Skip to main content
SpringerLink
Log in

Mechanistic and Structural Features of PROTAC Ternary Complexes

  • Protocol
  • First Online:
Targeted Protein Degradation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2365))

Abstract

The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol Lond 40:4–7

    Google Scholar 

  2. Labrijn AF, Janmaat ML, Reichert JM, Parren P (2019) Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18(8):585–608. https://doi.org/10.1038/s41573-019-0028-1

    Article  CAS  PubMed  Google Scholar 

  3. Profit AA, Lee TR, Lawrence DS (1999) Bivalent inhibitors of protein tyrosine kinases. J Am Chem Soc 121(2):280–283. https://doi.org/10.1021/ja983515n

    Article  CAS  Google Scholar 

  4. Tanaka M, Roberts JM, Seo HS, Souza A, Paulk J, Scott TG, DeAngelo SL, Dhe-Paganon S, Bradner JE (2016) Design and characterization of bivalent BET inhibitors. Nat Chem Biol 12(12):1089–1096. https://doi.org/10.1038/Nchembio.2209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Waring MJ, Chen HW, Rabow AA, Walker G, Bobby R, Boiko S, Bradbury RH, Callis R, Clark E, Dale I, Daniels DL, Dulak A, Flavell L, Holdgate G, Jowitt TA, Kikhney A, McAlister M, Mendez J, Ogg D, Patel J, Petteruti P, Robb GR, Robers MB, Saif S, Stratton N, Svergun DI, Wang WX, Whittaker D, Wilson DM, Yao Y (2016) Potent and selective bivalent inhibitors of BET bromodomains. Nat Chem Biol 12(12):1097–1104. https://doi.org/10.1038/Nchembio.2210

    Article  CAS  PubMed  Google Scholar 

  6. Che Y, Gilbert AM, Shanmugasundaram V, Noe MC (2018) Inducing protein-protein interactions with molecular glues. Bioorg Med Chem Lett 28(15):2585–2592. https://doi.org/10.1016/j.bmcl.2018.04.046

    Article  CAS  PubMed  Google Scholar 

  7. Sun X, Gao H, Yang Y, He M, Wu Y, Song Y, Tong Y, Rao Y (2019) PROTACs: great opportunities for academia and industry. Signal Transduct Target Ther 4:64. https://doi.org/10.1038/s41392-019-0101-6

    Article  PubMed  PubMed Central  Google Scholar 

  8. Petzold G, Fischer ES, Thomä NH (2016) Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532(7597):127–130. https://doi.org/10.1038/nature16979

    Article  CAS  PubMed  Google Scholar 

  9. Douglass EF Jr, Miller CJ, Sparer G, Shapiro H, Spiegel DA (2013) A comprehensive mathematical model for three-body binding equilibria. J Am Chem Soc 135(16):6092–6099. https://doi.org/10.1021/ja311795d

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mack ET, Perez-Castillejos R, Suo Z, Whitesides GM (2008) Exact analysis of ligand-induced dimerization of monomeric receptors. Anal Chem 80(14):5550–5555. https://doi.org/10.1021/ac800578w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Buxton BH (1905) Bacteriolytic power of immune serum and the theory of complement diversion. J Med Res 13(5):431–459

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Mulgrew K, Kinneer K, Yao XT, Ward BK, Damschroder MM, Walsh B, Mao SY, Gao C, Kiener PA, Coats S, Kinch MS, Tice DA (2006) Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, Abegrin. Mol Cancer Ther 5(12):3122–3129. https://doi.org/10.1158/1535-7163.MCT-06-0356

    Article  CAS  PubMed  Google Scholar 

  13. Bondeson DP, Mares A, Smith IE, Ko E, Campos S, Miah AH, Mulholland KE, Routly N, Buckley DL, Gustafson JL, Zinn N, Grandi P, Shimamura S, Bergamini G, Faelth-Savitski M, Bantscheff M, Cox C, Gordon DA, Willard RR, Flanagan JJ, Casillas LN, Votta BJ, den Besten W, Famm K, Kruidenier L, Carter PS, Harling JD, Churcher I, Crews CM (2015) Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat Chem Biol 11(8):611–617. https://doi.org/10.1038/nchembio.1858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gadd MS, Testa A, Lucas X, Chan KH, Chen W, Lamont DJ, Zengerle M, Ciulli A (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13(5):514–521. https://doi.org/10.1038/nchembio.2329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bondeson DP, Smith BE, Burslem GM, Buhimschi AD, Hines J, Jaime-Figueroa S, Wang J, Hamman BD, Ishchenko A, Crews CM (2018) Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem Biol 25(1):78–87. e75. https://doi.org/10.1016/j.chembiol.2017.09.010

