Discovery of songorine from Aconiti Radix lateralis praeparata as a novel Hadha inhibitor to alleviate doxorubicin-induced cardiomyopathy by PAMAM-modified high-density mitochondrial membrane chromatography

Radix Aconiti Lateralis Preparata (Fuzi in Chinese) is an efficient Chinese herbal medicine (CHM) against doxorubicin (DOX)-induced cardiomyopathy (DIC), but the cardioprotective compounds and targets of Fuzi remain unclear. In this work, a novel two-dimensional (2D) polyamidoamine (PAMAM)-modified cardiac mitochondrial membrane chromatography (CMMC) system, with the characteristic of high-density mitochondrial membrane coating and longer column lifespan, was developed for the identification of bioactive components from Fuzi to counteract DIC. Three compounds with strong retention were screened out to have cardioprotective effects against DOX-induced cardiotoxicity in different cardiomyocytes, and one promising compound songorine with the highest affinity was selected for further in vitro and in vivo pharmacodynamic validation and target identification. Hadha, involved in fatty acid oxidation (FAO) and apoptosis, was identified as the functional target of songorine by cardiac mitochondria proteomics, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) and molecular docking assays. siRNA experiments have also demonstrated that songorine can affect the function of Hadha. It was concluded that songorine could directly bind to the Hadha protein to alleviate the functional impairment of the mitochondrial FAO and oxidative phosphorylation (OXPHOS) and regulate the Bcl-2 family proteins in mice to treat DIC.