Guaianolide dimer KGA-1002 targets GRP94 and triggers unfolded protein response-associated degradation of SKP2/AKT axis as a novel antihepatoma agent

• Guaianolide dimer KGA-1002 exhibited significant antiHCC activity in vitro and in vivo . • KGA-1002 selectively bound to GRP94 among HSP90 isoforms. • Targeting GRP94 triggered UPR-associated degradation and disrupted GRP94-AKT interaction. • The inhibition of AKT promoted apoptosis through the SKP2/P21 axis. • The inhibition of AKT promoted reactive oxygen species production and induced ferroptosis. Hepatocellular carcinoma (HCC) remains a highly lethal malignant tumor with limited treatment options. The guaianolide dimer KGA-1002 exhibited significant inhibitory effects on HCC cells, but the specific target and mechanism remain unclear. To further evaluate the antiHCC efficacy of KGA-1002 and clarify its molecular target and mechanism. BrdU, flow cytometry, Transwell, and colony formation assays were utilized to evaluate the in vitro antiHCC efficacy of KGA-1002. Drug affinity responsive target stability (DARTS) and bioinformatics analyses were applied to identify its target. The binding site was confirmed through Mass spectra analysis and amino acid mutation. Knockdown cell lines were used to validate the GRP94-dependent effects of KGA-1002. Pulldown assays were performed to detect protein–protein interactions. Xenograft tumor models were employed to examine the in vivo efficacy and mechanism. KGA-1002 inhibited the proliferation and metastasis of HCC cells, induced apoptosis and cell-cycle arrest at the G0/G1 phase. GRP94 was identified as its direct target, and Ser 106 was confirmed as the covalent binding site. KGA-1002 targeting GRP94 triggered the unfolded protein response (UPR)-associated degradation and disrupted the GRP94-AKT interaction, resulting in AKT degradation. The degradation of AKT induced ferroptosis and reduced the stability of SKP2. The impairment of AKT and SKP2 promoted the nuclear accumulation of P21, which further induced apoptosis in HCC cells. In vivo , KGA-1002 (60 mg/kg) inhibited tumor growth by 61.6 %, and the effect was GRP94-dependent. KGA-1002 was identified as a highly promising lead compound for antiHCC. Its mechanism involved triggering UPR-associated degradation and disrupting the GRP94-AKT interaction. KGA-1002 simultaneously induced apoptosis and ferroptosis in HCC cells through the GRP94/AKT/SKP2/P21 signaling axis.