    Article  CAS  PubMed  Google Scholar 

  16. Roy RD, Rosenmund C, Stefan MI (2017) Cooperative binding mitigates the high-dose hook effect. BMC Syst Biol 11(1):74. https://doi.org/10.1186/s12918-017-0447-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Maniaci C, Hughes SJ, Testa A, Chen WZ, Lamont DJ, Rocha S, Alessi DR, Romeo R, Ciulli A (2017) Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat Commun 8(1):830. https://doi.org/10.1038/s41467-017-00954-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Steinebach C, Lindner S, Udeshi ND, Mani DC, Kehm H, Kopff S, Carr SA, Gutschow M, Kronke J (2018) Homo-PROTACs for the chemical knockdown of Cereblon. ACS Chem Biol 13(9):2771–2782. https://doi.org/10.1021/acschembio.8b00693

    Article  CAS  PubMed  Google Scholar 

  19. Saline M, Rodstrom KEJ, Fischer G, Orekhov VY, Karlsson BG, Lindkvist-Petersson K (2010) The structure of superantigen complexed with TCR and MHC reveals novel insights into superantigenic T cell activation. Nat Commun 1:119. https://doi.org/10.1038/ncomms1117

    Article  CAS  PubMed  Google Scholar 

  20. Andersen PS, Schuck P, Sundberg EJ, Geisler C, Karjalainen K, Mariuzza RA (2002) Quantifying the energetics of cooperativity in a ternary protein complex. Biochemistry 41(16):5177–5184. https://doi.org/10.1021/bi0200209

    Article  CAS  PubMed  Google Scholar 

  21. Choi JW, Chen J, Schreiber SL, Clardy J (1996) Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273(5272):239–242. https://doi.org/10.1126/science.273.5272.239

    Article  CAS  PubMed  Google Scholar 

  22. Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435(7041):446–451. https://doi.org/10.1038/nature03542

    Article  CAS  PubMed  Google Scholar 

  23. Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP center dot rapamycin center dot FRB ternary complex. J Am Chem Soc 127(13):4715–4721. https://doi.org/10.1021/ja043277y

    Article  CAS  PubMed  Google Scholar 

  24. Chamberlain PP, Cathers BE (2019) Cereblon modulators: low molecular weight inducers of protein degradation. Drug Discov Today Technol 31:29–34. https://doi.org/10.1016/j.ddtec.2019.02.004

    Article  PubMed  Google Scholar 

  25. Han T, Goralski M, Gaskill N, Capota E, Kim J, Ting TC, Xie Y, Williams NS, Nijhawan D (2017) Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356(6336):eaal3755. https://doi.org/10.1126/science.aal3755

    Article  CAS  PubMed  Google Scholar 

  26. Uehara T, Minoshima Y, Sagane K, Sugi NH, Mitsuhashi KO, Yamamoto N, Kamiyama H, Takahashi K, Kotake Y, Uesugi M, Yokoi A, Inoue A, Yoshida T, Mabuchi M, Tanaka A, Owa T (2017) Selective degradation of splicing factor CAPERalpha by anticancer sulfonamides. Nat Chem Biol 13(6):675–680. https://doi.org/10.1038/nchembio.2363

    Article  CAS  PubMed  Google Scholar 

  27. Bussiere DE, Xie LL, Srinivas H, Shu W, Burke A, Be C, Zhao JP, Godbole A, King D, Karki RG, Hornak V, Xu FM, Cobb J, Carte N, Frank AO, Frommlet A, Graff P, Knapp M, Fazal A, Okram B, Jiang SC, Michellys PY, Beckwith R, Voshol H, Wiesmann C, Solomon JM, Paulk J (2020) Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex. Nat Chem Biol 16(1):15–23. https://doi.org/10.1038/s41589-019-0411-6

