Primary Study Of Affinity Proteins In EA.hy926 Cell Of Farrerol Bio-active Derivatives

Objective:The discovery of drug target is original innovation for drug discovery.Previously,we have provided the first evidence that a Chinese medicine ingredient farrerol(DJS)and its derivatives(DJS-F,DJS-2Cl,DJS-NO2)have protective effect against hydrogen peroxide-induced apoptosis in EA.hy926 cell.However,the underline mechanism remains unclear.In this research,we aim to preliminarily use HSA as the model protein,discuss the function characteristics and intensity difference between farrerol bio-active compounds and biological macromolecules by the combination of DARTS-spectroscopic studies-molecular docking rtechnique.Furthermore,to discovery the affinity proteins of farrerol bio-active compounds in EA.hy926 by using hollow fiber cell capture technology,drug affinity responsive target stability(DARTS)technique and MALDI-TOF mass spectrometry.Our study will provide scientific basis for subsequent study to clear target protein and clarify the underline mechanism of these farrerol bioactive compunds.Methods:(1)In this study,the binding characteristics and intensity difference between farrerol bio-active derivatives and HSA were investigated using DARTS,spectroscopic studies and molecular docking.HSA was treated with selected compounds in DMSO or with DMSO alone,and incubated at room temperature for 1hour.Then each sample was proteolysed at room temperature for 30 min with Pronase.Finally,SDS loading buffer was added into each sample and heated to 95? for 5 min.Appropriate amounts of sample was then loaded onto 12% SDS-PAGE gels.Protein spots were visualized by using coomassiebrilliant blue G-250 staining.And analyzing the respective lanes of the gel for bands of HSA to estimate the difference of combination between HSA and farrerol bio-active derivatives;Spectroscopic studies were used to investigate the binding constants Ka of HSA and farrerol bio-active derivatives;the thermodynamic parameters were calculated to infer the main interaction force between HSA and farrerol bio-active derivatives;Three-dimensional fluorescence and circular dichroism(CD)spectroscopies were used to investgate the conformational changes in HSA on binding with farrerol bio-active derivatives.(2)Hollow fiber cell fishing technology(HFCF): EA.hy926 cell were cultured in vitro.HFCF-HPLC was utilized to preliminarily verify where the affinity proteins of farrerol bio-active derivatives are located in the EA.hy926 cell.The whole cell along with the cytomembrane and organelle of the EA.hy926 cell were employed to incubate with the farrerol bio-active derivatives via the carrier hollow fiber,then,the capture factors were calculated based on the peak area of HPLC peak;(3)The coupling technique of DARTS/SDS-PAGE/MALDI-TOF-MS: The cytoplasmic proteins and membrane proteins of EA.hy926 cell were extracted respectively.After farrerol bio-active derivatives treatment and subsequent Pronase mediated proteolytic digestion of cell lysates,the remaining proteins were separated and stained via SDS-PAGE with Coomassie blue.Finally,MALDI-TOF/TOF mass spectrometry and database retrieve were used to identification and retrieval the affinity proteins of these compounds in the EA.hy926 cell.Results:(1)DARTS/SDS-PAGE: With the concentration increase of DJS,the HSA band gradually deepened in a concentration dependent manner.The results showed that color depth of the HSA band of farrerol bio-active derivatives group were statistically deeper than that of the control group.Moreover,the HSA band of the farrerol derivatives were far deeper than that of farrerol.And the binding process had a certaindose-effect relationship.Fluorescence spectroscopies suggested that the binding constants Ka of DJS,DJS-F,DJS-2Cl and DJS-NO2 were 4.01×105 mol·L-1,4.39×105mol·L-1,8.83×105 mol·L-1,4.91×105 mol·L-1,respectively.The number of binding sites approximately were 1,which were in accordance with the result of molecular docking.Hydrogen bonds and hydrophobic interactions played an important role in the farrerol bio-active derivatives and HSA interactions,as indicated by the thermodynamic parameters.Three-dimensional fluorescence spectra and circular dichroism spectra experiments indicated that the addition of farrerol bio-active derivatives to the HSA produces a significant decrease in intensity along with a red shift in both peaks A and B.And circular dichroism spectra experiments showed the addition of farrerol bio-active derivatives to the HSA lead to a decreasement of?-helical structures.(2)The cell fishing factors of DJS,DJS-F,DJS-2Cl and DJS-NO2 were 1.20,3.28,1.56 and 3.66 respectively,which were greater than 1;The cell membrane fishing factors were 1.15,4.43,3.57,3.66 respectively,which were greater than 1;The cell organelles fishing factors were 0.94,0.28,0.74,0.94 respectively,which were less than 1.(3)Cytoplasmic protein of EA.hy926 treatment with farrerol bio-active derivatives resulted in enrichment of one main peptide band;Enriched band occurred around 70 k Da molecular weights.DARTS/Western blot technique showed that the differential protein bands was GRP78.In addition,membrane proteins of EA.hy926 treatment with farrerol bio-active derivatives also resulted in enrichment of several main peptide bands,which are under evaluation.Conclusions:(1)Using HSA as the model protein,the combination of DARTS-spectroscopic studies-molecular docking technique can be used as an effective method for investigating the affinity protein of active compounds in vitro.(2)Farrerol bio-active derivatives showed stronger binding affinity to HSA than DJS.(3)The results of HFCF-HPLC demonstrated that farrerol bio-active derivatives are both well combined with EA.hy926 cell.Farrerol bio-active derivatives showed stronger binding ability toEA.hy926 cell than DJS;The sequence of binding intensity happened to align perfectly with the protective effect against hydrogen peroxide-induced apoptosis in EA.hy926 cell,which indicating that the affinity proteins of farrerol bio-active derivatives in EA.hy926 cell were located on the cell membrane or in the cytosol.(4)The results of DARTS/Western Blot suggested that glucose regulated protein 78(GRP78)and Ezrin might be one of the affinity proteins of farrerol bio-active derivatives in EA.hy926 cell.it lays an experimental foundation and providing scientific basis for identifying the target proteins of these compounds and guiding the further structural modification.