Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Abstract

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise ‘undruggable’ targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

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: Properties and specificity of small-molecule USP7 inhibitors.
Figure 2: USP7–inhibitor complex structures.
Figure 3: Molecular basis of selective USP7 inhibition.
Figure 4: Effects of USP7 inhibitor FT671 in cell lines.
Figure 5: Physiological response to USP7 specific inhibitor FT671.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    Kruiswijk, F., Labuschagne, C. F. & Vousden, K. H. p53 in survival, death and metabolic health: a lifeguard with a licence to kill. Nat. Rev. Mol. Cell Biol. 16, 393–405 (2015)

    CAS  PubMed  Google Scholar 

  2. 2

    Wade, M ., Li, Y.-C. & Wahl, G. M. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat. Rev. Cancer 13, 83–96 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Khoo, K. H., Verma, C. S., Lane, D. P. & Lane, D. P. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 13, 217–236 (2014)

    CAS  PubMed  Google Scholar 

  4. 4

    Cummins, J. M. et al. Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature 428, http://dx.doi.org/10.1038/nature02501 (2004)

  5. 5

    Li, M., Brooks, C. L., Kon, N. & Gu, W. A dynamic role of HAUSP in the p53–Mdm2 pathway. Mol. Cell 13, 879–886 (2004)

    CAS  PubMed  Google Scholar 

  6. 6

    Li, M . et al. Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648–653 (2002)

    ADS  CAS  Google Scholar 

  7. 7

    Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401–1412 (2011)

    CAS  Google Scholar 

  8. 8

    Weinstock, J. et al. Selective dual inhibitors of the cancer-related deubiquitylating proteases USP7 and USP47. ACS Med. Chem. Lett. 3, 789–792 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Ritorto, M. S. et al. Screening of DUB activity and specificity by MALDI–TOF mass spectrometry. Nat. Commun. 5, 4763 (2014)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Colland, F . et al. Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells. Mol. Cancer Ther. 8, 2286–2295 (2009)

    ADS  CAS  PubMed  Google Scholar 

  11. 11

    Reverdy, C. et al. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem. Biol. 19, 467–477 (2012)

    CAS  Google Scholar 

  12. 12

    Chauhan, D. et al. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 22, 345–358 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Wang, L. et al. Ubiquitin-specific protease-7 inhibition impairs Tip60-dependent Foxp3+ T-regulatory cell function and promotes antitumor immunity. EBioMedicine 13, 99–112 (2016)

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Yamaguchi, M. et al. Spongiacidin C, a pyrrole alkaloid from the marine sponge Stylissa massa, functions as a USP7 inhibitor. Bioorg. Med. Chem. Lett. 23, 3884–3886 (2013)

    CAS  PubMed  Google Scholar 

  15. 15

    Dar, A., Shibata, E. & Dutta, A. Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway. Mol. Cell. Biol. 33, 3309–3320 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Fan, Y.-H. et al. USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis. Cell Death Dis. 4, e867 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Lee, G. et al. Small-molecule inhibitors of USP7 induce apoptosis through oxidative and endoplasmic reticulum stress in cancer cells. Biochem. Biophys. Res. Commun. 470, 181–186 (2016)

    CAS  PubMed  Google Scholar 

  18. 18

    An, T. et al. USP7 inhibitor P5091 inhibits Wnt signaling and colorectal tumor growth. Biochem. Pharmacol. 131, 29–39 (2017)

    CAS  PubMed  Google Scholar 

  19. 19

    Zhan, M. et al. Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway. Biochem. Biophys. Res. Commun. 484, 429–434 (2017)

    CAS  PubMed  Google Scholar 

  20. 20

    Hassiepen, U. et al. A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal. Biochem. 371, 201–207 (2007)

    CAS  PubMed  Google Scholar 

  21. 21

    Ioannidis, S . et al. Pyrrolo and pyrazolopyrimidines as ubiquitin-specific protease 7 inhibitors. International Patent Publication No. WO2016109515 (2016)

  22. 22

    Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002)

    CAS  Google Scholar 

  23. 23

    McGouran, J. F., Gaertner, S. R., Altun, M., Kramer, H. B. & Kessler, B. M. Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem. Biol. 20, 1447–1455 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Ye, Y., Scheel, H., Hofmann, K. & Komander, D. Dissection of USP catalytic domains reveals five common insertion points. Mol. Biosyst. 5, 1797–1808 (2009)