    Article  CAS  PubMed  Google Scholar 

  28. Chan KH, Zengerle M, Testa A, Ciulli A (2018) Impact of target warhead and linkage vector on inducing protein degradation: comparison of Bromodomain and extra-terminal (BET) degraders derived from Triazolodiazepine (JQ1) and Tetrahydroquinoline (I-BET726) BET inhibitor scaffolds. J Med Chem 61(2):504–513. https://doi.org/10.1021/acs.jmedchem.6b01912

    Article  CAS  PubMed  Google Scholar 

  29. Zorba A, Nguyen C, Xu Y, Starr J, Borzilleri K, Smith J, Zhu H, Farley KA, Ding W, Schiemer J, Feng X, Chang JS, Uccello DP, Young JA, Garcia-Irrizary CN, Czabaniuk L, Schuff B, Oliver R, Montgomery J, Hayward MM, Coe J, Chen J, Niosi M, Luthra S, Shah JC, El-Kattan A, Qiu X, West GM, Noe MC, Shanmugasundaram V, Gilbert AM, Brown MF, Calabrese MF (2018) Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc Natl Acad Sci U S A 115(31):E7285–E7292. https://doi.org/10.1073/pnas.1803662115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zoppi V, Hughes SJ, Maniaci C, Testa A, Gmaschitz T, Wieshofer C, Koegl M, Riching KM, Daniels DL, Spallarossa A, Ciulli A (2019) Iterative design and optimization of initially inactive proteolysis targeting chimeras (PROTACs) identify VZ185 as a potent, fast, and selective von Hippel-Lindau (VHL) based dual degrader probe of BRD9 and BRD7. J Med Chem 62(2):699–726. https://doi.org/10.1021/acs.jmedchem.8b01413

    Article  CAS  PubMed  Google Scholar 

  31. Yang JL, Li YB, Aguilar A, Liu ZM, Yang CY, Wang SM (2019) Simple structural modifications converting a Bona fide MDM2 PROTAC degrader into a molecular glue molecule: a cautionary tale in the design of PROTAC degraders. J Med Chem 62(21):9471–9487. https://doi.org/10.1021/acs.jmedchem.9b00846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ishoey M, Chorn S, Singh N, Jaeger MG, Brand M, Paulk J, Bauer S, Erb MA, Parapatics K, Muller AC, Bennett KL, Ecker GF, Bradner JE, Winter GE (2018) Translation termination factor GSPT1 is a phenotypically relevant off-target of heterobifunctional Phthalimide degraders. ACS Chem Biol 13(3):553–560. https://doi.org/10.1021/acschembio.7b00969

    Article  CAS  PubMed  Google Scholar 

  33. Zengerle M, Chan KH, Ciulli A (2015) Selective small molecule induced degradation of the BET Bromodomain protein BRD4. ACS Chem Biol 10(8):1770–1777. https://doi.org/10.1021/acschembio.5b00216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Baud MGJ, Lin-Shiao E, Cardote T, Tallant C, Pschibul A, Chan KH, Zengerle M, Garcia JR, Kwan TT, Ferguson FM, Ciulli A (2014) Chemical biology. A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes. Science 346(6209):638–641. https://doi.org/10.1126/science.1249830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Roy MJ, Winkler S, Hughes SJ, Whitworth C, Galant M, Farnaby W, Rumpel K, Ciulli A (2019) SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. ACS Chem Biol 14(3):361–368. https://doi.org/10.1021/acschembio.9b00092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pillow TH, Adhikari P, Blake RA, Chen J, Del Rosario G, Deshmukh G, Figueroa I, Gascoigne KE, Kamath AV, Kaufman S, Kleinheinz T, Kozak KR, Latifi B, Leipold DD, Sing Li C, Li R, Mulvihill MM, O'Donohue A, Rowntree RK, Sadowsky JD, Wai J, Wang X, Wu C, Xu Z, Yao H, Yu SF, Zhang D, Zang R, Zhang H, Zhou H, Zhu X, Dragovich PS (2020) Antibody conjugation of a chimeric BET degrader enables in vivo activity. ChemMedChem 15(1):17–25. https://doi.org/10.1002/cmdc.201900497

