle and treatment groups (n = 10 for each group). In drug treatment groups, mice were given TAK‐632 or its analogues with different concentrations by p.o. gavage 2 hr before TNF injection. mTNF‐α was diluted in endotoxin‐free PBS and injected i.v. (250 or 400 μg·kg−1) in a volume of 200 μl. Z‐VAD‐fmk was given i.p. (200 μg) 15 min before and 75 μg 1 hr after mTNF‐α treatment. Body temperature was monitored with an electric thermometer. Serum was collected at 6 hr after injection and IL‐6 was determined by an IL‐6 elisa kit (Dakewe Biology Co., Beijing, China). At the end of the experiment, mice were killed with an overdose of anaesthetic (i.v. injection of 1.2% avertin solution; 0.2 ml per 10 g).

2.6.8. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. Results are presented as means ± SEM. Student's t‐test and one‐way ANOVA were used for comparison among the different groups. The log‐rank (Mantel‐Cox) test was performed for survival curve analysis using GraphPad Prism 7.00 (RRID:SCR_002798). P < 0.05 was considered statistically significant.

2.9. Materials

TAK‐632 (CAS#1228591–30‐7) was purchased from MedChemExpress (Monmouth Junction, NJ). Recombinant mouse/human TNF‐α and z‐VAD‐fmk were purchased from R&D System (Minneapolis, MN). Protease inhibitor cocktail were purchased from Sigma‐Aldrich (St. Louis, MO). Smac mimetic (SM‐164) was a gift from Dr Zheng‐gang Liu (NCI, NIH). Antibodies were from commercial sources: anti‐RIPK1 (BD Biosciences Cat# 610458, RRID:AB_397831); anti‐human phospho‐RIPK1 (Cell Signaling Technology, Cat# 65746); anti‐mouse phospho‐RIPK1 (Abcam Cat# ab195117, RRID:AB_276815); anti‐mouse RIPK3 (Sigma‐Aldrich Cat# PRS2283, RRID:AB_1856303); anti‐human‐RIPK3 (Abcam Cat# ab56164, RRID:AB_2178667); anti‐human phospho‐RIPK3 (Abcam Cat# ab209384, RRID:AB_2714035); anti‐human MLKL (Abcam Cat# ab184718, RRID:AB_2755030); anti‐human phospho‐MLKL (Abcam Cat# ab187091, RRID:AB_2619685); anti‐mouse phospho‐RIPK3 (Abcam Cat# ab222320); anti‐mouse phospho‐MLKL (Abcam Cat# ab196436, RRID:AB_2687465); and anti‐actin (Sigma‐Aldrich Cat# A3853, RRID:AB_262137) from Sigma. pFLAG‐hRIPK1, pcDNA4/V5‐hRIPK3, and pCMV‐Tag2A/FLAG‐hMLKL were kindly provided by Zheng‐gang Liu (NCI, NIH).

2.10. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a, b).

3. RESULTS

3.4. Drug screening identifies the pan‐Raf inhibitor TAK‐632 as a necroptosis inhibitor

Fluorination is an important strategy in drug design, and fluorinated compounds constitute a substantial proportion of therapeutically useful drugs (Gillis, Eastman, Hill, Donnelly, & Meanwell, 2015; Meanwell, 2018; Richardson, 2016). To identify novel necroptosis inhibitors with potential clinical application, a panel of 500 compounds containing fluorine was screened for their ability to block TNF‐α‐induced necroptosis. HT‐29 cells were treated with TNF‐α, Smac mimetic, and caspase inhibitor z‐VAD‐FMK (TSZ) to induce necroptosis, and the cell survival was measured by MTT assay for each compound (Figure 1a). Among the drugs investigated, the pan‐Raf kinase inhibitor TAK‐632 (Okaniwa et al., 2013) clearly protected HT‐29 cells from TSZ‐induced necroptosis (Figure 1b). In addition, the necroptosis inhibitor dabrafenib was also identified in our screen, indicating the validity of our experimental screen (Figure 1b). Phase contrast microscopy further revealed that TAK‐632 prevented TSZ‐induced necrotic morphology, such as cell swelling and plasma membrane rupture (Figure 1c). Remarkably, TAK‐632 did not protect cells from TNF‐α plus Smac mimetic (TS) or cycloheximide (TC)‐induced apoptosis in both HT‐29 and MEF cells, suggesting that TAK‐632 specifically inhibited the necroptosis pathway (Figure 1d and Supporting Information Figure S1). We then performed a dose–response assay to quantitatively analyse the inhibitory potency of TAK‐632. TAK‐632 dose‐dependently inhibited TSZ or TNF‐α, cycloheximide, and z‐VAD‐FMK (TCZ)‐induced necroptosis (Figure 1e,f). We then tested the inhibitory effect of TAK‐632 on other human and murine cells. TAK‐632 efficiently inhibited TSZ‐induced necroptosis in both human THP‐1 and U937 cells (Figure 2a,b). In murine L929 and J774A.1 cells, TAK‐632 dose‐dependently protected against necroptosis induced by TNF‐α and z‐VAD‐FMK (TZ; Figure 2c,d). Furthermore, TAK‐632 also prevented Fas‐induced necroptosis in HT‐29 cells and RIPK1‐independent necroptosis induced by Poly (I:C) plus z‐VAD‐fmk in RIPK1‐deficient MEF cells (Dillon et al., 2014; Figure 2e,f). Thus, our results suggest that TAK‐632 is a potent and effective necroptosis inhibitor.

Figure 1.

