First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib

Abstract

The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).

Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors

“…We further speculate that PARG inhibitors may work better against cancer than PARP inhibitors (49, 53, 54). First, due to HuR’s established overabundance in cancer (19, 55–58), an HuR-dependent increase in PARG in tumor versus normal cells provides a therapeutic window.…”

“…Meanwhile, PARG is the primary enzyme for hydrolyzing PARylation, and thus inhibiting this enzyme in the context of the HuR regulated- DNA repair process (17) could potentially increase specificity and reduce toxicity compared to currently studied pan-PARP inhibitors. With increasing evidence for PARG’s role in the DDR pathway, future studies will aim to study PARG inhibition in PDA with small molecule inhibitors (54) and gene silencing methods. These studies will ultimately reveal whether targeting PARG is a better therapeutic strategy than targeting PARP in cancer cells.…”

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

“…This is the first such report of a screen for synthetic lethal interactions (or more correctly, induced cell death) with PARG and the first potential synthetic lethal use for the novel PARG inhibitor PDD00017273 [40]. It is worth noting that the PDD00017273 induced reduction in long-term survival seen here in MCF7 cells, is in contrast to that seen with the same agent in a short-term assay in HeLa cells [40], this could be due to cellular specificity or the long-term versus short-term effects of the drug.…”

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate

“…Recent efforts to develop more scalable PARG activity assays have been modestly successful, for example, a four-component FRET system to detect the interaction between a PARylated protein and XRCC1 (Kim et al, 2015; Stowell et al, 2016). While this approach has been implemented in microtiter format and utilized to discover a first-in-class PARG inhibitor, it suffers from an inability to accurately measure enzyme kinetics and cannot operate in cell lysate (James et al, 2016). There clearly is still an unmet need for a facile and continuous activity assay for PAR-degrading enzymatic activity.…”