Structure-based discovery of a novel small-molecule inhibitor of ATG4B that modulates autophagy in esophageal squamous cell carcinoma therapy.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with few treatment options and an unfavorable prognosis. Autophagy, a cellular recycling pathway frequently co-opted by tumors, supports ESCC progression and therapy resistance. Targeting its key executor, the cysteine protease ATG4B, represents a promising anticancer strategy. Here, we describe the discovery of compound a25, a novel small-molecule inhibitor of ATG4B, through structure-based virtual screening and systematic structural optimization. In ESCC cell lines, compound a25 effectively blocked autophagic flux, as shown by decreased LC3-II levels and increased p62 accumulation. Functionally, compound a25 significantly inhibited proliferation, migration, and clonogenic survival. Direct target engagement was confirmed by cellular thermal shift assay and surface plasmon resonance. In vivo, compound a25 administration robustly suppressed tumor growth in ESCC xenograft models without evident toxicity. Mechanistically, ATG4B inhibition by compound a25 disrupted autophagic homeostasis, leading to impaired autophagy flux and enhanced cell death. Our study identifies compound a25 as a novel ATG4B inhibitor with compelling preclinical efficacy against ESCC, providing a strategic foundation for targeting pro-tumor autophagy in gastrointestinal cancers.