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Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators

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The TNF Superfamily

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2248))

Abstract

Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor–receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor–receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.

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References

  1. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308

    Article  CAS  PubMed  Google Scholar 

  2. Wajant H, Scheurich P (2011) TNFR1-induced activation of the classical NF-kappaB pathway. FEBS J 278:862–876

    Article  CAS  PubMed  Google Scholar 

  3. Brenner D, Blaser H, Mak TW (2015) Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol 15:362–374

    Article  CAS  PubMed  Google Scholar 

  4. NIH (2005) Progress in autoimmune diseases research, Report to Congress, National Institutes of Health, The Autoimmune Diseases Coordinating Committee, March 2005, forward and pages i, 1, 2, 16, 17, 28, 29, 30, 32, 52

    Google Scholar 

  5. Sedger LM, McDermott MF (2014) TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants – past, present and future. Cytokine Growth Factor Rev 25:453–472

    Article  CAS  PubMed  Google Scholar 

  6. Tracey D, Klareskog L, Sasso EH et al (2008) Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther 117:244–279

    Article  CAS  PubMed  Google Scholar 

  7. Shakoor N, Michalska M, Harris CA et al (2002) Drug-induced systemic lupus erythematosus associated with etanercept therapy. Lancet 359:579–580

    Article  CAS  PubMed  Google Scholar 

  8. Wolfe F, Michaud K (2004) Lymphoma in rheumatoid arthritis: the effect of methotrexate and anti-tumor necrosis factor therapy in 18,572 patients. Arthritis Rheum 50(6):1740–1751

    Article  CAS  PubMed  Google Scholar 

  9. Steeland S, Libert C, Vandenbroucke RE (2018) A new venue of TNF targeting. Int J Mol Sci 19:1442

    Article  PubMed Central  CAS  Google Scholar 

  10. Fischer R, Kontermann R, Maier O (2015) Targeting sTNF/TNFR1 signaling as a new therapeutic strategy. Antibodies 4:48

    Article  CAS  Google Scholar 

  11. Zettlitz KA, Lorenz V, Landauer K et al (2010) ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody. mAbs 2:639–647

    Article  PubMed  PubMed Central  Google Scholar 

  12. Steeland S, Puimège L, Vandenbroucke RE et al (2015) Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1. J Biol Chem 290:4022–4037

    Article  CAS  PubMed  Google Scholar 

  13. Carter PH, Scherle PA, Muckelbauer JK et al (2001) Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha. Proc Natl Acad Sci U S A 98:11879–11884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen S, Feng Z, Wang Y et al (2017) Discovery of novel ligands for TNF-α and TNF Receptor-1 through structure-based virtual screening and biological assay. J Chem Inf Model 57:1101–1111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Deng G-M, Zheng L, Ka-Ming Chan F et al (2005) Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain of tumor necrosis factor receptors. Nat Med 11:1066–1072

    Article  CAS  PubMed  Google Scholar 

  16. Schon A, Lam SY, Freire E (2011) Thermodynamics-based drug design: strategies for inhibiting protein-protein interactions. Future Med Chem 3:1129–1137

    Article  PubMed  CAS  Google Scholar 

  17. Schon A, Madani N, Smith AB et al (2011) Some binding-related drug properties are dependent on thermodynamic signature. Chem Biol Drug Des 77(3):161–165

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wells JA, McClendon CL (2007) Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 450:1001

    Article  CAS  PubMed  Google Scholar 

  19. Lewis AK, Valley CC, Sachs JN (2012) TNFR1 signaling is associated with backbone conformational changes of receptor dimers consistent with overactivation in the R92Q TRAPS mutant. Biochemistry 51:6545–6555

    Article  CAS  PubMed  Google Scholar 

  20. Lewis Andrew K, James Zachary M, McCaffrey Jesse E et al (2014) Open and closed conformations of the isolated transmembrane domain of death receptor 5 support a new model of activation. Biophys J 106:L21–L24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Valley CC, Lewis AK, Mudaliar DJ et al (2012) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized. J Biol Chem 287:21265–21278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Valley CC, Lewis AK, Sachs JN (2017) Piecing it together: unraveling the elusive structure-function relationship in single-pass membrane receptors. Biochim Biophys Acta Biomembr 1859:1398–1416

    Article  CAS  PubMed  Google Scholar 

  23. Fricke F, Malkusch S, Wangorsch G et al (2014) Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers. Histochem Cell Biol 142:91–101

    Article  CAS  PubMed  Google Scholar 

  24. Wang Y, Bugge K, Kragelund BB et al (2018) Role of protein dynamics in transmembrane receptor signalling. Curr Opin Struct Biol 48:74–82

    Article  CAS  PubMed  Google Scholar 

  25. Vunnam N, Lo CH, Grant BD et al (2017) Soluble extracellular domain of death receptor 5 inhibits TRAIL-induced apoptosis by disrupting receptor–receptor interactions. J Mol Biol 429:2943–2953

    Article  CAS  PubMed  Google Scholar 

  26. Vunnam N, Campbell-Bezat CK, Lewis AK et al (2017) Death receptor 5 activation is energetically coupled to opening of the transmembrane domain dimer. Biophys J 113:381–392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lo CH, Schaaf TM, Grant BD et al (2019) Noncompetitive inhibitors of TNFR1 probe conformational activation states. Sci Signal 12:eaav5637

