Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes

• Published in: Biomedicine & pharmacotherapy Vol. 134; p. 111160
• Main Authors: Huang, Shuai, Wang, Fu-Jia, Lin, Hao, Liu, Tian, Zhao, Cheng-Xiao, Chen, Lian-Guo
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 01.02.2021; Elsevier
• Subjects: Antioxidants - pharmacology; Apoptosis - drug effects; Cell Line; CHAF1B; Chromatin Assembly Factor-1 - genetics; Chromatin Assembly Factor-1 - metabolism; Glucose - toxicity; Humans; Hydrogen Peroxide - toxicity; Isoflavones - pharmacology; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - pathology; Oxidative Stress - drug effects; Probe; Proteomics; Puerarin; Signal Transduction; Steric hindrance structural; Targets; Ubiquitin-Conjugating Enzymes - metabolism; CHAF1B; Targets; Steric hindrance structural; Puerarin; Probe
• ISSN: 0753-3322; 1950-6007
• DOI: 10.1016/j.biopha.2020.111160

[Display omitted] •Puerarin protects cardiomyocytes from oxidative stress damage.•Two isomers (syn and anti) unequivocally exist in the puerarin-based probe.•CHAF1B binding with puerarin is benefit for cardiomyocytes’ survival. Natural products are an important source of new drugs. Some of them may be used directly in clinical settings without further structural modification. One of these directly used natural products is puerarin (Pue), which protects cardiomyocytes against oxidative stress and high glucose stress. Although Pue has been used in clinics for many years, its direct binding targets involved in the protection of cardiomyocytes are not yet fully understood. Here, we reported that Pue could prevent cardiomyocytes from apoptosis under H2O2 and high glucose conditions. Based on affinity-based protein profiling methods, we synthesized an active Pue probe (Pue-DA) with a photosensitive crosslinker to initiate a biological orthogonal reaction. Because of the steric hindrance of Pue-DA, two conformational isomers (syn and anti) unequivocally existed in the probe, and these transformed into one isomer when the probe was heated at 60 °C. We confirmed that the alkylation was on the 7-position phenol group of Pue. Mass spectroscopy revealed that Pue-DA can bind with three proteins, namely CHAF1B, UBE2C, and UBE2T. Finally, cellular thermal shift assay showed that Pue has the ability to stabilize CHAF1B stabilization. The knock-down of CHAF1B reduced the protective effect of Pue on cardiomyocytes. In conclusion, Pue protects cardiomyocytes from apoptosis through binding with CHAF1B.