Abstract
After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.
Highlights
Cells use a varied array of post-translational protein modifications to regulate signalling pathways
PARylation is principally driven by PARPs 1–3 after DNA damage and alkylating agents are known to induce base excision repair (BER) pathways, intermediates of which lead to activation of PARPs12
Our preliminary data showed Hela cells that have been stably knocked down (KD) for poly(ADP ribose) glycohydrolase (PARG) were more sensitive to growth inhibition by the alkylating agent methylmethane sulfonate (MMS) (Supplemental Figure 1a)
Summary
Cells use a varied array of post-translational protein modifications to regulate signalling pathways. Cancer cells rely on DNA repair more heavily than normal cells and inhibitors of these pathways have been in preclinical and clinical evaluation for a number of years[2] The success of this strategy is exemplified by the inhibition of PARPs using olaparib that recently gained regulatory approval for use in ovarian cancers[3]. Many of these are large tannin-like molecules such as gallotannin which have been shown to have a number of effects unrelated to PARG inhibition (e.g. anti-oxidant properties[4]) Other compounds, such as APD-HPD and rhodamine-based PARG inhibitors (RBPIs), have shown good specificity for eukaryotic PARG but are either not cell permeable or have only been tested in biochemical assays[5,6,7]. We were mindful of the need to develop assays to detect cell-permeable inhibitors and the method development is contained
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