Pääkkönen, J. | Search bioRxiv

bioRxiv2025

Dimerization of human PARP15 is required for NAD+ binding and automodification

PARP proteins are enzymes catalyzing ADP-ribosylation, a well-conserved post-translational modification. In addition to the catalytic domain present in all PARPs, these proteins have a wide selection of other domains possessing different functions. Additional domains present in human PARP15 are macrodomains, capable of binding ADP-ribose. PARP15 is the least studied of the macrodomain-containing human PARPs, and it is linked to different diseases from infections to cancer. We show that the full-length canonical isoform 1 of PARP15 auto-ADP-ribosylates robustly on glutamate and aspartate residues, which can be hydrolyzed by known ADP-ribosylhydrolases. We have been able to locate several modification sites to the region of tandem macrodomains. In agreement with earlier reports regarding the catalytic domain dimerization being the requirement for enzyme activity, we show this in the full-length context with the recombinant isoform 1 of PARP15. We show that dimerization is required for the efficient substrate NAD+ binding, and we provide structural basis for this requirement with the help of a co-crystal structure of the catalytic domain dimer with an unhydrolyzable substrate analog.

biochemistry↗

bioRxiv2025

Discovery of Tankyrase scaffolding inhibitor specifically targeting the ARC4 peptide binding domain

In the past, development of tankyrase inhibitors has focused on the ADP-ribosyltransferase domain. Targeting tankyrases ability to interact with protein substrates through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this paper, we employed a FRET-based assay to identify ARC4-binding compounds by screening the EU-OPENSCREEN Pilot and Commercials Diversity libraries. We discovered an effective series of compounds with the same scaffold and through chemical synthesis we obtained the compound S8 (ARCher-142), which binds selectively to ARC4 with potency of 8 {micro}M. NMR analysis and X-ray crystallography allowed us to identify the binding site in ARC4 and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/{beta}-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4 inhibition. SignificanceTankyrases impact a variety of cellular processes by binding proteins through their ARC domains and the inhibition of these scaffolding functions represents an alternative therapeutic approach to catalytic inhibitors. With a FRET-based high-throughput screening of the EU-OPENSCREEN Pilot and Commercials Diversity libraries we discovered a pyrrolone-based scaffold that is interestingly selective towards ARC4, despite the high conservation of the ARC binding site. Our synthesized compound S8 (ARCher-142) displays an 8 {micro}M potency for TNKS2 ARC4. With NMR and X-ray crystallography we demonstrate that S8 (ARCher-142) competes with the peptide optimized for binding and extends to a unique hydrophobic sub-pocket of ARC4. The compound attenuates the WNT/{beta}-catenin signaling pathway in cells and interestingly offers the possibility to target specific protein-protein interactions mediated by ARC4, paving the way for the development of a pyrrolone-based class of tankyrase scaffolding inhibitors.

biochemistry↗