Ganoderic acid T, a novel activator of pyruvate carboxylase, exhibits potent anti-liver cancer activity

Abstract

Background: Ganoderic acid T (GAT), a lanostane triterpenoid isolated from the methanol extract of G. lucidum mycelia, has demonstrated potent antitumor activity against various human cancer types. However, the specific molecular targets of GAT in cancer cells remain largely unknown. Therefore, this study systematically investigates these targets using the in vitro and in vivo hepatocellular carcinoma (HCC) model.

Methods: The anti-tumor activities of GAT were validated in HCC cells, xenograft tumor models in nude mice, and patient-derived organoid models. The specific molecular target of GAT was identified through targeted fishing techniques. Experimental approaches such as proteomics, metabolomics, biotin pull-down assays, molecular docking studies, molecular dynamics simulations, DARTS, CETSA, and biolayer interferometry (BLI) were employed to confirm the binding between GAT and its molecular target as well as elucidate the underlying mechanism.

Results: We have identified pyruvate carboxylase (PC) as a direct target of GAT. GAT, through its binding to the pocket composed of Arg453, Thr457, and Ile459 of PC, enhances the activity of PC, consequently disrupting the anaplerotic flux mediated by PC into the tricarboxylic acid (TCA) cycle. This disruption leads to impaired mitochondrial oxidative phosphorylation (OXPHOS) via the induction of reactive oxygen species (ROS)-mediated JNK/p38 MAPK signaling pathways, ultimately inhibiting HCC cell proliferation. Furthermore, molecular dynamics simulation suggests that GAT binds to and interacts with the biotin carboxylase (BC) domain of PC. This interaction potentially induces conformational changes in the protein structure of PC, leading to a tighter arrangement within the BC domain and stabilizing formation of a catalytically competent tetramer configuration for PC. The mutation of these key sites resulted in the destabilization of the BC domain and a reduction in the cytotoxic effect of GAT on HCC cells.

Conclusion: Overall, these findings demonstrate that GAT directly targets PC through an allosteric mechanism, providing valuable insights into the anti-HCC properties of GAT.

Keywords: Allosteric activation; Ganoderic acid T; Hepatocellular carcinoma; Metabolic reprogramming; Pyruvate carboxylase.