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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Sirtuins (SIRTs) are enzymes that remove protein lysine acyl modifications, regulating vital cellular processes like metabolism, gene transcription, DNA repair, and stress response.
• Several SIRTs are implicated in cancer as non-oncogene addiction targets, making them promising candidates for anticancer drug development.
• Developing potent and isoform-selective SIRT inhibitors requires effective high-throughput screening (HTS) methods.

Purpose of the Study:

• To design and synthesize a novel fluorescent polarization (FP) tracer for sirtuin activity.
• To develop a robust, high-throughput FP competition assay for screening inhibitors of SIRT1-3.
• To validate the assay's performance and demonstrate its utility in accelerating SIRT inhibitor discovery.

Main Methods:

• Design and synthesis of a high-affinity fluorescent polarization (FP) tracer, KP-SC-1.
• Development of a high-throughput FP competition assay for SIRT1-3 inhibition screening.
• Validation of the assay using known SIRT1-3 inhibitors and assessment of its stability and performance in pilot library screening.

Main Results:

• A novel FP tracer, KP-SC-1, was successfully synthesized and utilized to establish a robust HTS assay for SIRT1-3.
• The assay demonstrated effectiveness in detecting both NAD+-dependent and NAD+-independent SIRT1-3 inhibitors.
• The developed FP assay requires significantly less SIRT1-3 enzyme compared to previous methods, enhancing its suitability for large-scale screening.

Conclusions:

• The newly developed FP competition assay is a stable and high-performing method for screening SIRT1-3 inhibitors.
• This assay's reduced enzyme requirement makes it particularly advantageous for high-throughput library screening.
• The assay is expected to accelerate the discovery and development of novel SIRT1-3 inhibitors for potential therapeutic applications, especially in cancer.