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


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.

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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).


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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).

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Corresponding authors

Correspondence to Daniel P Funeriu or Jörg Eppinger.

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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.

Supplementary information

Supplementary Fig. 1

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

Supplementary Fig. 2

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

Supplementary Fig. 3

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

Supplementary Fig. 4

Full kinetic modelling of Cathepsin C (PDF 56 kb)

Supplementary Fig. 5

Full kinetic modelling of Cathepsin H (PDF 58 kb)

Supplementary Fig. 6

Full kinetic modelling of Cathepsin K (PDF 56 kb)

Supplementary Fig. 7

Full kinetic modelling of Cathepsin L (PDF 57 kb)

Supplementary Fig. 8

Full kinetic modelling of Cathepsin S (PDF 56 kb)

Supplementary Fig. 9

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

Supplementary Table 1

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

Supplementary Table 2

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

Supplementary Table 3

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

Supplementary Methods (PDF 541 kb)

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Funeriu, D., Eppinger, J., Denizot, L. et al. Enzyme family–specific and activity-based screening of chemical libraries using enzyme microarrays. Nat Biotechnol 23, 622–627 (2005). https://doi.org/10.1038/nbt1090

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