    CAS  PubMed  Google Scholar 

  26. 26

    Faesen, A. C. et al. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase. Mol. Cell 44, 147–159 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Rougé, L. et al. Molecular understanding of USP7 substrate recognition and C-terminal activation. Structure 24, 1335–1345 (2016)

    PubMed  Google Scholar 

  28. 28

    Kim, R. Q., van Dijk, W. J. & Sixma, T. K. Structure of USP7 catalytic domain and three Ubl-domains reveals a connector α-helix with regulatory role. J. Struct. Biol. 195, 11–18 (2016)

    CAS  PubMed  Google Scholar 

  29. 29

    van der Knaap, J. A. et al. GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17, 695–707 (2005)

    CAS  PubMed  Google Scholar 

  30. 30

    Kim, R. Q. & Sixma, T. K. Regulation of USP7: a high incidence of E3 complexes. J. Mol. Biol. http://dx.doi.org/10.1016/j.jmb.2017.05.028 (2017)

  31. 31

    Barak, Y., Juven, T., Haffner, R. & Oren, M. mdm2 expression is induced by wild type p53 activity. EMBO J. 12, 461–468 (1993)

    Google Scholar 

  32. 32

    Wu, X., Bayle, J. H., Olson, D. & Levine, A. J. The p53–mdm-2 autoregulatory feedback loop. Genes Dev. 7, 1126–1132 (1993)

    CAS  PubMed  Google Scholar 

  33. 33

    Tavana, O. et al. HAUSP deubiquitinates and stabilizes N-Myc in neuroblastoma. Nat. Med. 22, 1180–1186 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Felle, M. et al. The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1. Nucleic Acids Res. 39, 8355–8365 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Du, Z. et al. DNMT1 stability is regulated by proteins coordinating deubiquitination and acetylation-driven ubiquitination. Sci. Signal. 3, ra80 (2010)

    PubMed  PubMed Central  Google Scholar 

  36. 36

    Clague, M. J. et al. Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315 (2013)

    CAS  Google Scholar 

  37. 37

    Cohen, P. & Tcherpakov, M. Will the ubiquitin system furnish as many drug targets as protein kinases? Cell 143, 686–693 (2010)

    CAS  PubMed  Google Scholar 

  38. 38

    Salami, J. & Crews, C. M. Waste disposal—an attractive strategy for cancer therapy. Science 355, 1163–1167 (2017)

    ADS  CAS  PubMed  Google Scholar 

  39. 39

    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 

  40. 40

    Clerici, M., Luna-Vargas, M. P. A., Faesen, A. C. & Sixma, T. K. The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange. Nat. Commun. 5, 5399 (2014)

    ADS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Fischer, R. & Kessler, B. M. Gel-aided sample preparation (GASP)—a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics 15, 1224–1229 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Winter, G. Xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Cryst. 43, 186–190 (2010)

    CAS  Google Scholar 

  43. 43

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

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

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

    CAS  Google Scholar 

  45. 45

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

    CAS  PubMed  Google Scholar 

  46. 46

    Avvakumov, G. V. et al. Amino-terminal dimerization, NRDP1–rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (USP8). J. Biol. Chem. 281, 38061–38070 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Li, H. et al. Allosteric activation of ubiquitin-specific proteases by β-propeller proteins UAF1 and WDR20. Mol. Cell 63, 249–260 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Diamond Light Source for access to beamlines I03 and I04. We thank S. M. Boyd for providing Extended Data Fig. 4, and S. Suhrawardy, V. Smith and Z. Yu for help with experiments. Work in the D.K. laboratory is supported by the Medical Research Council (U105192732), the European Research Council (309756), and the Lister Institute for Preventive Medicine. Work in the B.M.K. laboratory was supported by a John Fell Fund 133/075, the Wellcome Trust (097813/Z/11/Z) and the Engineering and Physical Sciences Research Council (EP/N034295/1). L.S. received a stipend from North West Cancer Research.

