Structure-Based Design of First Ndc80 Loop-Targeting Peptide Disrupting Ndc80-Nuf2 Interaction for Hepatocellular Carcinoma Therapy.

Ndc80 is essential for kinetochore-microtubule attachment, but its broad protein-protein interaction interface remains difficult to target. Here, we used structure-guided peptide optimization to develop Peptide-4, a loop-directed peptide designed to modulate the Ndc80-Nuf2 interaction. In contrast to reported Ndc80-directed compounds acting through the Hec1-Nek2 axis or the calponin homology domain, Peptide-4 addresses an underexplored loop region. Peptide-4 bound Ndc80 with a Kd of 0.45 ± 0.01 nM and inhibited the Ndc80-Nuf2 interaction with an IC50 of 0.76 ± 0.03 nM. In Huh7 cells, Peptide-4 altered microtubule organization, suppressed proliferation, clonogenic growth, migration, and invasion, and induced G2/M-phase accumulation and apoptosis. Ndc80 depletion attenuated its antiproliferative effect, consistent with a contribution of Ndc80 to its cellular activity. Peptide-4 also reduced tumor growth in Huh7 xenografts without apparent toxicity under the tested conditions. These findings support the Ndc80 loop as an underexplored peptide-addressable site and identify Peptide-4 as a lead for further optimization.