Structure‐Guided Synthetic Peptide Targeting Nucleolus and Neural Progenitor Protein Nuclear Import Impairs Ribosome Biogenesis and Cancer Cell Proliferation

Nucleolus and neural progenitor protein (NEPRO) is a nucleolar factor required for 40S ribosomal subunit maturation and is therefore essential for the high translational demand of proliferating cancer cells. Here, we identify a bipartite nuclear localization signal (NLS; aa 74-96) in NEPRO and show that residues in both basic clusters are required for nuclear targeting. A disease-associated mutation within the C-terminal cluster, R94C, abolished NEPRO nuclear localization and markedly reduced binding to importin-α1 in vitro and in cells. Importin-α1-NLS complexes revealed that R94 forms persistent hydrogen bonds, salt bridges, and hydrophobic contacts with importin-α1 residues (A269, W273, P308, T311, P312, N350), explaining its central role in NLS recognition. Guided by these insights, we designed a rational synthetic hexapeptide inhibitor (H₂N-AWPTPD-COOH) that is soluble, monodisperse, shows intrinsic fluorescence and is non-amyloidogenic. AWPTPD peptide binds wild-type NEPRO but not the R94C variant, and ab initio modeling shows peptide engagement of the R94 surface. Cellular delivery of the synthetic peptide significantly mislocalized NEPRO to the cytoplasm, reduced polysome abundance, decreased collagen secretion/deposition and clonogenicity, and induced cell-cycle arrest with upregulation of senescence markers: p16^INK4A and p21^WAF1/CIP1. These results validate R94 as a targetable hotspot in NEPRO's NLS and demonstrate a peptide-based approach to perturb ribosome biogenesis and suppress cancer cell growth.