Sodium Channel Blockers Demonstrate Binding Affinity for the Tandem Tudor Domain of the Epigenetic Hub UHRF1

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The tandem Tudor domain (TTD) of UHRF1 is a compelling epigenetic target for novel cancer therapeutics. Here, we integrate theoretical simulations, sophisticated biophysical evaluations and cellular assays to characterize potent TTD binders. Screening of small drug‐ and fragment‐like collections identifies several TTD ligands, with the most promising hit being the local anesthetic hydroxyprocaine. The ligand is characterized in terms of its binding requisites by calorimetry, affording a K d of 1.46 μM and a well‐balanced thermodynamic profile. Molecular dynamics simulations combined with heat capacity measurements and osmotic stress titrations confirm that hydroxyprocaine binds stably to the TTD by displacing approximately 26 interfacial water molecules upon complexation. A targeted follow‐up screen focusing on sodium channel blockers yields two additional, although less promising hits, mexiletine and triamterene. In the DU145 prostate cancer cell line, hydroxyprocaine treatment significantly up‐regulates key downstream targets including the tumor‐suppressor p53 and, to a lesser degree, the stress and inflammation regulators p38 and p65, respectively, while exhibiting very low cytotoxicity. Finally, a previously undocumented interdomain interaction between TTD and its N‐terminal adjacent Ubiquitin‐like domain is reported, introducing a novel, potentially druggable UHRF1 regulatory feature. Together, these findings establish hydroxyprocaine as a highly viable chemical scaffold for TTD‐targeted drug development.