Study On The Inhibition Of Cytochrome P450 3A By Traditional Chinese Medicine Via Visualization Of Enzyme Activity

CYP3A(CYP3A4/5)metabolizes more than 50%of commonly used western medicine in clinic,and is also an important target enzyme for the metabolism of traditional Chinese medicine(TCM)in the human body.Co-medication of TCM and western medicine often inhibits CYP3A activity,resulting in drug-drug interaction(DDI)and serious adverse drug reactions.With the continuous development of combination therapy,the adverse interaction caused by the combined use of TCM and western medicine has become increasingly prominent.As the main metabolic enzyme in the biotransformation of western medicine and TCM,the study of CYP3A-mediated interaction between TCM and western medicine has become important.However,there is still a lack of systematic research on the interaction between TCM(components)and CYP3A.Fluorescent probe is important molecular tool to study metabolic targets such as CYP3A,which can realize the visual high-throughput screening and evaluation of TCM(components)that regulate the activity of target enzymes.In this study,according to the interaction between small-molecule fluorophores and CYP3A,a highly selective fluorescent probe BN-1 for CYP3A was successfully developed.Through the reported highly selective fluorescent probe NEN and the developed BN-1 of CYP3A,the high-throughput screening systems of CYP3A reversible inhibitors and time-dependent inhibitors were constructed.Subsequently,the inhibitory effects and types of hundreds of TCM on CYP3A were systematically studied.According to the fingerprints and inhibition profiles of the target TCM,the main inhibitory monomer components of Ginkgo Folium and Selaginelle Herba were successfully identified.In addition,the research on the potential interaction between representative TCM and western medicine has been carried out,and the relevant results can provide useful guidance for the safe and rational use of TCM.Methods:1.Construction and evaluation of fluorescent tool molecules:By analyzing the characteristics of CYP3A active cavity structure and substrates structure,using molecular docking technology,BN was used as a fluorescent group to optimize and develop a highly selective two-photon fluorescent probe for CYP3A.Using the in vitro incubation system,the basic properties of BN-1 were analyzed through enzyme linearity experiments,single-enzyme selectivity experiments,metabolic stability experiments and kinetic experiments.The imaging properties of BN-1 were evaluated by HepaRG cells and rat liver slices.2.Using the tool molecules NEN and BN-1,carry out research on the regulation of CYP3A activity by TCM:High throughput screening systems for reversible inhibitors and time-dependent inhibitors of CYP3A were constructed with fluorescent probes NEN and BN-1.To evaluate the feasibility and reliability of NEN and BN-1 in detecting the effects of TCM on CYP3A inhibition.Evaluation of the reversible and time-dependent inhibition of hundreds of TCM on CYP3A.3.Identification of inhibitory monomer components of TCM:Determine the fingerprint of the target TCM,thereby collecting the TCM liquid fraction,and obtain the TCM liquid fraction inhibition spectrum.The combination of the two atlases confirms the inhibitory monomer components of TCM and reveals the core components of TCM that inhibit CYP3A.4.Systematic research on target components of TCM:Investigation the reversible inhibition kinetics or time-dependent inhibition kinetics of the target TCM active ingredients,and obtain the inhibition parameters Ki,KI and Kinact,etc.Rationally speculate the reactive intermediate of the target components by GSH trapping assay,and their metabolic activation pathways were speculated.Via cytotoxicity experiments and mouse hepatotoxicity experiments,the potential hepatotoxicity of the target TCM components was carried out.5.Study on the interaction between TCM and western medicine:Through in vitro metabolism experiments,combined with the screening results,the inhibitory effects of reversible inhibitory and time-dependent inhibitory components of CYP3A on western medicine were clarified.Results:1.Among nearly 100 kinds of TCM,Ginkgo Folium and Selaginelle Herba exhibit potent inhibitory effects on CYP3A4;7-demethylginkgetin,ginkgetin and isoginkgetin in Ginkgo Folium and amentoflavone in Selaginelle Herba are the main components that inhibit CYP3A4 metabolism.There are potential interactions between four bisflavonoids and western medicine.2.A series of fluorescent derivatives BN1-12 were designed and synthesized.The highly selective fluorescent probe BN-1 of CYP3A was successfully optimized.The biological properties of BN-1 were systematically evaluated,which can be applied to the visual detection of CYP3A at multiple levels such as tissue homogenates,cells and tissues.BN-1 has the feasibility of realizing efficient screening of CYP3A time-dependent inhibitors and evaluation of inhibitory effects in complex biological systems.3.Using BN-1,a high-throughput screening system for time-dependent inhibitors of CYP3A was successfully constructed,and hundreds of time-dependent inhibitory effects of TCM were screened efficiently.Finally,Euodiae Fructus,Ephedrae Herba and Homalomenae Rhizoma,which have time-dependent inhibitory effects on CYP3A,were successfully screened.4.Taking Euodiae Fructus as an example,combined with the chemical constituent fingerprint and inhibition profile,the time-dependent inhibitory monomer components in Euodiae Fructus were successfully identified as limonin,evodiamine and rutaecarpine.The time-dependent inhibition kinetic parameters of three components were characterized,and metabolic activation pathways of three components were speculated by GSH trapping.The hepatotoxicity study at the cellular and animal levels were performed.5.Conducted research on the interaction between dozens of TCM such as Homalomenae Rhizoma and western medicine.After pre-incubation of TCM with NADPH,the inhibitory effect of Homalomenae Rhizoma on the oxidative metabolism of ticagrelor and midazolam was significantly enhanced.The residual activities of CYP3A were 14.5%and 17.85%respectively.It revealed that these TCM could potentially interfere with the metabolism of clinical co-administered western medicine,even leading to adverse drug-drug interactions.Conclusion:In this study,a highly selective CYP3A fluorescent probe BN-1 was successfully developed,which provides a new research method for exploring the biological function of CYP3A in complex biological environment.Based on the fluorescent probes NEN and BN-1,the high-throughput screening systems for reversible inhibitors and time-dependent inhibitors of CYP3A were successfully constructed,which can be applied to high-throughput screening of compounds such as western medicine and TCM.Based on this,the inhibitory effect of hundreds of TCM on CYP3A has been systematically studied.Meanwhile,confirmation and evaluation of inhibitory monomer components in target TCM complex components.It lays a foundation for systematic and regular research on the inhibitory effect of TCM and its components on CYP3A.The interaction study between TCM inhibitory components of CYP3A and western medicine was carried out.The relevant research results provide a new scientific basis for guiding the rational use of TCM and western medicine.