A dual-target modulator of STK11 and PP2Acα from the flowers of Chrysanthemum indicum mitigates drug-induced liver injury

INTRODUCTION: Hormesis, characterized by low-dose stimulation and high-dose inhibition, is a biphasic regulatory phenomenon, and its underlying mechanisms remain elusive. Drug-induced liver injury (DILI) can progress to liver fibrosis, liver failure, and ultimately death, and natural products hold considerable promise for the treatment of DILI. OBJECTIVES: To identify the active constituents and underlying biphasic regulatory mechanism of Chrysanthemum indicum against DILI. METHODS: Structural elucidation of the new compounds was achieved through integrated interpretation of HRESIMS, 1D and 2D NMR, and ECD. Signaling pathway was determined by mitochondrial transplantation in vitro and in vivo and RNA sequencing. Target proteins were validated by the drug affinity responsive target stability-mass spectrometry analyses, isothermal titration calorimetry, cellular thermal shift assay, shRNA, and liver-specific knockdown mice. Protein sites were validated by truncation experiments, molecular dynamics simulations, and point mutations. RESULTS: A total of 21 guaianolide sesquiterpenoids, including 13 new ones, were isolated and identified from the flowers of C. indicum. Interestingly, the new compound chrysanthemolide I (CI) alleviated acetaminophen-induced liver injury in vitro and in vivo. Mitochondria isolated from CI-treated hepatocytes attenuated DILI. CI attenuated AMP-activated protein kinase (AMPK)-mediated mitochondrial oxidative stress while enhancing AMPK-dependent mitochondrial biogenesis and mitophagy. At low doses, CI binds directly to ALA-205 and ARG-301 of serine/threonine kinase 11 (STK11) with high affinity to activate AMPK; at medium doses, binding of CI to STK11 reaches saturation, leading to peak AMPK activity; at high doses, CI additionally binds to SER-261 of serine/threonine-protein phosphatase 2A catalytic subunit α isoform (PP2Acα) with low affinity to inhibit AMPK activation. Furthermore, liver-specific knockdown of both STK11 and PP2Acα largely diminished the protective effect of CI against DILI. CONCLUSION: Novel guaianolide sesquiterpenoid CI was identified as an affinity-dependent dual-target regulator of STK11 and PP2Acα to alleviate DILI.