Figure 1

Open in a new tab

Identification of TAK‐632 as a potent necroptosis inhibitor. (a) Schematic overview of drug screen workflow. (b) Identification of necroptosis inhibitor by cellular screen with fluorinated compounds library. HT‐29 cells were pretreated with each compound (20 μM) for 30 min and then stimulated with TSZ for 16 hr to induce necroptosis. Cell survival was determined by MTT assay and normalized to untreated control cells. (c) Representative images (10×) of HT‐29 cells pretreated with DMSO or TAK‐632 (20 μM) followed by stimulation with TNF‐α (20 ng·ml−1), Smac mimetic (10 nM), and z‐VAD‐fmk (20 μM; TSZ) for 16 hr. (d) HT‐29 cells were pretreated with DMSO or TAK‐632(20 μM) followed by stimulation with TSZ for 16 hr or TNF‐α (20 ng·ml−1) plus Smac mimetic (10 nM; TS) for 24 hr. The percentage of PI‐negative cells was determined by flow cytometry after PI staining. (e) HT‐29 cells were pretreated with DMSO or TAK‐632 at the indicated concentrations followed by stimulation with TSZ for 16 hr; the percentage of PI‐negative cells was determined by flow cytometry. The EC50 of TAK‐632 was calculated as 1.44 μM. (f) HT‐29 cells were treated with various concentrations of TAK‐632, as indicated, by stimulation with TNF‐α (20 ng·ml−1), cycloheximide (5 μg·ml−1), and z‐VAD‐fmk (20 μM; TCZ) for 16 hr, and the percentage of PI‐negative cells was determined by flow cytometry. Results shown are means ± SD from five independent experiments. *P < 0.05, significant effect of TAK‐632 treatment

Figure 2.

Figure 2

Open in a new tab

TAK‐632 blocks necroptosis in both human and murine cell lines. (a) THP‐1 or (b) U937 cells were pretreated with DMSO or TAK‐632 at the indicated concentrations followed by stimulation with TSZ for 16 hr. (c) L929 or (d) J774A.1 cells were pretreated with DMSO or TAK‐632 followed by stimulation with TZ for 16 hr. (e) HT‐29 cells were pretreated with DMSO or TAK‐632 followed by stimulation with SZ plus Fas ligand (50 ng·ml−1) for 16 hr. Cell death was determined by flow cytometry with PI staining. (f) RIPK1‐deficient MEF cells were treated with TAK‐632 followed by stimulation with Poly (I:C) 100 ng·ml−1 and z‐VAD‐fmk 40 μM for 48 hr. Cell viability was analysed by CellTiter‐Glo luminescent assay. Results shown are means ± SD from five independent experiments. *P < 0.05, significant effect of TAK‐632 treatment

3.5. TAK‐632 blocks necrosome formation by inhibiting the phosphorylation of RIPK1 and RIPK3

To investigate the inhibitory mechanism of TAK‐632 in necroptosis, we first examined whether TNF‐induced activation of NF‐κB was affected. As shown in Figure 3a, pretreatment with TAK‐632 did not influence the degradation of IκBα after TNF‐α stimulation, indicating that TAK‐632 had no effect on the TNF‐activated NF‐κB signalling pathway. However, TAK‐632 selectively inhibited the phosphorylation RIPK1, RIPK3, and MLKL when necroptosis was induced by TSZ in HT‐29 cells (Figure 3b). Similar results were also obtained in murine L929 cells (Figure 3c). As the phosphorylation of RIPK1 and RIPK3 is essential for RIPK1‐RIPK3 necrosome formation (Cho et al., 2009; He et al., 2009), we then examined the necrosome formation in TAK‐632‐treated cells and we found that TAK‐632 blocked TSZ‐induced necrosome formation (Figure 3d). In order to test whether TAK‐632 directly inhibited RIPK1‐RIPK3 or RIPK3‐MLKL interaction, we co‐expressed RIPK3‐V5 and FLAG‐RIPK1 or FLAG‐MLKL in HEK293T cells and did co‐immunoprecipitation assays with or without TAK‐632. We found that TAK‐632 not only prevented necrosome formation of RIPK1‐RIPK3 but also inhibited the interaction between RIPK3 and MLKL (Figure 3e,f). Thus, these results indicate that TAK‐632 inhibits TSZ‐induced phosphorylation of RIPK1/3 and disrupts the formation of the RIPK1‐RIPK3 and RIPK3‐MLKL complex.

Figure 3.

Figure 3

Open in a new tab

TAK‐632 inhibits phosphorylation of RIPK1/3 and blocks necrosome formation. (a) HT‐29 cells were pretreated with TAK‐632 (10 μM) for 30 min followed by stimulation with TNF‐α (20 ng·ml−1) at the indicated time points. Cells were lysed and immunoblotted with the anti‐IκBα antibody. (b) HT‐29 cells were pretreated with TAK‐632 (10 μM) followed by stimulation with TSZ at the indicated time points. Cells were lysed and immunoblotted with the indicated antibodies. (c) L929 cells were pretreated with TAK‐632 (5 μM) followed by stimulation with mTNF‐α (20 ng) plus z‐VAD‐FMK (20 μM; TZ) at the indicated time points. Cells were lysed and immunoblotted with the indicated antibodies. (d) HT‐29 cells were treated with DMSO or TAK‐632 (10 μM) for 4 hr. Cell lysates were immunoprecipitated with anti‐RIPK1 antibody (IP: RIPK1) and analysed by immunoblotting with indicated antibodies. (e) HEK293T cells were transfected with RIP3‐V5 and FLAG‐RIPK1 or (f) FLAG‐MLKL as indicated. After 24 hr, cell lysates were immunoprecipitated with anti‐FLAG antibody (IP: FLAG) and analysed by immunoblotting with the indicated antibodies. All western data are representative of five independent experiments

