Identification and functional analysis of chloride intracellular channel protein 1-binding peptide and its uses for ProTac-based cancer therapy

Targeted therapy using antibodies that treat only certain cancer cells is currently being studied because it causes less side effects on normal cells. Compared to antibodies, peptides are relatively smaller and have higher tissue penetration ability and lower immunogenicity. Chloride intracellular channel protein1 (CLIC1) is a metamorphic protein as it can exist in two forms: soluble globular protein in the cytoplasm or transmembrane protein. CLIC1 is related to cancer angiogenesis, cell migration and metastasis and is upregulated in various types of cancer, including hepatocellular carcinoma and lung cancer, presenting it as a promising therapeutic cancer target. In this study, we identified a peptide candidate (CLIC1pep) that binds to CLIC1 using phage display. The affinity and specific binding of CLIC1pep to CLIC1 was validated by SPR, ELISA, and pull-down assays. CLIC1pep linked to a cell-penetrating peptide (R7) (R7-CLIC1pep) showed an increased cell penetration and binding to CLIC1 in the cytoplasm, whereas it did not induce tumor cell death. However, R7-linked CLIC1pep inhibited the membrane insertion of CLIC1 and reduced the migration and invasion of cancer cells through reducing the p-ERK levels. Moreover, R7-CLIC1pep-linked E3 ligase peptide was synthesized for a ProTAC against CLIC1 and degraded CLIC1 in tumor cells. These results suggest that R7-CLIC1pep and R7-CLIC1pep-linked E3 ligase peptide hold a potential for a ProTac-based cancer therapy of CLIC1-high tumors.