USP7 small-molecule inhibitors interfere with ubiquitin binding

Lorna Kategaya, Paola Di Lello, Lionel Rougé, Richard Pastor, Kevin R. Clark, Jason Drummond, Tracy Kleinheinz, Eva Lin, John-Paul Upton, Sumit Prakash, Johanna Heideker, Mark McCleland, Maria Stella Ritorto, Dario R. Alessi, Matthias Trost, Travis W. Bainbridge, Michael C. M. Kwok, Taylur P. Ma, Zachary Stiffler, Bradley Brasher & 24 others Yinyan Tang, Priyadarshini Jaishankar, Brian R. Hearn, Adam R. Renslo, Michelle R. Arkin, Frederick Cohen, Kebing Yu, Frank Peale, Florian Gnad, Matthew T. Chang, Christiaan Klijn, Elizabeth Blackwood, Scott E. Martin, William F. Forrest, James A. Ernst, Chudi Ndubaku, Xiaojing Wang, Maureen H. Beresini, Vickie Tsui, Carsten Schwerdtfeger, Robert A. Blake, Jeremy Murray, Till Maurer, Ingrid E. Wertz

Research output: Contribution to journalLetter

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Abstract

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Original languageEnglish
Pages (from-to)534-538
Number of pages5
JournalNature
Volume550
Early online date18 Oct 2017
DOIs
Publication statusPublished - 18 Oct 2017

Fingerprint

Ubiquitin-Specific Proteases
Ubiquitin
Magnetic Resonance Spectroscopy
Tumor Suppressor Protein p53
Proteins
Ubiquitination
Protease Inhibitors
Eukaryota
Cysteine
Hydrogen
Neoplasms
Cell Survival

Keywords

  • Drug discovery
  • Mechanism of action
  • Ubiquitylation

Cite this

Kategaya, L., Di Lello, P., Rougé, L., Pastor, R., Clark, K. R., Drummond, J., ... Wertz, I. E. (2017). USP7 small-molecule inhibitors interfere with ubiquitin binding. Nature, 550, 534-538. https://doi.org/10.1038/nature24006
Kategaya, Lorna ; Di Lello, Paola ; Rougé, Lionel ; Pastor, Richard ; Clark, Kevin R. ; Drummond, Jason ; Kleinheinz, Tracy ; Lin, Eva ; Upton, John-Paul ; Prakash, Sumit ; Heideker, Johanna ; McCleland, Mark ; Ritorto, Maria Stella ; Alessi, Dario R. ; Trost, Matthias ; Bainbridge, Travis W. ; Kwok, Michael C. M. ; Ma, Taylur P. ; Stiffler, Zachary ; Brasher, Bradley ; Tang, Yinyan ; Jaishankar, Priyadarshini ; Hearn, Brian R. ; Renslo, Adam R. ; Arkin, Michelle R. ; Cohen, Frederick ; Yu, Kebing ; Peale, Frank ; Gnad, Florian ; Chang, Matthew T. ; Klijn, Christiaan ; Blackwood, Elizabeth ; Martin, Scott E. ; Forrest, William F. ; Ernst, James A. ; Ndubaku, Chudi ; Wang, Xiaojing ; Beresini, Maureen H. ; Tsui, Vickie ; Schwerdtfeger, Carsten ; Blake, Robert A. ; Murray, Jeremy ; Maurer, Till ; Wertz, Ingrid E. / USP7 small-molecule inhibitors interfere with ubiquitin binding. In: Nature. 2017 ; Vol. 550. pp. 534-538.
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abstract = "The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 {\AA} distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.",
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author = "Lorna Kategaya and {Di Lello}, Paola and Lionel Roug{\'e} and Richard Pastor and Clark, {Kevin R.} and Jason Drummond and Tracy Kleinheinz and Eva Lin and John-Paul Upton and Sumit Prakash and Johanna Heideker and Mark McCleland and Ritorto, {Maria Stella} and Alessi, {Dario R.} and Matthias Trost and Bainbridge, {Travis W.} and Kwok, {Michael C. M.} and Ma, {Taylur P.} and Zachary Stiffler and Bradley Brasher and Yinyan Tang and Priyadarshini Jaishankar and Hearn, {Brian R.} and Renslo, {Adam R.} and Arkin, {Michelle R.} and Frederick Cohen and Kebing Yu and Frank Peale and Florian Gnad and Chang, {Matthew T.} and Christiaan Klijn and Elizabeth Blackwood and Martin, {Scott E.} and Forrest, {William F.} and Ernst, {James A.} and Chudi Ndubaku and Xiaojing Wang and Beresini, {Maureen H.} and Vickie Tsui and Carsten Schwerdtfeger and Blake, {Robert A.} and Jeremy Murray and Till Maurer and Wertz, {Ingrid E.}",
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Kategaya, L, Di Lello, P, Rougé, L, Pastor, R, Clark, KR, Drummond, J, Kleinheinz, T, Lin, E, Upton, J-P, Prakash, S, Heideker, J, McCleland, M, Ritorto, MS, Alessi, DR, Trost, M, Bainbridge, TW, Kwok, MCM, Ma, TP, Stiffler, Z, Brasher, B, Tang, Y, Jaishankar, P, Hearn, BR, Renslo, AR, Arkin, MR, Cohen, F, Yu, K, Peale, F, Gnad, F, Chang, MT, Klijn, C, Blackwood, E, Martin, SE, Forrest, WF, Ernst, JA, Ndubaku, C, Wang, X, Beresini, MH, Tsui, V, Schwerdtfeger, C, Blake, RA, Murray, J, Maurer, T & Wertz, IE 2017, 'USP7 small-molecule inhibitors interfere with ubiquitin binding', Nature, vol. 550, pp. 534-538. https://doi.org/10.1038/nature24006