3.6. TAK‐632 is a dual kinase inhibitor for RIPK1 and RIPK3

TAK‐632 was previously identified as a pan‐Raf inhibitor. However, Raf kinase is not involved in necroptosis signalling pathway, suggesting that TAK‐632 protects cells from necroptosis, independently of Raf inhibition (Li et al., 2014; Martens et al., 2017). As TAK‐632 directly disrupts RIPK1‐RIPK3 necrosome formation, we then questioned whether TAK‐632 directly interacts with RIPK1 or RIPK3. We first employed a DARTS (Lomenick et al., 2009), which relies on the reduction of protease susceptibility of the target protein upon drug binding, to detect the potential interaction between TAK‐632 and RIPK1/3 kinases. As shown in Figure 4a, RIPK1 and RIPK3 were both protected from protease digestion in the extracts of TAK‐632 treated cells, whereas no protection of MLKL was detected in the same sample, suggesting that TAK‐632 may interact with both RIPK1 and RIPK3. We then synthesized a biotinylated TAK‐632 by substituting a long‐chain biotin for its cyclopropyl group (see Supporting Information). This biotinylated TAK‐632 inhibited TNF‐induced necroptosis at higher concentrations, although it showed cytotoxic effect on HT‐29 cells (Supporting Information Figure S2). In order to confirm the interactions between TAK‐632 and RIPK1/3, a pull‐down assay was performed by using the biotinylated TAK‐632 in unstimulated HT‐29 cell lysates. As shown in Figure 4b, RIPK1 and RIPK3 were both pulled down by the biotinylated TAK‐632, while MLKL was not in the same complex, suggesting the specificity of the interactions. Thus, our data demonstrate that TAK‐632 binds to RIPK1 and RIPK3 in TSZ‐unstimulated cells.

Figure 4.

Figure 4

Open in a new tab

TAK‐632 directly binds to RIPK1 and 3 and inhibits their kinase activity. (a) DARTS analysis for TAK‐632. HT‐29 cell lysates were incubated with or without TAK‐632 (50 μM) for 1 hr and then digested with 0.01% Pronase for 30 min. Cells were lysed and immunoblotted with the indicated antibodies. (b) Streptavidin pull down of biotinylated TAK‐632. Biotinylated TAK‐632 was added to HT‐29 cell lysate and pulled down with streptavidin agaroses. Both pull‐down samples and total lysates (Input) were analysed by SDS‐PAGE and immunoblotted with the indicated antibodies. (c) HEK293T cells were transfected with FLAG‐RIPK1 or (d) RIPK3‐V5. After 12 hr, the cells were treated with TAK‐632 at the indicated concentrations, for 6 hr. Cell lysates were then analysed by SDS‐PAGE and immunoblotted with the indicated antibodies. All western data are representative of five independent experiments. (e) K D values against recombinant RIPK1 and (f) RIPK3 kinase were determined by KINOMEscan™ assay using 10‐point dose range (5 to 2 nM, five repetitions for each concentration) of TAK‐632. (g) Quantification of ADP‐Glo kinase assays performed with recombinant hRIPK1 in the presence of increasing concentrations of TAK‐632. (h) Quantification of radiometric kinase assays ([γ‐32P]ATP) performed with recombinant hRIPK1 in the presence of increasing concentrations of TAK‐632

As TAK‐632 interacts with RIPK1 and RIPK3, we then questioned whether TAK‐632 inhibited kinase activities of RIPK1 and RIPK3. Previous studies demonstrated that RIPK1 and RIPK3 were auto‐phosphorylated when overexpressed in the cells. We found that TAK‐632 inhibited auto‐phosphorylation of RIPK1 and RIPK3 in HEK293T cells, indicating that TAK‐632 may inhibit kinase activities of both (Figure 4c,d). Furthermore, we tested whether TAK‐632 directly inhibited the kinase activities of RIPK1 or RIPK3. First, by incubating specific DNA‐tagged RIPK1 or RIPK3 with TAK‐632 in vitro, we found that TAK‐632 dose‐dependently inhibited the ligand binding of RIPK1 and RIPK3 with the equilibrium K Ds as 480 and 105 nM respectively (Figure 4e,f). Second, by performing in vitro kinase assays with recombinant RIPK1 and RIPK3 proteins, we found that TAK‐632 strongly blocked kinase activities of RIPK1 and RIPK3 with IC50 values of 326 and 90 nM respectively (Figure 4g,h). Thus, our data illustrate that the pan‐Raf inhibitor TAK‐632 is also a dual kinase inhibitor for RIPK1 and RIPK3.

3.7. TAK‐632 protects mice from TNF‐induced SIRS

To explore whether TAK‐632 protects against RIP kinase‐driven inflammation in vivo, we tested it in the TNF‐induced SIRS model (Duprez et al., 2011). TAK‐632 (100 mg·kg−1) given by intragastric gavage 2 hr before i.v. injection of mTNF‐α, protected mice from hypothermia and death (Figure 5a,b). Furthermore, when these mice were examined at 6 hr, the serum levels of IL‐6 were markedly decreased by TAK‐632 treatment (Figure 5c). Thus, these results demonstrate that TAK‐632 protects against TNF‐induced SIRS in vivo.

Figure 5.

