The E6 oncoprotein of high-risk human papillomaviruses (HPVs) promotes cervical carcinogenesis by forming a complex with the ubiquitin ligase E6AP to mediate p53 proteasomal degradation. A 15-amino-acid peptide (pep11) was shown to disrupt the HPV16 E6/E6AP complex, to restore p53 stability and to induce apoptosis in HPV16-positive cancer cells. In this study, a short cell-penetrating peptide (CPP) was conjugated to the pep11 N-terminus to improve its solubility and intracellular delivery in HPV-positive cancer cells.

CPP-pep11 was synthesized using Boc chemistry. The interactions of CPP-pep11 with HPV16 and HPV18 E6 were modelled using AlphaFold2, while peptide structures were characterised by NMR spectroscopy. HPV16-positive SiHa and HPV18-positive C4-I cervical cancer cells were treated with CPP-pep11 (0.5–20 µM) for 24–72 h. Effects on proliferation and clonogenicity were assessed using xCELLigence and colony formation assays. Peptide uptake and localization were analysed by confocal microscopy and differential cell fractionation, while p53 expression and viability were evaluated by Western blotting and cytotoxicity assays.

AlphaFold2 modelling suggested that CPP-pep11 can interact with both HPV16 E6 and HPV18 E6, with a more stable predicted binding to HPV16 E6. CPP-pep11 was soluble in water at 20 µM and, according to NMR analysis, predominantly adopted a disordered conformation. CPP-pep11 efficiently entered the SiHa and C4-I cell membranes, mainly localised to the cytoplasm at lower concentrations (0.5–5 µM) and to the nucleus at higher concentrations (10–20 µM). CPP-pep11 induced dose- and time-dependent inhibition of proliferation, with a significant reduction in colony formation in both cell lines. Treatment also restored p53 levels at 48 and 72 h, consistent with disruption of E6-mediated degradation.

In conclusion, CPP-pep11 is a novel peptide-based inhibitor of HPV16 E6 and HPV18 E6 with therapeutic potential in HPV-associated malignancies by restoring p53 levels and inhibiting cancer cell proliferation.