The Analysis Of Drug Target Proteins Based On Cell Membrane Chromatography And Affinity Probe Technology

Despite the rapid development of combinatorial chemistry,ultra-high throughput screening,post-genomic project initiation,and the combined use of computer aided technologies,there are still two bottlenecks in drug discovery and development.One is the identification and validation of target biological macromolecules;the other is the design and discovery of bioactive small molecules.This article focuses on the aspects of target analysis.1.Construction of an integrated cell membrane chromatography system and its application in target identification of VEGFR-2 receptor inhibitorsCell membrane chromatography(CMC)is a combination of biomembrane technology and chromatographic techniques.It preserves the biological characteristics of cell membranes to a certain extent and can mimic bioactive small molecules and their target proteins on the cell membrane under physiological conditions.The process of mutual integration.The integrated cell membrane chromatographic system consists of multiple cell membrane chromatographic models,high-performance liquid chromatography,and timeof-flight mass spectrometry.Among them,multiple cell membrane chromatographic models complement each other and verify each other,making up for the short life cycle and poor reproducibility of biofilms.Internal defects;high-performance liquid chromatography can effectively separate complex samples;time-of-flight mass spectrometry can accurately identify each of these components,making the conclusions more truthful,accurate,and reliable than conventional cell membrane chromatograms.In this section,the plasma membrane chromatographic models of SMMC-7721/CMC and HepG2/CMC two different types of primary liver cancers were used to construct an integrated cell membrane chromatographic analysis system and used in the study of the activity and target evaluation of 8 man-made compounds.Each quinazoline compound was designed and synthesized for vascular endothelial growth factor receptor-2(VEGFR-2).While using the integrated cell membrane chromatographic system for target validation,the inhibition of VEGFR-2 by the above eight compounds was also determined using classical kinase inhibition methods.The results showed that the retention behavior of the compounds in the integrated cell membrane chromatographic system was consistent with the order of the inhibitory rate of the kinases,that is,the above two methods were correlated.Compared with the traditional method of measuring each compound sample to be evaluated separately,the integrated cell membrane chromatography system can directly analyze complex systems containing multiple samples,and the conclusions are true and reliable.At the same time,it is more time and cost-saving and can be used in drug research.A practical analysis method.2.The synthesis of TCP-mat probe and its application in matrine target identificationThe integrated cell membrane chromatographic system can quickly and efficiently analyze the interaction between bioactive small molecules and cell membrane proteins.However,natural biofilms contain hundreds of active proteins,and small molecule drugs may cooperate with multiple receptors.Due to the interference of various non-specific binding factors,it is difficult to identify,confirm,or exclude the possibility of a certain membrane protein as a potential target without other clues that may bind target-related information.Therefore,the integrated cell membrane chromatography system is used in There are still difficulties in the accurate identification and verification of bioactive smallmolecule-binding targets,and there is still a need for more in-depth,precise,and direct target identification techniques to support and coordinate,such as target-based analysis of affinity probes.A trifunctional probe(TCP)is a kind of affinity probe,which is a kind of probe that has three functional groups: ligand group,photoreactive group and label group.Effective means for the identification of active small molecule targets.Matrine is a natural alkaloid that has been found to have anti-tumor activity,but its direct binding to target and related molecular mechanisms of action is not yet clear,which also hinders the further structural transformation and enhanced activity of matrine.The research and development of pharmaceutical dosage forms,etc.,eventually became one of the many obstacles to the clinical treatment of first-line drugs.Therefore,it is of great significance to look for and identify the direct target of matrine.The TC P-matrine probe is an affinity probe with both matrine,4-benzoate azide,and biotin,with matrine as a ligand group and 4-benzoate azide as a photoreactive group.As a labeling group,it forms a biomagnification system with streptavidin for the purification and analysis of target proteins.In this section,TCP-matrine affinity probes were used for the target identification of matrine.It was demonstrated for the first time that annexin A2(ANXA2)is the active ingredient in Chinese matrine(Matrine)and it is also a promising one.The direct-binding target protein of the anticancer drug)further confirmed that ANXA2 is a key molecule for matrine anti-migration efficacy through antibody blocking experiments and knockdown expression experiments.Matrine can effectively inhibit ANXA2-mediated fibrinolysis.The biotransformation of plasminogen(PLG)to plasmin(PN)inhibits the migration of tumor cells.3.The synthesis of TCP-MLN8237 probe and its application in MLN8237 target identificationMLN8237 is a small molecule selective inhibitor of Aurora Kinase A(AKA).It inhibits cell growth and development,induces apoptosis,inhibits cell migration,invasion,and other activities.Phase I clinical trials also showed that MLN8237 is Malignant tumors have a good therapeutic effect,and there are some degrees of adverse reactions,such as nausea,vomiting,fever,anemia,etc.In theory,MLN8237 may have other potential target proteins.The search for and identification of other direct targets of the small molecule MLN8237 has important implications for elucidating its mechanism of action,mastering the molecular characteristics more comprehensively,and ultimately achieving its extensive clinical application.Based on the structure-activity relationship of MLN8237,the structure of carboxyl moiety was modified.For the first time,a TCP-MLN8237 probe was synthesized.The probe contains three different functional groups: MLN8237,4-benzoate azide,and biotin.And there is no significant difference between the probe and the drug molecule itself in promoting apoptosis.Compared to the control group(the same backbone but not containing the ligand group MLN8237,containing only the 4-benzoyl azide,biotin group probe)as the control probe,it was enriched by ultraviolet irradiation,magnetic beads purification,After separation by gel electrophoresis and silver ion staining,only one differential protein band was isolated and identified by multi-level protein profiling.The protein is AKA,which is currently recognized as the strongest target of MLN8237,illustrating TCP-MLN8237.The probe is effective,but because the probe is a rigid structure,it may affect the binding process between the ligand group and its target protein,there are still some limitations,there is room for optimization and improvement.4.The synthesis of DNA-MLN8237 probe and its application in MLN8237 target identificationBased on the TCP affinity probe,the rigid skeleton of lysine is updated to the flexible backbone of the oligonucleotide chain,called DNA affinity probe,and the binding probe(BP)and capture probe are used.(Capture probe,CP)together.In this section,the BP containing MLN8237,the CP containing 4-benzoate azide,and fluorescein were synthesized using the DNA affinity probe method.After the binding of BP(MLN8237)to the target protein,CP(4-benzoic acid azide)was synthesized.The process of photocross-linking of the target with the target protein and the imaging of the CP(fluorescein)on the polyacrylamide electrophoresis gel finally identified seven potential target proteins in addition to the AKA.Among these potential target protein molecules,the p38 P38 mitogen-activated protein kinase(p38)and laminin receptor(LAMR)have been biologically validated.Among them,the affinity of MLN8237 and p38 is about 8 ?M,and the related biological functions(promoting cells Apoptosis may be obscured by AKA;the affinity of MLN8237 to LAMR is approximately 1.8 ?M,which may partly explain its biological function against tumor migration and invasion,and provide effective information for a deeper understanding of the efficacy,toxicity and side effects of MLN8237.Provide further evidence for the comprehensive evaluation of MLN8237 as a potential clinical candidate drug.At the same time,it also shows that using DNA affinity probes to find small molecule targets is a very effective analytical method.