Figure 5

Open in a new tab

TAK‐632 protects mice from TNF‐induced SIRS. (a) C57BL/6 J mice were pretreated with or without TAK‐632 (100 mg·kg−1) and then induced SIRS with mTNFα (250 μg·kg−1) and z‐VAD‐fmk. The body temperature (means ± SEM) and (b) survival curve of the vehicle control and TAK‐632‐treated mice (n = 10 for each group) are shown. (c) After SIRS induction for 6 hr, serum levels of IL‐6 were determined by elisa. *P < 0.05, significantly different from vehicle control group

3.8. Identification of selective RIPK1/3 inhibitors from TAK‐632 analogues

In order to make TAK‐632 more selective towards RIPK1 and/or RIPK3, we first applied molecular docking analysis to examine the binding mode of TAK‐632 with RIPK1 and RIPK3. Details of this analysis are provided in the Supporting Information. Similar to the pyrrole group of necrostatin‐4 (Supporting Information Figure S3a), the trifluorophenyl group of TAK‐632 occupies the hydrophobic pocket (Lys45, Leu90, and Met92) of RIPK1 (Supporting Information Figure S3b). The phenoxy‐benzo[d]thiazol moiety of TAK‐632 perfectly inserts into an “open” loop in RIPK1, and the cyclopropyl group is solvent‐exposed (Supporting Information Figure S4a). Then, we generated a docked model of human RIPK3 and TAK‐632 based on the co‐crystal structure of B‐Raf with TAK‐632 (PDB ID: 4KSP). TAK‐632 was docked into the ATP‐binding pocket of homology‐modelled RIPK3. Similarly, the trifluorophenyl group of TAK‐632 occupied the hydrophobic pocket of RIPK3 (Lys127, Ala146, and Asn125; Supporting Information Figure S4b). The cyclopropane carboxamide moiety of TAK‐632 forms hydrogen bonds between the carbonyl of amide and the NH of His85 (2.0 Å).

The syntheses of the series of compounds derived from the structure of TAK‐632 are described in the Supporting Information. Based on the molecular docking studies summarised above, we first split TAK‐632 into two fragments (SZM580 and SZM582; see Table 1) and found that these two fragments lost all inhibitory activity for necroptosis, suggesting that the scaffold of TAK‐632 is critical for necroptosis inhibition. We then tested whether possible substituents in the TAK‐632 molecule would make it more selective towards RIPK1 and/or RIPK3. It has been reported that the cyano group of TAK‐632 is critical for B‐Raf kinase inhibition (Okaniwa et al., 2013). We then generated an analogue lacking the cyano group (SZM594). As expected, SZM594 showed approximately half the inhibition for B‐Raf kinase, while its inhibitory activity against RIPK1 and RIPK3 was greatly increased compared with TAK‐632 (Table 1). Consistent with the increased inhibition of RIPK1/3 kinases in vitro, the analogue SZM594 inhibited necroptosis at approximately eightfold lower concentrations, compared with TAK‐632 (Table 1). We then examined effects of the linker region between the phenylamine and trifluoromethylphenyl group of TAK‐632 on inhibitory activity of necroptosis. We found that several substituents (SZM597, SZM607, and SZM 613) in this position lost inhibitory activities for necroptosis (Table 1). Interestingly, introduction of a methylene in the linker region (SZM603) displayed approximately fourfold lower activity against B‐Raf kinase while retaining activities against RIPK1 and RIPK3, compared with TAK‐632 (Table 1). Furthermore, the EC50 of SZM603 for necroptosis inhibition was decreased about twofold compared with its parent TAK‐632.

Table 1.

Development of TAK‐632 analogues and their inhibitory activities against B‐Raf, RIPK1 and RIPK3

Compounds Structure TSZ‐induced necroptosis assay (HT‐29 cell, EC50, μM)a B‐RAF (IC50, μM, Latha screen Assay)b RIPK1 (K D, μM, KINOMEscan™) RIPK3 (K D, μM, KINOMEscan™)
TAK632
(1).

chemical structure image

Open in a new tab
 1.44 ± 0.62 0.158 0.48 0.11
SZM580
(2).

chemical structure image

Open in a new tab
 >50 n.d. n.d. n.d.
SZM582
(3).

chemical structure image

Open in a new tab
 >50 n.d. n.d. n.d.
SZM594
(4).

chemical structure image

Open in a new tab
 0.17 ± 0.03 0.322 0.097 0.077
SZM603
(5).

chemical structure image

Open in a new tab
 0.65 ± 0.11 0.659 0.51 0.79
SZM607
(6).

chemical structure image

Open in a new tab
 >50 n.d. n.d. n.d.
SZM613
(7).

chemical structure image

Open in a new tab
 >50 n.d. n.d. n.d.
SZM597
(8).

chemical structure image

Open in a new tab
 >50 n.d. n.d. n.d.
Open in a new tab
a

EC50s of necroptotic inhibitory effects are presented as mean ± SD from five independent experiments.

b

Results of in vitro kinase assays are presented as means of two independent experiments. n.d.: not determined.

Similar to TAK‐632, SZM594 and SZM603 also prevented RIPK1‐independent necroptosis induced by Poly (I:C) plus z‐VAD‐fmk in RIPK1‐deficient MEF cells (Supporting Information Figure S5), but these analogues did not protect HT‐29 and MEF cells from TS‐induced apoptosis (Supporting Information Figure S6). We then examined the cytotoxicity of TAK‐632 and its analogues in HT‐29 cells. TAK‐632 and its optimized analogues did not induce significant apoptosis at concentrations from 1 to 10 μM using a 72 hr incubation (Supporting Information Figure S7). As RIPK3 inhibitors were reported to induce RIPK1‐dependent apoptosis (Mandal et al., 2014), we further examined the toxicity of TAK‐632 and its analogues in WT and RIPK3‐deficient MEF cells. Compared with previously reported RIPK1/3 dual inhibitors, ponatinib and rebastinib (Fauster et al., 2015; Najjar et al., 2015), TAK‐632 and its analogues exhibited lower toxicity in both WT and RIPK3‐deficient MEF cells, as determined by their IC50 values (Supporting Information Figure S8). We then tested the selectivity of TAK‐632 and its analogue SZM594 against a panel of kinases taken from the human kinome. TAK‐632 exhibited more than 65% inhibition of 13 other kinases (out of a panel of 97 kinases that did not include RIPK1/3) when used in vitro at 1 μM. Although SZM594 showed lower inhibitory activity against B‐Raf kinase, its overall selectivity was not significantly improved in the human kinome screen (Supporting Information Figure S9 and Table S1).

