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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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).
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.
Full kinetic modelling for Cathepsin B bovine (PDF 59 kb)
Full kinetic modelling for Cathepsin B, human (PDF 57 kb)
Full kinetic modelling for Cathepsin B, recombinant (PDF 55 kb)
Full kinetic modelling of Cathepsin C (PDF 56 kb)
Full kinetic modelling of Cathepsin H (PDF 58 kb)
Full kinetic modelling of Cathepsin K (PDF 56 kb)
Full kinetic modelling of Cathepsin L (PDF 57 kb)
Full kinetic modelling of Cathepsin S (PDF 56 kb)
Results of the influence of surfactant (SDS) and pH on the microarrayed enzyme's activity. (PDF 495 kb)
On-chip kinetic constants for the reaction between cathepsins and the FAL 1. (PDF 35 kb)
Literature values of inhibition constants of different cathepsins for the known inhibitors screened in the HTS setup. (PDF 41 kb)
Kiapp/nM values for different cathepsins, determined in evaluation experiment (PDF 27 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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