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Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

Nature Chemical Biology volume 17, pages 1157–1167 (2021)Cite this article

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Abstract

Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel–Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

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Fig. 1: Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader.
Fig. 2: Quantitative degradation, ubiquitination and mass spectrometry analyses reveal that SIM1 has preference for BRD2.
Fig. 3: Potent SIM1 results in more efficacious apoptosis-induced cytotoxicity in BET-sensitive cancer cell lines.
Fig. 4: SIM1 induces a conformation change in BRD4, intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL.
Fig. 5: SIM1 forms cooperative stable ternary complexes with enhanced cellular residence time and shows a favorable pharmacokinetic (PK) profile in mice.
Fig. 6: Models of trivalent ternary complex formation and advantages over monovalent and bivalent compounds.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary information files. Mass spectrometry proteomics data (Fig. 2e and Extended Data Fig. 2f) are provided in Supplementary Data Set 1. The study report of PKs of SIM1 following single i.v. and s.c. administrations to mice provided by Chempartner Co., Ltd., is provided in Supplementary Data Set 2. The study report of BROMOscan profiling service data (Fig. 5a) is provided as Supplementary Data Set 3. Source data are provided with this paper.

References

  1. Deshaies, R. J. Multispecific drugs herald a new era of biopharmaceutical innovation. Nature 580, 329–338 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Che, Y., Gilbert, A. M., Shanmugasundaram, V. & Noe, M. C. Inducing protein–protein interactions with molecular glues. Bioorg. Med. Chem. Lett. 28, 2585–2592 (2018).

    Article  CAS  PubMed  Google Scholar 

  3. Maniaci, C. & Ciulli, A. Bifunctional chemical probes inducing protein–protein interactions. Curr. Opin. Chem. Biol. 52, 145–156 (2019).

    Article  CAS  PubMed  Google Scholar 

  4. Pettersson, M. & Crews, C. M. PROteolysis TArgeting Chimeras (PROTACs)—past, present and future. Drug Discov. Today Technol. 31, 15–27 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  5. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Salami, J. et al. Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance. Commun. Biol. 1, 100 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Hu, J. et al. Discovery of ERD-308 as a highly potent proteolysis targeting chimera (PROTAC) degrader of estrogen receptor (ER). J. Med. Chem. 62, 1420–1442 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Qin, C. et al. Discovery of QCA570 as an exceptionally potent and efficacious proteolysis targeting chimera (PROTAC) degrader of the bromodomain and extra-terminal (BET) proteins capable of inducing complete and durable tumor regression. J. Med. Chem. 61, 6685–6704 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zoppi, V. et al. 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, 699–726 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Farnaby, W. et al. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 15, 672–680 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zorba, A. et al. Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl Acad. Sci. USA 115, E7285–E7292 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Popow, J. et al. Highly selective PTK2 proteolysis targeting chimeras to probe focal adhesion kinase scaffolding functions. J. Med. Chem. 62, 2508–2520 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Burslem, G. M. et al. The advantages of targeted protein degradation over inhibition: an RTK case study. Cell Chem. Biol. 25, 67–77 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. Bensimon, A. et al. Targeted degradation of SLC transporters reveals amenability of multi-pass transmembrane proteins to ligand-induced proteolysis. Cell Chem. Biol. 27, 728–739 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bondeson, D. P. et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25, 78–87 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Olson, C. M. et al. Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nat. Chem. Biol. 14, 163–170 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Tovell, H. et al. Design and characterization of SGK3–PROTAC1, an isoform specific SGK3 kinase PROTAC degrader. ACS Chem. Biol. 14, 2024–2034 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Testa, A. et al. 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, 9299–9313 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Han, X. et al. 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, 11218–11231 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Riching, K. M. et al. Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action. ACS Chem. Biol. 13, 2758–2770 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Daniels, D. L., Riching, K. M. & Urh, M. Monitoring and deciphering protein degradation pathways inside cells. Drug Discov. Today Technol. 31, 61–68 (2019).

    Article  PubMed  Google Scholar 

  25. Roy, M. J. et al. SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. ACS Chem. Biol. 14, 361–368 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nowak, R. P. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 14, 706–714 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mammen, M., Choi, S.-K. & Whitesides, G. M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. Engl. 37, 2754–2794 (1998).

