| The integrin family of cell adhesion molecules plays a vital role in angiogenesis during the neoplastic procedure. Recently, integrins have been suggested as potential targets for cancer research. In this thesis, we presented the synthesis of a class of novel cyclopeptides. These cyclopeptides were subsequently screened with SPR technology in order to investigate their binding affinities towards integrins. Molecular docking was then carried out to understand the binding mode between the cyclopeptides and integrin protein. The biological activities of the candidates were evaluated in vitro and in vivo. The present results suggested that c-Lys is a potential integrin ligand or inhibitor for cancer therapeutics. The thesis consists of four major sections.I. The design and synthesis of cyclopeptides based on the RGD integrin-binding site by click chemistry was investigated. At the cyclization step, the conditions (such as catalyst, time and temperature) of click chemistry reaction were optimized. Finally, a series of novel compounds containing RGD motif were obtained under optimized conditions (DCM, CuBr/DBU = 1:3, r.t.,4-6h). The results held promise for future work.Ⅱ. The interactions.between integrin avβ3 and cyclopeptides were determined by surface plasmon resonance biosensor (SPR) technology. Integrin avβ3 was first immobilized on the chip under optimized condition. The cyclopeptide ligands for integrin avβ3 receptor were then used for binding analysis and kinetic measurement. It was found that the cyclopeptides c-Lys, c-Gly and c-Pro have better affinity to integrin avβ3 respectively.III. The binding modes of the cyclopeptides within integrin protein were described using the program LigandFit. The cyclopeptide ligands were docked into the integrin active sites and then were ranked according to scores evaluated by scoring function. It was found that the cyclopeptide c-Lys was ranked on the top of the list, followed by c-Gly and c-Pro, while the cyclopeptide c-Leu was at the bottom. We have further analyzed the docking modes of cyclopeptides c-Lys and c-Leu binding to integrin avβ3 respectively, indicating that the integrin avβ3-c-Lys complex has strong multiple hydrophobic and hydrogen bond interactions. The cyclopeptide c-Lys could be used to further evaluation as candidates for integrin inhibitors.Ⅳ. The effects of candidate cyclopeptides on integrin-mediated cell behavior were investigated in vitro and in vivo. The adhesion, invasion and migration capacities of cocultured HUVEC cells treated with the cyclopeptides were evaluated for cell adhesion, migration and invasion assay in vitro, respectively. In the cell adhesion assay, it was found that the adhesion ability of HUVEC cells treated with cyclopeptide c-Lys was evidently inhibited (p<0.01), and those treated with c-Gly displayed weaker inhibitory effect in the same assay (p<0.05). In the cell migration assay, treatments with c-Lys and c-Gly were resulted the fewer migrating HUVEC cells (p<0.01). In the cell invasion assay, treatments with c-Lys for HUVEC cells resulted in prominent inhibition of cell invasion ability (p<0.01) while treatments with c-Gly was followed (p<0.05). In order to investigate the effect of cyclopeptides on F-actin cytoskeleton and focal adhesion, the immunofluorescence staining technique was employed. It was found that treatment with cyclopeptides resulted in a disrupted cytoskeleton with both F-actin and focal adhesion (vinculin) network in the cell body. Besides, angiogenesis assay in vitro revealed the inhibition of capillary-like tube formation of HUVEC cells by cyclopeptides c-Lys and c-Gly respectively. Furthermore, the potential tumor target of cyclopeptide c-Lys in vivo was investigated roughly. The time courses of its distribution and accumulation in xenograft tumor tissues were studied using a live animal fluorescence imaging system. The maximum concentration of fluorescence-labeled cyclopeptide was detected on the tumor site at 6h after injection.In addition, the recent research of antiangiogenic drugs was briefly introduced.
|
PDF Full Text Request