Next Generation Bicycle Scaffolds: Phage Selection With Added Functionality

BicycleTx有限公司是一家生物技术公司，旨在开发一种基于硫醚桥接双环肽（bicycle）的新治疗方式。他们的技术是基于筛选合成约束噬菌体编码的组合肽库M13噬菌体显示短肽库（9 - 20个残基），其中包括3个Cys残基，通过Cys- sh与活性官能团在c3对称小分子支架上的反应交联，得到在pIII外壳蛋白上表达的构象受限的双环肽。然后针对一系列生物靶点筛选这些Bicycle文库，以鉴定具有高亲和力和选择性的肽序列。这项强大的技术使BicycleTx能够开发新的先导物，以解决肿瘤学，免疫肿瘤学和抗菌治疗中的顽固性疾病和不可药物靶点。一个关键的限制是目前不可能筛选具有附加功能的Bicycle文库，例如细胞穿透肽，将Bicycle固定在膜上的脂质，或用于革兰氏菌主动摄取的铁载体。这将使筛选得到的bicycle偶联文库以结合其目标成为可能，其中选择过程固有地包括细胞渗透，膜表面定位或革兰氏菌的摄取。在这个项目中，学生将设计和合成新的双功能，c3对称支架承载(i)三个相同的基团共价连接到三个噬菌体Cys-SH和（ii）一个或三个“点击”手柄附加额外的功能。这些下一代c3对称自行车支架将被设计成具有烷基化基团，可以在温和的水条件下与噬菌体肽反应；点击手柄将允许脂质、多肽和铁载体的双正交附着。结构简单的支架将被用来避免与噬菌体肽的其他区域发生副反应。将合成和测试两种类型的支架：基于金刚烷核心的刚性支架，它将具有三个对称的烷基化基团和第四个附着点，用于附加功能的生物偶联；以及对称环六肽基支架，其中三个基团可与Cys-SH结合，另外三个用于连接附加基团。然后，学生将用噬菌体文库测试这些支架，以优化与支架结合的效率，然后筛选包含这些支架之一的文库，以结合治疗相关的靶标。将对高亲和脚踏车进行核磁共振/ x射线研究，以确定新支架对约束肽构象的影响。这些下一代自行车将结合生物的药理学特性与小分子的药代动力学和合成优势，使一系列治疗相关的目标得以解决，包括许多迄今为止无法用小分子药物治疗的目标。提议的工作显然适合BBSRC推进生物科学发现目标的前沿（特别是变革技术的优先事项）。它与药物和技术开发研究领域保持一致，并对对抗抗菌素耐药性的优先领域具有潜在影响。自然化学，2004,22 （2009）中国生物医学工程学报，2016,33(4):481 - 481。

BicycleTx Ltd is a biotech company founded to develop a new therapeutic modality based on thioether bridged bicyclic peptides (Bicycles). Their technology is based on screening synthetically constrained phage encoded combinatorial peptide libraries.1 M13 phage displaying short peptide libraries (9 - 20 residues) including three Cys residues are cross-linked through reaction of the Cys-SH with reactive functional groups on a C3-symmetrical small molecule scaffold to give conformationally constrained bicyclic peptides expressed on the pIII coat protein. These Bicycle libraries are then screened against a range of biological targets to identify peptide sequences with high affinity and selectivity. This powerful technology enables BicycleTx to develop novel leads to tackle intractable diseases and undruggable targets in oncology, immunoncology and antimicrobial therapeutics.2A critical limitation is that it is not currently possible to screen Bicycle libraries with additional functionality, such as cell penetrating peptides, lipids to anchor the Bicycles to membranes, or siderophores for active uptake to Gram -ve bacteria. This would make it possible to screen the resulting Bicycle-conjugated libraries for binding to their targets, where the selection process would inherently include cell penetration, membrane surface localisation, or uptake to Gram -ve bacteria.In this project, the student will design and synthesise novel bifunctional, C3-symmetric scaffolds bearing (i) three identical groups for covalent attachment to the three phage Cys-SH and (ii) one or three "click" handles to attach additional functionality. These Next Generation C3-symmetric Bicycle scaffolds will be designed with alkylating groups that can be reacted with the phage peptide under mild aqueous conditions; the click handles will allow biorthogonal attachment of lipids, peptides and siderophores. Structurally simple scaffolds will be used to avoid side-reactions with other regions of the phage peptide. Two types of scaffolds will be synthesised and tested: rigid scaffolds based on an adamantane core, which will have three symmetrical alkylating groups and a fourth point of attachment for bioconjugation of the additional functionality; and symmetrical cycle hexapeptide-based scaffolds with three groups that can be conjugated to the Cys-SH and three for attachment of the additional groups. The student will then test these scaffolds with phage libraries to optimise the efficiency of conjugation to the scaffolds, and may then screen a library containing one of these scaffolds for binding to a therapeutically relevant target. NMR/X-ray studies of high-affinity Bicycles will be carried out to determine the effects of the new scaffolds on the conformation of the constrained peptides.These next generation Bicycles will combine the pharmacological properties of a biologic with the pharmacokinetic and synthetic advantages of a small molecule, enabling a range of therapeutically relevant targets to be tackled, including many that have so far been undruggable with small molecules. The proposed work is a clear fit to the BBSRC Advancing the frontiers of bioscience discovery objective (in particular the transformative technologies priority). It is aligned to the Pharmaceuticals and Technology Development research areas, and has potential impact relating to the combatting antimicrobial resistance priority area.1. Nature Chem Biol 5, 502 (2009) 2. J Med Chem 63, 4107 (2020)