Abstract 4698: Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery
Authors: Pilo Caila , Awad Mohammad , Baron Veronique T. , Martins Fernando , Mikolosko Jonathan , Plouffe Steven , Tao Zhihua , Zientara-Rytter Katarzyna , Zhu Henry , Shashkin Pavel 
Published: March 2024

Inhibitors of poly (ADP-ribose) polymerases (PARPs) have revolutionized cancer therapy by highlighting the potential of synthetic lethal drugs to target the DNA damage response (DDR) network. More recently, the development of PARG inhibitors was spurred by the discovery that inhibiting PARG (poly (ADP-ribose) glycohydrolase) results in the accumulation of poly ADP-ribose on DDR proteins and induces cell death. Adding PARG or PARP inhibitors to a cell of interest and quantifying the resulting levels of PARylation can provide valuable insight on compound membrane permeability and target engagement in a cellular context. Nevertheless, assessing PARylation (poly-ADP-ribosylation in which multiple ribose units are added in linear or branched forms) in cells is difficult compared to biochemical assays, impeding the progress of candidate drug development. A sandwich ELISA-based assay was developed to analyze the total protein PARylation present in cellular extracts. Experimental protocols were optimized to discern differences in cellular PARylation levels resulting from activating the DDR and from exposure to PARP inhibitors and/or PARG inhibitors. It is worth noting that our assay specifically detects PARylation, measuring the effects associated with PARP family members 1 to 5, as other family members primarily catalyze MARylation (mono-ADP ribosylation in which a single ADP-ribose unit is added). Titration of known PARP inhibitors and PARG inhibitors was performed to validate the assay. 1) PARG inhibitor PDD00017273, used in combination with DNA damaging agent H2O2, increased the level of cellular PARylation, exhibiting a similar IC50 value across the four cell lines tested. This is consistent with the notion that PARG is the major de-PARylating enzyme in most cells. The potency of other known PARG inhibitors PDD00017272 and PDD00017238 ranked the compounds as expected from published results (PDD00017272&lt; PDD00017273&lt; PDD00017238). 2) Titration of the three PARP1/2 inhibitors Talazoparib, Olaparib and AZD5305, in combination with a DNA damage agent and PDD00017273 inhibitor, shows a dose-dependent reduction in cellular PARylation in HEK293 cells, as expected since PARP1 is activated in response to DNA damage and this activation is blocked by the inhibitors (IC50 values, Olaparib = 0.013 µM, AZD5305 = 0.0024 µM, Talazoparib = 0.0016 µM). These IC50 are consistent with published values and underscore the usefulness of the assay in measuring dose response effects, allowing accurate determination of compound IC50 in living cells. In summary, our optimized experimental cell treatment protocols and ELISA-based assay allow quantification of total cellular PARylation and accurately rank candidate drugs targeting PAR erasers or PAR writers in a high-throughput format, using living cells.

Citation Format: Caila Pilo, Mohammad Awad, Veronique T. Baron, Fernando Martins, Jonathan Mikolosko, Steven Plouffe, Zhihua Tao, Katarzyna Zientara-Rytter, Henry Zhu, Pavel Shashkin. Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4698.