A High-Throughput Fluorescence Polarization Assay for Screening Sirtuin Inhibitors.

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.