Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes

An Erratum to this article was published on 01 May 2009

This article has been updated

Abstract

High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the fluopol-ABPP assay.
Figure 2: Optimization and validation of the fluopol-ABPP assay for RBBP9.
Figure 3: Identification of RBBP9 primary hits.
Figure 4: Competitive ABPP in proteomes identifies emetine (1) as a selective inhibitor of RBBP9.
Figure 5: Mechanistic characterization of RBBP9 inhibitors.
Figure 6: Fluopol-ABPP identifies a selective inhibitor of GSTO1.

Similar content being viewed by others

Change history

  • 08 May 2009

    In the version of the article initially published, the IC50 value of cephaeline is given as 2.7 μM in Figure 3d. The correct value is 27 μM. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Inglese, J. et al. High-throughput screening assays for the identification of chemical probes. Nat. Chem. Biol. 3, 466–479 (2007).

    Article  CAS  Google Scholar 

  2. Shelat, A.A. & Guy, R.K. Scaffold composition and biological relevance of screening libraries. Nat. Chem. Biol. 3, 442–446 (2007).

    Article  CAS  Google Scholar 

  3. Ahn, K. et al. A novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity. Biochemistry 46, 13019–13030 (2007).

    Article  CAS  Google Scholar 

  4. Jo, E. et al. S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. Chem. Biol. 12, 703–715 (2005).

    Article  CAS  Google Scholar 

  5. Dolma, S., Lessnick, S.L., Hahn, W.C. & Stockwell, B.R. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3, 285–296 (2003).

    Article  CAS  Google Scholar 

  6. Galperin, M.Y. & Koonin, E.V. 'Conserved hypothetical' proteins: prioritization of targets for experimental study. Nucleic Acids Res. 32, 5452–5463 (2004).

    Article  CAS  Google Scholar 

  7. Woitach, J.T., Zhang, M., Niu, C.H. & Thorgeirsson, S.S. A retinoblastoma-binding protein that affects cell-cycle control and confers transforming ability. Nat. Genet. 19, 371–374 (1998).

    Article  CAS  Google Scholar 

  8. Rao, M. & Sockanathan, S. Transmembrane protein GDE2 induces motor neuron differentiation in vivo. Science 309, 2212–2215 (2005).

    Article  CAS  Google Scholar 

  9. Semba, S. et al. Biological functions of mammalian NIT1, the counterpart of the invertebrate NITFHIT rosetta stone protein, a possible tumor suppressor. J. Biol. Chem. 281, 28244–28253 (2006).

    Article  CAS  Google Scholar 

  10. Evans, M.J. & Cravatt, B.F. Mechanism-based profiling of enzyme families. Chem. Rev. 106, 3279–3301 (2006).

    Article  CAS  Google Scholar 

  11. Cravatt, B.F., Wright, A.T. & Kozarich, J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    Article  CAS  Google Scholar 

  12. Jessani, N., Liu, Y., Humphrey, M. & Cravatt, B.F. Enzyme activity profiles of the secreted and membrane proteome that depict cancer invasiveness. Proc. Natl. Acad. Sci. USA 99, 10335–10340 (2002).

    Article  CAS  Google Scholar 

  13. Jessani, N. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2, 691–697 (2005).

    Article  CAS  Google Scholar 

  14. Joyce, J.A. et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell 5, 443–453 (2004).

    Article  CAS  Google Scholar 

  15. Adam, G.C., Sorensen, E.J. & Cravatt, B.F. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat. Biotechnol. 20, 805–809 (2002).

    Article  CAS  Google Scholar 

  16. Greenbaum, D.C. et al. A role for the protease falcipain 1 in host cell invasion by the human malaria parasite. Science 298, 2002–2006 (2002).

    Article  CAS  Google Scholar 

  17. Blankman, J.L., Simon, G.S. & Cravatt, B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-Arachidonoylglycerol. Chem. Biol. 14, 1347–1356 (2007).

    Article  CAS  Google Scholar 

  18. Barglow, K.T. & Cravatt, B.F. Substrate mimicry in an activity-based probe that targets the nitrilase family of enzymes. Angew. Chem. Int. Edn. Engl. 45, 7408–7411 (2006).

    Article  CAS  Google Scholar 

  19. Leung, D., Hardouin, C., Boger, D.L. & Cravatt, B.F. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21, 687–691 (2003).

    Article  CAS  Google Scholar 

  20. Chiang, K.P., Niessen, S., Saghatelian, A. & Cravatt, B.F. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling. Chem. Biol. 13, 1041–1050 (2006).

    Article  CAS  Google Scholar 

  21. Li, W., Blankman, J.L. & Cravatt, B.F. A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases. J. Am. Chem. Soc. 129, 9594–9595 (2007).

    Article  CAS  Google Scholar 

  22. Owicki, J.C. Fluorescence polarization and anisotropy in high-throughput screening: perspectives and primer. J. Biomol. Screen. 5, 297–306 (2000).

    Article  CAS  Google Scholar 

  23. Vorobiev, S.M. et al. Crystal structure of human retinoblastoma binding protein 9. Proteins 74, 526–529 (2008).

    Article  Google Scholar 

  24. Patricelli, M.P., Giang, D.K., Stamp, L.M. & Burbaum, J.J. Direct visualization of serine hydrolase activities in complex proteome using fluorescent active site-directed probes. Proteomics 1, 1067–1071 (2001).

    Article  CAS  Google Scholar 

  25. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  Google Scholar 

  26. Hoover, H.S., Blankman, J.L., Niessen, S. & Cravatt, B.F. Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling. Bioorg. Med. Chem. Lett. 18, 5838–5841 (2008).

