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

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 (RIPK1inhibitor): 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.