Advertisement

Chemical Library Screens to Identify Pharmacological Modulators of Necroptosis

  • Danish Saleh
  • Alexei DegterevEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1857)

Abstract

Necroptosis is mediated by the formation of the detergent-insoluble necrosome complex between Ser/Thr kinases RIPK1 and RIPK3, which mediates RIPK3-dependent phosphorylation and activation of the critical necroptosis effector MLKL. Small molecule screens have been instrumental in the development of new chemical probes for this pathway. In this chapter, we describe several cellular assays that are readily amendable for the identification of new modulators of necroptosis as well as secondary assays to facilitate initial characterization of the mode of activity of small molecule hits.

Key words

Necroptosis Apoptosis Necrosome Complex 2b RIPK1 RIPK3 MLKL 

References

  1. 1.
    Galluzzi L, Kepp O, Chan FK, Kroemer G (2016) Necroptosis: mechanisms and relevance to disease. Annu Rev Pathol. https://doi.org/10.1146/annurev-pathol-052016-100247
  2. 2.
    Zhao H, Jaffer T, Eguchi S, Wang Z, Linkermann A, Ma D (2015) Role of necroptosis in the pathogenesis of solid organ injury. Cell Death Dis 6:e1975. https://doi.org/10.1038/cddis.2015.316CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Linkermann A, Green DR (2014) Necroptosis. N Engl J Med 370(5):455–465. https://doi.org/10.1056/NEJMra1310050CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Degterev A, Linkermann A (2016) Generation of small molecules to interfere with regulated necrosis. Cell Mol Life Sci 73(11–12):2251–2267. https://doi.org/10.1007/s00018-016-2198-xCrossRefPubMedGoogle Scholar
  5. 5.
    Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148(1–2):213–227. https://doi.org/10.1016/j.cell.2011.11.031CrossRefPubMedGoogle Scholar
  6. 6.
    Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1(2):112–119. https://doi.org/10.1038/nchembio711CrossRefPubMedGoogle Scholar
  7. 7.
    Ren Y, Su Y, Sun L, He S, Meng L, Liao D, Liu X, Ma Y, Liu C, Li S, Ruan H, Lei X, Wang X, Zhang Z (2016) Discovery of a highly potent, selective, and metabolically stable inhibitor of receptor-interacting protein 1 (RIP1) for the treatment of systemic inflammatory response syndrome. J Med Chem. https://doi.org/10.1021/acs.jmedchem.6b01196
  8. 8.
    Harris PA, King BW, Bandyopadhyay D, Berger SB, Campobasso N, Capriotti CA, Cox JA, Dare L, Dong X, Finger JN, Grady LC, Hoffman SJ, Jeong JU, Kang J, Kasparcova V, Lakdawala AS, Lehr R, McNulty DE, Nagilla R, Ouellette MT, Pao CS, Rendina AR, Schaeffer MC, Summerfield JD, Swift BA, Totoritis RD, Ward P, Zhang A, Zhang D, Marquis RW, Bertin J, Gough PJ (2016) DNA-encoded library screening identifies Benzo[b][1,4]oxazepin-4-ones as highly potent and Monoselective receptor interacting protein 1 kinase inhibitors. J Med Chem 59(5):2163–2178. https://doi.org/10.1021/acs.jmedchem.5b01898CrossRefPubMedGoogle Scholar
  9. 9.
    Berger SB, Harris P, Nagilla R, Kasparcova V, Hoffman S, Swift B, Dare L, Schaeffer M, Capriotti C, Ouellette M, King BW, Wisnoski D, Cox J, Reilly M, Marquis RW, Bertin J, Gough PJ (2015) Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase. Cell Death Discov (1):15009. https://doi.org/10.1038/cddiscovery.2015.9
  10. 10.
    Harris PA, Bandyopadhyay D, Berger SB, Campobasso N, Capriotti CA, Cox JA, Dare L, Finger JN, Hoffman SJ, Kahler KM, Lehr R, Lich JD, Nagilla R, Nolte RT, Ouellette MT, Pao CS, Schaeffer MC, Smallwood A, Sun HH, Swift BA, Totoritis RD, Ward P, Marquis RW, Bertin J, Gough PJ (2013) Discovery of small molecule RIP1 kinase inhibitors for the treatment of pathologies associated with necroptosis. ACS Med Chem Lett 4(12):1238–1243. https://doi.org/10.1021/ml400382pCrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mandal P, Berger SB, Pillay S, Moriwaki K, Huang C, Guo H, Lich JD, Finger J, Kasparcova V, Votta B, Ouellette M, King BW, Wisnoski D, Lakdawala AS, DeMartino MP, Casillas LN, Haile PA, Sehon CA, Marquis RW, Upton J, Daley-Bauer LP, Roback L, Ramia N, Dovey CM, Carette JE, Chan FK, Bertin J, Gough PJ, Mocarski ES, Kaiser WJ (2014) RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell 56(4):481–495. https://doi.org/10.1016/j.molcel.2014.10.021CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Li D, Li C, Li L, Chen S, Wang L, Li Q, Wang X, Lei X, Shen Z (2016) Natural product Kongensin a is a non-canonical HSP90 inhibitor that blocks RIP3-dependent necroptosis. Cell Chem Biol 23(2):257–266. https://doi.org/10.1016/j.chembiol.2015.08.018CrossRefPubMedGoogle Scholar
