Novel PARP Inhibitors Screening And Preliminary Research On Its Activity

PARP1is an important part of DNA repair machinery. Therefore, PARP1has beenexploited as a potential target for the development of pharmacological strategies to increasethe anti-cancer efficacy of chemotheraputic agents. Recently, many leading pharmaceuticalcompanies have opened clinical trials of PARP Inhibitors. Our aim is to establish a PARPinhibitor screening assay and develop a preliminary research on its pharmacodynamics.In this study, we inserted hPARP1gene into the pFastBacTM1, a baculovirus transfervector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. Afterco-transfecting the Bacmid-hPARP1into Sf9insect cells, the enzyme, hPARP1expressed bySf9insect cells, was purified by3-Aminobenzamide affinity chromatography. The enzyme,which migrates as a116kDa band on SDS-PAGE, was identified as hPARP1by Western Blot;the activity of expressed and purified hPARP1was confirmed by quantitation of the NAD+remaining after the PARP-catalyzed reation. Utilizing hPARP expressed by Sf9insect cell, wetested IC₅₀values of known PARP inhibitors including DPQ and6（5H）-phenanthridone bythis assay, which obtained an excellent correlation between NAD+quantitation method andliterature values. This assay detected PARP inhibitors in a screen using96-well plates，ofwhich IC₅₀values are determined by this assay. In addition, we have screened the mostsensitive cancer cell to PARP inhibitor6（5H）-phenathridone, using a BRCA1-deleted andBRCA2-mutated MX-1and a BRCA2-mutated Capan-1as positive control.pFast-hPARP1was constructed successfully and transformed into DH10Bac producingBacmid-hPARP1. Protein was obtained after the purification by3-Aminobezamide affinitycolumn and its specific activity is1.988U/μg. Relying on quantitation of the NAD+remainingafter the PARP-catalyzed reation,72compounds are to be screened and multiple IC₅₀valuesof41compounds, which exhibit PARP inhibiting activity, are to be determined. In thesecompounds, IC₅₀values of7compounds is below500nmol/L. Using colony formation assayand CCK8assay, we have selected a sensitive cell line called JF305, a pancreatic cancer cellline. In order to test this, HC-232（A），a screened PARP inhibitor，has been used to test JF305,which has exhibited growth inhibition.Our study obtained hPARP1enzyme by construction, expression and purificationdesigned by ourselves and established PARP inhibitor screening assay to determine the IC₅₀values of PARP inhibitors. Using cell assay, we firstly proposed a cell line called JF305whichwas sensitive to PHE. It facilitates PARP inhibitor screening studies especially developing ourown intellectual property PARP inhibitors.