Objective To explore the antitumor small molecules targeting the ubiquitin–proteasome system (UPS) on the basis of active molecules from traditional Chinese medicine.

Methods Ub^G76V-GFP stably expressing cell line was constructed to screen novel small molecule inhibitors targeting UPS. The fluorogenic substrates of Suc-LLVY-AMC, Z-LLE-AMC, and Boc-LRR-AMC were used to assess the effect of dioscin on the 20S proteasome hydrolase activity. The Ub-AMC substrate was used to evaluate the effect of dioscin on the intracellular deubiquitinating enzyme activity. Western blot was used to detect the effect of dioscin on intracellular ubiquitination levels. CCK-8 and colony formation assays were used to detect the inhibitory effect of dioscin on the tumor cell proliferation.

Results Dioscin is a UPS inhibitor discovered through the Ub^G76V-GFP reporter system. It enhances intracellular ubiquitination and inhibits tumor cell proliferation and colony formation by targeting deubiquitinating enzymes.

Conclusion Dioscin could significantly inhibit tumor cell proliferation by targeting ubiquitin–proteasome.