Synthesis and characterisation of amyloid fibre motifs and mimetics participating in cooperative binding with serum amyloid p component.

血浆糖蛋白，血清淀粉样蛋白p成分（SAP）结合淀粉样蛋白纤维沉积在病变组织。已知有超过20种不同的蛋白质可以形成纤维。SAP在其天然状态下不与任何蛋白质结合。因此，纤维识别基序必须产生于纤维形成过程中发生的结构转变。SAP可能参与促进纤维沉积，并且已知可以稳定纤维，保护它们免受蛋白水解攻击和吞噬细胞的消化。因此，这种相互作用似乎是淀粉样蛋白清除药物的一个很好的靶点。SAP是五聚体的，每个Mr 23500亚基携带一个双钙位点，被认为是淀粉样蛋白识别位点。磷酸乙醇胺或d -脯氨酸等小分子通过其酸性成分结合钙离子，以中等亲和力（Kd 20-40微摩尔）结合到该位点。实验药物CPHPC （Pepys et al Nature 2002）是一种二价化合物，由两个d -脯氨酸残基通过六碳脂肪链的N端连接而成。该化合物交联成对的SAP分子形成十聚体复合物，其中5个药物分子与10个SAP亚基相互作用。这种结合模式的协同性使相互作用的亲和力提高了3个数量级（Kd - 10纳米摩尔）。在体内产生的数十年的SAP可被肝脏迅速清除，但该化合物没有足够的亲和力来完全从纤维沉积物中剥离所有SAP。本项目的目的是合成一个面携带五个d -脯氨酸臂或十个分布在两面的d -脯氨酸臂的环肽。假设这些化合物应该以钙依赖的方式与SAP结合，具有增强的协同性和高亲和力，形成具有阻断纤维识别位点或交联十聚体的五聚体SAP。Broadbridge博士在制备各种不同的环肽方面拥有丰富的经验，这一阶段的培训将在他的实验室进行。肽蛋白研究有限公司这些产物与SAP的结合将通过UCL的量热法进行表征，并通过SAP的计算机图形调查和d -脯氨酸头基的结合位点来研究潜在的结合模式。材料将与SAP共结晶，并通过x射线分析研究相互作用的详细结构。我们还将研究可能暴露在纤维表面的肽序列。对β 1-42的结构研究和对甲状腺转甲状腺素和β 2 -微球蛋白的H/D交换测量表明，可能的区域暴露在纤维上。这项工作的结果将为研究纤维形成提供新的工具，并有助于设计更有效的阻滞剂。

The plasma glycoprotein, serum amyloid p component (SAP) binds to amyloid fibres wherever they are deposited in diseased tissues. More than twenty different proteins are known to form fibres. SAP does not bind to any of the proteins in their native state. Therefore the fibre recognition motif must arise from the structural transition that takes place during fibre formation. SAP may be involved in promoting fibre deposition and is known to stabilise fibres, protecting them against proteolytic attack and digestion by phagocytic cells. The interaction therefore appears to be a good target for amyloid clearing drugs. SAP is pentameric and each Mr 23500 subunit carries a double calcium site that is believed to be the amyloid recognition site. Small molecules such as phosphoethanolamine or D-proline bind to this site with modest affinity (Kd 20-40 micromolar) via their acidic components binding the calcium ions. The experimental drug CPHPC (Pepys et al Nature 2002) is a bivalent compound comprising two D-proline residues linked through their N termini via a six carbon aliphatic chain. This compound crosslinks pairs of SAP molecules forming a decameric complex where five drug molecules interact with ten SAP subunits. The cooperativity of this mode of binding enhances the affinity of the interaction by three orders (Kd 10 nano-molar). The resulting decamers of SAP are rapidly cleared by the liver in vivo but the compound has insuffient affinity to completely strip all SAP from fibre deposits. The aim of this project is to synthesise cyclic peptides carrying five D-proline arms on one face or ten D-proline arms distributed on both faces. The hypothesis is that these compounds should bind to SAP in a calcium dependent manner with enhanced cooperativity and high affinity, forming pentameric SAP with blocked fibre recognition sites or crosslinked decamers. Dr Broadbridge has extensive experience in preparing a wide spectrum of different cyclic peptides and this phase of the training will be carried out in his lab. at Peptide Protein Research Ltd. The binding of these products to SAP will be characterised by calorimetry at UCL and the potential mode of binding investigated by computer graphic investigation of SAP and the binding sites for D-proline head groups. The materials will be co-crystallised with SAP and the detailed structure of the interaction investigated by X-ray analysis. We will also investigate peptide sequences likely to be exposed on the surface of fibres. Structural studies on Abeta 1-42 and H/D exchange measurements on transthyretin and beta2 - microglobulin suggest likely regions that are exposed on fibres. The results of the work will provide new tools for investigating fibre formation and contribute to the design of more effective blockers of this process.