Target Proteins And Biodistribution Research Of Arctigenin,an Effective Compound Of Tranditional Chinese Medicine Based On Chemical Biology

Chemical biology has evolved into a global community of scientists dedicated to understanding science at the intersections of chemistry and biology. The past several decades have been a remarkable period for chemical biology, marked by major scientific advances and dramatic changes to the scope and sophistication of research at different displines. Now, the multiple intellectual and methodological threads that have contributed to our view of modern tranditional Chinese medicine (TCM). This is an important paradigm shift "from structure to biological activity" in natural products chemistry. In this review, natural products are renamed as naturally occurring ligands (NOLs) to underline their biological importance. However, the efficacy of traditional Chinese medicine ingredients in vivo absorption, metabolism and distribution with complexity, which in turn often have "multiple targets" feature in the body, which gives the chemical biology research of TCM is a great challenge. In order to establish the TCM chemical biology research platform of efficacient components, this paper chose arctigenin and glycyrrhetinic acid as examples, launched chemical biology research of targets confirmatory and metabolic distribution.Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of azide modified protein by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were1.25-6.31μm in size and48.53-73.05%in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). The quantity of BSA which was captured by the optimal MMs was67.07μg. It was close to the actual captured quantity (63.92±0.18μg), so we could conclude that the regression model was significant. To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group through1,3,4,6-0-acetyl-N-azide acetylmannosamine (Ac4ManNAz) in human lung adenocarcinoma A549cells. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3+2] cycloaddition reaction. Using this strategy,51μg A549cell surface glycoproteins could be isolated by1Ml MMs.The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.Subsequently, we prepared azide-modified MMs, and arctigenin were alkynyl modified.The target protein of arctigenin in human bronchial epithelial cells (BEAS-2B) were captured. According to the preliminary results of our laboratory by reverse docking, the target of arctigenin anti-inflammatory effects may be3-phosphoinositide-dependent protein kinase1(PDPK1). Then we verified PDPK1in captured proteins through western blotting. Molecular docking and molecular dynamics were performed to research the bingding between arctigenin and PDPK1. The results indicated that arctigenin was an effective PDPK1inhibitor and the inhabition activity was close to253which was a strong PDPK1inhibitor reported. In summary, the present study establishes a method which could enrich cancer cell surface glycoprotein and drug target protein based on magnetic sepatation and click chemistry. The results also confirmed the anti-inflammatory effect of arctigenin was related with PDPK1, it would be the basis for the further study of mechanism.Since there were somelimitations in the target capturing of MMs,a novel self-assembled poly-lysine fluorescent nanospheres was designed and modifiedby azide functional group to bind alkynyl arctigenin.Then the morphology and surface chemical properties were characterized by TEM, SEM, FT-IR and particle size analysis. In addition, the material was used to fluorescent tracingat the animal and cell levels.It confirmed that target protein of arctigeninwere mainly distributed in the cells and the binding was specific. Through capturing by nanospheres and reducted by disulfide bond, the target protein of arctigenin in BEAS-2B cells were obtained. Subsequently, the protein were identified by high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). After bioinformatics analysis, a total of19proteins were identified as specific binding target protein of arctigenin. There were16types of protein in the interaction network and were divided into four functional proteomics, then analyzed by KEGG function annotations.Furthermore, the functions were correspondence to the indications of arctigenin. Finally, we found four kinds of protein, Peptidyl-prolyl isomerase (PPIA), heat shock protein70(HSPA8), actin γ1(ACTG1) and3-glyceraldehyde phosphate dehydrogenase (GAPDH) could bind to arctigenin through virtual screening and close to the related indications, so they should be target protein of arctigenin.Finally, in order to explain scientific interpretation of the compatibility of "drug pairs" in traditional Chinese medicine, the classic "drug pairs"-Radix Platycodonis and Radix Glycyrrhizae for the therapy of respiratory system disease were adopted as the object for the study. After glycyrrhetinic acid was radiolabeled, the Positron Emission Tomography (PET) technology was used for investigating the distribution in vivo.It was found that the concentration of glycyrrhetinic acid in mice lungs was significantly increased. Subsequently two modes of administration of Platycodonis were compared and administration before the injection of the glycyrrhetinic acid displayed significant effect of guiding drug. The combination of traditional Chinese medicine guide theory and compatibility theory systematically interpret the mechanism of respiratory system disease therapy by the classic "drug pairs"-Radix Platycodonis and Radix Glycyrrhizae from a multidimensional perspective and explore the new thought and method for the study of the compatibility of "drug pairs" in traditional Chinese medicine.This paper applied some important chemical biology tools such as click chemistry, magnetic capturing, nano-assembly and PET imaging.arctigenin The distribution, cellular localization and the target protein of arctigenin and the mechanisms of licorice bellflower were researched. We initially established a strategy that screening and identification of target proteins within cells, and analyzed by bioinformatics, molecular docking and confirm the appropriate target protein. Finally, its biological function is calculated based on structural analysis tools, and related chemical biology approach to reveal the molecular mechanism of TCM at the molecular level.