Targeted affinity fishing of components from the n-butanol extract of Gualou-Xiebai-Banxia decoction for the FGF21/FGFR1/βKlotho-FRS2α pathway and verification of their activities

Explored eleven potential ligands in the n-butanol fraction of Gualou-Xiebai-Banxia decoction against FGFR1, βKlotho and FRS2α. • Revealed the interactions between part ligands and three protein targets. • Constructed the first interaction network to unveil the empirical use of the n-butanol fraction of Gualou-Xiebai-Banxia decoction. • Integrating AlphaFold3 predictions with molecular dynamics simulations for the FGFR1, βKlotho, and FRS2α protein complex. • Timosaponin BII exerts effects via the FGF21/FGFR1/βKlotho-FRS2α axis. To systematically deconvolute bioactive components of the n-butanol fraction of Gualou-Xiebai-Banxia decoction (GXB-N) mitigating myocardial ischemia, an integrated drug discovery strategy combining affinity ultrafiltration with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. This approach was designed to screen specific ligands from the complex matrix that bind to key proteins of the fibroblast growth factor 21 (FGF21) signaling complex, including fibroblast growth factor receptor 1 (FGFR1), βKlotho, and fibroblast growth factor receptor substrate 2α (FRS2α). The thermodynamic stability of identified ligands was evaluated using molecular docking and molecular dynamics simulations, while pharmacological efficacy was validated in an isoproterenol-induced zebrafish model. The screening strategy successfully identified Timosaponin BII as a lead compound with notable binding for the FGFR1/βKlotho-FRS2α complex. In silico simulations demonstrated that Timosaponin BII stably occupies the active pockets of these targets. In vivo assays revealed that Timosaponin BII treatment significantly reversed myocardial ischemia phenotypes, characterized by improved cardiac function and reduced pathological lesions, through the activation of the FGF21 signaling pathway. This study demonstrates the efficacy of the affinity ultrafiltration LC-MS/MS method for clarifying the molecular mechanisms of complex herbal matrices and supports the potential of Timosaponin BII as a therapeutic agent for coronary artery disease. Download: Download high-res image (314KB) Download: Download full-size image