Creating an intracellular screening platform for cyclic peptide drug discovery

我们提出了一个创新的、新颖的、高质量的研究想法来解决药物发现中的一个关键瓶颈。我们的想法是通过一个具有巨大潜力提供高额回报的令人兴奋的想法，在细胞内创造类似药物的环肽，从而促进一场发现革命。环肽具有很大的药物转译潜力，因为它们是超结构的，尽管它们尺寸小，不具免疫原性，可生物利用，但提供了巨大的细胞通透性潜力。然而，它们的化学合成通常是缓慢和繁琐的，产量很低，并且通过不可持续的和有毒的体外化学方法进行，使得它们的创造和药物发现的测试是一个缓慢和昂贵的反复试验过程。为了解决这个主要的瓶颈，我们将进行三个主要目标：1.一种绿色和可持续的环肽生产方法-OaAEP1是一种天冬酰胺基内肽酶(AEP)，它催化植物白花蛇舌草中的多肽循环，而工程C247A变体被用于我们最近建立的细胞内肽循环系统(Tang&Mason，Jacau，印刷：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套具有不同大小和结构的治疗相关环肽(例如CP1、STFI-1、MCoTI-II、Kalata B1)，从而验证新的生物合成方法用于环肽生产。众所周知，McoTI-II和Kalata B1等环半胱氨酸结肽的化学合成和折叠是具有挑战性的，细胞内的H2 T环化可以指导正确的环肽二硫化物桥联。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用于细胞内应用-我们将通过定向进化来改造OaAEP1，以提高I)溶解性II)连接酶活性和III)在细胞内发挥作用的能力。这将通过在暴露于溶剂的位置部分扰乱OaAEP1的氨基酸序列来生成589,824个成员的文库，该文库将使用分裂的二氢叶酸还原酶(MDHFR)进行活性筛选。只有连接mDHFR片段才能重建活性，导致细胞生长，随后的竞争选择丰富最可溶性和最活跃的OaAEP1文库成员。交付：一种优化的工程酶，用于细胞内催化。选择试验还允许通过定向进化对酶进行快速工程，以进一步修改属性，如底物专一性。

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.