Analyzing the Structure-Activity Relationship of Necrostain-1 and Its Analogues as RIPK1 Inhibitors on Necroptosis

Huilin Liu; Heng Wang; Haoyi Zheng; Xiuping Chen

doi:10.53941/ijddp.2025.100012

Open Access

Article

Analyzing the Structure-Activity Relationship of Necrostain-1 and Its Analogues as RIPK1 Inhibitors on Necroptosis

Huilin Liu¹

,

Heng Wang¹

,

Haoyi Zheng¹

,

Xiuping Chen^{1, 2, 3, *}

Author Information

Submitted: 6 Oct 2024 | Revised: 5 Nov 2024 | Accepted: 6 Nov 2024 | Published: 20 Jun 2025

Abstract

Necroptosis is a type of programmed cell death mediated by the RIPK1-RIPK3-MLKL axis. Receptor-interacting serine/threonine-protein kinase 1(RIPK1), the key upstream regulator of necroptosis, has been implicated in the pathogenesis of various diseases, and its inhibitors are being tested in clinical trials. Necrostatin-1 (Nec-1), the firstly identified RIPK1 inhibitor, effectively prevents necroptosis by specifically inhibiting the kinase activity of RIPK1. In our study, we compared the inhibitory efficiency of Nec-1 and its analogues on RIPK1 by using the TBZ-induced necroptosis model in HT29 cells and analyzing their structure-activity relationship (SAR). The results showed that Nec-1, Nec-1s, Nec-a1, Nec-a2, Nec-a3, Nec-a4, and Nec-a5 exhibited potent inhibition of TBZ-induced necroptosis and phosphorylation of RIPK1, RIPK3, MLKL. Molecular docking showed that there were two potential binding pockets between Nec-1 and its analogues with RIPK1. SAR analysis showed that the type and size of a substituent at the nitrogen atom in the 3-position of the imidazolidine ring markedly influenced the activity, indicating that a substituent at this position is essential for maintaining the biological function. This study helps to further elucidate how Nec-1 works and could lead to the development of more effective analogues.

Keywords

necroptosis | RIPK1 | Necrostatin-1 | molecular docking | SAR

References

1.

Bertheloot, D.; Latz, E.; Franklin, B.S. Necroptosis, pyroptosis and apoptosis: An intricate game of cell death. Cell. Mol. Immunol. 2021, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3.

2.

Degterev, A.; Huang, Z.; Boyce, M.; et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 2005, 1, 112–119. https://doi.org/10.1038/nchembio711.

3.

Peng, F.; Liao, M.; Qin, R.; et al. Regulated cell death (RCD) in cancer: Key pathways and targeted therapies. Signal Transduct. Target. Ther. 2022, 7, 286. https://doi.org/10.1038/s41392-022-01110-y.

4.

Zhang, D.W.; Shao, J.; Lin, J.; et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009, 325, 332–336. https://doi.org/10.1126/science.1172308.

5.

Cho, Y.S.; Challa, S.; Moquin, D.; et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009, 137, 1112–1123. https://doi.org/10.1016/j.cell.2009.05.037.

6.

He, X.Y.; Wang, F.; Suo, X.G.; et al. Compound-42 alleviates acute kidney injury by targeting RIPK3-mediated necroptosis. Br. J. Pharmacol. 2023, 180, 2641–2660. https://doi.org/10.1111/bph.16152.

7.

Vanlangenakker, N.; Vanden Berghe, T.; Vandenabeele, P. Many stimuli pull the necrotic trigger, an overview. Cell Death Differ. 2012, 19, 75–86. https://doi.org/10.1038/cdd.2011.164.

8.

Vandenabeele, P.; Galluzzi, L.; Vanden Berghe, T.; et al. Molecular mechanisms of necroptosis: An ordered cellular explosion. Nat. Rev. Mol. Cell Biol. 2010, 11, 700–714. https://doi.org/10.1038/nrm2970.

9.

Sun, L.; Wang, H.; Wang, Z.; et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012, 148, 213–227. https://doi.org/10.1016/j.cell.2011.11.031.

10.

Linkermann, A.; Green, D.R. Necroptosis. N. Engl. J. Med. 2014, 370, 455–465. https://doi.org/10.1056/NEJMra1310050.

11.

Chen, X.; Li, W.; Ren, J.; et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 2014, 24, 105–121. https://doi.org/10.1038/cr.2013.171.

12.

Yuan, J.; Amin, P.; Ofengeim, D. Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat. Rev. . Neurosci. 2019, 20, 19–33. https://doi.org/10.1038/s41583-018-0093-1.

13.

Shan, B.; Pan, H.; Najafov, A.; et al. Necroptosis in development and diseases. Genes Dev. 2018, 32, 327–340. https://doi.org/10.1101/gad.312561.118.

14.

Dong, L.; Liang, F.; Lou, Z.; et al. Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells. Cells 2022, 11, 3139. https://doi.org/10.3390/cells11193139.

15.

Mifflin, L.; Ofengeim, D.; Yuan, J. Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target. Nat. Rev. Drug Discov. 2020, 19, 553–571. https://doi.org/10.1038/s41573-020-0071-y.

16.

