Binding and ubiquitination-mediated degradation of ACKR3 by the novel Scutellarein derivative TBS6b potently suppresses hepatocellular carcinoma

Abstract

Scutellarein, a flavonoid compound from the traditional Chinese herb Scutellaria baicalensis, exhibits inhibitory effects against hepatocellular carcinoma (HCC), but its clinical application is limited by relatively weak potency. To enhance its antitumor activity, we synthesized a novel derivative, 5,6,7-trimethoxy-4′-benzimidazolyl scutellarein 6b (TBS6b), by introducing antitumor pharmacophores—trimethoxyphenyl and benzimidazole—into the scutellarein scaffold. TBS6b demonstrated significantly improved anti-HCC activity both in vitro and in vivo. Cell-based assays, including colony formation, EdU staining, wound healing, transwell migration, and western blot analysis, demonstrated that TBS6b significantly inhibits HCC cell proliferation, migration, and invasion. Mechanistically, we employed proteomic and transcriptomic sequencing, along with western blot and qRT-PCR experiments, to predict and validate atypical chemokine receptor 3 (ACKR3) as the target of TBS6b. Molecular docking studies confirmed that TBS6b binds tightly to the ACKR3 protein. Additionally, with the aid of pharmacological tools, we established that TBS6b promotes the ubiquitination and degradation of ACKR3. Tissue microarray analysis and queries of public databases revealed that ACKR3 expression is elevated in HCC tissues compared to adjacent non-cancerous tissues, correlating closely with patient survival. By constructing cell lines with either silenced or overexpressed ACKR3, we confirmed that ACKR3 promotes the proliferation, migration, and invasion of HCC. Finally, rescue experiments indicated that TBS6b exerts its anticancer effects primarily through targeting ACKR3. These findings establish ACKR3 as a critical target through which TBS6b mediates its anticancer activity against HCC.