Creating an intracellular screening platform for cyclic peptide drug discovery

创建用于环肽药物发现的细胞内筛选平台

• 批准号: EP/Z533002/1
• 负责人: Jody Mason
• 金额: $ 19.11万
• 依托单位: University of Bath
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FZ533002%2F1
• 关键词: Creating; intracellular; screening; platform; cyclic

We propose an innovative, novel, high quality research idea to address a key bottleneck in drug discovery. Our idea is to facilitate a discovery revolution by creating drug-like cyclic peptides inside cells via an exciting idea with significant potential to offer high reward. Cyclic peptides harbour significant potential for translation into drugs since they are ultra-structured despite their small size, non-immunogenic, bioavailable, while offering significant potential for cell permeability. However, their chemical synthesis is often slow and cumbersome, offering poor yields, and undertaken via unsustainable and toxic in vitro chemical means, making their creation and testing for drug discovery a slow and costly trial-and-error process.To address this major bottleneck, we will undertake three major aims:1. A green and sustainable approach to cyclic peptide production - OaAEP1 is an asparaginyl endopeptidase (AEP) that catalyses peptide cyclisation in the plant Oldenlandia affinis, and the engineered C247A variant is employed in our recently established intracellular peptide cyclisation system (Tang & Mason, JACS Au, In press: https://pubs.acs.org/doi/10.1021/jacsau.3c00591). we will apply this biosynthetic approach to generate a suite of therapeutically relevant cyclic peptides of diverse size and structure (e.g. CP1, STFI-1, MCoTI-II, Kalata B1), and therefore validate the novel biosynthetic approach to cyclic peptide production. Chemical synthesis and folding of cyclic cysteine knotted peptides such as McoTI-II and Kalata B1 are known to be challenging and in-cell H2T cyclisation can guide correct cyclotide disulphide bridging.Deliverable: A novel biosynthetic approach will offer a convenient, safe, sustainable, scalable and low-cost alternative to existing chemical methods.2. Optimising OaAEP1 for intracellular applications - We will engineer OaAEP1 by directed evolution to enhance i) solubility ii) ligase activity and iii) ability to function inside cells. This will be achieved by partially scrambling the amino acid sequence of OaAEP1 at solvent exposed positions to generate a 589,824-member library which will be screened for activity using split dihydrofolate reductase (mDHFR). Only ligation of mDHFR fragments will enable reconstitution of activity resulting in cell growth, with subsequent competition selection enriching the most soluble and active OaAEP1 library members.Deliverable: An engineered enzyme optimised for intracellular catalysis. The selection assay also allows rapid engineering of the enzyme by directed evolution to further modify attributes such as substrate specificity.

我们提出了一种创新的，新颖的高质量研究思想，以解决药物发现中的关键瓶颈。我们的想法是通过一个令人振奋的想法来提供高度奖励的巨大潜力，来促进发现革命的革命。循环肽具有将药物转化为巨大的潜力，因为它们的大小很小，不发育，可生物利用，同时具有显着的细胞渗透性潜力。但是，它们的化学合成通常很慢而笨重，产量较差，并通过不可持续的有毒体外化学方法进行，从而使它们的创建和对药物发现进行测试是缓慢且昂贵的试验过程。要解决这一主要瓶颈，我们将承担三个主要目标：1。绿色且可持续的环肽生产方法-OAAEP1是一种白素内肽酶（AEP），它在植物Oldenlandia affinis中催化肽环化，并且在我们最近建立的胞内肽循环系统中使用了工程化的C247A变体（Tang＆Mason，Tang＆Mason，Jacs Auu， https://pubs.acs.org/doi/10.1021/jacsau.3c00591）。我们将采用这种生物合成方法来生成一套具有多种大小和结构的治疗相关的环状肽（例如CP1，STFI-1，McOti-II，Kalata B1），因此可以验证新型的生物合成方法以生产环状肽的生产。众所周知，循环半胱氨酸打结的肽（例如McOTI-II和Kalata B1）的化学合成和折叠是具有挑战性的，并且在细胞内H2T循环化可以指导正确的旋转二硫化物硫化物桥接。可替代：可提供的：一种新颖的生物合成方法，可以提供便利的，可持续的，可持续的，可持续的，可持续的，可持续的，可用的，可衡量的，可提供的化学方法。针对细胞内应用优化OAAEP1-我们将通过定向进化来设计OAAEP1以增强i）溶解度ii）连接酶活性和III）在细胞内部功能。这将通过在溶剂裸露位置处部分争夺OAAEEP1的氨基酸序列以产生589,824成员的文库，该库将使用分裂二氢叶酸还原酶（MDHFR）进行筛选以进行活动。只有MDHFR片段的连接才能重新建立导致细胞生长的活性，随后的竞争选择丰富了最可溶和活跃的OAAEEP1库成员。可替代：一种针对细胞内催化优化的工程酶。选择分析还允许通过定向进化来快速对酶进行快速工程，以进一步修改诸如底物特异性之类的属性。