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July 17th, 2025
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Captor Therapeutics S.A., ul. Dunska 11, 54-247 Wroclaw, Poland
molecular biology
biorxiv

A Robust Crystallographic Platform for High-Throughput β-Catenin Ligand Discovery

Klejnot, M.Open in Google Scholar•Skowron, A. N.Open in Google Scholar•Szymanski, J.Open in Google Scholar•Ulatowski, F.Open in Google Scholar•Bista, M.Open in Google Scholar•Cottens, S.Open in Google Scholar•Dabrowiecka, G.Open in Google Scholar•Gajewska, D.Open in Google Scholar•Gorecka-Minakowska, K. M.Open in Google Scholar•Kotlarek, D.Open in Google Scholaret al.

This study presents a robust crystallographic platform for assessing {beta}-catenin protein-binding compounds. We developed a standardized protein production protocol for the armadillo domain of {beta}-catenin (BC-ARM) and performed biophysical screens using Surface Plasmon Resonance (SPR) and Differential Scanning Fluorimetry (DSF). These findings led to the successful determination of the co-crystal structure of BC-ARM with compound 1 binding to previously reported site but distinct from known transcription factor binding sites. To broaden the search for novel BC binding sites, we utilized FragLites library with a cyclic peptide-stabilized BC-ARM construct. This yielded two high-resolution co-crystal structures identifying a previously unreported binding hotspot. Recognizing the limitations of the cyclic peptide-bound construct for general screening, we designed a novel, truncated BC-ARM construct. This new construct eliminates unstructured regions, reliably producing high-quality, diffracting crystals suitable for high-throughput crystallographic studies. In conclusion, the ligand-bound {beta}-catenin structures and this novel, robust BC-ARM construct establish a powerful platform for further {beta}-catenin investigation.

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