Fluorescence-based Assay For Disease-related Liver Transport Protein And CYP3A4 Of Human Liver Microsome

Fluorescence is the emission of light from the singlet excited state of a molecule.It can be measured with great sensitivity.We know chromophore is a molecule that can absorb light(UV,visible,or near IR)to generate an electronically excited state.A fluorophore is a molecule that can absorb light to form an electronically excited state and then also re-emit some of that excited state energy as light.So we say fluorophore is a special example of a chromophore.Fluorescent probe is a kind of molecules that have characteristic fluorescence in the ultraviolet visible near infrared region,and the fluorescence properties(excitation and emission wavelength,intensity,lifetime,polarization,etc.)can be changed sensitively with the properties of the environment,such as polarity,refractive index,viscosity and so on.At present,fluorescent probes usually include fluorescein probes,inorganic ion fluorescent probes,fluorescent quantum dots,molecular beacons,etc.However,most molecules of interest are not fluorescent,and chemical processes do not usually result in the emission of light.This is why fluorescent probes are important.By interacting with an analyte of interest,or undergoing a chemical transformation that leads to a change in fluorescence,they allow light to be used as a tool to “spy” on molecular processes,the development of medical devices,and the analysis of environmental samples.Small-molecule fluorescent probes are molecules that change their fluorescence emission in response to a binding event,chemical reaction,or change in their immediate environment.This is very important for the study of biological systems,in drug discovery,cellular imaging,environmental analysis,and various medical applications.Numerous disease states are associated with having a fatty liver.These include obesity,atherosclerosis and insulin resistance.Because the liver can make its own fat,dietary restriction does not treat fatty liver diseases.The liver makes fatty acids from simple precursors,such as lactic acid.Lactate can enter the liver from the bloodstream,through the action of ATP-independent transport(“pump”)proteins.The driving force for the lactate pump is the release of protons.The lactate pump is a member of the monocarboxylate transporter family.Proteins in this family transport a range of monocarboxylic acids across cell membranes.So we have an idea to develop small-molecule inhibitors.Developing small-molecule inhibitors of lactate pumps could lead to drugs for treating fatty liver diseases.There are no known inhibitors of the pump.This is because there are no known assays for pump protein activity.There are crystal structures of prokaryotic lactate pump proteins.Through a collaborator-professor Youcai Zhang in our school,we have access to unique,stable human liver cell lines with overexpressed lactate pumps.Structural data are not available.This makes reliable structure-based drug design impossible.In a broader application of fluorescent probes to study metabolism,we hope our fluorescence molecules could make some progress on the CYP enzyme activity.Cytochrome P450(CYP)enzymes are large and important class of proteins involved in the biotransformation of many xenobiotic and endobiotic compounds.There are many different cytochrome P450 enzymes(CYPs).In humans,most of the CYPs are found in the liver,CYP3A4 contributes to the metabolism of more than 30% of drugs in clinical use,and responsible for “Phase I” metabolism of lipophilic(= greasy)organic molecules.Phase I metabolism involves oxidation of the organic molecules to form more polar products.So we choose CYP3A4 to do our luminescence assay.CYP probes are based on oxidative de-alkylation or de-benzylation.The ability of cytochrome P450 monooxygenases to catalyse a wide variety of C-H activation reactions makes them highly desirable biocatalysts.So,what others focus on is CYPs are often very good at oxidizing C-H bonds and making C-OH groups.We have a different idea and hope to make some different oxidiation,like phosphine oxidation,or sulfides oxidation.Our group is expert in turning oxidation reactions and hope it can turn into changes in fluorescence.We know that many of these reactions have never been coupled to a fluorescence assay.First we hope be able to turn phosphine to phospine oxide oxidation into a fluorescence response.At the same time we hope can turn the oxidation of sulfides to sulfoxides,and sulfoxides to sulfones,into changes in fluorescence.