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A High-Throughput Fluorescence Polarization Assay for Screening Sirtuin Inhibitors
Kewen Peng1,2, Suryadeep Chakraborty1,3, Yizhen Jin1,3
1Department of Medicine, The University of Chicago, Chicago, Illinois 60637, United States.
Abstract:
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.
Insights
Researchers developed a new high-throughput assay using fluorescent polarization to screen for sirtuin (SIRT1-3) inhibitors. This method accelerates the discovery of potential anticancer drugs targeting sirtuins.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Sirtuins (SIRTs) are enzymes that regulate crucial cellular processes like metabolism and DNA repair.
- Sirtuins are implicated in cancer development and are promising therapeutic targets.
- Developing selective sirtuin inhibitors requires efficient high-throughput screening (HTS) methods.
Purpose of the Study:
- To design and validate a novel, robust, and high-throughput fluorescent polarization (FP) competition assay for screening SIRT1-3 inhibitors.
- To utilize a newly synthesized high-affinity FP tracer (KP-SC-1) for assay development.
- To enable the identification of both NAD+-dependent and NAD+-independent SIRT1-3 inhibitors.
Main Methods:
- Design and synthesis of a fluorescent polarization (FP) tracer, KP-SC-1.
- Development of a high-throughput FP competition assay for SIRT1-3.
- Validation of the assay using known SIRT1-3 inhibitors and pilot library screening.
Main Results:
- A robust, high-throughput FP competition assay for SIRT1-3 inhibitors was successfully developed.
- The assay demonstrated stability and outstanding performance in pilot screening.
- The FP assay requires significantly less SIRT1-3 enzyme compared to previous methods, enhancing efficiency for HTS.
Conclusions:
- The developed FP assay is a valuable tool for accelerating the discovery and development of SIRT1-3 inhibitors.
- This assay facilitates the identification of novel anticancer drug candidates targeting sirtuins.
- The reduced enzyme requirement makes the assay particularly suitable for large-scale drug screening efforts.
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