Alessandra Feoli, Giulio Esposito, Manuela Grimaldi, Monica Viviano, Ida Pacilio, Josef Krátký, Anna Maria D'Ursi, Ciro Milite, Haitao Li, Mark T Bedford, Sabrina Castellano, Gianluca Sbardella
Spindlin1 (SPIN1) is an epigenetic reader involved in oncology, whose third Tudor domain remains largely underexplored. Here, we applied a minimalist pruning strategy to the 26-mer peptide DOCpep3 to identify the core pharmacophore for SPIN1 Tudor 3 domain binding. This yielded truncated linear peptide analogues (e.g., 1, 3, and 4) displaying low nanomolar affinities, significantly outperforming the parent peptide. Orthogonal biophysical validation (MST and SPR) confirmed competitive target engagement. Furthermore, circular dichroism spectroscopy revealed that these pruned ligands induce distinct structural rearrangements in SPIN1, establishing high-affinity chemical probes for drug discovery.