Ensembling and filtering: an effective and rapid in silico multitarget drug-design strategy to identify RIPK1 and RIPK3 inhibitors


Necroptosis, a programmed necrosis pathway, is witnessed in diverse human diseases and is primarily regulated by receptor-interacting serine/threonine protein kinase 1 (RIPK1) and RIPK3. Ablation or inhibition of these individual proteins, or both, has been shown to be protective in various in vitro and in vivo disease models involving necroptosis. In this study, we propose an effective and rapid virtual screening strategy to identify multitarget inhibitors of both RIPK1 and RIPK3. It involves ensemble pharmacophore-based screening (EPS) of a compound database, post-EPS filtration (PEPSF) of the ligand hits, and multiple dockings. Structurally diverse inhibitors were identified through ensemble pharmacophore features, and the speed of this process was enhanced by filtering out the compounds containing cross-features. The stability of these inhibitors with both of the proteins was verified by means of molecular dynamics (MD) simulation.

A generalized workflow employed in this study. Subsequent utilization of EPS and PEPSF might lead to reduced computational time and load.

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  1. 1.

    Thornton C, Rousset CI, Kichev A, Miyakuni Y, Vontell R, Baburamani AA, Fleiss B, Gressens P, Hagberg H (2012) Molecular mechanisms of neonatal brain injury. Neurol Res Int 506320:1–16. doi:10.1155/2012/506320

    Article  Google Scholar 

  2. 2.

    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 325:332–336. doi:10.1126/science.1172308

    Article  CAS  Google Scholar 

  3. 3.

    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 137:1100–1111. doi:10.1016/j.cell.2009.05.021

    Article  CAS  Google Scholar 

  4. 4.

    Fayaz SM, Suvanish Kumar V, Rajanikant GK (2014) Necroptosis: who knew there were so many interesting ways to die? CNS Neurol Disord Drug Targets 13:42–51. doi:10.2174/18715273113126660189

    Article  CAS  Google Scholar 

  5. 5.

    Linkermann A, Hackl MJ, Kunzendorf U, Walczak H, Krautwald S, Jevnikar AM (2013) Necroptosis in immunity and ischemia reperfusion injury. Am J Transplant 13:2797–2804. doi:10.1111/ajt.12448

    Article  CAS  Google Scholar 

  6. 6.

    Mehta SL, Manhas N, Raghubir R (2007) Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Res Rev 54:34–66. doi:10.1016/j.brainresrev.2006.11.003

    Article  CAS  Google Scholar 

  7. 7.

    Meylan E, Burns K, Hofmann K, Blancheteau V, Martinon F, Kelliher M, Tschopp J (2004) RIP1 is an essential mediator of Toll-like receptor3-induced NF-kB activation. Nat Immunol 5:503–507. doi:10.1038/ni1061

    Article  CAS  Google Scholar 

  8. 8.

    Linkermann A, Green DR (2014) Necroptosis. N Engl J Med 370:455–465. doi:10.1056/NEJMra1310050

    Article  CAS  Google Scholar 

  9. 9.

    Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11:700–714. doi:10.1038/nrm2970

  10. 10.

    Chan FK, Shisler J, Bixby JG, Felices M, Zheng L, Appel M, Orenstein J, Moss B, Lenardo MJ (2003) A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J Biol Chem 278:51613–51621. doi:10.1074/jbc.M305633200

    Article  CAS  Google Scholar 

  11. 11.

    Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1:489–495. doi:10.1038/82732

    Article  CAS  Google Scholar 

  12. 12.

    Lin Y, Choksi S, Shen HM, Yang QF, Hur GM, Kim YS, Tran JH, Nedospasov SA, Liu ZG (2004) Tumor necrosis factor induced non-apoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem 279:10822–10828. doi:10.1074/jbc.M313141200

    Article  CAS  Google Scholar 

  13. 13.

    Ch’en IL, Tsau JS, Molkentin JD, Komatsu M, Hedrick SM (2011) Mechanisms of necroptosis in T cells. J Exp Med 208:633–641. doi:10.1084/jem.20110251

    Article  Google Scholar 

  14. 14.

    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 1:112–119. doi:10.1038/nchembio711

    Article  CAS  Google Scholar 

  15. 15.

    Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM (2007) Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther 21:227–233. doi:10.1007/s10557-007-6035-1

    Article  CAS  Google Scholar 

  16. 16.

    Xu X, Chua CC, Kong J, Kostrzewa RM, Kumaraguru U, Hamdy RC, Chua BH (2007) Necrostatin-1 protects against glutamate induced glutathione depletion and caspase-independent cell death in HT-22 cells. J Neurochem 103:2004–2014. doi:10.1111/j.1471-4159.2007.04884.x

    Article  CAS  Google Scholar 

  17. 17.

    Bao L, Li Y, Deng SX, Landry D, Tabas I (2006) Sitosterol containing lipoproteins trigger free sterol-induced caspase-independent death in ACAT-competent macrophages. J Biol Chem 281:33635–33649. doi:10.1074/jbc.M606339200

    Article  CAS  Google Scholar 

  18. 18.

