An HTS-compatible HTRF assay measuring the glycohydrolase activity of human PARG.

Abstract

Poly(ADP-ribose)(PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses, and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly-described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed, and from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.