Notably, SZM594 blocked necroptosis with higher efficiency than other classic known RIPK1 (Nec‐1), RIPK3 (GSK‐872), or MLKL (NSA‐1) inhibitors in HT‐29 cells (Figure 6a). We then compared the dose‐related responses to TAK‐632 and its analogue SZM594 in our TNF‐induced SIRS model. In this model, both compounds were given orally at 25 and 50 mg·kg−1, respectively, to investigate the differential protective effects. We found that SZM594 was more effective than TAK‐632 in protecting mice from death and hypothermia (Figure 6b,c). Thus, these results indicate that SZM594, an analogue of TAK‐632, exhibited increased protective activity against TNF‐induced SIRS model in vivo.

Figures 6.

Figures 6

Open in a new tab

SZM594 efficiently blocks necroptosis. (a) HT‐29 cells were pretreated with TAK‐632, TAK‐632 analogue SZM594 or SZM603, Nec‐1 (RIPK1 inhibitor), GSK‐872 (RIPK3 inhibitor), or NSA‐1 (MLKL inhibitor) as indicated concentrations and then followed by stimulation with TSZ for 16 hr. Cell viability was analysed by CellTiter‐Glo luminescent assay, and the results were presented as mean ± SD from five independent experiments. (b) C57BL/6 J mice were pretreated with or without SZM594 or TAK‐632 (50 or 25 mg·kg−1, n = 10 for each group) and then SIRS was induced with mTNFα (400 μg·kg−1) and z‐VAD‐fmk. The survival curve and (c) the body temperature changes (means ± SEM) for each group are shown. *P < 0.05, significantly different from vehicle control group

4. DISCUSSION

Although many compounds blocking necroptotic cell death have been synthesised that target the core components of necroptosis, RIPK1, RIPK3, and MLKL, few of them are in clinical use to date (Degterev & Linkermann, 2016). As fluorinated compounds constitute a substantial proportion of clinically used compounds (Gillis et al., 2015; Richardson, 2016), we screened our in‐house fluorinated compounds library and identified TAK‐632 as a potent necroptosis inhibitor. TAK‐632 was initially identified as a selective pan‐Raf inhibitor and inhibited the growth of B‐Raf mutated melanoma (Nakamura et al., 2013; Okaniwa et al., 2013). As the kinase domain of B‐Raf protein shares strong similarity to the kinase domains of RIPK1 and RIPK3 (Degterev et al., 2008; Xie et al., 2013), Raf inhibitors may suppress necroptosis by targeting RIPK1 or RIPK3. For example, the B‐RafV600E inhibitor dabrafeninb inhibits necroptosis by targeting RIPK3 (Li et al., 2014). The multi‐kinase inhibitor sorafenib was originally designed as a Raf1 inhibitor and it was recently shown to inhibit RIPK1 and RIPK3 (Martens et al., 2017). The kinome screen assay demonstrated that TAK‐632 targeted an additional 13 kinases other than RIPK1 and RIPK3 (Supporting Information Figure S9 and Table S1). Its inhibitory activity for necroptosis cannot be explained by these identified targets, as low MW compounds like vandetanib and vemurafenib with similar target profiles do not inhibit necroptosis (Fauster et al., 2015; Li et al., 2014). In addition, compounds that selectively target the known targets of TAK‐632, including Raf, VEGFR, PDGFR, and Src kinase (Supporting Information Table S1), did not inhibit necroptosis (Li et al., 2014; Martens et al., 2017). Furthermore, we showed that TAK‐632 inhibited the kinase activities of RIPK1 and RIPK3 by directly binding to these kinases. Thus, TAK‐632 is a dual kinase inhibitor for RIPK1 and RIPK3 which leads it to inhibit necroptosis.

It is worth noting that dabrafenib only displays selective inhibition of RIPK3 over RIPK1, while TAK‐632 and sorafenib exhibit inhibition of both RIPK1/3 kinases (Martens et al., 2017). As dabrafenib, sorafenib, and TAK‐632 are distinct from each other in chemical structure, structure–activity relationship (SAR) studies in Raf kinase inhibitors may help to differentiate the functional groups that contribute to Raf inhibition from those that contribute to inhibition of RIP kinases. In this regard, we performed a preliminary SAR study on the TAK‐632 structure and found that the cyano group and the linker region between phenylamine and trifluoromethylphenyl group of TAK‐632 were critical for inhibitory activities against B‐Raf, RIPK1, and RIPK3. As suggested already, the loss of cyano group will decrease the affinity for B‐Raf kinase (Okaniwa et al., 2013). We therefore generated a new analogue (SZM594) which lacked the cyano group, and found it to display an eightfold increased anti‐necroptosis effect with twofold less inhibitory activity for B‐Raf, compared with TAK‐632, representing a promising lead compound for further development of potent and selective RIPK1 or RIPK3 inhibitors (Table 1). Furthermore, we found that the linker region between the phenylamine and trifluoromethylphenyl groups must be a long‐chain structure, and any group inserted in this region would significantly decrease the inhibitory activities for RIPK1 and RIPK3, suggesting that the binding site of this region might be a long and narrow pocket on the surface of RIPK1 and RIPK3 kinases.

Recently, several clinically used drugs have been identified as necroptosis inhibitors. However, the high cytotoxicity and moderate inhibitory potential for necroptosis limit further application of these compounds to anti‐necroptotic drug discovery. For example, the anti‐cancer drugs ponatinib and sorafenib inhibited necroptosis and also showed high‐cytotoxic effect (Fauster et al., 2015; Martens et al., 2017). Phenytoin was recently identified as a necroptosis inhibitor, but it was much less potent than specific RIPK1 inhibitor Nec‐1 (von Massenhausen et al., 2018). In addition, several compounds in clinical trials are RIPK1 inhibitors but, so far, none have reached Phase III clinical trials (Harris et al., 2016; Harris et al., 2017; Li et al., 2017). Thus, characterization of novel, selective, highly efficient, and less toxic, low MW inhibitors of necroptosis not only contributes to basic research but also holds great promise for the treatment of clinical disorders associated with necroptotic cell death (Degterev & Linkermann, 2016).