  13. 13.
    Fauster A, Rebsamen M, Huber KV, Bigenzahn JW, Stukalov A, Lardeau CH, Scorzoni S, Bruckner M, Gridling M, Parapatics K, Colinge J, Bennett KL, Kubicek S, Krautwald S, Linkermann A, Superti-Furga G (2015) A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis. Cell Death Dis 6:e1767. https://doi.org/10.1038/cddis.2015.130CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Najjar M, Suebsuwong C, Ray SS, Thapa RJ, Maki JL, Nogusa S, Shah S, Saleh D, Gough PJ, Bertin J, Yuan J, Balachandran S, Cuny GD, Degterev A (2015) Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1. Cell Rep 10(11):1850–1860. https://doi.org/10.1016/j.celrep.2015.02.052CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Li JX, Feng JM, Wang Y, Li XH, Chen XX, Su Y, Shen YY, Chen Y, Xiong B, Yang CH, Ding J, Miao ZH (2014) The B-Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury. Cell Death Dis 5:e1278. https://doi.org/10.1038/cddis.2014.241CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, Sehon CA, Marquis RW, Bertin J, Mocarski ES (2013) Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem 288(43):31268–31279. https://doi.org/10.1074/jbc.M113.462341CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Schworer SA, Smirnova II, Kurbatova I, Bagina U, Churova M, Fowler T, Roy AL, Degterev A, Poltorak A (2014) Toll-like receptor-mediated down-regulation of the deubiquitinase cylindromatosis (CYLD) protects macrophages from necroptosis in wild-derived mice. J Biol Chem 289(20):14422–14433. https://doi.org/10.1074/jbc.M114.547547CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Dillon CP, Weinlich R, Rodriguez DA, Cripps JG, Quarato G, Gurung P, Verbist KC, Brewer TL, Llambi F, Gong YN, Janke LJ, Kelliher MA, Kanneganti TD, Green DR (2014) RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. Cell 157(5):1189–1202. https://doi.org/10.1016/j.cell.2014.04.018CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Upton JW, Kaiser WJ, Mocarski ES (2012) DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe 11(3):290–297. https://doi.org/10.1016/j.chom.2012.01.016CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsiao YS, Damko E, Moquin D, Walz T, McDermott A, Chan FK, Wu H (2012) The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 150(2):339–350. https://doi.org/10.1016/j.cell.2012.06.019CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Moquin DM, McQuade T, Chan FK (2013) CYLD deubiquitinates RIP1 in the TNFalpha-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS One 8(10):e76841. https://doi.org/10.1371/journal.pone.0076841CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Najjar M, Saleh D, Zelic M, Nogusa S, Shah S, Tai A, Finger JN, Polykratis A, Gough PJ, Bertin J, Whalen MJ, Pasparakis M, Balachandran S, Kelliher M, Poltorak A, Degterev A (2016) RIPK1 and RIPK3 kinases promote cell-death-independent inflammation by toll-like receptor 4. Immunity 45(1):46–59. https://doi.org/10.1016/j.immuni.2016.06.007CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ofengeim D, Ito Y, Najafov A, Zhang Y, Shan B, DeWitt JP, Ye J, Zhang X, Chang A, Vakifahmetoglu-Norberg H, Geng J, Py B, Zhou W, Amin P, Berlink Lima J, Qi C, Yu Q, Trapp B, Yuan J (2015) Activation of necroptosis in multiple sclerosis. Cell Rep 10(11):1836–1849. https://doi.org/10.1016/j.celrep.2015.02.051CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, Dong MQ, Han J (2009) RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325(5938):332–336. https://doi.org/10.1126/science.1172308CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Medical Scientist Training Program, Program in Neuroscience, Sackler School of Graduate Biomedical SciencesTufts University School of MedicineBostonUSA
  2. 2.Department of Developmental, Molecular and Chemical BiologyTufts University School of MedicineBostonUSA

Personalised recommendations