    Article  PubMed  Google Scholar 

  28. Kiessling, L. L., Gestwicki, J. E. & Strong, L. E. Synthetic multivalent ligands as probes of signal transduction. Angew. Chem. Int. Ed. Engl. 45, 2348–2368 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wu, Q. et al. A chemical toolbox for the study of bromodomains and epigenetic signaling. Nat. Commun. 10, 1915 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Gilan, O. et al. Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation. Science 368, 387–394 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Winter, G. E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. 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) α subunit with in vitro nanomolar affinities. J. Med. Chem. 57, 8657–8663 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhu, X. F. et al. Knockdown of heme oxygenase-1 promotes apoptosis and autophagy and enhances the cytotoxicity of doxorubicin in breast cancer cells. Oncol. Lett. 10, 2974–2980 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Maniaci, C. et al. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat. Commun. 8, 830 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Klein, V. G. et al. Understanding and improving the membrane permeability of VH032-based PROTACs. ACS Med. Chem. Lett. 11, 1732–1738 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Runcie, A. C. et al. Optimization of a ‘bump-and-hole’ approach to allele-selective BET bromodomain inhibition. Chem. Sci. 9, 2452–2468 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Smith, B. E. et al. Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase. Nat. Commun. 10, 131 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Hughes, S. J. & Ciulli, A. Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem 61, 505–516 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Testa, A., Hughes, S. J., Lucas, X., Wright, J. E. & Ciulli, A. Structure-based design of a macrocyclic PROTAC. Angew. Chem. Int. Ed. Engl. 59, 1727–1734 (2020).

    Article  CAS  PubMed  Google Scholar 

  45. Fisher, S. L. & Phillips, A. J. Targeted protein degradation and the enzymology of degraders. Curr. Opin. Chem. Biol. 44, 47–55 (2018).

    Article  CAS  PubMed  Google Scholar 

  46. Jiang, B. et al. Discovery and resistance mechanism of a selective CDK12 degrader. Nat. Chem. Biol. 17, 675–683 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lucas, X., Van Molle, I. & Ciulli, A. Surface probing by fragment-based screening and computational methods identifies ligandable pockets on the von Hippel–Lindau (VHL) E3 ubiquitin ligase. J. Med. Chem. 61, 7387–7393 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zheng, M. et al. Rational design and synthesis of novel dual PROTACs for simultaneous degradation of EGFR and PARP. J. Med. Chem. 64, 7839–7852 (2021).

    Article  CAS  PubMed  Google Scholar 

  49. Yamazoe, S. et al. Heterobifunctional molecules induce dephosphorylation of kinases-A proof of concept study. J. Med. Chem. 63, 2807–2813 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Siriwardena, S. U. et al. Phosphorylation-inducing chimeric small molecules. J. Am. Chem. Soc. 142, 14052–14057 (2020).

    Article  CAS  PubMed  Google Scholar 

  51. Baud, M. G. J. et al. A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes. Science 346, 638–641 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Munshi, A., Hobbs, M. & Meyn, R. E. Clonogenic cell survival assay. Methods Mol. Med. 110, 21–28 (2005).

    PubMed  Google Scholar 

  53. Van Molle, I. et al. Dissecting fragment-based lead discovery at the von Hippel–Lindau protein:hypoxia inducible factor 1α protein–protein interface. Chem. Biol. 19, 1300–1312 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. de Castro, G. V. & Ciulli, A. Spy vs. spy: selecting the best reporter for 19F NMR competition experiments. Chem. Commun. 55, 1482–1485 (2019).

    Article  Google Scholar 

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Acknowledgements

We thank A. Bond for the synthesis of ARV-771, G. Liu, Q. Gu, N. Li, C. Ji, S. Liao, C. Wang, H. Li and B. Guan at ChemPartner for in vivo pharmacokinetic studies and W. Farnaby and T. Kirkland for discussions and critical reading of the manuscript. Research reported in this publication was supported by the European Research Council (ERC, starting grant ERC-2012-StG-311460 DrugE3CRLs to A.C.), the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under grant agreement no. 875510 (EUbOPEN project) and Ono Pharma (visiting scientist fund to S.I.). K.-H. C. was funded by a Marie Skłodowska-Curie Actions Individual Fellowship from the European Commission (H2020-MSCA-IF-2014-655516). C.C. was funded by a PhD studentship from the UK Medical Research Council (MRC) under the doctoral training programme in Quantitative and Inter-disciplinary approaches to biomedical science (QI Biomed) (MR/N0123735/1). Biophysics and drug discovery activities were supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). K.M.R., S.D.M., N.M., M.U. and D.L.D. are funded by Promega Corporation.

