High-Throughput Platform for Real-Time Monitoring of ATP-Generating Enzymes in Living Cells Based on a Lanthanide Probe.

ABSTRACT

Remarkable variation between cell-free and cellular measurements of enzyme activity triggered the unmet need to develop tools for monitoring enzyme activity in living cells. Such tools will advance our understanding of the biological functions of enzymes and their potential impact on drug discovery. We report in this study a universal assay for monitoring ATP-generating enzymes in living cells using a self-assembled Tb3+ complex probe. Modulation of the rheological properties of cell culture media enabled shifting the lifetime of the Tb3+ complex in the presence of ATP from micro-to-millisecond range. Based on the response of the Tb3+ complex to ATP, cellular assays for 5 ATP-generating enzymes were developed. Remarkably, assessment of the activity of these enzymes in living cells is made possible for the first time. The pyruvate kinase M2 (PKM2) assay has been optimized for high-throughput screening (HTS) and further implemented in the identification of novel scaffolds as PKM2 inhibitors.