    Article  CAS  PubMed  Google Scholar 

  37. Testa A, Lucas X, Castro GV, Chan KH, Wright JE, Runcie AC, Gadd MS, Harrison WTA, Ko EJ, Fletcher D, Ciulli A (2018) 3-Fluoro-4-hydroxyprolines: synthesis, conformational analysis, and Stereoselective recognition by the VHL E3 ubiquitin ligase for targeted protein degradation. J Am Chem Soc 140(29):9299–9313. https://doi.org/10.1021/jacs.8b05807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Han X, Zhao L, Xiang W, Qin C, Miao B, Xu T, Wang M, Yang CY, Chinnaswamy K, Stuckey J, Wang S (2019) Discovery of highly potent and efficient PROTAC degraders of androgen receptor (AR) by employing weak binding affinity VHL E3 ligase ligands. J Med Chem 62(24):11218–11231. https://doi.org/10.1021/acs.jmedchem.9b01393

    Article  CAS  PubMed  Google Scholar 

  39. Testa A, Hughes SJ, Lucas X, Wright JE, Ciulli A (2020) Structure-based Design of a Macrocyclic PROTAC. Angew Chem Int Edit 59(4):1727–1734. https://doi.org/10.1002/anie.201914396

    Article  CAS  Google Scholar 

  40. Soumana DI, Yilmaz NK, Prachanronarong KL, Aydin C, Ali A, Schiffer CA (2016) Structural and thermodynamic effects of macrocyclization in HCV NS3/4A inhibitor MK-5172. ACS Chem Biol 11(4):900–909. https://doi.org/10.1021/acschembio.5b00647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Farnaby W, Koegl M, Roy MJ, Whitworth C, Diers E, Trainor N, Zollman D, Steurer S, Karolyi-Oezguer J, Riedmueller C, Gmaschitz T, Wachter J, Dank C, Galant M, Sharps B, Rumpel K, Traxler E, Gerstberger T, Schnitzer R, Petermann O, Greb P, Weinstabl H, Bader G, Zoephel A, Weiss-Puxbaum A, Ehrenhofer-Wolfer K, Wohrle S, Boehmelt G, Rinnenthal J, Arnhof H, Wiechens N, Wu MY, Owen-Hughes T, Ettmayer P, Pearson M, McConnell DB, Ciulli A (2019) BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat Chem Biol 15(7):672–680. https://doi.org/10.1038/s41589-019-0294-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Vangamudi B, Paul TA, Shah PK, Kost-Alimova M, Nottebaum L, Shi X, Zhan YA, Leo E, Mahadeshwar HS, Protopopov A, Futreal A, Tieu TN, Peoples M, Heffernan TP, Marszalek JR, Toniatti C, Petrocchi A, Verhelle D, Owen DR, Draetta G, Jones P, Palmer WS, Sharma S, Andersen JN (2015) The SMARCA2/4 ATPase domain surpasses the Bromodomain as a drug target in SWI/SNFMutant cancers: insights from cDNA rescue and PFI-3 inhibitor studies. Cancer Res 75(18):3865–3878. https://doi.org/10.1158/0008-5472.Can-14-3798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Albrecht B, Cote A, Crawford T, Duplessis M, Good A, Leblanc Y, Magnuson S, Nasveschuk C, Romero F, Tang Y (2016) Therapeutic pyridazine compounds and uses thereof WIPO patent WO2016138114

    Google Scholar 

  44. Soares P, Gadd MS, Frost J, Galdeano C, Ellis L, Epemolu O, Rocha S, Read KD, Ciulli A (2018) Group-based optimization of potent and cell-active inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin ligase: structure-activity relationships leading to the chemical probe (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyI)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyppyrrolidine-2-carboxamide) (VH298). J Med Chem 61(2):599–618. https://doi.org/10.1021/acs.jmedchem.7b00675

    Article  CAS  PubMed  Google Scholar 

  45. Lebakken CS, Riddle SM, Singh U, Frazee WJ, Eliason HC, Gao Y, Reichling LJ, Marks BD, Vogel KW (2009) Development and applications of a broad-coverage, TR-FRET-based kinase binding assay platform. J Biomol Screen 14(8):924–935. https://doi.org/10.1177/1087057109339207