Author information

Affiliations

Authors

Contributions

This study was directed by T.R.H., S.K., S.I., C.J.D., K.W.B., J.A.E., S.B.M., T.M.R., M.S., S.U., M.J.C., B.M.K. and D.K. S.I. led the chemistry, supported by C.J.D. and K.W.B. Synthetic routes and approaches were devised and carried out by B.C.F., A.C.T. and A.J.B., with computational chemistry support by D.R.L., J.A.C., M.W., C.L.M. and A.V.T. USP7 biochemical assays were developed and performed by C.L.M., A.C.L.M., L.M.T., C.A and F.S. USP10 and USP47 assays were performed by C.L.M. A.P.-F. carried out compound specificity studies and quantitative mass spectrometry using methods established by T.M.C., H.C. and J.F.M. under the guidance of B.M.K. Biophysical studies were performed as follows; surface plasmon resonance: A.C.L.M. and L.M.T.; circular dichroism: L.M.T.; kinact/Ki: A.C.L.M. using methods established by F.S. A.P.T. and W.W.K. performed the structural studies and analysis with input from D.R.L., A.V.T., N.J.E., M.G. and D.K. M.S.P. and L.M.T. produced protein for all experiments with input from L.M.D.S. Biological studies were designed and performed by C.H. with the help of L.S. under the guidance of S.U. and M.J.C., or performed independently by C.G.M., E.C.T., S.M.B., J.L., E.W. and M.S. under the guidance of S.I. and S.K. V.V.Z. carried out antiproliferative assay in MM.1S cells. J.L. and E.W. designed and supervised in vivo animal studies performed at Pharmaron, China. D.K., B.M.K., A.P.T. and S.I. wrote the manuscript, and all authors commented on the text. Authors S.I., C.J.D., T.R.H., S.K., S.U., M.J.C., B.M.K. and D.K. are current steering committee members of the DUB Alliance.

Corresponding authors

Correspondence to Andrew P. Turnbull or Stephanos Ioannidis or Benedikt M. Kessler or David Komander.

Ethics declarations

Competing interests

A.P.T., W.W.K., A.C.L.M., L.M.T., N.J.E., C.A., F.S., M.S.P., T.M.R., L.M.D.S., S.B.M. and T.R.H. are employees of CRUK Therapeutic Discovery Laboratories. S.I., E.C.T., S.M.B., D.R.L., J.A.C., A.V.T., J.L., E.W., B.C.F., V.V.Z., A.C.T., A.J.B., M.W., C.L.M., C.G.M., M.S., J.A.E., K.W.B., C.J.D. and S.K. are employees of FORMA Therapeutics. This work was performed by the DUB Alliance and funded by FORMA Therapeutics.

Additional information

Reviewer Information Nature thanks W. Gu and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Compound characterization.

a, Schematic diagram of the USP7 constructs used in this study. b, Representative SPR sensorgrams of USP7CD and Akt Pleckstrin homology (PH) domain (negative control) to measure affinity parameters (Kd values) of FT671 and FT827. Compounds were tested in twofold dilutions from 50 μM FT827) or 1.56 μM (FT671). Sensorgrams are plotted showing two technical replicates. Data are representative of three biological replicates. Values in parentheses represent s.e.m. range. c, IC50 curves for FT671 against USP7C-term (open triangles) and USP7CD (filled squares) in the ubiquitin-rhodamine assay. The graph displays three biological replicates, each point representing the mean of three technical replicates. d, Time course of the inhibition of USP7C-term by the covalent inhibitor FT827 used to derive kobs values at each concentration (left). kobs plot for covalent inhibitor FT827 used to derive the kinetic parameters (right). 18 biological replicates were run individually, a representative biological replicate is shown. Source data

Extended Data Figure 2 USP7 target engagement and specificity in MCF7 cells.