Author information

Author notes

  1. Satomi Imaide

    Present address: Discovery Technology Research Laboratories, Ono Pharmaceutical Co., Ltd., Osaka, Japan

  2. Scott J. Hughes & Andrea Testa

    Present address: Amphista Therapeutics Ltd., Newhouse, UK

  3. Kwok-Ho Chan

    Present address: GlaxoSmithKline R&D, Stevenage, UK

  4. Chiara Maniaci

    Present address: School of Natural and Environmental Sciences, Chemistry, Newcastle University, Newcastle upon Tyne, UK

  5. These authors contributed equally: Satomi Imaide, Kristin M. Riching, Nikolai Makukhin.

Authors and Affiliations

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

    Satomi Imaide, Nikolai Makukhin, Vesna Vetma, Claire Whitworth, Scott J. Hughes, Nicole Trainor, Angus D. Cowan, Kwok-Ho Chan, Conner Craigon, Andrea Testa, Chiara Maniaci & Alessio Ciulli

  2. Promega Corporation, Madison, WI, USA

    Kristin M. Riching, Sarah D. Mahan, Nancy Murphy, Marjeta Urh & Danette L. Daniels

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Contributions

A.C. and D.L.D. conceived the idea, directed and supervised the project and have overall project responsibility. S.I. optimized synthetic routes and synthesized all compounds, performed degradation and cell viability assays, expressed and purified proteins and performed SEC, AlphaLISA, ITC and SPR assays. K.M.R. performed kinetic degradation assays, washout and NanoBRET cellular ternary complex, ubiquitination, target engagement and residence time assays. N.M. designed and synthesized (R,S)-SIM1, scaled up synthesis of SIM1 for in vivo experiments and performed FP and SPR binding assays. V.V. performed PARP cleavage, clonogenic assays and flow cytometry analysis of apoptosis with assistance from C.C. C.W. performed degradation and Caspase-Glo assays. S.J.H. performed FP displacement assays. N.T. performed mass spectrometry proteomics assays. S.D.M. performed cellular cMyc and viability assays. N.M. performed the NanoBRET biosensor and mutant ternary complex assays. K.-H. C. designed and cloned tandem BRD4 WT and mutant constructs. A.D.C. expressed and purified tandem BET protein constructs. A.T., C.M. and A.C. designed compounds. A.T. and C.M. designed synthetic routes. M.U. discussed strategy and analyzed data. S.I., K.M.R, D.L.D. and A.C. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Danette L. Daniels or Alessio Ciulli.

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Competing interests

The authors declare the following competing financial interest(s): the Ciulli laboratory receives or has received sponsored research support from Almirall, Amphista therapeutics, Boehringer Ingelheim, Eisai, Nurix therapeutics and Ono Pharmaceuticals. A.C. is a scientific founder, shareholder and consultant of Amphista therapeutics, a company that is developing targeted protein degradation therapeutic platforms. Promega Corporation is the commercial owner by assignment of patents of the HaloTag, NanoLuc, NanoBRET target engagement and HiBiT technologies and their applications, and K.M.R., S.D.M., N.M., M.U. and D.L.D. are employees of Promega Corporation. S.I. is an employee of Ono Pharmaceuticals. S.J.H. and A.T. are employees of Amphista therapeutics. K.-H.C. is an employee of GlaxoSmithKline.

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Peer review information Nature Chemical Biology thanks Stefan Knapp and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Structure-based designed trivalent PROTAC SIM1 is the most potent degrader.

a) Inspection of tertiary complex crystal structures BRD4BD1:Bi-BET:BRD4BD1 (PDB: 5ad3) shows the bivalent inhibitor buried inside the protein interface, suggesting derivatization would impair the binding mode. b) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD4 HEK293 cells following treatment with 1 μM compounds or DMSO. Luminescence (RLU) was continuously monitored in 5 min intervals over a 24 h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) Immunoblot of BRD2, BRD3, BRD4 levels in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 and MZ1 for 4 h. Quantification of BET protein levels was done relative to DMSO control and plotted in Fig. 1e along with calculated DC50 values. The blot shown is representative of two independent experiments. Full blots are supplied as Source Data Files. d) Cell viability of A549 lung carcinoma cell line or HL-60 AML cell line following treatment with PROTACs or DMSO for 48 h in three replicates for each concentration point.

