Structural basis of PROTAC cooperative recognition for selective protein degradation

Abstract

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase–PROTAC–target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The crystal structure of the Brd4BD2–MZ1–VHL–ElonginC–ElonginB complex.
Figure 2: Brd4BD2 and VHL form a stable, cooperative complex in the presence of MZ1.
Figure 3: The molecular basis of MZ1-induced compact complex formation between Brd4BD2 and VHL.
Figure 4: Structure-guided design and characterization of Brd4-selective degrader AT1.
Figure 5: Schematic model of selective PROTAC-induced target degradation.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Huang, X. & Dixit, V.M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Petzold, G., Fischer, E.S. & Thomä, N.H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase. Nature 532, 127–130 (2016).

    CAS  Google Scholar 

  3. 3

    Matyskiela, M.E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 535, 252–257 (2016).

    CAS  Google Scholar 

  4. 4

    Lai, A.C. & Crews, C.M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. 5

    Sakamoto, K.M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Winter, G.E. et al. Drug development. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  Article  Google Scholar 

  7. 7

    Zengerle, M., Chan, K.-H. & Ciulli, A. Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem. Biol. 10, 1770–1777 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Bondeson, D.P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Deshaies, R.J. Protein degradation: prime time for PROTACs. Nat. Chem. Biol. 11, 634–635 (2015).

    CAS  PubMed  Google Scholar 

  11. 11

    Toure, M. & Crews, C.M. Small-molecule PROTACS: new approaches to protein degradation. Angew. Chem. Int. Edn Engl. 55, 1966–1973 (2016).

    CAS  Google Scholar 

  12. 12

    Lai, A.C. et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew. Chem. Int. Edn Engl. 55, 807–810 (2016).

    CAS  Google Scholar 

  13. 13

    Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    CAS  Article  Google Scholar 

  14. 14

    Galdeano, C. et al. Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J. Med. Chem. 57, 8657–8663 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Frost, J. et al. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat. Commun. 7, 13312 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Zuber, J. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Nicodeme, E. et al. Suppression of inflammation by a synthetic histone mimic. Nature 468, 1119–1123 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Hon, W.-C. et al. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature 417, 975–978 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Min, J.-H. et al. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 296, 1886–1889 (2002).

    CAS  PubMed  Google Scholar 

  20. 20

    Filippakopoulos, P. et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149, 214–231 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Tanaka, M. et al. Design and characterization of bivalent BET inhibitors. Nat. Chem. Biol. 12, 1089–1096 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Whitty, A. Cooperativity and biological complexity. Nat. Chem. Biol. 4, 435–439 (2008).

    CAS  PubMed  Google Scholar 

  23. 23

    Douglass, E.F. Jr., Miller, C.J., Sparer, G., Shapiro, H. & Spiegel, D.A. A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 135, 6092–6099 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Eglen, R.M. et al. The use of AlphaScreen technology in HTS: current status. Curr. Chem. Genomics 1, 2–10 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Roberts, J.M. & Bradner, J.E. A bead-based proximity assay for BRD4 ligand discovery. Curr. Protoc. Chem. Biol. 7, 263–278 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Stebbins, C.E., Kaelin, W.G. Jr. & Pavletich, N.P. Structure of the VHL-ElonginC–ElonginB complex: implications for VHL tumor suppressor function. Science 284, 455–461 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Duda, D.M. et al. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Zhou, M., Li, Q. & Wang, R. Current experimental methods for characterizing protein–protein interactions. ChemMedChem 11, 738–756 (2016).

    CAS  PubMed  Google Scholar 

  29. 29

    Raina, K. et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl. Acad. Sci. USA 113, 7124–7129 (2016).

    CAS  PubMed  Google Scholar 

  30. 30

    Bulatov, E. & Ciulli, A. Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation. Biochem. J. 467, 365–386 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Fischer, E.S., Park, E., Eck, M.J. & Thomä, N.H. SPLINTS: small-molecule protein ligand interface stabilizers. Curr. Opin. Struct. Biol. 37, 115–122 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Tan, X. et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007).

    CAS  PubMed  Google Scholar 

  34. 34

    Sheard, L.B. et al. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468, 400–405 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

    CAS  PubMed  Google Scholar 

  37. 37

    Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

    PubMed  Google Scholar 

  38. 38

    Chamberlain, P.P. et al. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 21, 803–809 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Pommier, Y. & Marchand, C. Interfacial inhibitors: targeting macromolecular complexes. Nat. Rev. Drug Discov. 11, 25–36 (2011).

