An optimized fluorescence assay for screening novel PARP-1 inhibitors

Poly(ADP-ribose) polymerase 1 (PARP-1) is an enzyme that catalyzes the formation of poly(ADP-ribose) (PAR) polymer chains from nicotinamide adenine dinucleotide (NAD + ) onto target proteins. PARP-1 plays an important role as a first responder in DNA damage repair and has been an attractive target for the development of anti-cancer therapeutics. This has led to high interest in the development of novel small molecule therapeutics by researchers to target PARP-1 and other members of the PARP-family of enzymes. However, identifying ideal drug candidates can be problematic as current commercial PARP-1 screening assays are cost-prohibitive for many small drug discovery laboratories (particularly for academic groups) and may require specialized equipment or techniques that many labs are not equipped for. Herein, we present a robust, low-cost and optimized PARP-1 assay that quantifies leftover NAD + substrate as a key readout for PARP-1 inhibition via conversion to a fluorophore, while simultaneously optimizing the assay for molecules that have high background fluorescence. The assay procedure has been validated with known PARP inhibitors and has proven to be a cost-effective fluorescence assay for use as a robust preliminary PARP-1 screen.