Rational design and synthesis of TBC1D2 inhibitors: Augmenting autophagy to improve sorafenib sensitivity in hepatocellular carcinoma.

Drug resistance is a major barrier to effective hepatocellular carcinoma therapy, and autophagy targeting holds great potential for overcoming this issue. Using binding energy data from molecular docking with TBC1 domain family member 2 (TBC1D2) as the target, we rationally designed compound G2 featuring a piperazine moiety. Target binding was validated via a competitive immunofluorescence assay. The binding affinity of G2 was determined by surface plasmon resonance, yielding a dissociation constant (KD) of 0.4 μM. Functional evaluation of G2 determined its aqueous solubility to be 0.3 mg/mL, with a half-maximal inhibitory concentration value of 80 ± 20 nM and a selectivity index of 23.1 in HCCLM3 cells. Subsequent mechanistic investigations revealed that this selectivity arose from the heightened responsiveness of TBC1D2 expression to G2 in HCCLM3 cells, thereby inducing selective autophagic cell death. In HCCLM3 xenograft mouse models, G2 showed excellent hepatic retention. G2 monotherapy (58.2% tumor growth inhibition) and its combination with sorafenib (70.9%) exerted superior antitumor activity versus sorafenib monotherapy (52.8%), with favorable safety. Collectively, our findings establish G2 as a promising therapeutic candidate for surmounting sorafenib resistance, characterized by selective antitumor activity against malignant hepatocellular carcinoma.