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).
Highlights
I t is estimated that the average cell receives 10 000 DNA damaging events every day.[1]
While agents targeting the ataxia telangiectasia and Rad3related protein (ATR) and ataxia telangiectasia mutated (ATM) kinases involved in DNA double strand break (DSB) repair are currently undergoing clinical evaluation,[3−5] considerably more effort has been directed toward developing inhibitors of the enzyme poly(ADP-ribose) polymerase (PARP), shown to be synthetic lethal in homologous recombination-deficient (HR-deficient) cancers, such as those having lost the BRCA1/2 genes.[6]
poly(ADP-ribose) glycohydrolase (PARG) inhibition would be expected to reduce PARP DNA binding and enzymatic activity,[32,33] we show that these novel PARG inhibitors offer overlapping yet distinct pharmacology to that observed through inhibition of their PARP counterproteins
Summary
I t is estimated that the average cell receives 10 000 DNA damaging events every day.[1]. PARP recognizes sites of DNA single strand breaks (SSBs) and acts as a recruitment hub for the apparatus required to effect repair of SSBs, such as XRCC1, LIG3, and POLB.[7] Upon recognition of the SSB, PARP autoribosylates to form branched poly(ADP-ribose) (PAR) chains, often containing up to 200 ADP-ribose units.[8] These act as a cellular marker of DNA damage and facilitate the recruitment of repair factors Inhibition of this process, leads to replication fork disruption, conversion of the SSBs to DNA DSBs, and, in the absence of DSB repair, cell death.[9] Exploitation of this mechanism has led to a plethora of PARP inhibitors entering clinical trials,[10] of which olaparib (Lynparza) was the first to gain FDA regulatory approval in 2015.11 emergence of resistance to PARP inhibitors, possibly through positive selection of rare BRCA2 revertant clones, has already been found in patients. As ADP-ribose is “locked in” to the PAR chains, NAD levels drop after DNA damage,[15] perhaps due to NAD recycling impairment, potentially impeding both the ability of the cell to effectively control levels of oxidative stress and leading to metabolic catastrophy.[16]
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