    Article  CAS  PubMed  Google Scholar 

  46. Glickman JF, Wu X, Mercuri R, Illy C, Bowen BR, He Y, Sills M (2002) A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. J Biomol Screen 7(1):3–10. https://doi.org/10.1177/108705710200700102

    Article  CAS  PubMed  Google Scholar 

  47. Beaudet L, Rodriguez-Suarez R, Venne M-H, Caron M, Bédard J, Brechler V, Parent S, Bielefeld-Sévigny M (2008) AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery. Nat Methods 5(12):A10

    Article  Google Scholar 

  48. Winter GE, Mayer A, Buckley DL, Erb MA, Roderick JE, Vittori S, Reyes JM, di Iulio J, Souza A, Ott CJ, Roberts JM, Zeid R, Scott TG, Paulk J, Lachance K, Olson CM, Dastjerdi S, Bauer S, Lin CY, Gray NS, Kelliher MA, Churchman LS, Bradner JE (2017) BET Bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol Cell 67(1):5–18.e19. https://doi.org/10.1016/j.molcel.2017.06.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wurz RP, Dellamaggiore K, Dou H, Javier N, Lo M-C, McCarter JD, Mohl D, Sastri C, Lipford JR, Cee VJ (2018) A “click chemistry platform” for the rapid synthesis of bispecific molecules for inducing protein degradation. J Med Chem 61(2):453–461. https://doi.org/10.1021/acs.jmedchem.6b01781

    Article  CAS  PubMed  Google Scholar 

  50. Simonetta KR, Taygerly J, Boyle K, Basham SE, Padovani C, Lou Y, Cummins TJ, Yung SL, von Soly SK, Kayser F, Kuriyan J, Rape M, Cardozo M, Gallop MA, Bence NF, Barsanti PA, Saha A (2019) Prospective discovery of small molecule enhancers of an E3 ligase-substrate interaction. Nat Commun 10(1):1402. https://doi.org/10.1038/s41467-019-09358-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Nowak RP, DeAngelo SL, Buckley D, He Z, Donovan KA, An J, Safaee N, Jedrychowski MP, Ponthier CM, Ishoey M, Zhang T, Mancias JD, Gray NS, Bradner JE, Fischer ES (2018) Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat Chem Biol 14(7):706–714. https://doi.org/10.1038/s41589-018-0055-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hsu JH-R, Rasmusson T, Robinson J, Pachl F, Read J, Kawatkar S, O’Donovan DH, Bagal S, Code E, Rawlins P, Argyrou A, Tomlinson R, Gao N, Zhu X, Chiarparin E, Jacques K, Shen M, Woods H, Bednarski E, Wilson DM, Drew L, Castaldi MP, Fawell S, Bloecher A (2020) EED-targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex. Cell Chem Biol 27(1):41–46.e17. https://doi.org/10.1016/j.chembiol.2019.11.004

    Article  CAS  PubMed  Google Scholar 

  53. Ciulli A (2013) Biophysical screening for the discovery of small-molecule ligands. In: Williams MA, Daviter T (eds) Protein-ligand interactions: methods and applications. Humana Press, Totowa, NJ, pp 357–388. https://doi.org/10.1007/978-1-62703-398-5_13

    Chapter  Google Scholar 

  54. Fisher SL, Phillips AJ (2018) Targeted protein degradation and the enzymology of degraders. Curr Opin Chem Biol 44:47–55. https://doi.org/10.1016/j.cbpa.2018.05.004

    Article  CAS  PubMed  Google Scholar 

  55. Hann MM, Simpson GL (2014) Intracellular drug concentration and disposition–the missing link? Methods 68(2):283–285. https://doi.org/10.1016/j.ymeth.2014.05.009

    Article  CAS  PubMed  Google Scholar 

  56. Swinney DC (2004) Biochemical mechanisms of drug action: what does it take for success? Nat Rev Drug Discov 3(9):801–808. https://doi.org/10.1038/nrd1500

