Chemical proteomics and phenotypic profiling identify αFOD as a podophyllotoxin-derived PGK1 inhibitor that suppresses glycolysis and proliferation in hepatocellular carcinoma.

HCC remains one leading cause of cancer-related mortality worldwide, but effective systemic therapeutic options are limited. PGK1 has emerged as a promising therapeutic target in HCC, yet PGK1 inhibitors with anti-HCC activity are rarely reported. This study was sought to identify the target of a novel podophyllotoxin derivative αFOD and evaluate its anti-HCC efficacy, glycolytic inhibition, and preliminary safety profile. αFOD preferentially inhibited tumor cells over non-malignant cells and displayed potent activity against HCC cell lines, with IC50 of 0.80 ± 0.18  μM in HepG2, 2.57 ± 0.75  μM in Huh7, and 1.82 ± 1.04  μM in SNU-387 cells. In two HCC patient-derived organoids, αFOD exhibited IC50 of 8.76 ± 0.79 and 3.70 ± 0.25  μM, with efficacy comparable to that of oxaliplatin and lenvatinib and superior over that of etoposide. Using chemical proteomics and phenotypic profiling, we identified PGK1 as a target of αFOD. Surface plasmon resonance assays revealed a Kd of 11.50  μM, and molecular interaction analyses based on our in-house apo-PGK1 crystal structure predicted that αFOD occupies the 3-phosphoglycerate-binding pocket of PGK1 through interactions with Arg38, Arg65, Thr167, and Ala376. In consistency with target engagement, αFOD inhibited PGK1 enzymatic activity and suppressed glycolytic flux in HepG2 cells. In a HepG2 xenograft model, αFOD inhibited 66.7% tumor growth and outperformed etoposide while exhibiting reduced systemic toxicity compared to oxaliplatin. Collectively, these findings establish αFOD as a podophyllotoxin-derived PGK1 inhibitor that suppresses glycolysis and HCC proliferation, supporting the rational development of Podophyllum-derived scaffolds and highlighting PGK1 inhibition as a promising therapeutic strategy for HCC.