Study On The Metabolism And Targets Of Doxorubicin In Tumor Cells

Pharmacological research is one of the main contents of drug registration.With the development of molecular pharmacology,the interpretation of drug mechanism has shifted from understanding the basic functions,pharmacokinetics and toxicology of drugs to the functional analysis of drug targets.This research focuses on the target cells and organs of the drug.Taking the anti-tumor anthracycline adriamycin(ADM),which has been used clinically for decades,as an example,and using drug target cells as research materials to develop drug disposal analysis methods for target proteins and cells.And then supplement the existing pharmacological research methods,quickly and comprehensively understand the drug mechanism.The main results are as follows:1.Take ADM as an example to establish a method for studying intracellular drug metabolites.Classical pharmacokinetics can obtain the basic information of the bodys disposal of drugs,including data on the absorption,distribution,metabolism and excretion of drugs in the body.However,the lack of understanding of drug treatment target cells/target organs cannot explain the behavioral characteristics of drug molecules.In this paper,the target cells of ADM-breast cancer ductal epithelial cells(MCF7)and its drug-resistant cells(MCF7/ADM)are used as research materials.After being treated by ADM,the ADM and its metabolites in the cells are organically extracted,the extract components are separated by UPLC,and some components are analyzed by tandem mass spectrometry.Based on the characteristic absorption of ADM and its metabolites at 480 nm and the stable structure of its mother nucleus m/z 321,three ADM structural analogues derived from MCF7/ADM were selected for multi-stage mass spectrometry identification.Three unreported metabolites of ADM were found and their chemical structures were deduced.2.Take ADM as an example to establish a drug target screening method based on protein microarray.The identification of drug targets is an important part of pharmacological research.Classical and reverse pharmacology both adopt the strategy of verifying the target of drug molecules from the pharmacodynamics and toxicological phenomena,leading to the need for decades or longer to analyze the drug mechanism,and the level of understanding is very single and gradual.In this paper,biotinylated ADM was designed and synthesized.With the help of the super affinity between avidin labeled with CY5 fluorophore and biotin,a drug molecule tracer probe was constructed.The ADM binding protein was screened on a chip containing more than 21,000 human proteins,and the ADM/biotinylated-ADM competitive binding reaction was used to identify the ADM-specific binding protein.After counting the fluorescence signal values,401 proteins with SNR greater than 4.0 were obtained as potential targets of ADM,including 30 high-affinity proteins with SNR greater than 10.0.By optimizing the methodology,a feasible and quality controllable protein microarray drug target screening method was established.3.Determine HRAS as the target of ADM.It is common knowledge that the complexity of the spatial structure of drug molecules leads to numerous binding targets.Collecting the functional interpretations of these target proteins,combined with the study of the drug effects on the function of target proteins,on the one hand,can analyze the mechanism of drug action and potential therapeutic indications at the molecular level;on the other hand,it can also do prospective research on the side effects of drugs,and predict how target cells/target organs will dispose of drugs.This paper uses GO clustering,protein interaction and other bioinformatics methods to analyze the functions of 401ADM potential binding proteins,and finds that they mainly focus on cell adhesion,intracellular metabolism and signal transduction.Further study of the modification effect of related enzyme proteins on ADM is expected to analyze the mechanism of target cells producing new metabolites of ADM.These results suggest that ADM can act on multiple functional pathways of target cells and has the characteristics of multiple targets,far exceeding the currently known therapeutic effects of ADM drugs.The systematic disease association analysis also found that some binding proteins are highly correlated with the cardiotoxic side effects of ADM.Based on the combination of cluster analysis,protein interaction network and SNR value,it is proposed that the ADM binding protein HRAS with high signal-to-noise ratio has an important role in the target cells of ADM.Using BLI technology,the equilibrium dissociation constant(K_D)of HRAS and doxorubicin binding was determined to be 10.2 n M.4.Doxorubicin improves the drug sensitivity of tumor cells to doxorubicin by activating the downstream pathways of HRAS.Based on the knowledge that HRAS and RAF combine to conduct signals,an in vitro HRAS-RAF binding experiment under the intervention of ADM was designed,and it was found that ADM can promote the formation of HRAS-RAF complex.The results of bio-computational simulation also show that the structure of the ternary complex formed by ADM is more stable,and the binding site of ADM is predicted to be near the binding site of HRAS and RAF.Using bladder cancer cells RT4 and J82 with high expression of HRAS as research materials,it was verified that ADM activates the relevant pathways of cell proliferation by promoting the combination of HRAS and RAF,and accelerates the cell cycle process.Compared with the classical experimental evidence that ADM interferes with DNA replication to trigger cell cycle arrest to promote cell apoptosis,it suggests that ADM has a bidirectional pharmacological effect of activating cell proliferation and accelerating cell death.It is proposed that in the process of anti-tumor therapy,ADM can kill fast-growing tumor cells,and it is also expected to promote the G0-phase cells in the heterogeneous tumor cell population that are not sensitive to drugs to enter the proliferation phase,and enhance the killing effect of ADM.Based on target cell drug disposal and drug action targets,this study established a research method for intracellular intervention drugs and their binding molecules,discovered new ADM metabolites and targets,and enriched the understanding of ADM action mechanisms.The established intracellular drug metabolite analysis method and protein microarray-based drug binding protein screening method have universal applicability and provide new tools for pharmacological research.