Creating an intracellular screening platform for cyclic peptide drug discovery

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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.