Metal Ions Based On Inorganic Compounds With In Vitro Inhibitory Activity Of Matrix Metalloproteinase 13

Matrix metalloproteinase(MMPs)are zinc-dependent protein and peptide hydrolases,which constitute a separate family within the "metzincin" clan of metallopeptidases(MMPs).MMPs are capable of degrading extracellar matrix(ECM)components to participate in physiological processes.The pocket in the core domain is hydrophobic in nature,but variable in depth depending on the MMPs,representing a determining factor of substrate specificity of MMPs.The depth and the length of the S1' pocket and amino acids all represent critical foundations for the design and synthesis of matrix metalloproteinase inhibitors(MMPIs).MMPs play a key role not only in invasion processes,angiogenesis,and metastasis,but also in cancer cell transformation,growth,apoptosis,signal transduction and immune regulation.It has been shown before that the secretion of MMPs by microvascular endothelial cells represents a critical step in the formation of new blood vessels and most of the MMPs may initiate and promote angiogenesis.One of the therapeutic strategies for these diseases is the design of drugs and inhibitors to target their catalytic domain.In this thesis,the catalytic domain of MMP-13(cdMMP-13)was expressed in E.coli through recombinant technology.In addition,the metal ions which was join the refolded buffer have influenced on the purity of cdMMP-13 and also impact on the secondary structure.Then,screening the different metal ions impact on cdMMP-13 and research the new inhibitors of MMP-13 were investgated.The thesis includes the following contents:1.Preparation of cdMMP-13(cdMMP-13)with pet-28a and screening of crystallization conditions.The cdMMP-13 with pet-28a was induced in E.coli BL21.The purification conditions of cdMMP-13 was used the dynamic adsorption.One time in the dynamic adsorption method,19-22mg protein samples can be loaded,and 13mg cdMMP-13 with purification of 95%can be recovered.In addition,cdMMP-13 with pet-28a can be refolded by desalting exclusion chromatography.For investigation and found that the stability of the target protein has a slight degradation phenomenon.At last,crystallization conditions of the complex of refolded cdMMP-13 and its inhibitor were screened at 4?,but none crystal formed after 384 conditions screened.2.Preparation of cdMMP-13(cdMMP-13)with pet-22b and screening of crystallization conditions.Molecular cloning and genetic mapping of the pet-22b gene to cdMMP-13.The cdMMP-13 with pet-22b was induced in E.coli BL21.The expression product of BL21 was indentified and demonstrated that the cdMMP-13 was expressed as an inclusion body in BL21.The purification conditions of cdMMP-13 was used the dynamic adsorption.One time in the dynamic adsorption method,17-20mg protein samples can be loaded,and 13mg cdMMP-13,with purification of 99%can be recovered.3.The influence of different metal ions of cdMMP-13-pet-22b and research the inhibitory mechanisms of the new inhibitor.The target protein was obtained by refolding the recombinant histidine-tagged catalytic domain of MMP-13.Circular dichroism(CD)analysis confirmed that cdMMP-13,desalted with the metal ions,increases the stability of the secondary structure of the protein.Including these metal ions,trivalent metal ions has the strongest influence on its stability,this may be the trivalent metal ions will replaced the zinc ions of the active center and make it closer to combine with the site around the active center.By comparison of the inhibitory activity of compounds containing different valence metal ions with a positive control inhibitor(i.e.CL-82198)on cdMMP-13,the half maximal inhibitory concentration value(IC50)showed that trivalent metal compounds in particular exhibit a strong cdMMP-13 inhibition.Compared to other compounds,potassium hexacyanoferrate(?)(K3[Fe(CN)6])has been shown to exhibit the best inhibitory effect.Furthermore,the binding event between K3[Fe(CN)6]and cdMMP-13 has been confirmed by isothermal titration calorimetry(ITC)and an endothelial cell tube formation test provided evidence for the notion that this interaction may have a potential effect on anti-angiogenesis.The crystallization codition of cdMMP-13 was optimized:the concentration of cdMMP-13 was 18 mg/ml,the concentration of ammonium sulfate was 1.8mol/L,the temperature was 16?.