Design and Synthesis of Diyne Girder a-Helix Peptides: Novel tools for the Regulation of Protein-Protein interactions

蛋白质-蛋白质相互作用(PPI)在几乎所有生物过程中都是必不可少的。因此，使用化学探针调节PPI的能力对于研究这些分子识别事件的性质和开发新的治疗方法非常重要。然而，由于PPI界面的相对大而动态的性质，调节PPI的细胞渗透性化学探针的开发仍然是化学生物学中的主要挑战之一。Jamieson Group最近的研究集中在开发合成策略来从构象上限制多肽采用其生物活性的α-螺旋构象，并克服与多肽相关的许多糟糕的物理化学和药理学问题。假设：本项目的中心假设是，高度刚性的侧链到侧链的1，3-二炔桥将作为一个极其有效的构象约束，从而提供具有高度α-螺旋结构的多肽以及改善的结合亲和力和物理化学性质。以Glasser-Hay反应为关键步骤，开发1，3-二炔桥联α-螺旋肽的合成方法。应用1，3-二炔大梁多肽来调节重要的PPI，包括Gpx3/Mia40(与Kostas Tokatlidis教授合作(MVLS))、螺毒素/VGSC(与DSTL合作)和泛素蛋白质复合体(与Helen Walden教授(MVLS)合作)本专业将专注于一项新技术的开发和应用，即1，3-二炔桥联α-螺旋多肽。我们计划将这些新的化学工具应用于调控芋螺毒素/VGSC(DSTL：DSTL/AGR/R/CBRN/01)、Gpx3/Mia40(BBSRC：BB/R009031/1)蛋白质-蛋白质相互作用。我最近还与Helen Walden教授(2017年8月加入UOG MVLS)启动了一项新的合作，专注于泛素蛋白质复合体的调控，这些目标是对我的团队目前在脱泛素酶方面的研究(EPSRC：EP/N034260/1)的补充。除了研究现有项目中的新途径，这名学生所做的工作将提供数据，以支持未来EPSRC关于这些新泛素蛋白质复合体目标的拨款申请(2018年夏季)。与此项研究相关的具体工作包通过开发新的化学方法来制备非天然炔基氨基酸，并随后使用先进的微波辅助固相合成方法将其合并到特定的肽序列中，从而补充了这些补助金中描述的那些内容。如果合适，学生将有机会在Dstl Porton Down花费时间，使用先进的核磁共振和分子动力学来确定多肽的3D结构。这种多肽设计的独特卖点之一是有可能使用共聚焦拉曼成像来可视化细胞中的多肽，而不需要耦合荧光团。这一性质将有助于研究螺旋多肽穿透细胞膜的物理化学要求，这是多肽药物发现的最后主要前沿之一。这些分子的生物活性将通过基于荧光的抑制分析(使用Tokatlidis和Walden)和微流控膜片钳系统(使用Dstl Proton Down)来评估。化学生物学是一个重要的交叉学科领域，在国内外都具有相当重要的地位，如EPSRC、BBSRC和MRC的研究战略所强调的那样。这个特殊的项目非常符合RCUK技术接触生命倡议的化学生物学主题，因为它寻求开发在生物化学和医学中应用的新型工具化合物。重要的是，这项工作还将提供有可能加强我的REF2021影响案例研究的数据，该案例研究目前正在与DSTL共同开发：影响政府关于生物武器的知识和立法。

Protein-protein interactions (PPIs) are essential in almost every biological process. The ability to regulate PPIs using chemical probes is therefore extremely important for studying the nature of these molecular recognition events and the development of novel therapeutics. However, the development of cell-permeable chemical probes that regulate PPIs remains one of the major challenges in chemical biology due to the relatively large, dynamic nature of the PPI interface.Recent research in the Jamieson Group has focused on developing synthetic strategies to conformationally constrain peptides to adopt their bioactive a-helix conformation and also overcome many of the poor physicochemical and pharmacological issues associated with peptides.Hypothesis: The central hypothesis of this project is that a highly rigid side-chain to side-chain 1,3-diyne bridge will act as an extremely effective conformational constraint and thus provide peptides with high degrees of alpha-helical structure and improved binding affinity and physicochemical properties.Specific objectives1. Develop the synthesis of 1,3-diyne-bridged alpha-helix peptides using the Glasser-Hay reaction as a key step.2. Application of 1,3-diyne girder peptides to the regulation of important PPIs including Gpx3/Mia40 (in collaboration with Prof Kostas Tokatlidis(MVLS)), conotoxin/VGSC (in collaboration with Dstl) and Ubiquitin protein complexes (in collaboration with Prof Helen Walden (MVLS))This studentship will focus on the development and application of a novel technology, namely 1,3-diyne-bridged alpha-helix peptides. We plan to apply these new chemical tools to the regulation of conotoxin/VGSC (Dstl: DSTL/AGR/R/CBRN/01), Gpx3/Mia40 (BBSRC: BB/R009031/1) protein-protein interactions. I also recently initiated a new collaboration with Prof Helen Walden (joined UoG MVLS in August 2017) to focus on the regulation of Ubiquitin protein complexes, targets that are complimentary to current research in my group on Deubiquitinase enzymes (EPSRC: EP/N034260/1). As well as investigating new avenues in existing projects, the work done by this student will provide data to underpin a future EPSRC grant application (summer 2018) on these new ubiquitin protein complex targets.The specific work package associated with this studentship compliments those described in these grants through developing new chemistries to prepare non-native alkyne amino acids and their subsequent incorporation into specific peptide sequences using advanced microwave assisted solid phase synthesis methods. If appropriate, the student will have the opportunity to spend time at Dstl Porton Down in determine the 3D structures of the peptides using advanced NMR and molecular dynamics. One of the unique selling points of this peptide design is the potential to use confocal Raman imaging to visualise the peptides in cells without the need to couple a fluorophore. This property will facilitate investigations into the physicochemical requirements of helix peptides to penetrate the cell membrane, one of the last major frontiers in peptide drug discovery. The biological actively of these molecules will be assessed using fluorescence based inhibition assays (with Tokatlidis and Walden) and a microfluidic patch clamp system (with Dstl Proton Down).Chemical Biology, a key interdisciplinary area of considerable and growing importance both nationally and internationally as highlighted in the research strategies of EPSRC, BBSRC and MRC. This particular project fits extremely well with the Chemical Biology theme of the RCUK Technology Touching Life initiative as it seeks to develop novel tool compounds with application in Biochemistry and medicine.Importantly, this work will also provide data that has the potential to enhance my REF2021 Impact Case Study currently in development with Dstl 'Influencing Government knowledge and legislation on Biological Weapons'.