Disrupting the tumor-associated TNKS-USP25 protein-protein interface in cancer: structural basis, druggability, and therapeutic opportunities.

Tankyrases (TNKS1/2) are multi-domain poly(ADP-ribose) polymerases that regulate Wnt/β-catenin signaling and broader cellular programs through both catalytic activity and extensive protein-protein interaction (PPI) networks. While most tankyrase-directed drug discovery has focused on inhibiting the PARP catalytic site, an emerging alternative is to target tankyrase stability by disrupting its interaction with the deubiquitinase USP25. USP25 functions as a positive regulator of tankyrase abundance by counteracting ubiquitin-dependent turnover; consequently, blocking the TNKS-USP25 PPI can reduce tankyrase levels, stabilize pathway antagonists such as AXIN, and dampen Wnt transcriptional output. In this review, we discuss current understanding of tankyrase domain architecture with emphasis on ankyrin repeat clusters (ARC1/2/4/5) that recognize short tankyrase-binding motifs (TBMs), and we highlight why ARC5 is a particularly actionable node for intervention in the TNKS-USP25 axis. We summarize the structural basis of USP25 recruitment via a C-terminal TBM-like element, discuss ARC hotspot features that support ligandability, and provide a practical chemical-biology framework for validating PPI disruption using orthogonal assays (co-immunoprecipitation/proximity ligation, biophysics such as SPR/ITC, cellular target engagement, and displacement formats including FP/FRET). We then evaluate reported small-molecule disruptors, including C44 and UAT-B, as proof-of-concept agents that link ARC5-centered binding to tankyrase destabilization and antitumor phenotypes in prostate cancer and multidrug-resistant colorectal cancer models. Finally, we outline key challenges-selectivity across ARCs, off-target risk, and context-dependent biology-and propose future directions, including structure-guided optimization, improved cell-active chemotypes, and dual-mechanism strategies that combine PPI disruption with catalytic inhibition or targeted degradation approaches.