    PubMed  Google Scholar 

  40. 40

    Thiel, P., Kaiser, M. & Ottmann, C. Small-molecule stabilization of protein–protein interactions: an underestimated concept in drug discovery? Angew. Chem. Int. Edn Engl. 51, 2012–2018 (2012).

    CAS  Google Scholar 

  41. 41

    Illendula, A. et al. Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice. Science 347, 779–784 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Waring, M.J. et al. Potent and selective bivalent inhibitors of BET bromodomains. Nat. Chem. Biol. 12, 1097–1104 (2016).

    CAS  PubMed  Google Scholar 

  43. 43

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  46. 46

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  Article  Google Scholar 

  48. 48

    Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Schüttelkopf, A.W. & van Aalten, D.M.F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    PubMed  Google Scholar 

  50. 50

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

  51. 51

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Laskowski, R.A. & Swindells, M.B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

    CAS  Google Scholar 

  53. 53

    Phillips, J.C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Brooks, B.R. et al. CHARMM: the biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Vanommeslaeghe, K. & MacKerell, A.D. Jr. Automation of the CHARMM general force field (CGenFF) I: bond perception and atom typing. J. Chem. Inf. Model. 52, 3144–3154 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J.Mol.Graph. 14, 33–38 (1996).

    CAS  PubMed  Google Scholar 

  57. 57

    Glykos, N.M. Software news and updates. Carma: a molecular dynamics analysis program. J. Comput. Chem. 27, 1765–1768 (2006).

    CAS  PubMed  Google Scholar 

  58. 58

    Manza, L.L., Stamer, S.L., Ham, A.-J.L., Codreanu, S.G. & Liebler, D.C. Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5, 1742–1745 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).

    CAS  PubMed  Google Scholar 

  60. 60

    Gilar, M., Olivova, P., Daly, A.E. & Gebler, J.C. Orthogonality of separation in two-dimensional liquid chromatography. Anal. Chem. 77, 6426–6434 (2005).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the European Research Council (ERC-2012-StG-311460 DrugE3CRLs Starting Grant to A.C.); the UK Biotechnology and Biological Sciences Research Council (BBSRC grant BB/J001201/2 to A.C.); the European Commission (H2020-MSCA-IF-2014-655516 Marie Skłodowska-Curie Actions Individual Fellowship to K.-H.C. and H2020-MSCA-IF-2015-806323 Marie Skłodowska-Curie Actions Individual Fellowship to X.L.); and the Wellcome Trust (Strategic Awards 100476/Z/12/Z for biophysics and drug discovery and 094090/Z/10/Z for structural biology and X-ray crystallography to the Division of Biological Chemistry and Drug Discovery). We are thankful to P. Fyfe for support with the in-house X-ray facility; L. Finn for support with tissue culture facility (MRC-PPU); the Ferguson lab for access to LI-COR equipment; T. Cardote (Ciulli lab, BCDD, SLS, Dundee) for the gift of full-length Cul2-Rbx1 and A. Knebel (MRC-PPU/DSTT) for the gift of E1 and E2 enzymes; the Division of Computational Biology for support with computational cluster; and to Diamond Light Source for beamtime (BAG proposal MX10071) and beamline support at beamline I04-1.

Author information

Affiliations

Authors

Contributions

A.C. conceived the idea and directed the project. M.S.G., X.L., A.T., K.-H.C. and A.C. designed the experiments and interpreted results. M.S.G., X.L., A.T., and K.-H.C. performed experiments. A.T. and M.Z. contributed to compound design and synthesized compounds. W.C. performed MS proteomics experiments under the supervision of D.J.L. M.S.G., X.L. and A.C. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Alessio Ciulli.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–12 (PDF 4451 kb)

Supplementary Note

Chemistry (PDF 2033 kb)

Supplementary Data Set

Proteomic analysis of relative protein abundance in HeLa cells. Results are graphically represented in Figure 4f,g (XLSX 7377 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gadd, M., Testa, A., Lucas, X. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514–521 (2017). https://doi.org/10.1038/nchembio.2329

Download citation

Further reading