| Pharmaceutical analysis is the eyes of pharmaceutical science.Pharmaceutical analysis has been used in each processes of drug discovery as an important research tool.As the rapid development of analysis technology,the research concept of pharmaceutical analysis has been greatly changed.The research area of pharmaceutical analysis is not only restricted in quantitative analysis and drug quality control,but shifts from focusing on drug quality to the combination with life science.The research of pharmaceutical analysis is converted from chemical analysis-directed to biological activity analysis-directed.Under this back ground,drug activity screening has been included in the research area of pharmaceutical analysis.Establishing novel analytical method with high analysis rapid,high throughput and high accuracy to find active compound is the new task of pharmaceutical analysis.Drug screening methods based on the drug-target interaction is an important research direction of drug activity screening.Screening active compounds targeting on specific protein could effectively facilitate the progress of drug development.However,the current methods could not match all the requirement simultaneously,including specificity,sensitivity,throughput and maintenance of natural structure of protein.Hence,it is urgent to establish novel screening method to meet the need of drug development.Surface plasmon resonance(SPR)biosensor could be used to characterize the interaction between biomolecules,including the interaction between small molecules and protein targets.The past decades has witnessed the rapid development of drug screening methods based on SPR biosensor.SPR biosensor has many advantages,including labelfree manner,real-time monitoring and high specificity.Currently,SPR biosensor has been used in the screening of active compounds from Chinese herbal medicines.The conventional screening method of SPR requires the isolation and purification of each compounds in medicinal herbs.Then the affinities between compounds and protein were detected one by one.This strategy is labor-intensive and has a low throughput.In this project,we have established a SPR-based active ingredients recognition system(SPRAIRS).The system does not need prior isolation of compounds in herbal extracts and could be used to screen active compounds targeting on specific protein from herbal extracts directly.Compared with the conventional strategy,SPR-AIRS has many advantages,including high throughput,high enrichment ability and clear structure information.The research of this project could provide technical support to the development of drug screening method targeting on specific protein.The major research contents of this project could be divided into four following parts:1.The construction and methodological performance evalutation of surface plasmon resonance biosensor-based active ingredients recognition systemIn this chapter,a surface plasmon resonance biosensor-based active ingredients recognition system was constructed.We evaluated the methodological performance of SPR-AIRS in the following four aspects:(1)specificity,the evaluation results indicated that Signal transducer and activator of transcription 3(STAT3)small molecular ligand could have good response signal on STAT3-immobilized chip,while other compounds have no response signal.The KD between active compound stattic and STAT3 is detected as 26.97 ?M,which validated that the chip has good specific and could be used to characterize the affinity of STAT3 ligands;(2)limit of detection and linearity,the evaluation results indicated that it needs at least five recovery cycles for the precision identification of STAT3 small molecular ligand in recovery sample,linearity evaluation results indicated that from 5 to 100 recovery cycles,there is a good linear relationship between the amount of recovered stattic and the number of recovery cycles(R2=0.9917);(3)saturability of the chip,the results indicate that it is necessary to select a proper concentration based on the compound's KD value;(4)robustness of the system,it indicates that inactive compounds in the matrix could not interfere with active compounds in the process of screening.The evaluation results of methodological performance demonstrated that SPR-AIRS has high sensitivity,specity and robustness,which could be used to screen active compounds from herbal medicines.2.STAT3 small molecular ligands screening for medicinal herbs by applying surface plasmon resonance biosensor-based active ingredients recognition systemIn this chapter,we applied SPR-AIRS in the screening of STAT3 small molecular ligands screening for medicinal herbs.Totally,32 medicinal herbs were selected for prescreening.Nine medicinal herbs with good response signal on STAT3-immobilized chip were selected for further ligand fishing.Nine candidate compounds were screened out from herbal extracts.Then SPR affinity assays were performed to validate the interaction between STAT3 and candidate compounds.It was found that there were 7 compounds having good affinities with STAT3.The results of molecular docking indicated that five compounds could effectively bind to the SH2 domain of STAT3.These five compounds were regarded as potential STAT3 small molecular ligands.Apoptosis assays results indicated that the five compounds could induce the apoptosis of Hep G2 and MCF-7 cell lines.luciferase report assay results indicated that five compounds could inhibit the STAT3-driven transcriptional activity.Western blot assay results indicated that neobaicalein and polydatin could inhibit the phosphorylation of STAT3 and regulate the expression of apoptosis-related proteins.In conclusion,the research demonstrated the feasibility of SPR biosensor-based screening method applying to complex drug systems,and our findings suggest that SPR-AIRS could be a sensitive and effective solution for the discovery of active compounds from a complex matrix.3.Identification of eupatilin and ginkgolide B as MAPK14 ligands from medicinal herbs by surface plasmon resonance biosensor-based active ingredients recognition systemIn this chapter,SPR-AIRS was applied to screen MAPK14 mitogen-activated protein kinase(MAPK14)ligands from herbal extracts.After MAPK14 protein was immobilized on a SPR chip,the suitability of SPR-AIRS was validated,five medicinal herbs were screened by the system.Two active compounds,eupatilin(EPT)and ginkgolide B(GKB),were identified as potential MAPK14 ligands.The KD values of EPT and GKB were measured as 21.68±2.21 and 44.71±1.80 ?M,respectively.They can inhibit MAPK14 activities significantly and bind to the ATP binding site on MAPK14.Competitive assays demonstrate that EPT and GKB could bind to the same site of SB 203580 on p38.Furthermore,EPT and GKB can inhibit cell proliferation(IC50=30.31±6.84 and 42.97± 0.83 ?M),induce apoptosis and G2/M cell cycle arrest against K562 cell line.The results proved that EPT and GKB are effective MAPK14 ligands.These results prove that SPRAIRS could be an effective method to screen active compounds acting on a specific protein from complex systems.4.An SPR-based membrane protein-targeted active ingredients recognition system: construction and implementation in ligand screening from herbal medicinesMembrane proteins(MPs)are important drug targets and play important roles in several biological processes.Due to the instablity of transmembrane domain of MPs,it is still a challenging task to detect the interaction between MPs and their small moleclar ligands.In this study,an SPR-based membrane protein-targeted active ingredients recognition system was constructed.This system contains two major modules: affinity detection module and ligand screening module.Through the combination of these two functional modules,it is feasible to screen small molecular ligands targeting MPs from herbal medicines.First,we have constructed high/low comparative C-X-C chemokine receptor type 4(CXCR4)-expressed lentiviral particles(LVPs)models and characterized the expression levels.Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module.The KD of AMD3100 was 32.48±3.17 n M.Furthermore,the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound.Subsequently,this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts.Senkyunolide I was screened out from Chuanxiong extract.The affinity constant between senkyunolide I and CXCR4 was 2.94±0.36 ?M.Molecular docking revealed that senkyunolide I could effectively bind to CXCR4.Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process.In conclusion,a SPR-based small molecular ligand screening system combined with a virus-based stabilization strategy was constructed.The SPR-based membrane protein-targeted active ingredients recognition system will be an effective tool to screen target components from complex systems acting on MPs. |
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