In our experiments, TAK‐632 and its analogues exhibited very low cytotoxicity over the concentration range showing blockade of necroptosis in cells. Furthermore, unlike the RIPK1 inhibitor Nec‐1 or Nec‐1s that require i.v. administration in vivo (Takahashi et al., 2012), TAK‐632 has high metabolic stability after oral administration (Nakamura et al., 2013). We found that oral administration of TAK‐632 protected mice against TNF‐induced SIRS in vivo. Moreover, SZM594 was a much better inhibitor than TAK‐632 in the same model of SIRS, suggesting its potential for therapeutic application in necroptosis‐associated diseases.

However, several important issues concerning the in vivo use of TAK‐632 and its analogues as necroptosis inhibitors should be taken into account. First, TAK‐632 and SZM594 also target several kinases other than RIPK1 and RIPK3 (Supporting Information Figure S9). The existence of off‐target effects of TAK‐632 may affect its protective activity in necroinflammation in vivo. For example, Src kinase was identified as a target of TAK‐632 and could be activated during inflammation (Byeon et al., 2012). The double effects of TAK‐632 on RIPK and Src kinases may have important in vivo implications. As low MW compounds specifically targeting RIPK1 or RIPK3 have important therapeutic potential in pathological conditions, it is necessary to generate more derivatives of TAK‐632 in order to separate the specificity for RIPK1 and RIPK3 from that for other kinases. Second, the metabolites of TAK‐632 and its analogues, generated in vivo, may also affect necroinflammation. Thus, it would be worthwhile to investigate the metabolic stability and profile of TAK‐632 and its analogues.

In conclusion, our study not only identified that TAK‐632 was a dual kinase inhibitor for RIPK1 and RIPK3 but also revealed the possibility of increasing selectivity for RIPK1 or RIPK3 from the TAK‐632 structure. As necroptosis is involved in a wide spectrum of human disorders, this study provides an important basis for optimization in the development of necroptosis inhibitors that could be used as drugs.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Z.C., X.C., and C.Z. conceived and designed experiments. X.C., C.Z., Y.R., H.Z., X.Q., and L.H. performed experiments. Z.C., X.C., and C.Z. interpreted the data and wrote the original manuscript. J.F., Z.M., Y.C., Z.L., H.Z., and H.W. provided helpful discussions and refined the paper.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1

Supporting info item

Click here for additional data file. (3MB, pdf)

ACKNOWLEDGEMENTS

This work was supported by grants from China's 1000 Young Talents Program (Z. C.); the National Natural Science Foundation of China (81773075 to Z. C., 81503039 to X. C., and 81872791 to C. Z.); Shanghai International Cooperation and Exchange Project to Z. C. (18410720600); the General Financial Grant from China Postdoctoral Science Foundation (2017M613314 to X. C.); the “Yang‐Fan” project of Science and Technology Commission of Shanghai Municipality (15YF1400200 to X. C.); the Shanghai “Chen‐Guang” Project (16CG42 to C. Z.); the Shanghai Municipal Commission of Health and Family Planning (2017YQ052 to C. Z.); the Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (2017QNRC061 to C. Z.); and the Key Research and Development Program of Ningxia (2018BFH02001‐01 to C. Z.).

Chen X, Zhuang C, Ren Y, et al. Identification of the Raf kinase inhibitor TAK‐632 and its analogues as potent inhibitors of necroptosis by targeting RIPK1 and RIPK3. Br J Pharmacol. 2019;176:2095–2108. 10.1111/bph.14653

Contributor Information

Chunlin Zhuang, Email: zclnathan@163.com.

Zhenyu Cai, Email: drcaizhenyu@126.com.

Hong‐yang Wang, Email: hywangk@vip.sina.com.

