Nature Biotechnology23, 622 - 627 (2005)
Published online: 10 April 2005; | doi:10.1038/nbt1090
Enzyme family−specific and activity-based screening of chemical libraries using enzyme microarrays
Daniel P Funeriu1, Jörg Eppinger2, Lucile Denizot1, Masato Miyake1
& Jun Miyake1
1
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakouji, Amagasaki, Hyogo, 661-0974, Japan.
2
ForschungsDozentur Molekulare Katalyse, Lehrstuhl für Anorganische Chemie, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany.
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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