Identification of UQCRB as an oxymatrine recognizing protein using a T7 phage display screen

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• 作者：Yan-hui Sun^a ; ¹ ; ^{19652459@qq.com" class="auth_mail" title="E-mail the corresponding author} ; Xiao-yuan Zhang^a ; ¹ ; ^{396373192@qq.com" class="auth_mail" title="E-mail the corresponding author} ; Wei-qun Xie^a ; ¹ ; ^{913918715@qq.com" class="auth_mail" title="E-mail the corresponding author} ; Guang-jian Liu^b ; ^{guangjian1984@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Xi-xin He^c ; ^{mark07@gzucm.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Ya-li Huang^a ; ^{huangyali@gzucm.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Guang-xian Zhang^a ; ^{zhangguangxian@gzucm.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Jian Wang^a ; ^{zswj@gzucm.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Zao-yuan Kuang^a ; ^{zykuang@gzucm.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Ren Zhang^a ; ^{zhangrenn@foxmail.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Oxymatrine (PubChem CID ; 114850)
• 刊名：Journal of Ethnopharmacology
• 出版年：2016
• 出版时间：4 December 2016
• 年：2016
• 卷：193
• 期：Complete
• 页码：133-139
• 全文大小：1306 K

文摘

Sophora flavescens Aiton (Radix Sophorae Flavescentis, Kushen) is used in traditional Chinese medicine to treat chronic hepatitis B (CHB), and has the ability to clear heat and dampness from the body. Oxymatrine is one of the major bioactive compounds extracted from Sophora flavescens Aiton and constitutes more than 90% of the oxymatrine injection commonly used for CHB treatment in clinics in China.

Aim of the study

We aim to analyze the protein binding target of oxymatrine in treating CHB by screening a T7 phage display cDNA library of human CHB and examine the biochemistry of protein-ligand binding between oxymatrine and its ligands.

Materials and methods

A T7 phage cDNA library of human CHB was biopanned by affinity selection using oxymatrine as bait. The interaction of oxymatrine with its candidate binding protein was investigated by affinity assay, molecular docking, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR).

Results

A library of potential oxymatrine binding peptides was generated. Ubiquinol-cytochrome c reductase binding protein (UQCRB) was one of the candidate binding proteins of oxymatrine. UQCRB-displaying T7 phage binding numbers in the oxymatrine group were significantly higher than that in the control group, biotin group, and matrine group (p<0.05 or p<0.01). Three-dimensional structure modeling of the UQCRB with oxymatrine showed that their binding interfaces matched and oxymatrine inserted into a deeper pocket of UQCRB, which mainly involved amino acid residues Tyr21, Arg33, Tyr83, Glu84, Asp86, Pro88, and Glu91. The binding affinity constant (Kb) from SPR was 4.2 mM. The Kb from ITC experiment was 3.9 mM and stoichiometry was fixed as 1, which fit very well with the result of SPR. The binding of oxymatrine to UQCRB was driven by strong enthalpy forces such as hydrogen bonds and polar interactions as the heat released was about 157 kcal/mol and ΔG was less than zero.

Conclusions

In this study, using the T7 phage display system, we have identified UQCRB as a direct binding protein of oxymatrine. Furthermore, the specificity and molecular interaction of oxymatrine with UQCRB were also determined. The binding of UQCRB to oxymatrine suggests that UQCRB is a potential target of oxymatrine in treating CHB. These results provide new understanding into the mechanism of oxymatrine and insights into the strategy on the treatment of CHB.