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.

我们提出了一个创新的，新颖的，高质量的研究思路，以解决药物发现的关键瓶颈。我们的想法是通过一个令人兴奋的想法，在细胞内创造类似药物的环状肽，从而促进一场发现革命，这个想法有很大的潜力，可以提供高回报。环肽具有转化为药物的巨大潜力，因为尽管它们体积小，但它们具有超结构，非免疫原性，生物可利用性，同时具有显著的细胞渗透性潜力。然而，它们的化学合成通常缓慢而繁琐，产量很低，而且是通过不可持续和有毒的体外化学手段进行的，这使得它们的创造和药物发现的测试成为一个缓慢而昂贵的试错过程。为解决这一重大瓶颈，我们将采取三个主要目标：一种绿色和可持续的方法来生产环肽- OaAEP1是一种天冬酰胺内肽酶（AEP），在植物亲和叶蕨中催化肽环化，工程C247A变体被用于我们最近建立的细胞内肽环化系统（Tang & Mason, JACS Au, in press: https://pubs.acs.org/doi/10.1021/jacsau.3c00591）。我们将应用这种生物合成方法来产生一系列不同大小和结构的治疗相关的环肽（例如CP1， STFI-1, MCoTI-II, Kalata B1），从而验证这种新的生物合成方法来生产环肽。环半胱氨酸打结肽如McoTI-II和Kalata B1的化学合成和折叠是具有挑战性的，细胞内H2T环化可以指导正确的环酰二硫桥接。可交付：一种新的生物合成方法将为现有的化学方法提供一种方便、安全、可持续、可扩展和低成本的替代方法。优化OaAEP1在细胞内的应用-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iii)细胞内功能的能力。这将通过在溶剂暴露位置部分打乱OaAEP1的氨基酸序列来实现，以产生589,824个成员的文库，该文库将使用分裂二氢叶酸还原酶（mDHFR）筛选其活性。只有连接mDHFR片段才能实现活性的重建，从而导致细胞生长，随后的竞争选择丰富了最可溶性和活性的OaAEP1文库成员。可交付：一种优化的用于细胞内催化的工程酶。选择试验还允许通过定向进化对酶进行快速工程，以进一步修改底物特异性等属性。