Identification of AIF1 as a protein target of notoginsenosides in brain tissue of mice with intracerebral hemorrhage based on hapten immunoaffinity fishing technique.

Background: Intracerebral hemorrhage (ICH) is a deadly stroke with high mortality or disability. Although recent research has demonstrated the efficacy of Methods: First, we generated polyclonal antibodies against notoginsenosides by immunizing rats with a ginsenoside Rh1-mcKLH conjugate. Second, the potential target proteins of notoginsenosides in brain tissue of ICH mice were identified using LC-MS-based hapten immunoaffinity fishing (HIAF). Third, the disease target databases and these proteins intersected. Fourth, biolayer interferometry (BLI), molecular docking, and site-directed mutagenesis were performed to validate allograft inflammatory factor 1 (AIF1) as a protein target of notoginsenosides. Last, bioinformatics analysis was performed to examine AIF1's biological characteristics. Results: A potential protein target of notoginsenosides, AIF1, was found by intersecting the identified protein targets with the disease target databases via LC-MS-based HIAF. BLI analysis revealed that Compound K (CK) and AIF1 had the highest direct interaction, with an average shift value of 0.1091 nm. Subsequently, site-directed mutagenesis, molecular docking, and BLI kinetic analysis demonstrated that CK specifically bound to AIF1 with an affinity value of 4.33 ± 0.17E-6 M, with a significant reliance on residues L122 and E125. Bioinformatics analysis showed that AIF1 and its directly interacting proteins were associated with microglial activation. Conclusion: Our study proposed a new technology for screening natural small molecule protein targets, and successfully identified AIF1 as a protein target of notoginsenosides, providing a chemical and biological basis for further research into targeting AIF1 to treat ICH.