Structure-Based Virtual Screening for KHK-A Inhibitors with Anti-Hepatocellular Carcinoma Activity

Background: Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor worldwide and is associated with a poor prognosis. Oxidative stress is a key factor in the occurrence and progression of HCC. KHK-A, a key protein in the oxidative stress pathway, plays an important role in various cancers. This study aimed to discover small-molecule inhibitors targeting KHK-A through structure-based virtual screening, evaluate their therapeutic effects on HCC, and explore the potential of KHK-A as a therapeutic target for HCC. Methods: Based on the crystal structure of KHK-A, potential small-molecule inhibitors (HK1 to HK-24) were screened from the SPECS database using the Discovery Studio (DS) 2019 software. The effects of these compounds were evaluated through molecular docking and cellular experiments. Results: The screened compound HK-4 significantly inhibited HCC cell proliferation, migration, and invasion ex vivo. The half-maximal inhibitory concentrations (IC₅₀) of HK-4 in HepG2, PLC/PRF/5, and HuH7 cells were 22.54 µM, 23.91 µM, and 23.38 µM, respectively. HK-4 induced G1 phase arrest and apoptosis, and reduced the protein levels of p-AKT and p-mTOR in the PI3K-AKT signaling pathway. Conclusions: Through structure-based virtual screening, this study identified HK-4, a small-molecule inhibitor of KHK-A with anti-HCC activity. Its mechanism of action is closely related to the regulation of the PI3K-AKT signaling pathway. This finding provides experimental evidence supporting KHK-A as a therapeutic target for HCC and offers a new direction for the development of novel anti-HCC drugs.