Source data

Extended Data Fig. 2 SIM1 shows delayed and minimal hook effect and has preference for BRD2.

a) Identical live cell degradation SIM1 dose response graphs presented in Fig. 2a (right) of luminescence (RLU) plotted against time with a zoom-in of initial degradation within the first 3 h. b) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with serially diluted ARV-771. Luminescence (RLU) was continuously monitored in 5–15 min intervals over a 24 h time period and fractional RLU was determined by normalization to DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) Comparison of BRD3 and BRD4 degradation rates, degradation maximum (Dmax), and Dmax50 values from kinetic profiles of SIM1 (Fig. 2a), ARV-771 (Extended Data Fig. 2b), and the previously determined MZ1 (Riching et.al. ACS Chem. Biol. 2018). d) NanoBRET ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 100 nM SIM1 or MZ1 or DMSO treatment in quadruplicate. Values are expressed as fold increase over DMSO control. e) Quantification of representative proteins of mass spectrometry proteomics on Fig. 2e. Data are presented as mean values with error bars representing the SD from triplicates. All t-tests performed were two-tailed assuming equal variances. Exact P values: SIM1 (BRD2 P = 0.00001, BRD3 P = 0.00024, BRD4 P = 0.00004, MYC P = 0.00072, HMOX1 P = 0.00082); cis-SIM1 (BRD2 P = 0.45, BRD3 P = 0.11, BRD4 P = 0.05, MYC P = 0.01, HMOX1 P = 0.04).

Extended Data Fig. 3 SIM1 shows potent Myc-driven activity in BET-sensitive AML and prostate cancer lines.

a) Loss in CRISPR cMyc-HiBiT protein levels (left quadrant) and correlative cell viability (right quadrant) in MV4;11 cells treated with 3 nM, 10 nM, 50 nM and 100 nM concentration of the indicated compounds. Luminescence and cell viability by CellTiter-Glo were measured at various time points over 24 h and normalized to the DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) (upper graphs) Quantified expression levels of endogenous BRD3 and BRD4 in 22Rv1 prostate cancer cell line treated with compounds for 4 h. Curves are a best fit of means from two biologically independent experiments. (lower image) Representative images of Western blots for Fig. 3b and upper graphs. Full blots are supplied as Source Data Files. c) Immunoblots of endogenous BRD2, BRD3 and BRD4 in 22Rv1 cells treated with SIM1 (100 nM) or cis-SIM1 (100 nM) with/without the addition of proteasome inhibitor (MG132, 3μM) or VHL inhibitor (VH298, 10μM) for 4 h. Blot is representative from two biologically independent experiments. Full blots are supplied as Source Data Files.

Source data

Extended Data Fig. 4 SIM1 induces pronounced apoptosis in prostate cancer 22Rv1 cell line.

a) Immunoblot of PARP-cleavage in 22Rv1 cells with SIM1 or cis-SIM1 at 10 nM for the indicated time point with/without the addition of caspase inhibitor (QVD-OPh, 20 μM) or necroptosis inhibitor (Necrostatin-1, 20 μM). b) Immunoblot of PARP-cleavage in 22Rv1 cells with MT1 (left) or MZ1 (right) at 10 nM or 1 μM for the indicated time point with/without the addition of caspase inhibitor (QVD-OPh, 20 μM) or necroptosis inhibitor (Necrostatin-1, 20 μM). Full blots for panels (a-b) are supplied as Source Data Files. c) Caspase-Glo 3/7 assays in 22Rv1 cells treated in presence of BET degrader or inhibitor either in the absence (white bars) or presence of VHL inhibitor VH298 (orange bars) or caspase inhibitor QVD-OPh (red bars) for 24 h. Data are presented as mean values with error bars representing the SD from three biologically independent experiments. SIM1, cis-SIM1 and MT1 were used at 100 nM. MZ1 and ARV-771 were used at 1 μM. VH298 was used at 10 μM. QvD-OPh was used at 20 μM.

Extended Data Fig. 5 SIM1 shows potent induction of early and late apoptosis in MV4;11 cells.

Images represent raw data-containing dot plots generated in FlowJo software by analysing 10000 events per condition. Staining for viability (DAPI) and phosphatidyl serine (FITC:Apotracker Green) is shown on the x-axis and y-axis, respectively. Different quadrants represent differently stained population of cells: Q1 (DAPI-/FITC + ), Q2 (DAPI + /FITC + ), Q3 (DAPI + /FITC-), Q4 (DAPI-/FITC-). Percentage of events per quadrant is denoted on individual graphs. Data shown are representative of n = 3 independent experiments. Gating was performed on a DMSO control and applied unchanged to other conditions, as shown in Supplementary Fig. 1.

