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

Abstract

Background and Purpose: Necroptosis is a form of programmed, caspaseindependent, 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 necroptosisassociated 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. Abbreviations: DARTS, drug affinity responsive target stability assay; Nec-1, necrostatin-1; SAR, structure-activity relationship; SIRS, systemic inflammatory response syndrome Xiaofei Chen, Chunlin Zhuang and Yibin Ren are contributed equally to this work.

Ligand-based substituent-anchoring design of selective receptor-interacting protein kinase 1 necroptosis inhibitors for ulcerative colitis therapy

“…Our previous results have suggested that the benzyl part (R) of TAK-632 might be the critical fragment for the selectivity of RIP3 over RIP1 29 . We also predicted that the nitrogen of benzothiazole ring might form hydrogen-bonding interactions with the RIP1 and RIP3 kinases, however, the contribution to the kinase activity has not been confirmed yet 28 , 29 . On the other hand, the hydrogen-bonding interactions might be disrupted by changing the conformation of the benzothiazole ring, probably leading to different activity toward RIP1 and RIP3.…”

“…The ant-inecroptosis activity was measured using a chemiluminescence assay following our previous studies 28 , 29 , 31 , 32 , 33 . Different substitutions (R 1 ) at the C-7-position were introduced in the first round of optimizations ( Table 1 ).…”

“…In 2018, we identified TAK-632 ( 5 , Fig. 2 ) from our fluorinated-compound library that effectively blocking necroptosis (HT-29, EC 50 = 1.44 μmol/L) by dual-targeting RIP1 ( K d = 0.48 μmol/L) and RIP3 ( K d = 0.11 μmol/L), respectively 28 . Further optimizations by removing the cyano group on benzothiazole ring obtained compound 6 (SZM-594), which exhibited ∼8-fold improved anti-necroptotic activity (HT-29, EC 50 = 0.17 μmol/L) and higher dual-targeting activity toward RIP1 ( K d = 0.1 μmol/L) and RIP3 ( K d = 0.08 μmol/L) kinases 28 .…”

“…2 ) from our fluorinated-compound library that effectively blocking necroptosis (HT-29, EC 50 = 1.44 μmol/L) by dual-targeting RIP1 ( K d = 0.48 μmol/L) and RIP3 ( K d = 0.11 μmol/L), respectively 28 . Further optimizations by removing the cyano group on benzothiazole ring obtained compound 6 (SZM-594), which exhibited ∼8-fold improved anti-necroptotic activity (HT-29, EC 50 = 0.17 μmol/L) and higher dual-targeting activity toward RIP1 ( K d = 0.1 μmol/L) and RIP3 ( K d = 0.08 μmol/L) kinases 28 . In addition, the carbamide replacement of the amide in the terminal trifluoromethylbenzyl group resulted in compound 7 (SZM-630) with good anti-necroptotic activity (HT-29, EC 50 = 0.44 μmol/L) and high selectivity for RIP3 ( K d = 0.08 μmol/L) over RIP1 ( K d > 5 μmol/L) 29 .…”

“…These results suggested that the benzyl part of TAK-632 might be the critical fragment for the selectivity of RIP3 over RIP1.

Figure 2 Our previous optimizations of TAK-632 28 , 29 .

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