Abstract

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered ‘undruggable’1,2. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes4,5,6,7,8,9,10, including those that can access regions of proteins that are difficult to target through binding affinity alone5,10,11. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand–protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Fragment screening to predict druggability (ligandability) and lead discovery success. Drug Discov. Today 16, 284–287 (2011)

  2. 2.

    & The druggable genome. Nature Rev. Drug Discov. 1, 727–730 (2002)

  3. 3.

    , , & Fragment-based approaches in drug discovery and chemical biology. Biochemistry 51, 4990–5003 (2012)

  4. 4.

    et al. Site-directed ligand discovery. Proc. Natl Acad. Sci. USA 97, 9367–9372 (2000)

  5. 5.

    et al. Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein–protein interaction. Protein Sci. 21, 1885–1896 (2012)

  6. 6.

    , & Kinetic template-guided tethering of fragments. ChemMedChem 7, 2082–2086 (2012)

  7. 7.

    , & A fragment-based method to discover irreversible covalent inhibitors of cysteine proteases. J. Med. Chem. 57, 4969–4974 (2014)

  8. 8.

    , , , & Promiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragments. J. Med. Chem. 57, 7590–7599 (2014)

  9. 9.

    , , , & Electrophilic fragment-based design of reversible covalent kinase inhibitors. J. Am. Chem. Soc. 135, 5298–5301 (2013)

  10. 10.

    , , , & K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013)

  11. 11.

    et al. Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov. 6, 316–329 (2016)

  12. 12.

    et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010)

  13. 13.

    , , & A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nature Methods 11, 79–85 (2014)

  14. 14.

    , , & A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Edn Engl. 41, 2596–2599 (2002)

  15. 15.

    et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 20, 146–159 (2013)

  16. 16.

    et al. Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew. Chem. Int. Ed. Engl. 53, 199–204 (2014)

  17. 17.

    et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nature Chem. Biol. 6, 900–906 (2010)

  18. 18.

    et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 27, 4702–4711 (2008)

  19. 19.

    et al. Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2). J. Med. Chem. 46, 49–63 (2003)

  20. 20.

    et al. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nature Chem. Biol. 10, 760–767 (2014)

  21. 21.

    et al. Covalent docking of large libraries for the discovery of chemical probes. Nature Chem. Biol. 10, 1066–1072 (2014)

  22. 22.

    , , , & Action at a distance: allostery and the development of drugs to target cancer cell metabolism. Chem. Biol. 21, 1143–1161 (2014)

  23. 23.

    et al. Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach. Mol. Cell. Proteomics 9, 1400–1410 (2010)

  24. 24.

    , & Selective detection and inhibition of active caspase-3 in cells with optimized peptides. J. Am. Chem. Soc. 135, 12869–12876 (2013)

  25. 25.

    , & Life and death in peripheral T cells. Nature Rev. Immunol. 7, 532–542 (2007)

  26. 26.

    et al. Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk. PLoS ONE 5, e13638 (2010)

  27. 27.

    et al. Activation and specificity of human caspase-10. Biochemistry 49, 8307–8315 (2010)

  28. 28.

    , & Genetic disorders of programmed cell death in the immune system. Annu. Rev. Immunol. 24, 321–352 (2006)

  29. 29.

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

  30. 30.

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

  31. 31.

    , & Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes. Nature Protocols 2, 1414–1425 (2007)

  32. 32.

    et al. The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase. Proc. Natl Acad. Sci. USA 111, 14924–14929 (2014)

  33. 33.

    et al. Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors. Nat. Chem. Biol. 7, 469–478 (2011)

  34. 34.

    ; UniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 43, D204–D212 (2015)

  35. 35.

    et al. DrugBank 4.0: shedding new light on drug metabolism. Nucleic Acids Res. 42, D1091–D1097 (2014)

  36. 36.

    et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000)

  37. 37.

    et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009)

  38. 38.

    The PyMOL Molecular Graphics System (Delano Scientific, 2002)

  39. 39.

    et al. Open Babel: an open chemical toolbox. J. Cheminform. 3, 33 (2011)

  40. 40.

    et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009)

  41. 41.

    , & Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996)

  42. 42.

    , & NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675 (2012)

  43. 43.

    et al. The I-TASSER Suite: protein structure and function prediction. Nature Methods 12, 7–8 (2015)

Download references

Acknowledgements

This work was supported by the National Institutes of Health (CA087660 (B.F.C.), GM090294 (B.F.C.), GM108208 (K.M.B.), GM069832 (S.F., A.J.O.)). We thank J. Cisar, K. Mowen, C. Wang, M. Suciu, M. Dix, G. Simon, M. Carrillo, and J. Hulce for experimental assistance, M. Lenardo, L. Zheng and R. Siegel for helpful suggestions, the Marletta and Vogt laboratories at The Scripps Research Institute for sharing instrumentation, and Iterative Threading ASSEmbly Refinement (I-TASSER) for the structural modelling of IMPDH2.