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Lewis AK, Valley CC, Peery SL et al (2016) Death receptor 5 networks require membrane cholesterol for proper structure and function. J Mol Biol 428:4843–4855

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Murali R, Cheng X, Berezov A et al (2005) Disabling TNF receptor signaling by induced conformational perturbation of tryptophan-107. Proc Natl Acad Sci U S A 102:10970–10975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Banner DW, D'Arcy A, Janes W et al (1993) Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex: implications for TNF receptor activation. Cell 73:431–445

    Article  CAS  PubMed  Google Scholar 

  31. Naismith JH, Devine TQ, Brandhuber BJ et al (1995) Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor. J Biol Chem 270(22):13303–13307

    Article  CAS  PubMed  Google Scholar 

  32. Lo CH, Vunnam N, Lewis AK et al (2017) An innovative high-throughput screening approach for discovery of small molecules that inhibit TNF receptors. SLAS Discov 22:950–961

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Lo CH, Huber EC, Sachs JN (2020) Conformational states of TNFR1 as a molecular switch for receptor function. Protein Sci 29. https://doi.org/10.1002/pro.3829; Published 20 Jan 2020

  34. Petersen KJ, Peterson KC, Muretta JM et al (2014) Fluorescence lifetime plate reader: resolution and precision meet high-throughput. Rev Sci Instrum 85:113101

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Gruber SJ, Cornea RL, Li J et al (2014) Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells. J Biomol Screen 19(2):215–222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Muretta JM, Kyrychenko A, Ladokhin AS et al (2010) High-performance time-resolved fluorescence by direct waveform recording. Rev Sci Instrum 81:103101–103101

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Schaaf TM, Peterson KC, Grant BD et al (2017) High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA. SLAS Discov 22:262–273

    CAS  PubMed  Google Scholar 

  38. Schaaf TM, Peterson KC, Grant BD et al (2017) Spectral unmixing plate reader: high-throughput, high-precision FRET assays in living cells. SLAS Discov 22:250–261

    CAS  PubMed  Google Scholar 

  39. Schaaf TM, Li A, Grant BD et al (2018) Red-shifted FRET biosensors for high-throughput fluorescence lifetime screening. Biosensors 8:99

    Article  CAS  PubMed Central  Google Scholar 

  40. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

    Article  CAS  PubMed  Google Scholar 

  41. Birmingham A, Selfors LM, Forster T et al (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6:569–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. McAllister R, Schofield C, Pettman G et al (2002) Adaptation of recombinant HEK-293 cells to growth in serum free suspension. In: Bernard A, Griffiths B, Noé W, Wurm F (eds) Animal cell technology: products from cells, cells as products: proceedings of the 16th ESACT meeting April 25–29, 1999, Lugano, Switzerland. Springer Netherlands, Dordrecht, pp 367–369

    Google Scholar 

  43. Cornea RL, Gruber SJ, Lockamy EL et al (2013) High-throughput FRET assay yields allosteric SERCA activators. J Biomol Screen 18:97–107

    Article  PubMed  CAS  Google Scholar 

  44. Rebbeck RT, Essawy MM, Nitu FR et al (2016) High-throughput screens to discover small-molecule modulators of ryanodine receptor calcium release channels. SLAS Discov 22:176–186

    PubMed  PubMed Central  Google Scholar 

  45. Stroik DR, Yuen SL, Janicek KA et al (2018) Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells. Sci Rep 8:12560–12560

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Lo CH, Lim CK-W, Ding Z et al (2019) Targeting the ensemble of heterogeneous tau oligomers in cells: a novel small molecule screening platform for tauopathies. Alzheimers Dement 15:1489–1502

    Article  PubMed  PubMed Central  Google Scholar 

  47. Braun AR, Liao EE, Horvath M et al (2020) Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor. bioRxiv:2020.01.09.900845

    Google Scholar 

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Acknowledgements

We thank Samantha Yuen and Prachi Bawaskar from the Thomas group and Benjamin Grant from Fluorescence Innovations for technical discussions. The pRH132 plasmid was a gift from the Reuben Harris lab at UMN. Flow cytometry and FACS were performed at the UMN Lillehei Heart Institute, confocal fluorescence microscopy was conducted at the UMN Imaging Center, compound dispensing at the UMN Institute of Therapeutics Discovery and Development, and spectroscopy measurements at the UMN Biophysical Technology Center. This study was supported by U.S. NIH grants to J.N.S. (R01 GM107175 and R35 GM131814) and D.D.T. (R01 GM27906, R37 AG26260, R42 DA03762). C.H.L. was supported by a Doctoral Dissertation Fellowship from the UMN.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA

    Chih Hung Lo & Jonathan N. Sachs

  2. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

    Tory M. Schaaf & David D. Thomas

  3. Photonic Pharma LLC, Minneapolis, MN, USA

    David D. Thomas

Authors
  1. Chih Hung Lo
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  2. Tory M. Schaaf
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  3. David D. Thomas
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  4. Jonathan N. Sachs
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Corresponding author

Correspondence to Jonathan N. Sachs .

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Editors and Affiliations

  1. INSERM, Sorbonne Université, Paris, France

    Jagadeesh Bayry

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Lo, C.H., Schaaf, T.M., Thomas, D.D., Sachs, J.N. (2021). Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators. In: Bayry, J. (eds) The TNF Superfamily. Methods in Molecular Biology, vol 2248. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1130-2_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1130-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1129-6

  • Online ISBN: 978-1-0716-1130-2

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