    Hong Q, Hsu LJ, Schultz L, Pratt N, Mattison J, Chang NS (2007) Zfra affects TNF-mediated cell death by interacting with death domain protein TRADD and negatively regulates the activation of NF-kappaB, JNK1, p53 and WOX1 during stress response. BMC Mol Biol 8:50. doi:10.1186/1471-2199-8-50

    Article  Google Scholar 

  19. 19.

    Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 5:313–321. doi:10.1038/nchembio.83

    Article  Google Scholar 

  20. 20.

    Ch’en IL, Beisner DR, Degterev A, Lynch C, Yuan J, Hoffmann A, Hedrick SM (2008) Antigen-mediated T cell expansion regulated by parallel pathways of death. Proc Natl Acad Sci USA 105:17463–17468. doi:10.1073/pnas.0808043105

  21. 21.

    Ting AT, Pimentel-Muinos FX, Seed B (1996) RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis. EMBO J 15:6189–6196

    CAS  Google Scholar 

  22. 22.

    Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D, Daley-Bauer LP, Hakem R, Caspary T, Mocarski ES (2011) RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471:368–372. doi:10.1038/nature09857

    Article  CAS  Google Scholar 

  23. 23.

    Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C, Hakem R, Salvesen GS, Green DR (2011) Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471:363–367. doi:10.1038/nature09852

    Article  CAS  Google Scholar 

  24. 24.

    Vanlangenakker N, Bertrand MJ, Bogaert P, Vandenabeele P, VandenBerghe T (2011) TNF induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members. Cell Death Dis 2:e230. doi:10.1038/cddis.2011.111

  25. 25.

    Zou J, Xie HZ, Yang SY, Chen JJ, Ren JX, Wei YQ (2008) Towards more accurate pharmacophore modeling: multicomplex based comprehensive pharmacophore map and most-frequent feature pharmacophore model of CDK2. J Mol Graph Model 27:430–438. doi:10.1016/j.jmgm.2008.07.004

    Article  CAS  Google Scholar 

  26. 26.

    Fayaz SM, Rajanikant GK (2014) Ensemble pharmacophore meets ensemble docking: a novel screening strategy for the identification of RIPK1 inhibitors. J Comput Aided Mol Des 28:779–794. doi:10.1007/s10822-014-9771-x

    Article  CAS  Google Scholar 

  27. 27.

    Nair SB, Fayaz SM, Rajanikant GK (2013) A novel multi-target drug screening strategy directed against key proteins of DAPk family. Comb Chem High Throughput Screen 16:449–457. doi:10.2174/1386207311316060005

    Article  CAS  Google Scholar 

  28. 28.

    Darden T, York D, Pedersen L (1993) Particle mesh Ewald—an N.log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10093. doi:10.1063/1.464397

    Article  CAS  Google Scholar 

  29. 29.

    Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47:1739–1749. doi:10.1021/jm0306430

    Article  CAS  Google Scholar 

  30. 30.

    Salam NK, Nuti R, Sherman W (2009) Novel method for generating structure-based pharmacophores using energetic analysis. J Chem Inf Model 49:2356–2368. doi:10.1021/ci900212v

    Article  CAS  Google Scholar 

  31. 31.

    Irwin JJ, Shoichet BK (2005) ZINC—a free database of commercially available compounds for virtual screening. J Chem Inf Model 45:177–182. doi:10.1021/ci049714+

    Article  CAS  Google Scholar 

  32. 32.

    Bender A, Glen RC (2005) A discussion of measures of enrichment in virtual screening: comparing the information content of descriptors with increasing levels of sophistication. J Chem Inf Model 45:1369–1375. doi:10.1021/ci0500177

    Article  CAS  Google Scholar 

  33. 33.

    Jain AN, Nicholls A (2008) Recommendations for evaluation of computational methods. J Comput Aided Mol Des 22:133–139. doi:10.1007/s10822-008-9196-5

    Article  CAS  Google Scholar 

  34. 34.

    Hamza A, Wei NN, Zhan CG (2012) Ligand-based virtual screening approach using a new scoring function. J Chem Inf Model 52:963–974. doi:10.1021/ci200617d

    Article  CAS  Google Scholar 

  35. 35.

    van Aalten DM, Bywater R, Findlay JB, Hendlich M, Hooft RW, Vriend G (1996) PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules. J Comput Aided Mol Des 10:255–262. doi:10.1007/BF00355047

    Article  Google Scholar 

  36. 36.

    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38. doi:10.1016/0263-7855(96)00018-5

    Article  CAS  Google Scholar 

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Correspondence to G. K. Rajanikant.

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This study was funded by the Department of Biotechnology, Government of India via the Bioinformatics Infrastructure Facility for Biology Teaching through Bioinformatics (BIF-BTBI) (grant number: BT/BI/25/001/2006, dated 25/03/2011).

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Fayaz, S.M., Rajanikant, G.K. Ensembling and filtering: an effective and rapid in silico multitarget drug-design strategy to identify RIPK1 and RIPK3 inhibitors. J Mol Model 21, 314 (2015). https://doi.org/10.1007/s00894-015-2855-2

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  • Ensemble pharmacophore
  • Ensemble docking
  • Dual ensemble screening (DES)
  • Ensemble pharmacophore-based screening (EPS)
  • Post-EPS filtration (PEPSF)
  • Dual inhibitors