REFERENCES

  1. Alexander, S. P. H. , Fabbro, D. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , … CGTP Collaborators . (2017a). The Concise Guide to PHARMACOLOGY 2017/18: Catalytic receptors. British Journal of Pharmacology, 174(Suppl 1), S225–S271. 10.1111/bph.13876 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander, S. P. H. , Fabbro, D. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , … CGTP Collaborators (2017b). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Enzymes. British Journal of Pharmacology, 174(Suppl 1), S272–S359. 10.1111/bph.13877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Byeon, S. E. , Yi, Y. S. , Oh, J. , Yoo, B. C. , Hong, S. , & Cho, J. Y. (2012). The role of Src kinase in macrophage‐mediated inflammatory responses. Mediators of Inflammation, 2012, 512926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cai, Z. , Jitkaew, S. , Zhao, J. , Chiang, H. C. , Choksi, S. , Liu, J. , … Liu, Z. G. (2014). Plasma membrane translocation of trimerized MLKL protein is required for TNF‐induced necroptosis. Nature Cell Biology, 16(1), 55–65. 10.1038/ncb2883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai, Z. , & Liu, Z. G. (2014). Execution of RIPK3‐regulated necrosis. Molecular & Cellular Oncology, 1(2), e960759 10.4161/23723548.2014.960759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen, X. , Li, W. , Ren, J. , Huang, D. , He, W. T. , Song, Y. , … Han, J. (2014). Translocation of mixed lineage kinase domain‐like protein to plasma membrane leads to necrotic cell death. Cell Research, 24(1), 105–121. 10.1038/cr.2013.171 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cho, Y. S. , Challa, S. , Moquin, D. , Genga, R. , Ray, T. D. , Guildford, M. , & Chan, F. K. M. (2009). Phosphorylation‐driven assembly of the RIP1‐RIP3 complex regulates programmed necrosis and virus‐induced inflammation. Cell, 137(6), 1112–1123. 10.1016/j.cell.2009.05.037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Degterev, A. , Hitomi, J. , Germscheid, M. , Ch'en, I. L. , Korkina, O. , Teng, X. , … Yuan, J. (2008). Identification of RIP1 kinase as a specific cellular target of necrostatins. Nature Chemical Biology, 4(5), 313–321. 10.1038/nchembio.83 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Degterev, A. , Huang, Z. , Boyce, M. , Li, Y. , Jagtap, P. , Mizushima, N. , … Yuan, J. (2005). Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nature Chemical Biology, 1(2), 112–119. 10.1038/nchembio711 [DOI] [PubMed] [Google Scholar]
  10. Degterev, A. , & Linkermann, A. (2016). Generation of small molecules to interfere with regulated necrosis. Cellular and molecular life sciences: CMLS, 73(11‐12), 2251–2267. 10.1007/s00018-016-2198-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dillon, C. P. , Weinlich, R. , Rodriguez, D. A. , Cripps, J. G. , Quarato, G. , Gurung, P. , … Green, D. R. (2014). RIPK1 blocks early postnatal lethality mediated by caspase‐8 and RIPK3. Cell, 157(5), 1189–1202. 10.1016/j.cell.2014.04.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dondelinger, Y. , Declercq, W. , Montessuit, S. , Roelandt, R. , Goncalves, A. , Bruggeman, I. , … Vandenabeele, P. (2014). MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Reports, 7(4), 971–981. 10.1016/j.celrep.2014.04.026 [DOI] [PubMed] [Google Scholar]
  13. Duprez, L. , Takahashi, N. , Van Hauwermeiren, F. , Vandendriessche, B. , Goossens, V. , Vanden Berghe, T. , … Vandenabeele, P. (2011). RIP kinase‐dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity, 35(6), 908–918. 10.1016/j.immuni.2011.09.020 [DOI] [PubMed] [Google Scholar]
  14. Fauster, A. , Rebsamen, M. , Huber, K. V. , Bigenzahn, J. W. , Stukalov, A. , Lardeau, C. H. , … Superti‐Furga, G. (2015). A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis. Cell Death & Disease, 6, e1767 10.1038/cddis.2015.130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Galluzzi, L. , Kepp, O. , Chan, F. K. , & Kroemer, G. (2017). Necroptosis: Mechanisms and relevance to disease. Annual Review of Pathology, 12, 103–130. 10.1146/annurev-pathol-052016-100247 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gillis, E. P. , Eastman, K. J. , Hill, M. D. , Donnelly, D. J. , & Meanwell, N. A. (2015). Applications of Fluorine in Medicinal Chemistry. Journal of Medicinal Chemistry, 58(21), 8315–8359. 10.1021/acs.jmedchem.5b00258 [DOI] [PubMed] [Google Scholar]
  17. Grootjans, S. , Vanden Berghe, T. , & Vandenabeele, P. (2017). Initiation and execution mechanisms of necroptosis: An overview. Cell Death and Differentiation, 24(7), 1184–1195. 10.1038/cdd.2017.65 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harding, S. D. , Sharman, J. L. , Faccenda, E. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucleic Acids Research, 46(D1), D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harris, P. A. , Berger, S. B. , Jeong, J. U. , Nagilla, R. , Bandyopadhyay, D. , Campobasso, N. , … Bertin, J. (2017). Discovery of a first‐in‐class receptor interacting protein 1 (RIP1) kinase specific clinical candidate (GSK2982772) for the treatment of inflammatory diseases. Journal of Medicinal Chemistry, 60(4), 1247–1261. 10.1021/acs.jmedchem.6b01751 [DOI] [PubMed] [Google Scholar]
  20. Harris, P. A. , King, B. W. , Bandyopadhyay, D. , Berger, S. B. , Campobasso, N. , Capriotti, C. A. , … Gough, P. J. (2016). DNA‐encoded library screening identifies benzo[b][1,4]oxazepin‐4‐ones as highly potent and monoselective receptor interacting protein 1 kinase inhibitors. Journal of Medicinal Chemistry, 59(5), 2163–2178. 10.1021/acs.jmedchem.5b01898 [DOI] [PubMed] [Google Scholar]
  21. He, S. , Wang, L. , Miao, L. , Wang, T. , Du, F. , Zhao, L. , & Wang, X. (2009). Receptor interacting protein kinase‐3 determines cellular necrotic response to TNF‐alpha. Cell, 137(6), 1100–1111. 10.1016/j.cell.2009.05.021 [DOI] [PubMed] [Google Scholar]