USP7 small-molecule inhibitors interfere with ubiquitin binding. / Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R.; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R.; Trost, Matthias; Bainbridge, Travis W.; Kwok, Michael C. M.; Ma, Taylur P.; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R.; Renslo, Adam R.; Arkin, Michelle R.; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T.; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E.; Forrest, William F.; Ernst, James A.; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H.; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A.; Murray, Jeremy; Maurer, Till (Lead / Corresponding author); Wertz, Ingrid E. (Lead / Corresponding author).

In: Nature, Vol. 550, 18.10.2017, p. 534-538.

Research output: Contribution to journalLetter

TY - JOUR

T1 - USP7 small-molecule inhibitors interfere with ubiquitin binding

AU - Kategaya, Lorna

AU - Di Lello, Paola

AU - Rougé, Lionel

AU - Pastor, Richard

AU - Clark, Kevin R.

AU - Drummond, Jason

AU - Kleinheinz, Tracy

AU - Lin, Eva

AU - Upton, John-Paul

AU - Prakash, Sumit

AU - Heideker, Johanna

AU - McCleland, Mark

AU - Ritorto, Maria Stella

AU - Alessi, Dario R.

AU - Trost, Matthias

AU - Bainbridge, Travis W.

AU - Kwok, Michael C. M.

AU - Ma, Taylur P.

AU - Stiffler, Zachary

AU - Brasher, Bradley

AU - Tang, Yinyan

AU - Jaishankar, Priyadarshini

AU - Hearn, Brian R.

AU - Renslo, Adam R.

AU - Arkin, Michelle R.

AU - Cohen, Frederick

AU - Yu, Kebing

AU - Peale, Frank

AU - Gnad, Florian

AU - Chang, Matthew T.

AU - Klijn, Christiaan

AU - Blackwood, Elizabeth

AU - Martin, Scott E.

AU - Forrest, William F.

AU - Ernst, James A.

AU - Ndubaku, Chudi

AU - Wang, Xiaojing

AU - Beresini, Maureen H.

AU - Tsui, Vickie

AU - Schwerdtfeger, Carsten

AU - Blake, Robert A.

AU - Murray, Jeremy

AU - Maurer, Till

AU - Wertz, Ingrid E.

N1 - © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

PY - 2017/10/18

Y1 - 2017/10/18

N2 - The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

AB - The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

KW - Drug discovery

KW - Mechanism of action

KW - Ubiquitylation

U2 - 10.1038/nature24006

DO - 10.1038/nature24006

M3 - Letter

VL - 550

SP - 534

EP - 538

JO - Nature

JF - Nature

SN - 0028-0836

ER -

Kategaya L, Di Lello P, Rougé L, Pastor R, Clark KR, Drummond J et al. USP7 small-molecule inhibitors interfere with ubiquitin binding. Nature. 2017 Oct 18;550:534-538. https://doi.org/10.1038/nature24006