Characterization of the specific proteins to which HH-A, a drug that alleviates ischemic brain injury, binds in rat plasma.

Stroke, a destructive and burdensome disease, continues to affect an increasing number of families. HH-A, also known as (S)-6-((3',5-diallyl-2,4'-dihydroxy-[1,1'-biphenyl]-3-yl)amino)-6-oxohexane-1,5-diaminium chloride, has attracted attention for its ability to increase the oxygen-carrying capacity of hemoglobin and strongly alleviates ischemic brain injury in rat models of hypoxia. Owing to its low recovery rate after radioactive drug extraction, HH-A is thought to covalently bind to rat plasma proteins. In this study, intact protein, peptide, and amino acid analyses were performed on plasma samples via deconvolution of the intact protein, protein modification via proteomics, and protein mass spectrometry. HH-A was found to covalently bind to Lys413 of rat albumin. Molecular docking technology was used to simulate the binding mode of HH-A to rat albumin, the amino group of lysine is most likely to bind to the ortho-phenolic hydroxyl group judging from the reaction distance and the number of hydrogen bonds. This study demonstrated for the first time that honokiol and its derivatives (HH-A) covalently bind to rat plasma proteins and that the formation of new α,β-unsaturated aldehyde and ketone moieties in the drug is the key to these binding interactions. This experiment explains the binding mechanism of traditional Chinese medicine honokiol with plasma albumin at the molecular level, support the clinical research and development for HH-A. SIGNIFICANCE STATEMENT: This study demonstrated that honokiol and its derivatives (a new drug HH-A, also known as (S)-6-((3',5-diallyl-2,4'-dihydroxy-[1,1'-biphenyl]-3-yl)amino)-6-oxohexane-1,5-diaminium chloride) covalently bind to rat plasma proteins via deconvolution of the intact protein, protein modification via proteomics, and protein mass spectrometry. This work supports the clinical research and development for HH-A.