A Bespoke Screening Platform To Study Mono(Adp-Ribosylation)

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are post-translational modifications deposited on multiple amino acids by PARP enzymes using nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate. While there are approved drugs and clinical trials on-going for inhibitors of the polyPARP enzymes that deposit poly(ADP-ribose) (specifically PARP1 and PARP2 inhibitors), monoPARP enzymes that deposit mono(ADP-ribose) are only recently gaining recognition for their role in cellular stress signaling, inflammation and cancer. However, there is a lack of chemical probes to study their function in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop screening assays to enable determination of potency and selectivity of inhibitors during the hit finding and lead optimization phases. The development of enzyme assays is complicated by the fact that the substrates for the majority of the monoPARPs are unknown, and even for those with identified substrates, it is uncertain how they engage their substrates. Here we describe the development of robust high-throughput biochemical and cellular monoPARP assays that overcome the lack of knowledge around the substrates and construction of a family-wide screening panel. We highlight derivatized microplates that activate the enzymes to self-MARylate in dissociation enhanced lanthanide fluorescence assays (DELFIA), antibodies that recognize MARylation in in-cell western (ICW) and immunofluorescence (IF) assays, and NAD+-competitive molecular probes that are used to develop in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) and cellular NanoLuc bioluminescence resonance energy transfer (NanoBRET) probe displacement assays. Additionally, we employ several methods to characterize inhibitor binding kinetics. These assays have been used in high-throughput screening campaigns of up to 500,000 compounds, as well as in the development of potent and selective inhibitors of multiple monoPARP enzymes including RBN012759, a tool compound for PARP14 that inhibits in vitro with an IC50 of 3 nM and in cells using with an IC50 of 9 nM, and is 300-fold selective over all other PARP enzymes. Citation Format: Tim J. Wigle, Danielle J. Blackwell, Laurie B. Schenkel, Yue Ren, William D. Church, Hetvi J. Desai, Kerren K. Swinger, Andrew G. Santospago, Christina R. Majer, Alvin Z. Lu, Mario Niepel, Nicholas R. Perl, Melissa M. Vasbinder, Heike Keilhack, Kevin W. Kuntz. A bespoke screening platform to study mono(ADP-ribosylation) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 506.