  22. Kaczmarek, A. , Vandenabeele, P. , & Krysko, D. V. (2013). Necroptosis: The release of damage‐associated molecular patterns and its physiological relevance. Immunity, 38(2), 209–223. 10.1016/j.immuni.2013.02.003 [DOI] [PubMed] [Google Scholar]
  23. Kilkenny, C. , Browne, W. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: The ARRIVE guidelines. British Journal of Pharmacology, 160(7), 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li, J. X. , Feng, J. M. , Wang, Y. , Li, X. H. , Chen, X. X. , Su, Y. , … Miao, Z. H. (2014). The B‐Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen‐induced liver injury. Cell Death & Disease, 5, e1278 10.1038/cddis.2014.241 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li, Y. , Qian, L. , & Yuan, J. (2017). Small molecule probes for cellular death machines. Current Opinion in Chemical Biology, 39, 74–82. 10.1016/j.cbpa.2017.05.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lomenick, B. , Hao, R. , Jonai, N. , Chin, R. M. , Aghajan, M. , Warburton, S. , … Huang, J. (2009). Target identification using drug affinity responsive target stability (DARTS). Proceedings of the National Academy of Sciences of the United States of America, 106(51), 21984–21989. 10.1073/pnas.0910040106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mandal, P. , Berger, S. B. , Pillay, S. , Moriwaki, K. , Huang, C. , Guo, H. , … Kaiser, W. J. (2014). RIP3 induces apoptosis independent of pronecrotic kinase activity. Molecular Cell, 56(4), 481–495. 10.1016/j.molcel.2014.10.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martens, S. , Jeong, M. , Tonnus, W. , Feldmann, F. , Hofmans, S. , Goossens, V. , … Vandenabeele, P. (2017). Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury. Cell Death & Disease, 8(6), e2904 10.1038/cddis.2017.298 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meanwell, N. A. (2018). Fluorine and fluorinated motifs in the design and application of bioisosteres for drug design. Journal of Medicinal Chemistry, 61(14), 5822–5880. 10.1021/acs.jmedchem.7b01788 [DOI] [PubMed] [Google Scholar]
  30. Najjar, M. , Suebsuwong, C. , Ray, S. S. , Thapa, R. J. , Maki, J. L. , Nogusa, S. , … Degterev, A. (2015). Structure guided design of potent and selective ponatinib‐based hybrid inhibitors for RIPK1. Cell Reports, 10(11), 1850–1860. 10.1016/j.celrep.2015.02.052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakamura, A. , Arita, T. , Tsuchiya, S. , Donelan, J. , Chouitar, J. , Carideo, E. , … Yoshida, S. (2013). Antitumor activity of the selective pan‐RAF inhibitor TAK‐632 in BRAF inhibitor‐resistant melanoma. Cancer Research, 73(23), 7043–7055. 10.1158/0008-5472.CAN-13-1825 [DOI] [PubMed] [Google Scholar]
  32. Okaniwa, M. , Hirose, M. , Arita, T. , Yabuki, M. , Nakamura, A. , Takagi, T. , … Ishikawa, T. (2013). Discovery of a selective kinase inhibitor (TAK‐632) targeting pan‐RAF inhibition: Design, synthesis, and biological evaluation of C‐7‐substituted 1,3‐benzothiazole derivatives. Journal of Medicinal Chemistry, 56(16), 6478–6494. 10.1021/jm400778d [DOI] [PubMed] [Google Scholar]
  33. Richardson, P. (2016). Fluorination methods for drug discovery and development. Expert Opinion on Drug Discovery, 11(10), 983–999. 10.1080/17460441.2016.1223037 [DOI] [PubMed] [Google Scholar]
  34. Sarhan, M. , Land, W. G. , Tonnus, W. , Hugo, C. P. , & Linkermann, A. (2018). Origin and consequences of necroinflammation. Physiological Reviews, 98(2), 727–780. 10.1152/physrev.00041.2016 [DOI] [PubMed] [Google Scholar]
  35. Sun, L. , Wang, H. , Wang, Z. , He, S. , Chen, S. , Liao, D. , … Wang, X. (2012). Mixed lineage kinase domain‐like protein mediates necrosis signaling downstream of RIP3 kinase. Cell, 148(1‐2), 213–227. 10.1016/j.cell.2011.11.031 [DOI] [PubMed] [Google Scholar]
  36. Takahashi, N. , Duprez, L. , Grootjans, S. , Cauwels, A. , Nerinckx, W. , DuHadaway, J. B. , … Vandenabeele, P. (2012). Necrostatin‐1 analogues: Critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death & Disease, 3, e437 10.1038/cddis.2012.176 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Teng, X. , Degterev, A. , Jagtap, P. , Xing, X. , Choi, S. , Denu, R. , … Cuny, G. D. (2005). Structure‐activity relationship study of novel necroptosis inhibitors. Bioorganic & Medicinal Chemistry Letters, 15(22), 5039–5044. 10.1016/j.bmcl.2005.07.077 [DOI] [PubMed] [Google Scholar]
  38. von Massenhausen, A. , Tonnus, W. , Himmerkus, N. , Parmentier, S. , Saleh, D. , Rodriguez, D. , … Linkermann, A. (2018). Phenytoin inhibits necroptosis. Cell Death & Disease, 9(3), 359 10.1038/s41419-018-0394-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wang, H. , Sun, L. , Su, L. , Rizo, J. , Liu, L. , Wang, L. F. , … Wang, X. (2014). Mixed lineage kinase domain‐like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Molecular Cell, 54(1), 133–146. 10.1016/j.molcel.2014.03.003 [DOI] [PubMed] [Google Scholar]
  40. Weinlich, R. , Oberst, A. , Beere, H. M. , & Green, D. R. (2017). Necroptosis in development, inflammation and disease. Nature Reviews. Molecular Cell Biology, 18(2), 127–136. 10.1038/nrm.2016.149 [DOI] [PubMed] [Google Scholar]
  41. Xie, T. , Peng, W. , Liu, Y. , Yan, C. , Maki, J. , Degterev, A. , … Shi, Y. (2013). Structural basis of RIP1 inhibition by necrostatins. Structure, 21(3), 493–499. 10.1016/j.str.2013.01.016 [DOI] [PubMed] [Google Scholar]
  42. Zhao, J. , Jitkaew, S. , Cai, Z. , Choksi, S. , Li, Q. , Luo, J. , & Liu, Z. G. (2012). Mixed lineage kinase domain‐like is a key receptor interacting protein 3 downstream component of TNF‐induced necrosis. Proceedings of the National Academy of Sciences of the United States of America, 109(14), 5322–5327. 10.1073/pnas.1200012109 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Supporting info item

Click here for additional data file. (3MB, pdf)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES