Abstract 4237: Homogenous detection of O-Acetyl-ADP-ribose as a generic HTS assay method for SIRTs

Abstract Sirtuins (SIRTs) are deacetylases that catalyze removal of acetyl groups from histones and other proteins; they are important components of the epigenetic regulatory landscape and have been targeted for diverse indications, including cancer. SIRTs deacetylate in an NAD-dependent reaction that yields O-acetyl-ADP-ribose (OAADPr) and nicotinamide as products along with the deacetylated protein. Screening and hit-to-lead efforts targeting SIRTs has been hampered by a lack of robust HTS assays methods for detection of SIRT enzymatic activity, a situation that is exacerbated by the diversity of deacetylated protein products and the inherent instability of the OAADPr product. To address this unmet need, we developed a generic activity assay for SIRTs based on enzymatic conversion of OAADPr to AMP in real time, which is then detected using the Transcreener AMP/GMP assay with a fluorescence polarization (FP) or time resolved Förster resonance energy transfer (TR-FRET) readout. Using recombinant full length Sirt1 and Sirt2, we determined optimal NAD, acetylated peptide substrate, and enzyme concentration for initial velocity detection of deacetylase activity. For both enzymes, a robust signal was observed using concentrations in the nanomolar range, and there was good concordance in activity measured using FP and TR-FRET readouts. The assays were validated for HTS using a 1,280 collection of bioactive compounds. Selected hits were further profiled in dose-response assays. The assay Z’ value was above 0.7, supporting its robustness for screening and hit-to-lead/SAR for Sirt1 and Sirt2 antagonists. To our knowledge, the Transcreener OAADPr assay is the first SIRT activity assay that relies on detection of OAADPr rather than the deacetylated protein product. By enabling homogenous detection of SIRT activity with native acetylated substrates, it should simplify screening and profiling of inhibitors and facilitate drug discovery efforts targeting SIRTs. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos-Martinez, Robert Lowery. Homogenous detection of O-Acetyl-ADP-ribose as a generic HTS assay method for SIRTs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4237.