Letter | Published:

Enzyme family–specific and activity-based screening of chemical libraries using enzyme microarrays

Subjects

Abstract

The potential of protein microarrays1 in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments2,3. In the particular case of enzyme microarrays3, low-molecular-weight fluorescent affinity labels4,5,6,7,8,9,10 (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process11. Relying on this principle3, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, Kiapp values and inhibitor profiles were confirmed and refined.

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

    Kambhampati, D. (ed.). Protein microarray technology (Wiley-VCH, Heidelberg, 2004).

  2. 2

    Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).

  3. 3

    Eppinger, J., Funeriu, D.P., Miyake, M., Denizot, L. & Miyake, J. Enzyme microarrays: on-chip determination of inhibition constants based on affinity-label detection of enzymatic activity. Angew. Chem. Int. Ed. 43, 3806–3810 (2004).

  4. 4

    Chen, G.Y., Uttamchandani, M., Zhu, Q., Wang, G. & Yao, S.Q. Developing a strategy for activity-based detection of enzymes in a protein microarray. ChemBioChem 4, 336–339 (2003).

  5. 5

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

  6. 6

    Campbell, D.A. & Szardenings, A.K. Functional profiling of the proteome with affinity labels. Curr. Opin. Chem. Biol. 7, 296–303 (2003).

  7. 7

    Speers, A.E. & Cravatt, B.F. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 5, 535–546 (2004).

  8. 8

    Greenbaum, D.C. et al. Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design. Chem. Biol. 9, 1085–1094 (2002).

  9. 9

    Jessani, N. & Cravatt, B.F. The development and application of methods for activity-based protein profiling. Curr. Opin. Chem. Biol. 8, 54–59 (2004).

  10. 10

    Goulet, B. et al. A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor. Mol. Cell 14, 207–219 (2004).

  11. 11

    Gosalia, D.N. & Diamond, S.L. Printing chemical libraries on microarrays for fluid phase nanoliter reactions. Proc. Natl. Acad. Sci. USA 100, 8721–8726 (2003).

  12. 12

    Berdowska, I. Cysteine proteases as disease markers. Clin. Chim. Acta 342, 41–69 (2004).

  13. 13

    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).

  14. 14

    Lecaille, F., Kaleta, J. & Brömme, D. Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem. Rev. 102, 4459–4488 (2002).

  15. 15

    Kang, K. & Kim, W. Recent developments of cathepsin inhibitors and their selectivity. Exp. Opin. Therap. Pat. 12, 419–432 (2002).

  16. 16

    Powers, J.C., Asgian, J.L., Ekici, Ö.D. & James, K.E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750 (2002).

  17. 17

    Otto, H.-H. & Schirmeister, T. Cysteine proteases and their inhibitors. Chem. Rev. 97, 133–171 (1997).

  18. 18

    Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260–273 (1996).

  19. 19

    Carrillo, A., Gujraty, K.V. & Kane, R.S. Surfaces and substrates. in Microarray Technology and Its Applications (eds. Mueller, U.R. & Nicolau, D.V.) 45–61 (Springer GmbH, Berlin, 2005).

  20. 20

    Kuzmic, P. et al. High-throughput screening of enzyme inhibitors: automatic determination of tight-binding inhibition constants. Anal. Biochem. 281, 62–67 (2000).

  21. 21

    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).

  22. 22

    Sasaki, T. et al. Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins. J. Enzyme Inhib. 3, 195–201 (1990).

  23. 23

    Yamashita, D.S. et al. Structure and design of potent and selective cathepsin K inhibitors. J. Am. Chem. Soc. 119, 11351–11352 (1997).

  24. 24

    Rydzewski, R.M. et al. Peptidic 1-cyanopyrrolidines: synthesis and SAR of a series of potent, selective cathepsin inhibitors. Bioorg. Med. Chem. 10, 3277–3284 (2002).

  25. 25

    Kirschke, H., Barrett, A.J. & Rawlings, N.D. Proteinases 1: lysosomal cysteine proteases. Protein Profile 2, 1581–1643 (1995).

  26. 26

    Gilbert, B.A. & Rando, R.R. Modular design of biotinylated photoaffinity probes: synthesis and utilization of a biotinylated pepstatin photoprobe. J. Am. Chem. Soc. 117, 8061–8066 (1995).

  27. 27

    Hagenstein, M.C. et al. Affinity-based tagging of protein families with reversible inhibitors: a concept for functional proteomics. Angew. Chem. Int. Ed. 42, 5635–5638 (2003).

  28. 28

    Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M. & Cravatt, B.F. Activity-based probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci. USA 101, 10000–10005 (2004).

Download references

Acknowledgements

We thank Robert Menard for providing recombinant cathepsin B and recombinant cathepsin L. This work was supported by the National Institute of Advanced Industrial Science and Technology of Japan and by the Stifterverband für die Deutsche Wissenschaft (Projekt-Nr. 11047: ForschungsDozentur Molekulare Katalyse).

Author information

Competing interests

The authors are currently involved in the founding process of the biotechnology company Alceis Sarl., which might, among others, use the technology described in the article.

Correspondence to Daniel P Funeriu or Jörg Eppinger.

Supplementary information

  1. Supplementary Fig. 1

    Full kinetic modelling for Cathepsin B bovine (PDF 59 kb)

  2. Supplementary Fig. 2

    Full kinetic modelling for Cathepsin B, human (PDF 57 kb)

  3. Supplementary Fig. 3

    Full kinetic modelling for Cathepsin B, recombinant (PDF 55 kb)

  4. Supplementary Fig. 4

    Full kinetic modelling of Cathepsin C (PDF 56 kb)

  5. Supplementary Fig. 5

    Full kinetic modelling of Cathepsin H (PDF 58 kb)

  6. Supplementary Fig. 6

    Full kinetic modelling of Cathepsin K (PDF 56 kb)

  7. Supplementary Fig. 7

    Full kinetic modelling of Cathepsin L (PDF 57 kb)

  8. Supplementary Fig. 8

    Full kinetic modelling of Cathepsin S (PDF 56 kb)

  9. Supplementary Fig. 9

    Results of the influence of surfactant (SDS) and pH on the microarrayed enzyme's activity. (PDF 495 kb)

  10. Supplementary Table 1

    On-chip kinetic constants for the reaction between cathepsins and the FAL 1. (PDF 35 kb)

  11. Supplementary Table 2

    Literature values of inhibition constants of different cathepsins for the known inhibitors screened in the HTS setup. (PDF 41 kb)

  12. Supplementary Table 3

    Kiapp/nM values for different cathepsins, determined in evaluation experiment (PDF 27 kb)

  13. Supplementary Methods (PDF 541 kb)

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

  • Received

  • Accepted

  • Published

  • Issue Date

DOI

https://doi.org/10.1038/nbt1090

Further reading

Figure 1: Schematic explanation of TEMA technology.
Figure 2: Results of the kinetic setup experiments.
Figure 3: Results of the HTS setup experiment.
Figure 4: Results of the validation setup experiment for two inhibitors (CNI, reversible and E-64, irreversible).