a, Schematic representation of DUB activity-based profiling of small-molecule inhibitors. Compounds were incubated with either intact MCF7 cells (in situ) or crude cell extracts, followed by labelling with the active site DUB probe HA–UbC2Br. DUBs, either targeted by compounds or labelled by the DUB probe, were visualized by western blotting or subjected to immunoprecipitation for enrichment and analysis by quantitative mass spectrometry7,23. be, FT671 and FT827 selectively target USP7 and disallow HA–UbC2Br binding, but do not notably interact with other DUBs as assessed by anti-HA immunoblotting. One out of two independent biological replicates is shown. b, MCF7 cell extracts incubated with FT671. c, Intact MCF7 cells incubated with FT671. d, MCF7 cell extracts incubated with FT827. e, Intact MCF7 cells incubated with FT827. f, Immunoprecipitation of cellular DUBs captured by HA–UbC2Br labelling for 10 min after incubation of MCF7 cell extracts with DMSO, 1 μM or 10 μM of FT671 or FT827, or 50 μM P22077 for 60 min. Immunoprecipitated material was separated by SDS–PAGE and analysed by western blotting using the indicated antibodies. g, FT671, FT827 and P22077 were incubated with MCF7 cell extracts at the indicated concentrations for 60 min, followed by labelling with HA–UbC2Br for either 5 min (left) or 60 min (right). Samples were separated by SDS–PAGE and analysed by western blotting using the indicated antibodies. Uncropped images for gels are shown in Supplementary Fig. 1.

Extended Data Figure 3 Electron density for FT827 and FT671.

ac, Stereo representation of 2|Fo| − |Fc| electron density maps, contoured at 1σ, covering all atoms of the inhibitors in each of the two independent USP7 molecules of the asymmetric unit. Protein is shown as in Fig. 2a, and compound carbon atoms are in yellow. a, FT671; b, FT827; c, FT827, in an orientation to show the covalent linkage between inhibitor and Cys223.

Extended Data Figure 4 Schematic of FT671 and FT827 interactions with USP7.

a, b, Compound interactions and surrounding residues are labelled, hydrogen bonds indicated, and residues shown according to their chemical properties (see key). The images were generated with Molecular Operating Environment (MOE) (v.2013.08, Montreal, Canada). a, FT671; b, FT827.

Extended Data Figure 5 Molecular basis for USP7 compound specificity.

a, Superposition of the USP7–FT671 complex with indicated DUBs. Shown are the isolated cartoon structures that are shown superimposed in Fig. 3a, b. USP7 apo and USP7–FT671 (top left) show an identical switching loop position, whereas the corresponding region in other apo USP structures is in a distinct conformation that resembles the USP7~Ub complex (see Fig. 3a–c). Structures displayed: USP7 apo (purple, PDB code 1NB824); USP4 apo (PDB code 2Y6E40); yUBP6 apo (PDB code 1VJV); USP8 apo (PDB code 2GFO46); USP12 apo (PDB code 5K1647); USP14 apo (PDB code 2AYN48). b, Binding site detail, showing the interactions between palm subdomain Tyr residues (Tyr465 and Tyr514 in USP7) and the thumb subdomain and switching loop backbone present in all USP apo structures. The USP7 apo switching loop conformation disallows the Tyr465 interaction, which rotates away from the thumb subdomain, and allows Tyr514 to adopt a buried conformation (Tyr514 ‘down’ position). This generates space for compound binding. In other USP apo structures, the equivalent Tyr residues form hydrogen bonds with the thumb subdomain and occupy the compound binding site.

Extended Data Figure 6 Characterization of compound resistant USP7 mutants.

ac, SPR-based binding of FT671 (a), FT827 (b) and ubiquitin (c) to indicated USP7 mutants. For compound binding to wild-type USP7CD and Akt-PH controls, see Extended Data Fig. 1b. Sensorgrams are plotted showing two technical replicates. Data are representative of three biological replicates. Values in parentheses represent s.e.m. range. d, Summary table of binding constants observed for experiments in ac and Extended Data Fig. 1b. Biological repeats are indicated (n = 9 for USP7CD wild type, n = 3 for USP7CD mutants), and values in parentheses indicate s.e.m. e, Circular dichroism profiles of wild-type USP7CD (red), USP7CD(F291N) (orange), USP7CD(Q297A) (green), and USP7CD(Y465N) (blue); each experiment was performed in triplicate. f, Tabulated values for experiments shown in Fig. 3f. g, Structure of activated USP7. The switching loop in USP7 is a point of intrinsic allosteric regulation, provided by a C-terminal module of five ubiquitin-like domains (termed HUBL1 to HUBL5) followed by an activation peptide (amino acids 1084–1102)26 (see Extended Data Fig. 1a). In full-length USP7, the activation peptide stabilizes the active conformation of the switching loop in the ubiquitin-bound state26,27,28, and this can be modulated by USP7-activating proteins, such as GMPS26,29. Shown is the structure of activated USP7 bound to its C-terminal activation peptide. The model was generated from PDB code 5JTV27, and shown are the catalytic domain with ubiquitin in the S1 site (coloured as in Fig. 2a) of molecule 1 in the asymmetric unit and the HUBL5 domain and activation peptide (orange) of molecule 2 in the asymmetric unit, which in the crystal binds to molecule 1 in trans. Source data

