ATG4 targeting peptide inhibits autophagy and elevates reactive oxygen species to trigger cancer cell death

ATG4, an autophagy protease, cleaves ATG8 family proteins to activate autophagy, a process linked to cancer progression. This study aimed to design α-helical peptides targeting ATG4 to assess their potential as autophagy inhibitors for cancer therapy. Three α-helical peptides (H1–H3) derived from GBRL2 (a human ATG8 homolog) were synthesized. Their effects on ATG4B activity and binding stability were evaluated using reporter assays and molecular dynamics simulations. Autophagy modulation was analyzed by immunoblotting and confocal microscopy. Anticancer effects were assessed using flow cytometry, fluorescence imaging, tumorsphere models, and xenograft studies. Among the peptides, the second helical peptide, GBRL2H2, significantly inhibited ATG4B/A enzymatic activity in vitro. The ATG4 Targeting Peptide (GBRL2H2, termed ATP) fused with the cell-penetrating Tat peptide internalized into cells and reduced intracellular ATG4 activity and autophagic flux, whereas Tat or ATP alone elicited minimal to modest effects. Consistently, Tat-ATP treatment decreased cancer cell viability and colony formation. Additionally, Tat-ATP significantly increased the population of dead cancer cells concomitant with decreased mitophagy and mitochondrial membrane potential, elevated reactive oxygen species (ROS) and caspase-3/7 activity. The observed cell death induced by Tat-ATP was substantially reversed by treatment with ROS scavengers and caspase inhibitors, indicating the involvement of ROS and caspase-mediated cell death pathways. Furthermore, Tat-ATP impaired cancer cell motility and reduced the viability of cancer cell line- and patient-derived tumorspheres. Administration of Tat-ATP also led to a significant reduction in tumor size in a xenograft mouse model. These results indicate the Tat-ATP is a novel ATG4 inhibitor, highlighting its potential as an anticancer agent. The ATG4B Targeting Peptide (ATP) suppresses ATG4B proteolytic activity (IC₅₀ = 0.7 µM). Cell-permeable Tat-ATP reduces intracellular ATG4B activity and autophagy. Tat-ATP triggers cancer cell death in patient-derived tumorspheres and xenografted tumors. Tat-ATP emerges as a potential ATG4B inhibitor for targeted cancer therapy.