    Article  CAS  PubMed  Google Scholar 

  57. Bantscheff M, Hopf C, Savitski MM, Dittmann A, Grandi P, Michon A-M, Schlegl J, Abraham Y, Becher I, Bergamini G, Boesche M, Delling M, Dümpelfeld B, Eberhard D, Huthmacher C, Mathieson T, Poeckel D, Reader V, Strunk K, Sweetman G, Kruse U, Neubauer G, Ramsden NG, Drewes G (2011) Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol 29(3):255–265. https://doi.org/10.1038/nbt.1759

    Article  CAS  PubMed  Google Scholar 

  58. Jessani N, Niessen S, Wei BQ, Nicolau M, Humphrey M, Ji Y, Han W, Noh D-Y, Yates JR, Jeffrey SS, Cravatt BF (2005) A streamlined platform for high-content functional proteomics of primary human specimens. Nat Methods 2(9):691–697. https://doi.org/10.1038/nmeth778

    Article  CAS  PubMed  Google Scholar 

  59. Médard G, Pachl F, Ruprecht B, Klaeger S, Heinzlmeir S, Helm D, Qiao H, Ku X, Wilhelm M, Kuehne T, Wu Z, Dittmann A, Hopf C, Kramer K, Kuster B (2015) Optimized chemical proteomics assay for kinase inhibitor profiling. J Proteome Res 14(3):1574–1586. https://doi.org/10.1021/pr5012608

    Article  CAS  PubMed  Google Scholar 

  60. AS H, Marjanovic J, Lewandowski TM, Marin V, Patterson M, Miesbauer L, Ready D, Williams J, Vasudevan A, Lin Q (2016) 2-Aryl-5-carboxytetrazole as a new photoaffinity label for drug target identification. J Am Chem Soc 138(44):14609–14615. https://doi.org/10.1021/jacs.6b06645

    Article  CAS  Google Scholar 

  61. Molina DM, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y, Nordlund P (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141):84–87

    Article  CAS  Google Scholar 

  62. Klaeger S, Heinzlmeir S, Wilhelm M, Polzer H, Vick B, Koenig PA, Reinecke M, Ruprecht B, Petzoldt S, Meng C, Zecha J, Reiter K, Qiao H, Helm D, Koch H, Schoof M, Canevari G, Casale E, Depaolini SR, Feuchtinger A, Wu Z, Schmidt T, Rueckert L, Becker W, Huenges J, Garz AK, Gohlke BO, Zolg DP, Kayser G, Vooder T, Preissner R, Hahne H, Tõnisson N, Kramer K, Götze K, Bassermann F, Schlegl J, Ehrlich HC, Aiche S, Walch A, Greif PA, Schneider S, Felder ER, Ruland J, Médard G, Jeremias I, Spiekermann K, Kuster B (2017) The target landscape of clinical kinase drugs. Science 358(6367):eaan4368

    Article  Google Scholar 

  63. Dart ML, Machleidt T, Jost E, Schwinn MK, Robers MB, Shi C, Kirkland TA, Killoran MP, Wilkinson JM, Hartnett JR, Zimmerman K, Wood KV (2018) Homogeneous assay for target engagement utilizing bioluminescent thermal shift. ACS Med Chem Lett 9(6):546–551

    Article  CAS  Google Scholar 

  64. Stoddart LA, Vernall AJ, Denman JL, Briddon SJ, Kellam B, Hill SJ (2012) Fragment screening at adenosine-A3 receptors in living cells using a fluorescence-based binding assay. Chem Biol 19(9):1105–1115

    Article  CAS  Google Scholar 

  65. Dubach JM, Kim E, Yang K, Cuccarese M, Giedt RJ, Meimetis LG, Vinegoni C, Weissleder R (2017) Quantitating drug-target engagement in single cells in vitro and in vivo. Nat Chem Biol 13(2):168–173

    Article  CAS  Google Scholar 

  66. Goyet E, Bouquier N, Ollendorff V, Perroy J (2016) Fast and high resolution single-cell BRET imaging. Sci Rep 6(1):28231

