The Study And Application Of The Molecular Biochromatographic Screening Methods For Inhibitors Of Matrix Metalloproteinases

Backgrounds: Malignancy can infiltrate other tissues and form tumor, it has become one of great diseases that could cause mankind death. It is indicated that proteolytic cleavage of BM and ECM by the proteinases released by the tumor cells is a primary step in the cascading nature of the process by the extensive study. Matrix metalloproteinases (MMPs), especially Gelatinase A and B play a key role in these proteinases. They can participate in mediating the refreshment of ECM and keep the stability of cells. It has been proved to play key roles in the processes of tumor growth, invasion, metastasis and angiogenesis. Therefore, it is believed that the growth and metastasis can be controlled by gelatinase inhibitors which are expected to be useful for the treatment of cancer. Then gelatinases have received considerable attention as targets for cancer drug development, and the inhibitors of grlatinases have been hot spot of development.The screening methods of MMPIs major on detecting the activity of gelatinases at present. But some orthodox methods have several shortcomings to be impossible avoided and are not fit for screening multicomponent and a great quantity of compounds, such as gelatin zymography, immunizing analytical method and fluorescence-substrate method. At the same time, combinatorial chemistry technology accelerates synthesis speed of new drugs and leading compounds. So it is very necessary to develop a feasible and rapid screening platform.Owing to above reasons, molecular biochromatogry about immobilized gelatinase was developed in the thesis. And it has been applied for the preliminary screening of a batch of compounds.Methods: Immobilized gelatinase reactors with chromatpgraphic silica and monolithic column as supports respectively were prepared.The preparation of immobilized gelatinase reactors with chromatpgraphic silica as supports: The prepared conditions were investigated by L₈(2⁷) orthogonal experiment. Investigated factors included enzyme concentration, enzyme amount, buffer concentration and pH, and analytical index were bonding rate and enzyme activity. Then the optimized condition of gelatinase activity was determined. Gelatinase was bonded to epoxydic or aldehyde silica supports after bond, flushing and saturation. Substrate was injected, and product was determined by two methods. One was shade selection of effluent, the other was the change of product peak area by adding inhibitor. The influence to product from flow-rate was considered. The value of Km was measured, and the stability of column was also considered.The preparation of immobilized gelatinase reactors with monolithic column as supports: Silica monolithic column was prepared by collosol-gel, using TMOS as material, PEG as phase separation accelerator, acetic acid, ammonia and DMF as hydrolyzed catalyst, the agent to prepare mesopores and dryer. The sizes of skeleton and macropores were measured by SEM, the sizes of mesopores were by nitrogen absorption, and the amount of Si-OH was by acid-base titration. TMOS/PEG, the agent concentration to prepare mesopares and aging time were considered. Cladding by PTFE, monolithic column was fixed on chromatographic system. Interval porosity was determined. Gelatinase reacted with epoxydic or aldehyde columns. Substrate was injected, and product was determined by two methods. One was shade selection of effluent, the other was the change of product peek area by adding inhibitor. The influence to product from flow-rate was considered. The value of Km was measured, and the stability of column was also considered.Screening compounds: Substrate and compounds with different concentrations were injected with LY52 as positive control. Its IC50 value was measured by product peak area. Compared with the value in literature and the one measured by colourimetry, validity of screening mode was verified. The IC50 values of other 40 compounds were determined by these two chromatography. Results: The prepared conditions of immobilized gelatinase by L₈(2⁷) orthogonal experiment were: enzyme concentration 0.5mg/mL, buffer concentration 0.05mol/L, pH6.0. Optimized conditions of gelatinase activity were substrate 300μl,pH8.5,42℃. The bonding rates were 28.6mg/g (epoxy group), 68.4 mg/g (aldehyde group). The peak in 40.854min was product'. The optimum flow-rate was 0.2mL/min. The Km were 0.63±0.006mmol/L (epoxy group), 0.54±0.012mmol/L(aldehyde group). The column was stable in a week. In comparison with bonding rate, Km and stability, it is proved that immobilized gelatinase reactor prepared by aldehyde group is better.The prepared conditions of monolith were TMOS/PEG3.77, 120℃1.0mol/L ammonia treatment, aging a day. The properties of monolith obtained by collosol-gel were the size of skeleton 1.3μm, the size of macropores 2.5μm, the size of mesopores 14nm, specific surface area 264m²/g, the amount of Si-OH 0.2378 mmol/g and interval porosity 81%. Reacted with gelatinase, enzyme concentration was 0.3mg/mL, and bonding rates were 88.6mg/g (epoxy group), 90.4 mg/g (aldehyde group) respectively. The peak in 37.409min was product'. The optimum flow-rate was 0.2mL/min. The Km were 0.28±0.005mmol/L (epoxy group), 0.21±0.008mmol/L (aldehyde group) respectively. The column was stable in a month. There was almost no difference in two activated group in comparison with bonding rate, Km and stability. Finally immobilized gelatinase reactor prepared by epoxy group was employed.The IC₅₀ values of LY52 were 0.018±0.0001μmol/L (silica), 0.021±0.0001μmol/L (monolith). And the values measured by colourimetry and from literature were 0.023±0.0004μmol/L,0.027±0.0004μmol/L respectively. The three values kept in one accord basically, and proved screening model was feasible. On the whole, the results of other 40 compounds maintained in the same level by three methods.Conclusion: The screening methods developed in the thesis are accurate and feasible. The rapid screen of multicompnent and a great quantity of compounds can come true.