Abstract 4184: Development of a homogenous poly(ADP-ribose) assay for screening and profiling PARP inhibitors

Abstract Poly(ADP-ribose) polymerases (PARPs) initiate DNA damage repair (DDR) by binding to double strand breaks and creating a scaffold of poly(ADP-ribose) (pADPr) chains for assembly of other DDR proteins. The use of PARP inhibitors for ovarian tumors with BRCA-1 mutations was the first clinical exploitation of synthetic lethality, and PARP inhibitors are now used for breast, prostate, pancreatic, and ovarian cancers. New types of PARP inhibitors are being sought to broaden the scope of indications. To facilitate this effort, we developed a high throughput screening (HTS) assay for PARPs that enables quantitative detection of pADPr formation in a homogenous format. The assay relies on enzymatic conversion of pADPr to AMP, 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. To minimize interference from screening compounds, we first optimized the concentration of coupling enzymes to ensure that the pADPr will be completely converted to AMP even if the conversion reaction is inhibited by as much as 80%. Using this optimized system, the assay generated a robust signal (Z’ > 0.8) using 0.8 nM PARP1 with sub-saturating NAD (100 μM) and saturated sheared salmon sperm DNA (0.25 mg/mL) under initial velocity conditions. The assay was validated for HTS using a 1, 280 compound bioactive library, resulting in 18 hits, 5 of which proved to be interference. We also screened a 3, 200 compound diversity library, resulting in 44 hits, 3 of which were identified as interference. Four known PARP1 inhibitors, Olaparib, PJ 34 HCl, Veliparib, and Rucaparib Camsylate, were detected in the bioactive screen and tested in dose response mode with both FP and TR-FRET readout; the measured IC50 values (0.45 nM, 29 nM, 1.4 nM and 0.4 nM, respectively) were consistent with published values. By enabling sensitive, quantitative detection of pADPr formation in a simple, mix-and-read format, the Transcreener pADPr assay should facilitate efforts to develop more effective PARP inhibitors. Citation Format: Mahbbat Ali, Ha Pham, Anibal Ramos-Martinez, Robert Lowery. Development of a homogenous poly(ADP-ribose) assay for screening and profiling PARP inhibitors [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 4184.