| Malignancies can infiltrate other tissues and form tumors, it has become one of great diseases that could cause mankind death. In recent years, through the extensive study of tumor infiltration and metastasis, matrix metalloproteinases(MMPs), especially gelatinase A and B, are found to have an important effect on tumor infiltration and metastasis; Under pathological condition, on the one hand, MMPs can facilitate invasion and metastasis of tumors through degradation of basal membrane and matrix, and breakthrough of matrix barrier, on the other hand MMPs facilitate growth and diffusion of tumors through endogenic vessels in hair cell and new angiogenesis. Therefore MMPs have received considerable attention as targets for cancer drug development, and the inhibitors of MMPs have been hot spot of development at present.However, there are few methods to screen inhibitors of MMPs. Some orthodox methods of measuring activity of MMPs may be used to screen their inhibitors, but these methods have some shortcomings to be impossible avoided and aren't fit for screening a great quantity of compounds. For example, gelatin zymography is perhaps the most commonly used technique for detecting MMP-2 and -9. Although sensitive and specific, it is cumbersome to perform, is only marginally quantifiable with a narrow linear range and is not suitable for rapid screening for evaluation of inhibitors of these enzymes. For the purpose of developing high throughout assays for screening inhibitors of gelatinases, several synthetic peptide substrates designed to mimic the natural substrate have been developed. These substrates, however, lack specificity and are often used to assay many different MMPs.1At the same time, the appearance of combinatorial chemistry technology develops new space for drugs, accelerates synthesis speed of new drugs and lead compounds. However, analysis and screening methods for these compounds commonly expend more time, can't satisfy the need of rapid screening. So it is very necessary to develop a feasible and rapid screening method.On account of these factors, in the thesis two methods were developed, each technology was studied, and was used to assay and screen some new compounds.The first method is a versatile assay using succinylated gelatin. In the method, the effect of reaction time, amount of substrate, buffer solution, pH of buffer, ionic strength(Ca2\ Zn2+) and reaction temperature were investigated; linear range, limit of quantitation and precision were detected. Before measuring the compounds of the inhibiting activity for gelatinases, the effect of amount of gelatinases, Ca2+and different reaction time in detecting procedure were investigated. Three kinds of compounds were assayed by this method, the relation of structure and activity for better compounds were simply analyzed.The second method is gelatinase biochromatography. Among the method, some factors affecting immobilized gelatinases: immobilized time, pH, concentration of ammonium sulfate were studied. Reproducibility of immobilization, tolerance of pH and organic solvent, retention of different compounds in different collumns were detected; then pH and flow rate of mobile phase, concentration of phosphate, different modifiers and concentration of methanol in mobile phase and so on were studied in detecting procedure. When all conditions were definited, we determined the stability and reproducibility of gelatinase column, detected the retention of different compounds, and compared inhibiting activity of these compounds.By comparing the assay results of two methods and analyzing the relation of structure and activity of compounds, we found that the results were anastomotic. It is thus clear that the developed methods can be used to screen the inhibitors of gelatinases in the thesis.Compared with orthodox zymography, and immunization, the two methods are both rapid, easy, and can save samples; It can realize high throughout screening withadvanced equipments; but it also has some shortages and needs further reseach. |
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