Author information

Author notes

    • Keriann M. Backus
    •  & Bruno E. Correia

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemical Physiology, The Scripps Research Institute. La Jolla, California 92307, USA

    • Keriann M. Backus
    • , Bruno E. Correia
    • , Kenneth M. Lum
    • , Benjamin D. Horning
    • , Bryan R. Lanning
    •  & Benjamin F. Cravatt
  2. Department of Integrative Structural and Computational Biology, The Scripps Research Institute. La Jolla, California 92307, USA

    • Stefano Forli
    •  & Arthur J. Olson
  3. Department of Molecular and Experimental Medicine, The Scripps Research Institute. La Jolla, California 92307, USA

    • Gonzalo E. González-Páez
    • , Sandip Chatterjee
    •  & Dennis W. Wolan
  4. Department of Immunology and Microbial Science, The Scripps Research Institute. La Jolla, California 92307, USA

    • John R. Teijaro

Authors

  1. Search for Keriann M. Backus in:

  2. Search for Bruno E. Correia in:

  3. Search for Kenneth M. Lum in:

  4. Search for Stefano Forli in:

  5. Search for Benjamin D. Horning in:

  6. Search for Gonzalo E. González-Páez in:

  7. Search for Sandip Chatterjee in:

  8. Search for Bryan R. Lanning in:

  9. Search for John R. Teijaro in:

  10. Search for Arthur J. Olson in:

  11. Search for Dennis W. Wolan in:

  12. Search for Benjamin F. Cravatt in:

Contributions

B.F.C. and K.M.B. conceived of the project. K.M.B. performed MS experiments and data analysis. B.E.C. wrote software, compiled and analysed MS data. S.F. wrote software and conducted reactive docking. K.M.B. cloned, overexpressed and purified proteins, and conducted inhibition studies in vitro and in situ. S.C. cloned and purified IDH1. K.M.B., K.M.L., B.D.H. and B.R.L. synthesized compounds. G.E.G.-P. expressed and purified recombinant caspases and TEV protease. D.W.W. provided assistance with the caspase studies. J.R.T assisted with the T-cell studies. A.J.O. provided technical advice. K.M.B., B.E.C. and B.F.C. designed experiments and analysed data. K.M.B., B.E.C. and B.F.C. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Keriann M. Backus or Benjamin F. Cravatt.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains Supplementary Text and Data, including a Supplementary Discussion, Supplementary Methods and additional references (see Contents list for more details).

Excel files

  1. 1.

    Supplementary Data

    This file contains Supplementary Table 1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature18002

Further reading

  • 1.

    Kinase inhibitors: the road ahead

    • Fleur M. Ferguson
    •  & Nathanael S. Gray

    Nature Reviews Drug Discovery (2018)

  • 2.

    Expanding the medicinal chemistry synthetic toolbox

    • Jonas Boström
    • , Dean G. Brown
    • , Robert J. Young
    •  & György M. Keserü

    Nature Reviews Drug Discovery (2018)

  • 3.

    Let K-Ras activate its own inhibitor

    • Alexander V. Statsyuk

    Nature Structural & Molecular Biology (2018)

  • 4.

    A high throughput screen for next-generation leads targeting malaria parasite transmission

    • Michael J. Delves
    • , Celia Miguel-Blanco
    • , Holly Matthews
    • , Irene Molina
    • , Andrea Ruecker
    • , Sabrina Yahiya
    • , Ursula Straschil
    • , Matthew Abraham
    • , María Luisa León
    • , Oliver J. Fischer
    • , Ainoa Rueda-Zubiaurre
    • , Jochen R. Brandt
    • , Álvaro Cortés
    • , Anna Barnard
    • , Matthew J. Fuchter
    • , Félix Calderón
    • , Elizabeth A. Winzeler
    • , Robert E. Sinden
    • , Esperanza Herreros
    • , Francisco J. Gamo
    •  & Jake Baum

    Nature Communications (2018)

  • 5.

    Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018

    • Lorenzo Galluzzi
    • , Ilio Vitale
    • , Stuart A. Aaronson
    • , John M. Abrams
    • , Dieter Adam
    • , Patrizia Agostinis
    • , Emad S. Alnemri
    • , Lucia Altucci
    • , Ivano Amelio
    • , David W. Andrews
    • , Margherita Annicchiarico-Petruzzelli
    • , Alexey V. Antonov
    • , Eli Arama
    • , Eric H. Baehrecke
    • , Nickolai A. Barlev
    • , Nicolas G. Bazan
    • , Francesca Bernassola
    • , Mathieu J. M. Bertrand
    • , Katiuscia Bianchi
    • , Mikhail V. Blagosklonny
    • , Klas Blomgren
    • , Christoph Borner
    • , Patricia Boya
    • , Catherine Brenner
    • , Michelangelo Campanella
    • , Eleonora Candi
    • , Didac Carmona-Gutierrez
    • , Francesco Cecconi
    • , Francis K.-M. Chan
    • , Navdeep S. Chandel
    • , Emily H. Cheng
    • , Jerry E. Chipuk
    • , John A. Cidlowski
    • , Aaron Ciechanover
    • , Gerald M. Cohen
    • , Marcus Conrad
    • , Juan R. Cubillos-Ruiz
    • , Peter E. Czabotar
    • , Vincenzo D’Angiolella
    • , Ted M. Dawson
    • , Valina L. Dawson
    • , Vincenzo De Laurenzi
    • , Ruggero De Maria
    • , Klaus-Michael Debatin
    • , Ralph J. DeBerardinis
    • , Mohanish Deshmukh
    • , Nicola Di Daniele
    • , Francesco Di Virgilio
    • , Vishva M. Dixit
    • , Scott J. Dixon
    • , Colin S. Duckett
    • , Brian D. Dynlacht
    • , Wafik S. El-Deiry
    • , John W. Elrod
    • , Gian Maria Fimia
    • , Simone Fulda
    • , Ana J. García-Sáez
    • , Abhishek D. Garg
    • , Carmen Garrido
    • , Evripidis Gavathiotis
    • , Pierre Golstein
    • , Eyal Gottlieb
    • , Douglas R. Green
    • , Lloyd A. Greene
    • , Hinrich Gronemeyer
    • , Atan Gross
    • , Gyorgy Hajnoczky
    • , J. Marie Hardwick
    • , Isaac S. Harris
    • , Michael O. Hengartner
    • , Claudio Hetz
    • , Hidenori Ichijo
    • , Marja Jäättelä
    • , Bertrand Joseph
    • , Philipp J. Jost
    • , Philippe P. Juin
    • , William J. Kaiser
    • , Michael Karin
    • , Thomas Kaufmann
    • , Oliver Kepp
    • , Adi Kimchi
    • , Richard N. Kitsis
    • , Daniel J. Klionsky
    • , Richard A. Knight
    • , Sharad Kumar
    • , Sam W. Lee
    • , John J. Lemasters
    • , Beth Levine
    • , Andreas Linkermann
    • , Stuart A. Lipton
    • , Richard A. Lockshin
    • , Carlos López-Otín
    • , Scott W. Lowe
    • , Tom Luedde
    • , Enrico Lugli
    • , Marion MacFarlane
    • , Frank Madeo
    • , Michal Malewicz
    • , Walter Malorni
    • , Gwenola Manic
    • , Jean-Christophe Marine
    • , Seamus J. Martin
    • , Jean-Claude Martinou
    • , Jan Paul Medema
    • , Patrick Mehlen
    • , Pascal Meier
    • , Sonia Melino
    • , Edward A. Miao
    • , Jeffery D. Molkentin
    • , Ute M. Moll
    • , Cristina Muñoz-Pinedo
    • , Shigekazu Nagata
    • , Gabriel Nuñez
    • , Andrew Oberst
    • , Moshe Oren
    • , Michael Overholtzer
    • , Michele Pagano
    • , Theocharis Panaretakis
    • , Manolis Pasparakis
    • , Josef M. Penninger
    • , David M. Pereira
    • , Shazib Pervaiz
    • , Marcus E. Peter
    • , Mauro Piacentini
    • , Paolo Pinton
    • , Jochen H.M. Prehn
    • , Hamsa Puthalakath
    • , Gabriel A. Rabinovich
    • , Markus Rehm
    • , Rosario Rizzuto
    • , Cecilia M.P. Rodrigues
    • , David C. Rubinsztein
    • , Thomas Rudel
    • , Kevin M. Ryan
    • , Emre Sayan
    • , Luca Scorrano
    • , Feng Shao
    • , Yufang Shi
    • , John Silke
    • , Hans-Uwe Simon
    • , Antonella Sistigu
    • , Brent R. Stockwell
    • , Andreas Strasser
    • , Gyorgy Szabadkai
    • , Stephen W.G. Tait
    • , Daolin Tang
    • , Nektarios Tavernarakis
    • , Andrew Thorburn
    • , Yoshihide Tsujimoto
    • , Boris Turk
    • , Tom Vanden Berghe
    • , Peter Vandenabeele
    • , Matthew G. Vander Heiden
    • , Andreas Villunger
    • , Herbert W. Virgin
    • , Karen H. Vousden
    • , Domagoj Vucic
    • , Erwin F. Wagner
    • , Henning Walczak
    • , David Wallach
    • , Ying Wang
    • , James A. Wells
    • , Will Wood
    • , Junying Yuan
    • , Zahra Zakeri
    • , Boris Zhivotovsky
    • , Laurence Zitvogel
    • , Gerry Melino
    •  & Guido Kroemer

    Cell Death & Differentiation (2018)

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.