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Abstract The 17-member poly (ADP-ribose) polymerase (PARP) family of proteins mediate a number of cellular processes including gene transcription, cell-division and DNA-repair. PARPs catalyze the addition of poly- or mono- ADP-ribose chains on target proteins, modifying their function and/or recruitment in various pathways. Poly (ADP-ribose) Glycohydrolase (PARG) catabolizes PAR units and facilitates the recycling of PARP and other modified effector proteins. A number of PARPS are implicated in human disease, and PARP inhibitors (PARPi’s) have seen major clinical success, however, many PARPs (especially mono PARPs) are understudied and chemical tools for understanding cellular PARPi selectivity and potency across the entire family are lacking. Similarly, several PARG inhibitors have been developed to date, but tools for directly assessing target engagement in a cellular context are sorely needed. Here we describe the development and application of target engagement assays for characterizing PARP and PARG inhibitor selectivity and affinity in live cells. This method is based on the NanoBRET™ assay platform, which is a proximity-based probe displacement assay that operates in live cells. Using NanoBRET™ technology, we have developed a single probe that covers 12 out of 17 PARP family members. We explored the family-wide selectivity of a diverse panel of clinical and preclinical PARP inhibitors in live cells, representing the first family-wide, cell-based method for profiling PARP selectivity. We also describe the development of a second probe that reports on PARG target engagement. Target engagement of PARG was observed for three out of five tested inhibitors, suggesting a potential indirect mode of action for certain inhibitors on PARG glycohydrolase activity. These assays offer a complementary approach for studying target engagement within the PARylation cycle. Citation Format: Ani Michaud, Kelly Teske, Jennifer Wilkinson, Chad Zimprich, James Vasta, Cesear Corona, Min Zhou, Elizabeth Dominguez, Kaitlin Dunn-Hoffman, Matthew Robers. Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays 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 (6Suppl): Abstract nr 7114.
Michaud et al. (Fri,) studied this question.