Silke, J.; Rickard, J.A.; Gerlic, M. Erratum: The diverse role of RIP kinases in necroptosis and inflammation. Nat. Immunol. 2015, 16, 889. https://doi.org/10.1038/ni0815-889b.

17.

Ju, E.; Park, K.A.; Shen, H.M.; et al. The resurrection of RIP kinase 1 as an early cell death checkpoint regulator-a potential target for therapy in the necroptosis era. Exp. Mol. Med. 2022, 54, 1401–1411. https://doi.org/10.1038/s12276-022-00847-4.

18.

Zhang, L.; Li, Y.; Tian, C.; et al. From Hit to Lead: Structure-Based Optimization of Novel Selective Inhibitors of Receptor-Interacting Protein Kinase 1 (RIPK1) for the Treatment of Inflammatory Diseases. J. Med. Chem. 2024, 67, 754–773. https://doi.org/10.1021/acs.jmedchem.3c02102.

19.

Tompson, D.J.; Davies, C.; Scott, N.E.; et al. Comparison of the Pharmacokinetics of RIPK1 Inhibitor GSK2982772 in Healthy Western and Japanese Subjects. Eur. J. Drug Metab. Pharmacokinet. 2021, 46, 71–83. https://doi.org/10.1007/s13318-020-00652-2.

20.

Bai, Y.; Qiao, Y.; Li, M.; et al. RIPK1 inhibitors: A key to unlocking the potential of necroptosis in drug development. Eur. J. Med. Chem. 2024, 265, 116123. https://doi.org/10.1016/j.ejmech.2024.116123.

21.

Chen, J.; Kos, R.; Garssen, J.; et al. Molecular Insights into the Mechanism of Necroptosis: The Necrosome As a Potential Therapeutic Target. Cells 2019, 8, 1486. https://doi.org/10.3390/cells8121486.

22.

Vasamsetti, S.B.; Karnewar, S.; Kanugula, A.K.; et al. Metformin inhibits monocyte-to-macrophage differentiation via AMPK-mediated inhibition of STAT3 activation: Potential role in atherosclerosis. Diabetes 2015, 64, 2028–2041. https://doi.org/10.2337/db14-1225.

23.

Weisel, K.; Scott, N.E.; Tompson, D.J.; et al. Randomized clinical study of safety, pharmacokinetics, and pharmacodynamics of RIPK1 inhibitor GSK2982772 in healthy volunteers. Pharmacol. Res. Perspect. 2017, 5. https://doi.org/10.1002/prp2.365.

24.

Martens, S.; Hofmans, S.; Declercq, W.; et al. Inhibitors Targeting RIPK1/RIPK3: Old and New Drugs. Trends Pharmacol. Sci. 2020, 41, 209–224. https://doi.org/10.1016/j.tips.2020.01.002.

25.

Xu, L.; Zhang, W.; Zhuang, C. Receptor-interacting protein kinase 1 (RIPK1) inhibitor: A review of the patent literature (2018-present). Expert Opin. Ther. Pat. 2023, 33, 101–124. https://doi.org/10.1080/13543776.2023.2195548.

26.

Rojas-Rivera, D.; Delvaeye, T.; Roelandt, R.; et al. When PERK inhibitors turn out to be new potent RIPK1 inhibitors: Critical issues on the specificity and use of GSK2606414 and GSK2656157. Cell Death Differ. 2017, 24, 1100–1110. https://doi.org/10.1038/cdd.2017.58.

27.

Son, J.Y.; Ju, J.S.; Kim, Y.M.; et al. TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats. Int. J. Mol. Sci. 2022, 23, 506. https://doi.org/10.3390/ijms23010506.

28.

Meng, H.; Wu, G.; Zhao, X.; et al. Discovery of a cooperative mode of inhibiting RIPK1 kinase. Cell Discov. 2021, 7, 41. https://doi.org/10.1038/s41421-021-00278-x.

29.

Li, Z.; Hao, Y.; Yang, C.; et al. Design, synthesis, and evaluation of potent RIPK1 inhibitors with in vivo anti-inflammatory activity. Eur. J. Med. Chem. 2022, 228, 114036. https://doi.org/10.1016/j.ejmech.2021.114036.

30.

Moriwaki, K.; Bertin, J.; Gough, P.J.; et al. Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death. Cell Death Dis. 2015, 6, e1636. https://doi.org/10.1038/cddis.2015.16.

31.

Gonçalves, J.; Amaral, J.D.; Capela, R.; et al. Necroptosis induced by ruthenium (II) complexes as mitochondrial disruptors. Cell Death Discov. 2024, 10, 261. https://doi.org/10.1038/s41420-024-02033-z.

32.

Lecoeur, H. Nuclear apoptosis detection by flow cytometry: Influence of endogenous endonucleases. Exp. Cell Res. 2002, 277, 1–14. https://doi.org/10.1006/excr.2002.5537.

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DOI

10.53941/ijddp.2025.100012

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Volume 4, Issue 2

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Liu, H., Wang, H., Zheng, H., & Chen, X. (2025). Analyzing the Structure-Activity Relationship of Necrostain-1 and Its Analogues as RIPK1 Inhibitors on Necroptosis. International Journal of Drug Discovery and Pharmacology, 4(2), 100012. https://doi.org/10.53941/ijddp.2025.100012

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