Poly(ADP-ribose) polymerase 3 (PARP3) is an important member of the PARP family and shares high structural similarities with both PARP1 and PARP2. The biological roles of PARP3 are currently under investigation; however, several key reports indicate the integral roles of PARP3 in DNA damage repair, and thus it has been investigated as a novel target in oncology. It is clear that the identification of selective PARP3 inhibitors would further advance the understanding of the biological roles of PARP3. Herein, we describe a modified PARP3 screening assay using biotinylated NAD+ as the specialized substrate. This method relies on the activity of PARP3 to transfer the biotinylated NAD+ onto a histone protein to form ADP-ribosylated histone. The biotin label on this histone protein is then detected and quantifies the intrinsic enzymatic activity of PARP3. We optimized the assay with respect to the histone, NAD+/biotinylated NAD+ mixture, DNA, and PARP3. Our developed screening system was then validated with a reported selective PARP3 inhibitor, ME0328, as well as utilizing five other clinically available PARP1/2 inhibitors. We demonstrated that our assay system was sensitive, efficient, and economical, and we reason that it could be useful for the development of highly selective PARP3 inhibitors in the future.
Keywords: ELISA; PARP1/2 inhibitor; PARP3; biotinylated NAD+.