    Article  CAS  Google Scholar 

  67. Robers MB, Dart ML, Woodroofe CC, Zimprich CA, Kirkland TA, Machleidt T, Kupcho KR, Levin S, Hartnett JR, Zimmerman K, Niles AL, Ohana RF, Daniels DL, Slater M, Wood MG, Cong M, Cheng YQ, Wood KV (2015) Target engagement and drug residence time can be observed in living cells with BRET. Nat Commun 6:10091

    Article  CAS  Google Scholar 

  68. Chessum NEA, Sharp SY, Caldwell JJ, Pasqua AE, Wilding B, Colombano G, Collins I, Ozer B, Richards M, Rowlands M, Stubbs M, Burke R, McAndrew PC, Clarke PA, Workman P, Cheeseman MD, Jones K (2018) Demonstrating in-cell target engagement using a pirin protein degradation probe (CCT367766). J Med Chem 61(3):918–933

    Article  CAS  Google Scholar 

  69. Whitworth C, Farnaby W, Koegl M, Schnitzer R, Steurer S, Ettmayer P, Ciulli A (2018) PO-449 optimisation of an AlphaLISA assay for the characterisation of PROTAC-induced ternary complexes within cell lysates. ESMO Open 3(Suppl 2):A198

    Article  Google Scholar 

  70. Riching KM, Mahan S, Corona CR, McDougall M, Vasta JD, Robers MB, Urh M, Daniels DL (2018) Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action. ACS Chem Biol 13(9):2758–2770

    Article  CAS  Google Scholar 

  71. Chung C-I, Zhang Q, Shu X (2018) Dynamic imaging of small molecule induced protein–protein interactions in living cells with a fluorophore phase transition based approach. Anal Chem 90(24):14287–14293

    Article  CAS  Google Scholar 

  72. Kaji T, Koga H, Kuroha M, Akimoto T, Hayata K (2020) Characterization of cereblon-dependent targeted protein degrader by visualizing the spatiotemporal ternary complex formation in cells. Sci Rep 10(1):3088

    Article  CAS  Google Scholar 

  73. Paiva S-L, Crews CM (2019) Targeted protein degradation: elements of PROTAC design. Curr Opin Chem Biol 50:111–119

    Article  CAS  Google Scholar 

  74. Chu T-T, Gao N, Li Q-Q, Chen P-G, Yang X-F, Chen Y-X, Zhao Y-F, Li Y-M (2016) Specific knockdown of endogenous tau protein by peptide-directed ubiquitin-proteasome degradation. Cell Chem Biol 23(4):453–461

    Article  CAS  Google Scholar 

  75. Khan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, Liu X, Thummuri D, Yuan Y, Wiegand JS, Pei J, Zhang W, Sharma A, McCurdy CR, Kuruvilla VM, Baran N, Ferrando AA, Kim YM, Rogojina A, Houghton PJ, Huang G, Hromas R, Konopleva M, Zheng G, Zhou D (2019) A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity. Nat Med 25(12):1938–1947. https://doi.org/10.1038/s41591-019-0668-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Ottis P, Toure M, Cromm PM, Ko E, Gustafson JL, Crews CM (2017) Assessing different E3 ligases for small molecule induced protein ubiquitination and degradation. ACS Chem Biol 12(10):2570–2578

    Article  CAS  Google Scholar 

  77. Kerres N, Steurer S, Schlager S, Bader G, Berger H, Caligiuri M, Dank C, Engen JR, Ettmayer P, Fischerauer B (2017) Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep 20(12):2860–2875

    Article  CAS  Google Scholar 

  78. Savitski MM, Zinn N, Faelth-Savitski M, Poeckel D, Gade S, Becher I, Muelbaier M, Wagner AJ, Strohmer K, Werner T, Melchert S, Petretich M, Rutkowska A, Vappiani J, Franken H, Steidel M, Sweetman GM, Gilan O, Lam EYN, Dawson MA, Prinjha RK, Grandi P, Bergamini G, Bantscheff M (2018) Multiplexed proteome dynamics profiling reveals mechanisms controlling protein homeostasis. Cell 173(1):260–274. e25

