Screening And Activity Evaluation Of PARP Inhibitors

Poly (ADP-ribose) polymerase-1is a post-translational modification enzyme involvingin the DNA repair pathway. It becomes a novel target for cancer treatments in recent years.Developing and verifying efficient and effective evaluation methods for potent PARPinhibitors is critical for clinical application. In this study we had established and evaluated anenzymatic assay capable for HTS screening, cellular models for confirming hits, and a novelcell model for both in vitro and in vivo anti-tumor evaluation of PARP inhibitors.Firstly, an enzymatic assay of PARP-1was established based on the measure ofremaining substrate: with15μg·mL-1DNA as activator,12.5nmol·L-1NAD+was used assubstrate. With6.25×10-4U PARP-1, a96-microwell screening method was set up for PARPinhibitors and assessed with high throughput technical parameters and validated with awell-known PARP inhibitor AZD2281. Through this method, there are52of82compoundsshowed50%or more PARP-1inhibition efficiency, and11compounds with IC50lower than0.5μmol·L-1.Than, two cellular assays of PARPs in Hela cell were established indirectly and directly:DNA damage agent MNNG was used to activate PARPs, ATP depletion was carried out asstimulated with100μmol·L-1MNNG for15min; PAR formation was determined after5minMNNG treatment. AZD2281was applied to validate the methods, and8of17compoundsshowed the EC50under3μmol·L-1-9μmol·L-1by ATP depletion assay; and compounds25and47inhibited PARPs activities in a dose-dependent manner by PAR assay.Besides, cells sensitive to PARP inhibitors were used to evaluate the anti-tumor activityof compound47. Sensitivity of JF-305cells to AZD2281was validated and AZD2281profoundly induced DNA DSBs and subsequent cell cycle arrest in S and G2/M phasefollowed by apoptosis in JF-305cells. Compound47showed cell toxicity to MDA-MB-436and JF-305cells by cell viability assay and clonogenic assay and it delayed the JF-305tumorgrowth in primary experiment.In summary, we established an enzymatic assay of PARP-1and two cellular models todetermine PARPs activities indirectly or directly. We confirmed a Chinese pancreatic cancercell line JF-305was hypersensitivity to PARP inhibitors and can be explored for theassessment of anti-tumor effect of PARP inhibitors. These can be effectively applied tohigh-throughput screening for PARP inhibitors and explore the possibilities of treatments forpancreatic cancer in Chinese population．...