Extended Data Figure 7 USP7 knockdown or inhibitors affect USP7 substrates in cell lines.

a, HCT116 cells were transfected with non-targeting control (NT1) or three different USP7-targeting siRNAs for 72 h, harvested and probed with indicated antibodies. A representative experiment from two biological replicates is shown. b, U2OS cells were transfected with a pool of four independent siRNAs targeting USP7 or a non-targeting control siRNA and processed as in a. A representative experiment from two biological replicates is shown. c, HCT116 cells were treated with 10 μM FT671 for 24 h and RNA was extracted for qPCR measurements with primer sets against indicated transcripts (see Methods). Data were analysed with the ΔCt method. Experiments were performed in triplicate. d, HCT116 cells were treated with FT671 (10 μM) for longer periods, harvested and probed with indicated antibodies. A representative experiment from two biological replicates is shown. e, Experiment as in Fig. 4d, with higher FT671 concentration. p53 levels are upregulated upon FT671 treatment in cells expressing GFP or GFP-tagged wild-type USP7, but not in cells expressing compound resistant mutants. USP7 expression was induced for 7 h by 1 μg ml−1 doxycycline, and cells were treated with 3 μM FT671 for the last 4 h before lysis. f, IMR-32 neuroblastoma cells33 were treated with indicated concentrations of FT671, and effects on p53 and N-Myc levels were tested by western blotting. A representative experiment from two biological replicates is shown. g, h, HCT116 (g) or MM.1S (h) cells were treated for indicated times with FT671, and western blotted for UHRF1 and DNMT1. Vinculin and β-actin serve as loading controls. A representative experiment from two biological replicates is shown. Uncropped images for gels are shown in Supplementary Fig. 1. Source data

Extended Data Figure 8 Characterization of FT671 effects in vivo.

a, MM.1S cells were treated for the indicated times with 10 μM FT671, and proteins detected by western blotting with indicated antibodies (see Methods). A representative experiment from two biological replicates is shown. b, Meso Scale analysis (see Methods) measuring the ratio of ubiquitinated versus non-ubiquitinated MDM2 in MM.1S cells upon FT671 treatment and proteasome inhibition (25 μM MG132). c, MM.1S cells were treated with 10 μM FT671 for the indicated times and RNA was extracted for qPCR measurements with primer sets against indicated transcripts (see Methods). Data were analysed with the ΔCt method. Experiments were performed in duplicate. d, qPCR analysis as in c, using a single time point against MCL1 and MYC, which in this setting are not p53 target genes after 24 h of 10 μM FT671. Experiments were performed in triplicate. e, MM.1S tumour xenograft tissues were analysed for p53 expression by western blotting and normalized to the β-actin loading control. Uncropped images for gels are shown in Supplementary Fig. 1. Source data

Extended Data Table 1 FT671 and FT827 specificity
Extended Data Table 2 Data collection and refinement statistics

Supplementary information

Reporting Summary (PDF 69 kb)

Supplementary Information

This file contains chemical synthesis of USP7 compounds. (PDF 630 kb)

Supplementary Figure 1

This file contains annotated full gel blots for all figures in the main manuscript. (PDF 5083 kb)

Supplementary Table 1

This file contains DUBs identified by mass spectrometry after HA-UbC2Br labelling of MCF7 cell extracts. (XLSX 178 kb)

Supplementary Table 2

This file contains all proteins identified by mass spectrometry after HA-UbC2Br labelling of MCF7 cell extracts. (XLSX 3099 kb)

Supplementary Table 3

This file contains the sequences for any RNAi/small RNA constructs used in this study. (XLSX 10 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Turnbull, A., Ioannidis, S., Krajewski, W. et al. Molecular basis of USP7 inhibition by selective small-molecule inhibitors. Nature 550, 481–486 (2017). https://doi.org/10.1038/nature24451

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing