第一部分:以細胞篩選合成五圓Iminocyclitols作為抗C型肝炎的研究第二部分: 利用螢光標記醣類之方法定量金奈米粒子上醣分子濃度

Abstract

Part I Replication of Hepatitis C virus (HCV) depends on host protein synthesis system. In this system, glycosyltransferases and glycosidases are involved in the processing of N-linked glycoproteins in the endoplasmic reticulum. Inhibition of these enzymes may have profound effects on maturation, transport, secretion of glycoproteins, and even alter cell-virus recognition processes. The iminocyclitol 2,5-dideoxy-2,5-imino-D- mannitol is a powerful inhibitor of a large range of α- and β-glucosidase. Based on this structure, compound with a cell-permeable alkyl chain, was synthesized and coupled with acid library in the microtiter plate. Without purification, >100 analogues were directly subjected to ex vivo anti-HCV infection cell based assay and showed good inhibitive ability despite most of them were of low inhibitive ability inα-glucosidase in vitro screening. Compared to the proven anti-HCV inhibitor NB-DNJ, several compounds were identified as better inhibitors. Five compounds with fused aromatic ring, thiopyridine or trifluoromethylbenzne showed good anti-HCV ability and were potent to become new drugs. These compounds were re-synthesized and purified to get their IC50 and Ki values for glucosidase and anti-HCV inhibition assay. Although the alkylated iminocyclitols showed only mildα-glucosidase inhibitory compared to corresponding non alkylated iminocyclitols, in cell-based assay they indeed showed good inhibition effect on anti-HCV inhibition. In brief, this work demonstrates the effectiveness of the simple combinatorial strategy for rapidly and efficiently discovering potent inhibitors by using cell-based assay system. Part II Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide a great platform for protein recognition research. Among the nanomaterials, spheral nanoparticle has been widely used as a model of cell, in which glyconanoparticle mimics the glycans on the cell surface. This property makes glyconanoparticle as an excellent material to study the multivalent interactions between glycan and protein. However, to study such interaction, the glycan density on nanoparticle surface is an important issue and a straightforward quantitative analysis of the interaction is rarely reported. In the study, direct measurement of the amount of fluorescent glycans on nanoparticles is affected by the property of nanoparticle. The standard curve of fluorescent glycans concentration to the fluorescence intensity should be modified with the existing of nanoparticles. Co-existence of two fluorescent compounds, coumarin and texas red, may influence the fluorescence of each other and is not ideal for study. Two modified experiments, enzyme - linked immunosorbent assay (ELISA) and fluorescence competition binding assay, are proposed here to determine the binding affinity of fluorescently labeled glyconanoparticles with corresponding lectins.