    Article  CAS  Google Scholar 

  79. Popow J, Arnhof H, Bader G, Berger H, Ciulli A, Covini D, Dank C, Gmaschitz T, Greb P, Karolyi-Özguer J, Koegl M, McConnell DB, Pearson M, Rieger M, Rinnenthal J, Roessler V, Schrenk A, Spina M, Steurer S, Trainor N, Traxler E, Wieshofer C, Zoephel A, Ettmayer P (2019) Highly selective PTK2 proteolysis targeting chimeras to probe focal adhesion kinase scaffolding functions. J Med Chem 62(5):2508–2520

    Article  CAS  Google Scholar 

  80. Buckley DL, Raina K, Darricarrere N, Hines J, Gustafson JL, Smith IE, Miah AH, Harling JD, Crews CM (2015) HaloPROTACS: use of small molecule PROTACs to induce degradation of HaloTag fusion proteins. ACS Chem Biol 10(8):1831–1837

    Article  CAS  Google Scholar 

  81. Tovell H, Testa A, Maniaci C, Zhou H, Prescott AR, Macartney T, Ciulli A, Alessi DR (2019) Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader. ACS Chem Biol 14(5):882–892

    Article  CAS  Google Scholar 

  82. Winter GE, Buckley DL, Paulk J, Roberts JM, Souza A, Dhe-Paganon S, Bradner JE (2015) Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348(6241):1376–1381

    Article  CAS  Google Scholar 

  83. Sun X, Wang J, Yao X, Zheng W, Mao Y, Lan T, Wang L, Sun Y, Zhang X, Zhao Q, Zhao J, Xiao RP, Zhang X, Ji G, Rao Y (2019) A chemical approach for global protein knockdown from mice to non-human primates. Cell Discov 5:10

    Article  Google Scholar 

  84. Mullard A (2019) Arvinas's PROTACs pass first safety and PK analysis. Nat Rev Drug Discov 18(12):895. https://doi.org/10.1038/d41573-019-00188-4

    Article  CAS  PubMed  Google Scholar 

  85. Nabet B, Ferguson FM, Seong BKA, Kuljanin M, Leggett AL, Mohardt ML, Robichaud A, Conway AS, Buckley DL, Mancias JD, Bradner JE, Stegmaier L, Gray NS. (2020) Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules bioRxiv 2020.03.13.980946. https://doi.org/10.1101/2020.03.13.980946

Download references

Acknowledgements and Funding

We would like to thank many members of the Ciulli group for helpful discussions. We apologise to the authors of many studies in the field which could not be mentioned due to space restriction.

The Ciulli laboratory’s work on PROTACs has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007-2013) as a Starting Grant to A.C. (grant agreement No. ERC-2012-StG-311460 DrugE3CRLs). R.C. is funded by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio doctoral training programme (BB/M010996/1). A.B. is funded by a PhD studentship from the Medical Research Scotland (MRS) (1170-2017). C.C. is funded by a PhD studentship from the UK Medical Research Council (MRC) under the doctoral training programme in Quantitative and Interdisciplinary approaches to biomedical science (QI Biomed) (MR/N0123735/1). The Ciulli laboratory receives or has received sponsored research support from Boehringer Ingelheim, Eisai, Nurix, Ono Pharmaceuticals and Amphista Therapeutics.

Conflict of interest statement: A.C. is the scientific founder, shareholder, nonexecutive director and consultant of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. The remaining author reports no competing interests.

Author information

Author notes

  1. Ryan Casement, Adam Bond and Conner Craigon contributed equally to this work.

Authors and Affiliations

  1. Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, UK

    Ryan Casement, Adam Bond, Conner Craigon & Alessio Ciulli

Authors
  1. Ryan Casement
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adam Bond
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Conner Craigon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alessio Ciulli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alessio Ciulli .

Editor information

Editors and Affiliations

  1. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Angela M. Cacace

  2. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Christopher M. Hickey

  3. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Miklós Békés

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Casement, R., Bond, A., Craigon, C., Ciulli, A. (2021). Mechanistic and Structural Features of PROTAC Ternary Complexes. In: Cacace, A.M., Hickey, C.M., Békés, M. (eds) Targeted Protein Degradation. Methods in Molecular Biology, vol 2365. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1665-9_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1665-9_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1664-2

  • Online ISBN: 978-1-0716-1665-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation