| As the mainly effective means to prevent and treat diseases, innovative drug discovery has become one of the hot topics in pharmaceutics. However, one of the bottle necks of the innovative drug development is the screening of bioactive compounds. The construction of effecient methodologies for bioactive compounds screening has become a key issue in this field. Taking voltage dependent anion channel-1 (VDAC-1) and β2-adrenoceptor (β2-AR) as examples, we constructed membrane protein chromatographic models in this dissertation. The VDAC-1 and β2-AR chromatographic models were used to investigate the interactions between VDAC-1 and three drugs and the binding studies of β2-AR and seven drugs, respectively. Fluorescence spectrometry, molecular docking and dynamic simulation were carried out to evaluate the chromatographic method. In addition, the VDAC-1 chromatographic column was used to screen the bioactive compounds from water extract of rheum palmatum which targeted to VDAC-1. The membrane protein chromatographic method is expected to provide new means for drug-memmbrane protein interaction and bioactive compounds screening from traditional Chinese medicine (TCM), it also plays a vital role in high throughput screening from complex systems. The dissertation was drafted as four chapters among which the author contributed greatly in following contents:1. By combining the specificity of membrane recognizing drugs and the high separation capacity of chromatographic technologies, we have constructed immobilized VDAC-1 and β2-AR chromatographic models in this dissertation, which extended the application of affinity chromatography.2. VDAC-1 chromatographic models were used to investigate the interactions between VDAC-1 and ATP, NADH and NADPH. Fluorescence spectrometry, molecular docking and dynamic simulation were also carried out to evaluate the chromatographic results. The data showed that the three drugs bound to VDAC-1 in equamolar amounts through nucleotide binding site. The association constants were decreased while the numbers of binding sites were increased with the increasing temperatures in the range of 10 to 45℃. Electrostatic interactions were the main driven forces for the binding of the three drugs and VDAC-1. β2-AR chromatographic models were applied to reveal the interactions between β2-AR and seven drugs. The rank order for the association constants of the seven drugs binding to β2-AR was bambuterol> clenbuterol> tulobuterol> clorprenaline> salbutamol> terbutaline> methoxyphenamine. Hydrogen bonds played important role during the binding. These results showed highly consistent with the data in literatures, which confirmed that the proposed VDAC-1 and β2-AR chromatographic models could be used to investigate the interaction between immobilized membrane proteins and drugs and it would probably become a powerful alternative for the high throughput determination of drug-protein interactions.3. The VDAC-1 chromatographic column was used to screen the bioactive compounds from rheum palmatum. Five ingredients including aloe emodin, rhein, emodin, rhubarb and catechin were identified to be the bioactive compounds in rheum palmatum which targeted to VDAC-1. These results showed that the proposed chromatographic model could be used to screen the bioactive compounds from TCM and it provided new methodology for high throughput screening of bioactive compounds from complex systems.4. In terms of chromatographic theory, we derived a series of novel formulas based on the classic frontal analysis, from which the association constant, numbers of binding site and the numbers of ligands binding to a protein immobilized on the column can be obtained simultaneously. The formulas have the advantages of informative and can overcome the lack of binding information in classic frontal analysis, zonal elution and injection amount dependent analysis. It provides theoretical bases for the comprehensive determination of drug-protein binding parameters by affinity chromatography. |
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