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

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Summary 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 µM. 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/β-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4 inhibition. Significance Tankyrases 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 µM 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/β-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.