Mechanistic design of cell-penetrating disruptors for phospho-dependent TACC3-CHC interaction

Mitotic spindle stability is enhanced through microtubule crosslinking by a complex formed when an α helix in transforming acidic coiled-coil 3 (TACC3) binds to the helical repeats of the heavy chain of clathrin (CHC). Here, we show that the phosphorylation of TACC3 at S558 modulates the interaction by overcoming the electrostatic repulsion between K507 of CHC and basic residues in TACC3. Leveraging this insight, we optimized the sequence using peptide arrays to develop a hydrocarbon-stapled peptide (SP TACC3) that binds CHC with over 400-fold higher affinity than the native sequence, disrupting the interaction. The crystal structure of the SP TACC3-CHC complex reveals the contribution of additional polar and hydrophobic contacts to the enhanced interaction. SP TACC3 penetrates cells and displaces TACC3 from the mitotic spindle, causing a delay in mitotic progression in two out of three cancer cell lines. This work showcases a strategy for targeting the TACC3-CHC interaction with hydrocarbon-stapled peptides in a cellular context for potential cancer therapies.