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生物活性环肽，将羊毛硫氨酸和胱硫醚作为二硫键的代谢稳定、不可还原的替代物。通过研究这些二硫键取代物的构象和生物学性质，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中可能的线索。蛋白质-蛋白质相互作用（PPI）和转录因子，这难以用小分子治疗剂或生物制剂解决。将构象约束应用于肽前导的常规方法是通过在两个Cys残基之间引入二硫键来环化。然而，这样的连接在体内被还原并且不是代谢稳定的;因此，替代地，在制备具有硫醚连接（来自掺入羊毛硫醚或胱硫醚）的环肽方面存在相当大的兴趣。事实上，CRB经常被他们的客户要求提供含有这些非天然连接的环状肽类似物，用于早期药物发现项目。塔博尔小组开创了固相肽合成（SPPS）方法，用于合成含镧系元素的肽。1，2，3这涉及正交保护的镧系元素的立体选择性合成，将该残基掺入线性肽中，选择性除去N-氨基酸部分上的保护基，在树脂上环化和扩链。这是目前世界范围内用于化学合成羊毛硫抗生素的方法，羊毛硫抗生素是一类新兴的抗菌肽。然而，这种方法尚未被广泛用于制备其他生物活性肽的不可还原的、构象受限的类似物，3、4、5，并且引入这种桥对这些肽的构象和生物学性质的影响也没有得到很好的理解。因此，本项目将集中于应用SPPS方法来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置换对肽构象的影响，特别是为了确定肽是否可以采用生物活性构象，将与Mate Erdelyi教授（乌普萨拉大学）合作，使用NAMFIS分析6，通过NMR分析这些肽的结构特性。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：第1年1 - 6个月LIDo培训课程（SysMIC，生物工业）。正交保护的羊毛硫氨酸和胱硫醚氨基酸结构单元的小规模合成。7 - 12月合成两种加压素类似物，7一种用羊毛硫氨酸取代，另一种用胱硫醚取代天然二硫键。2月13 - 18环肽的NMR研究：NAMFIS技术培训（University of Uppsala，Sweden：Prof Mate Erdelyi）19 - 24个月（第5节）第3年第25 - 32月加压素、生长抑素5和在位置1中合成的客户肽的所有变体硫醚桥长和α-立体化学的合成：核磁共振分析，生物学评价（钙通量FLIPR测定）.第33 - 38个月CRB安置2（第5节）第4年第39 - 42个月核磁共振分析肽合成安置2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