Extended Data Fig. 6 Complex formation, and cellular target engagement of SIM1.

a) Size exclusion chromatography of complex formation after incubation of SIM1 (red, 25 μM), MZ1 (orange, 25 μM), MT1 (green, 25 μM) or DMSO (cyan, 25 μM) with 25 μM BRD4 BD1-BD2 tandem protein (left panel: N433F mutant, right panel: N140F mutant with VCB protein). Intensity of peaks is absorbance 280 nM. b) NanoBRET target engagement assays with CRISPR HiBiT-BRD4 HEK293 cells stably expressing LgBiT performed in permeabilized and live cell formats. Cells were treated with a fluorescent BET tracer, then incubated with the indicated compounds at various concentrations to measure competitive displacement. IC50 values for each compound are shown for both permeabilized and live cells. Data are presented as mean values with error bars representing the SD of technical quadruplicates. c) NanoBRET ternary complex kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-VHL following 100 nM SIM1 or MZ1 or DMSO treatment. Values are expressed as fold increase over DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates.

Extended Data Fig. 7 SPR kinetics and cellular residence times of SIM1 ternary complexes.

a) Representative SPR sensorgrams for SIM1 (binary) and SIM1:BET tandem complexes (ternary) binding to immobilized VCB. Sensorgrams shown are for multi-cycle kinetic (MCK) experiments as representative of at least two independent experiments. Data were fitted globally to a 1:1 Langmuir model including a component for mass transfer effects. Fitted SPR parameters are in Supplementary Table 1. b) Live cell kinetic residence time experiments with BRD2 and BRD4 as measured by NanoBRET target engagement. CRISPR HiBiT-BRD2 or HiBiT-BRD4 cells were incubated with each of the indicated compounds in triplicate at their pre-determined EC80 values followed by addition of a competitive fluorescently-labeled BET tracer. NanoBRET was measured and is plotted as fractional occupancy over time. From these graphs, residence time rates (Kobs (h−1) and half-life (t½ (h)) were determined and are shown. Data are presented as mean values with error bars representing the SD of technical triplicates.

Extended Data Fig. 8 Cellular degradation and mechanistic analysis of CRBN-based trivalent degrader SIM4.

a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM4 over concentration range of 1nM-1mM. Luminescence (RLU) was continuously monitored over a 20 h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. (b,c) NanoBRET live cell assay of HiBiT-BET HEK293 cells expressing LgBiT with either HaloTag-CRBN for ternary complex studies (b) or HaloTag-Ubiquitin (c) and treated with DMSO, 0.1 mM SIM4, or 1 mM SIM4 treatment for 2.5 h. NanoBRET ratios were calculated at each time point and are graphed as fold increase over DMSO control. Data are presented as mean values with error bars representing the SD of technical quadruplicates. SIM4 shows only partial degradation (Dmax between 40–60%) for all BET family members. While ternary complex is observed with CRBN for BRD2, BRD3, and BRD4, minimal increase in ubiquitination is observed for all after treatment with SIM4.

Supplementary information

Supplementary Information

Supplementary Fig. 1, Supplementary Tables 1 and 2, Supplementary Note (synthetic procedures) and NMR spectra.

Reporting Summary

Supplementary Data 1

Supplementary data set: proteomic analysis of relative protein abundance in MV4;11 cells.

Supplementary Data 2

Supplementary data set: pharmacokinetics of SIM1 following single i.v. and s.c. administrations to male C57BL/6 mice.

Supplementary Data 3

BROMOscan profiling service report and data.

Source data

Source Data Fig. 1

Unprocessed western blots and/or gels for Fig. 1d,i.

Source Data Fig. 3

Unprocessed western blots and/or gels for Fig. 3d and Extended Data Fig. 4a,b.

Source Data Extended Data Fig. 1

Unprocessed western blots and/or gels for Extended Data Fig. 1b.

Source Data Extended Data Fig. 3

Unprocessed western blots and/or gels for Extended Data Fig. 3b,c.

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Imaide, S., Riching, K.M., Makukhin, N. et al. Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity. Nat Chem Biol 17, 1157–1167 (2021). https://doi.org/10.1038/s41589-021-00878-4

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