A High-Throughput Fluorescence Polarization Assay for Screening Sirtuin Inhibitors

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes including metabolism, transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered as nononcogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate their therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening of SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We postulate that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.