Synthesis and structural properties of cyclic peptides incorporating novel non-reducible cystine mimics

该项目的目的是开发一系列具有生物活性的环肽，其中含有硫氨酸和胱胱甘肽，作为代谢稳定，不可还原的二硫键替代品。通过研究这些二硫代物的构象和生物学特性，我们的目标是了解它们如何最好地用作研究和改变生物过程的工具，并评估它们作为药物发现计划的可能线索。对环肽的兴趣的复苏是由于它们与治疗相关靶点（如受体、蛋白-蛋白相互作用（PPI）和转录因子）结合时的效力和选择性，这些靶点难以用小分子疗法或生物制剂解决。对肽导联施加构象约束的传统方法是通过在两个Cys残基之间引入二硫桥来环化。然而，这种联系在体内会减少，并且在代谢上不稳定；因此，有相当大的兴趣制造具有硫醚连接的环肽（通过结合硫代氨酸或胱硫代氨酸）。事实上，CRB经常被客户要求为早期药物发现项目提供含有这些非自然联系的环肽类似物。Tabor小组开创了一种固相肽合成（SPPS）方法来合成含有硫氨酸的肽1,2,3这包括立体选择性合成正交保护的镧氨酸，将该残基结合到线性肽中，选择性去除w-氨基酸部分的保护基团，树脂上的环化和链延伸。这种方法现在被世界各地的研究小组用于化学合成抗生素，这是一种新兴的抗菌肽。然而，这种方法尚未广泛用于制备其他生物活性肽的不可还原、构象受限的类似物3,4,5，并且引入该桥对这些肽的构象和生物学特性的影响也不清楚。因此，该项目将专注于应用SPPS方法合成参与受体结合的关键肽的硫醚桥接构象约束类似物。为了了解这些二硫键替换对肽构象的影响，特别是确定肽是否可以采用生物活性构象，这些肽的结构特性将通过NMR进行分析，使用NAMFIS-分析，6与Prof Mate Erdelyi （Uppsala大学）合作。学生还将花3 - 4个月的时间在乌普萨拉接受这项技术的培训。工作计划：1年1 - 6个月LIDo培训课程（SysMIC, Bio-Industry）。小规模合成正交保护硫代氨酸和半胱硫代氨酸氨基酸构建块。第2个月13 - 18环肽的核磁共振研究：NAMFIS技术训练（瑞典乌普萨拉大学）；第3年第25 - 32个月合成所有变异硫醚桥长度和血管加压素的a-立体化学，7生长抑素5和在第1个位置合成的客户肽：核磁共振分析，生物评价（钙通量FLIPR测定）。第33 - 38个月CRB实习2（第5部分）第4年第39 - 42个月在实习中合成肽的核磁共振分析2个月第43 - 48个月论文写作

The aim of this project is to develop a range of bioactive cyclic peptides incorporating lanthionine and cystathione as metabolically stable, non-reducible replacements for disulfide linkages. By studying the conformational and biological properties of these disulfide replacements, we aim to understand how they could best be used as tools to study and alter biological processes, and to evaluate them as possible leads in drug discovery programmes.The resurgence of interest in cyclic peptides has been driven by their potency and selectivity when binding to therapeutically relevant targets, such as receptors, protein-protein interactions (PPI) and transcription factors, which are difficult to address with small molecule therapeutics or with biologics. The conventional approach to applying a conformational constraint to a peptide lead is to cyclise by introducing a disulfide bridge between two Cys residues. However, such linkages are reduced in vivo and are not metabolically stable; thus there is considerable interest in making cyclic peptides which have a thioether linkage (from incorporation of lanthionine or cystathionine) instead. Indeed, CRB are frequently asked by their clients to provide cyclic peptide analogues containing these unnatural linkages for early stage drug discovery programmes.The Tabor group have pioneered a solid-phase peptide synthesis (SPPS) approach to lanthionine- containing peptides.1,2,3 This involves the stereoselective synthesis of orthogonally protected lanthonine, the incorporation of this residue in a linear peptide, selective removal of the protecting groups on the w-amino acid moiety, cyclisation on-resin and chain extension. This is now the method used by groups worldwide for the chemical synthesis of lantibiotics, an emerging class of antimicrobial peptides. However, this method has not yet been widely used to prepare non-reducible, conformationally constrained analogues of other biologically active peptides,3,4,5 and the effects of introducing this bridge on the conformation and biological properties of these peptides is also not well understood. This project will therefore focus on applying the SPPS approach to synthesising thioether bridged conformationally constrained analogues of key peptides involved in receptor binding. In order to understand the effect that these disulphide bond replacements have on the peptide conformation, and in particular to determine whether the peptides can adopt the biologically active conformation, the structural properties of these peptides will be analysed by NMR, using NAMFIS- analysis,6 in collaboration with Prof Mate Erdelyi (University of Uppsala). The student will also spend 3 - 4 months receiving training in this technique at Uppsala. Workplan:Year 1 Months 1 - 6 LIDo training courses (SysMIC, Bio-Industry). Small-scale synthesis of orthogonally protected lanthionine and cystathionine amino acid building blocks.Months 7 - 12 Synthesis of two analogues of vasopressin,7 one with with lanthionine and one with cystathionine replacements for the native disulfide bondYear 2 Months 13 - 18 NMR studies of cyclic peptides: training in NAMFIS technique (University of Uppsala, Sweden: Prof Mate Erdelyi)Months 19 - 24 CRB Placement 1 (Section 5)Year 3 Months 25 - 32 Synthesis of all variants thioether bridge lengths and a-stereochemistry of vasopressin,7 somatostatin5 and of the client peptide synthesised in Placement 1: NMR analysis, biological evaluation (calcium flux FLIPR assay).Months 33 - 38 CRB Placement 2 (Section 5)Year 4 Months 39 - 42 NMR analysis of peptides synthesised in Placement 2 Months 43 - 48 Thesis writing