Biology subjects

Bosetti, C.

Publications and source records attributed to Bosetti, C..

3 recordsLinked to original sources

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↗

bioRxiv2024

Substitutions at the C-8 position of quinazolin-4-ones improve the potency of nicotinamide site binding tankyrase inhibitors

Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and some small molecule inhibitors have already been developed. These inhibitors typically interact with the nicotinamide binding site and extend along the NAD+ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as promising scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both nitro- and diol-substituents exhibit intriguing inhibition of TNKS2, with compound 49 displaying an IC50 of 65 nM, while compound 40s IC50 value is 14 nM. Both analogues show efficacy in cell assays and attenuate the tankyrase-controlled Wnt/{beta}-catenin signaling with sub-micromolar IC50. When tested against a wider panel of enzymes, compound 40 displayed high selectivity towards tankyrases, whereas 49 also inhibited other PARPs. The results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.

biochemistry↗

bioRxiv2022

An evolutionary perspective on the origin, conservation and binding partner acquisition of tankyrases

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species exist. In this work we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identified the single-celled choanoflagellates as earliest origin of tankyrases. We further show that sequence and structural aspects of TNKSs are well conserved even between highly diverged species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners vary greatly between species, indicating that tankyrases have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, although their regulatory functions in cells have likely changed considerably throughout evolution.

bioinformatics↗