Localization Of Adrenoceptor Using Ligand-QD And Its Application In Drug Screening

In chapter one, several major drug screening methods are reviewed briefly, including high specificity of screening, high-throughput screening and high content screening. Drug screening is the first process and critical step in the new drug research. Secondly, the adrenergic receptor (AR) is reviewed. Phenylephrine (PE) and Prazosin are introducted in detail. Thirdly, the fluorescent dyes were reviewed, we focus on the semiconductor fluorescent dye——quantum dots (QD). Owing to its many unique advantages, QD has made considerable progress in the application of drug screening. Last, we make a prospect on drug screening based on fluorescence.In chapter two,α_1A-AR was located by prazosin-QDs. We first developed a method for the location ofα_1A-AR on HEK293 cell membrane by TIRFM with prazosin-QDs. In the experiment, we performed our study on many factors which effect the location ofα_1A-AR, including proportion, concentration and incubation time. The optimum condition ofα_1A-AR location was determined. A series of experiments demonstrated the specificity between QD-streptavidin and Prazosin-biotin. The specificity between Prazosin-QD andα_1A-AR was also proved. Obtained results indicated that the QD-labeled prazosin molecules still had pharmacological activity. In the experiment, we observed that Prazosin-QDs in the solution were captured byα_1A-AR on the cell membrane. Through visualizing QD labeled to Prazosin, it demonstrated thatα_1A-AR was present on the cell membrane. This is the foundation of drug screening with QD.In chapter three,α_1A-AR was located by PE-QDs. Comparisons are made on the binding abilities respectively betweenα_1A-AR on the cell surface and four biotinylated phenylephrines with different chain lengths. It is found that the phenylephrine with longer chain between biotin and phenylephrine can bind toα_1A-AR on the cell surface more easily. Correspondingly, more fluorescent spots would appear under the same condition. It demonstrated that PE with longer strand had less steric hindrance and higher affinity. Therefore, we chose PE(D) which had longer strand as the model drug to locateα_1A-AR. Several methods have adopted to support the binding specificity of prazosin-QD and phenylephrine-QD toα_1A-AR. We also observed the function difference when agonist-phenylephrine and antagonist-prazosin were respectively bound toα_1A-AR. It was found that the fluorescence labeled agonist was distributed throughout the cells, whereas the antagonist remained completely on the cell surface. Furthermore, TIRFM is applied for tracking the internalization and the endosomes ofα_1A-AR induced by PE(D) for living HEK293 cells. Obtained results indicated that the QD-labeled phenylephrine molecules still had pharmacological activity. Thus, we can real time differentiateα_1A-AR agonist and antagonist by the distribution of receptor after induced by drugs.In chapter four, we first developed a method for drug screening based on QD labelled antagonist. Theα_1A-ARs on the plasma membrane were labelled by Prazosin-QD primarily. Then, other drugs acted on the HEK293 and competed with Prazosin-QD. Compare the degree of the fluorescence spots on the plasma membrane, we could evaluate the affinity between drug andα_1A-AR. The result demonstrated that the affinity sequence was Urapidil > Terazosin > Doxazosin > Phentolamine > Prazosin > Tamsulosin. It is consistent with the classical radioligand method, so we could research the interaction between drug and receptor and screen the drug which had selectivity with receptor subtype.