    Article  CAS  Google Scholar 

  27. Saario, S.M. et al. Characterization of the sulfhydryl-sensitive site in the enzyme responsible for hydrolysis of 2-arachidonoyl-glycerol in rat cerebellar membranes. Chem. Biol. 12, 649–656 (2005).

    Article  CAS  Google Scholar 

  28. Feng, B.Y., Shelat, A., Doman, T.N., Guy, R.K. & Shoichet, B.K. High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol. 1, 146–148 (2005).

    Article  CAS  Google Scholar 

  29. Feng, B.Y. et al. A high-throughput screen for aggregation-based inhibition in a large compound library. J. Med. Chem. 50, 2385–2390 (2007).

    Article  CAS  Google Scholar 

  30. Feng, B.Y. & Shoichet, B.K. A detergent-based assay for the detection of promiscuous inhibitors. Nat. Protoc. 1, 550–553 (2006).

    Article  CAS  Google Scholar 

  31. Boon-Unge, K. et al. Emetine regulates the alternative splicing of Bcl-x through a protein phosphatase 1-dependent mechanism. Chem. Biol. 14, 1386–1392 (2007).

    Article  CAS  Google Scholar 

  32. Keiser, M.J. et al. Relating protein pharmacology by ligand chemistry. Nat. Biotechnol. 25, 197–206 (2007).

    Article  CAS  Google Scholar 

  33. Grollman, A.P. Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics. Proc. Natl. Acad. Sci. USA 56, 1867–1874 (1966).

    Article  CAS  Google Scholar 

  34. Gupta, R.S. & Siminovitch, L. The molecular basis of emetine resistance in Chinese hamster ovary cells: alteration in the 40S ribosomal subunit. Cell 10, 61–66 (1977).

    Article  CAS  Google Scholar 

  35. Monks, T.J. & Jones, D.C. The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers. Curr. Drug Metab. 3, 425–438 (2002).

    Article  CAS  Google Scholar 

  36. Hayes, J.D., Flanagan, J.U. & Jowsey, I.R. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51–88 (2005).

    Article  CAS  Google Scholar 

  37. Yan, X.D., Pan, L.Y., Yuan, Y., Lang, J.H. & Mao, N. Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines. J. Proteome Res. 6, 772–780 (2007).

    Article  CAS  Google Scholar 

  38. Board, P.G. et al. Identification, characterization, and crystal structure of the omega class glutathione transferases. J. Biol. Chem. 275, 24798–24806 (2000).

    Article  CAS  Google Scholar 

  39. Whitbread, A.K. et al. Characterization of the omega class of glutathione transferases. Methods Enzymol. 401, 78–99 (2005).

    Article  CAS  Google Scholar 

  40. Zhang, K. & Wong, K.P. Glutathione conjugation of chlorambucil: measurement and modulation by plant polyphenols. Biochem. J. 325, 417–422 (1997).

    Article  CAS  Google Scholar 

  41. Adam, G.C., Sorensen, E.J. & Cravatt, B.F. Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes. Mol. Cell. Proteomics 1, 828–835 (2002).

    Article  CAS  Google Scholar 

  42. Liu, S., Cerione, R.A. & Clardy, J. Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity. Proc. Natl. Acad. Sci. USA 99, 2743–2747 (2002).

    Article  CAS  Google Scholar 

  43. Board, P.G. et al. S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1–1. Anal. Biochem. 374, 25–30 (2008).

    Article  CAS  Google Scholar 

  44. Torta, F., Usuelli, V., Malgaroli, A. & Bachi, A. Proteomic analysis of protein S-nitrosylation. Proteomics 8, 4484–4494 (2008).

    Article  CAS  Google Scholar 

  45. Poole, L.B. & Nelson, K.J. Discovering mechanisms of signaling-mediated cysteine oxidation. Curr. Opin. Chem. Biol. 12, 18–24 (2008).

    Article  CAS  Google Scholar 

  46. Vila, A. et al. Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol. 21, 432–444 (2008).

    Article  CAS  Google Scholar 

  47. Hafner, M. et al. Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature 444, 941–944 (2006).

    Article  CAS  Google Scholar 

  48. Antczak, C., Radu, C. & Djaballah, H. A profiling platform for the identification of selective metalloprotease inhibitors. J. Biomol. Screen. 13, 285–294 (2008).

    Article  CAS  Google Scholar 

  49. Patricelli, M.P. et al. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46, 350–358 (2007).

    Article  CAS  Google Scholar 

  50. Chandonia, J.M. & Brenner, S.E. The impact of structural genomics: expectations and outcomes. Science 311, 347–351 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Cravatt laboratory for helpful discussions. We are grateful to S. Schürer and P. Baillargeon for help purchasing emetine analogs, to S. Tully for assistance with the synthesis of 4NPG and to J. Garfunkle for assembly of the Boger chemical library. Full-length cDNA encoding human RBBP9 was a gift of the Cheresh laboratory (UCSD). This work was supported by the National Institutes of Health (CA132630, MH084512), a National Science Foundation Predoctoral Fellowship (D.A.B.), and the Skaggs Institute for Chemical Biology.

Author information

Authors and Affiliations

Authors

Contributions

D.A.B. performed experiments. D.A.B., S.J.B., H.R. and B.F.C. designed experiments and analyzed data. D.A.B. and B.F.C. wrote the paper.

Corresponding author

Correspondence to Benjamin F Cravatt.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–3 and Supplementary Methods (PDF 5868 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bachovchin, D., Brown, S., Rosen, H. et al. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol 27, 387–394 (2009). https://doi.org/10.1038/nbt.1531

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1531

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing