1
High Throughput Discovery of Novel Peptides with Biological Function F. Billwiller, G. Poloni, B. Marshall, X. Pan, X. Ni and A. Pitt Orbit Discovery Ltd., Oxford, United Kingdom © Orbit Discovery Ltd. 2019 Introduction ORBIT Peptide Display Platform Summary 1. One piece of DNA from a randomised or focused library binds to each bead. 2. DNA amplification on beads within emulsion droplet. 3. Generation of peptides on beads within emulsion droplet. Genotype is linked to phenotype. 4. Screening library is incubated with target protein or cells. 5. DNA sequencing informs of the amino acid sequence of hits. Bioinformatics reveals recurring motifs. www.orbitdiscovery.com PS005 ORBIT Peptide Display - Targeting HIV * Huang, L.-C., et al. (2013). Linking genotype to phenotype on beads: high throughput selection of peptides with biological function. Scientific Reports, 3, 1–9. The ORBIT peptide display platform possesses the positive attributes of traditional methods with the ability to address cell surface targets for the discovery of functional peptide therapeutic leads. Comparison With Other Methods Highly ranked peptides obtained by ORBIT bead-library screening on HIV-1 viral coat protein GP120 were synthesised and tested by ELISA for GP120 binding. Four peptides with the highest affinity to GP120 were tested in a HIV-1 replication assay at three concentrations with or without streptavidin conjugation. Three HIV-1 strains (BAL, IIIB, clade C) were tested (positive control: T20, Enfuvirtide). Surface plasmon resonance (Biacore) was used to measure the KD of the candidate peptides and GP120. The peptides were determined to have KD values between 39 and 80 μM, the Kd calculation for GP16 peptide is shown as an example. • Orbit Discovery is a peptide display technology company founded in 2016 as an Oxford University spin-out. • Peptides are highly active biological molecules with great drug potential, high specificity, low toxicity and low manufacturing costs which currently constitute 2-5% of the drug market. • The ORBIT peptide display platform is highly adaptable allowing for enhancements by the adoption of cutting-edge technologies. • The ORBIT Peptide Display Platform has all the positive attributes of phage display combined with the added flexibility of in vitro display methods. • ORBIT beads can display cyclic peptides and incorporate unnatural amino acids. • The platform is highly adaptable allowing for the adoption of cutting-edge technologies such as next generation DNA sequencing, microfluidics and FACS. • ORBIT peptide display can be utilised for the in situ screening of cell surface targets to identify not just peptides that specifically bind to the target but also those peptides that bind and elicit a functional response from the target. Whole Cell Screening Cells expressing a target linked to a reporter system allow for functional peptide hit identification in the primary screen. 1. Individual cells are encapsulated in droplets utilizing microfluidics technologies. 2. Fluorescent cells indicate GPCR activation by peptides displayed on ORBIT beads. 1. Target + negative control 2. Target + Orbit beads Via FACS we are able to identify and sort specific binding peptides to one or multiple in solution targets. 1. Soluble target with negative control (scaffold beads) 2. Soluble target with ORBIT beads. Positive binders showing increased fluorescence are indicated in green. Incorporation of Unnatural Amino Acids Codon reassignment was used to incorporate fluorescent amino acids into ORBIT displayed peptides. 1. Synthetic tRNAs loaded with BODIPY-labelled cysteine and azido phenylalanine (AzF) were added to an in vitro transcription/translation reaction depleted of endogenous tRNAs. TAMRA-DIBO was used to label AzF via click chemistry. 2. SDS-PAGE images of target proteins containing either one (left lane) or the two (right lane) fluorescent amino acids are shown. In Solution Screening A: Merged image B: 488nm channel for green fluorescence labelled non-natural amino acid 1 C: 546nm channel for blue fluorescence labelled non-natural amino acid 2 2. 1. 1. 2.
