Design and Synthesis of DNA-Encoded Libraries of Unnatural Peptides to Discover Specific Chemical Probes

Project Summary
Vast libraries of encoded peptides, including cyclic systems, can be synthesised using biological methods, such as ribosomal expression systems, and can be screened by affinity selection to produce high affinity protein ligands. However, the scope of the aforementioned biological methods is usually limited to proteinogenic amino acids and their close analogues. The incorporation of unnatural moieties would allow for the more specific targeting of selected proteins classes, potentially leading to compounds with greater proteolytic stability and cell permeability. The aim of the proposed project is to develop synthetic approaches to realise this goal by developing chemical, rather than biological, techniques for peptide library synthesis. These techniques lack the requirement of identification by biological machinery and can, therefore, incorporate a far wider range of building blocks. In a second strand, hybrid methodology will be developed which allows the incorporation of unnatural warheads for selected protein classes into biologically displayed peptide libraries.

Chemical Synthesis of Encoded Peptide Libraries
DELs are a technology for the synthesis and screening of a large number of molecules. Each DEL is a collection of these molecules, which are comprised of an organic moiety linked to a DNA oligonucleotide. The "genotype" (the DNA tag) serves as an amplifiable identification bar code for the displayed "phenotype" (the small organic molecule).

Peptoids are oligomers of N-alkylated glycines; a peptoid monomer is an amino acid with the side chain connected to the nitrogen of the backbone, rather than the alpha carbon. Peptoids offer certain advantages to peptides as drugs, incl. resistance to proteolytic degradation due to the tertiary amide, improved cell-permeability relative to peptides due to the absence of the amide NH for H-bonding, and side chain diversity as they can be theoretically synthesised from any given primary amine.

Peptoids are most often synthesised via a solid-supported submonomer method, using bromoacetic acid and then a primary amine. This is an issue, as an ?-halo carbonyl is a powerful electrophile, so will alkylate the DNA, so this method is incompatible with DEL synthesis.

Initial work will focus on the off-DNA synthesis of peptoid monomers and the optimisation of multiple routes towards these building blocks. Consequently, optimised conditions will be applied to a variety of amines (to afford a range of peptoid monomers) and the scope of the reactions elucidated. Concurrently, the on-DNA coupling of peptoid monomers will be established using micellar conditions. This will be developed to realise the scope of the couplings by testing the coupling of the aforementioned synthesised peptoid monomers on-DNA.

This will develop, in further work, to the synthesis of a range of peptoid-containing libraries, beginning with a small linear peptoid library, comprised of a range of the synthesised monomers. Extension of this will lead to the incorporation of building blocks that allow for the control of peptoid conformation, developing into the inclusion of conformational locks and cyclic structures.

Hybrid Biological and Chemical Synthesis
Biological techniques allow for the synthesis of even larger libraries than DELs, pushing the limit of library size to near 1013. The associated work in this strand will revolve around the incorporation of unnatural motifs into cyclic peptide libraries from ribosomal expression. These unnatural motifs, such as a single bromophenylalanine residue, will allow for the coupling of partners, e.g. boronates for Suzuku coupling, with known motifs that bind to certain target proteins. The work will involve the synthesis of said peptide libraries, the coupling of the specific binding motifs, and the subsequent testing of the diverse cyclic peptide libraries.

The CDT has five primary beneficiaries:
The CDT cohort
Our students will receive an innovative training experience making them highly employable and equipping them with the necessary knowledge and skillset in science and enterprise to become future innovators and leaders. The potential for careers in the field is substantial and students graduating from the CDT will be sought after by employers. The Life Sciences Industrial strategy states that nearly half of businesses cite a shortage of graduates as an issue in their ability to recruit talent. Collectively, the industrial partners directly involved in the co-creation of the proposal have identified recruitment needs over the next decade that already significantly exceed the output of the CDT cohort.
Life science industries
The cohort will make a vital contribution to the UK life sciences industry, filling the skills gap in this vital part of the economy and providing a talented workforce, able to instantly focus on industry relevant challenges. Through co-creation, industrial partners have shaped the training of future employees. Additional experience in management and entrepreneurship, as well as peer-to-peer activities and the beginning of a professional network provided by the cohort programme will enable graduates to become future leaders. Through direct involvement in the CDT and an ongoing programme of dissemination, stakeholders will benefit from the research and continue to contribute to its evolution. Instrument manufacturers will gain new applications for their technologies, pharmaceutical and biotech companies will gain new opportunities for drug discovery projects through new insight into disease and new methods and techniques.
Health and Society
Research outputs will ultimately benefit healthcare providers and patients in relevant areas, such as cancer, ageing and infection. Pathways to such impact are provided by involvement of industrial partners specialising in translational research and enabling networks such as the Northern Health Science Alliance, the First for Pharma group and the NHS, who will all be partners. Moreover, graduates of the CDT will provide future healthcare solutions throughout their careers in pharmaceuticals, biotechnology, contract research industries and academia.
UK economy
The cohort will contribute to growth in the life sciences industry, providing innovations that will be the vehicle for economic growth. Nationally, the Life Sciences Industrial Strategy Health Advanced Research Programme seeks to create two entirely new industries in the field over the next ten years. Regionally, medicines research is a central tenet of the Northern Powerhouse Strategy. The CDT will create new opportunities for the local life sciences sector, Inspiration for these new industries will come from researchers with an insight into both molecular and life sciences as evidenced by notable successes in the recent past. For example, the advent of Antibody Drug Conjugates and Proteolysis Targeting Chimeras arose from interdisciplinary research in this area, predominantly in the USA and have led to significant wealth and job creation. Providing a cohort of insightful, innovative and entrepreneurial scientists will help to ensure the UK remains at the forefront of future developments, in line with the aim of the Industrial Strategy of building a country confident, outward looking and fit for the future.
Institutions
Both host institutions will benefit hugely from hosting the CDT. The enhancement to the research culture provided by the presence of a diverse and international cohort of talented students will be beneficial to all researchers allied to the theme areas of the programme, who will also benefit from attending many of the scientific and networking events. The programme will further strengthen the existing scientific and cultural links between Newcastle and Durham and will provide a vehicle for new collaborative research.