13TSB_SynBio Enhanced discovery and scalable synthesis of therapeutic cyclic peptides

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry

Abstract

Natural products often have very desirable biological and material properties. In the future there will be immense pressure
to produce economically valuable materials with much reduced environmental impact. This means the Industrial
Biotechnology sector (which is related to, but distinct from the Pharmaceutical Industry) in the UK has a golden opportunity
to harness world class science. This proposal links first class academic science with unique expertise available in an
innovative small company, Ingenza. The main aim of the project is to develop a bacterial system (cell factory) for the
production of novel customizable and highly modified cyclic peptides in significant quantities. Cyclic peptides are found as
antibiotics, anticancer agents, in hormone therapy and in immune system modifying agents. In addition to their direct
medicinal role, they are also very useful tools in studying biological processes, this second role, as tools, is
underdeveloped simply because natural products are hard to make in sufficient amounts. The proposed work will solve
these problems by providing a plug 'n' play system in which changes can be made simply and quickly to the 'cell factory' to
produce a vast array of cyclic peptides at a useful scale.

Technical Summary

Natural products often have very desirable biological and material properties. In the future there will be immense pressure
to produce economically valuable materials with much reduced environmental impact. This means the Industrial
Biotechnology sector (which is related to, but distinct from the Pharmaceutical Industry) in the UK has a golden opportunity
to harness world class science. This proposal links first class academic science with unique expertise available in an
innovative small company, Ingenza. The main aim of the project is to develop a bacterial system (cell factory) for the
production of novel customizable and highly modified cyclic peptides in significant quantities. Cyclic peptides are found as
antibiotics, anticancer agents, in hormone therapy and in immune system modifying agents. In addition to their direct
medicinal role, they are also very useful tools in studying biological processes, this second role, as tools, is
underdeveloped simply because natural products are hard to make in sufficient amounts. The proposed work will solve
these problems by providing a plug 'n' play system in which changes can be made simply and quickly to the 'cell factory' to
produce a vast array of cyclic peptides at a useful scale.

Planned Impact

Who might benefit from this research? In terms of economic impact the main beneficiaries will be the UK Industrial
Biotechnology sector. A direct link to an innovative UK biotechnology company, and a letter of support from a large
pharmaceutical company (Astra Zeneca) are clear indications of this. Ingenza's investment shows that the biotechnology
we are developing is of importance and utility to the UK industrial biotechnology sector. We see the technology as enabling benefit by the production of new pharmaceutical lead molecules for diseases which are hard to treat using small molecule
therapeutics.
How might they benefit from this research? The Industrial Biotechnology sector will benefit from adopting new but de-risked
technology. They will benefit from exchange of people and of ideas. The cell based process for the production of complex
cyclic peptides will give rise to new materials which we will test for bioactivity in a number of disease targeted screens. We
have extant screening collaborations with Merck in the US and academic groups in the UK and Europe. These compounds
can then be developed for commercial application and can be licensed or co-developed by industry.
Cyclic peptides are now recognised to be particularly powerful molecules in modulating protein-protein interactions

Publications

10 25 50
 
Description Brief description of feasibility study
This project aimed to develop an in vivo platform to increase the level of patellamide-like compounds production using engineered E.coli, to allow access to > 100 mg of material and thus demonstrate that a patellamide production process could be made economical and feasible to meet future manufacturing needs for pharmaceutically relevant patellamides. The native gene cluster that produces both the PatE precursor peptide itself, and the enzymes necessary for PatE processing and maturation, was used as the source of genetic material. Genes of the cluster were cloned and expressed in E.coli and their individual and concerted activities assessed. We aimed to 1) show novel, scalable patellamide production that could overcome yield limitations, 2) demonstrate the ability to introduce gene orthologues to modify patellamide structure using synthetic biology, 3) demonstrate potential to generate libraries of modified cyanobactins incorporating unnatural amino acids. The project aimed to provide a basis to expect that a class of bioactive molecules, of very high interest to the pharmaceutical industry, could serve as viable leads for new drugs, able to be adapted by enzymatic modification and produced using a platform manufacturing method with a cost of goods acceptable to allow progression to clinical evaluation and launch.
Outcome of study
What did you achieve and is it what you set out to do? If not why? Where are the gaps? What are the deliverables of the study?
Important feasibility demonstrations below, all key deliverables of the project, were achieved during the project.
1. Genes of the Pat cluster were cloned and a series of single and dual cassette expression vectors designed and revised as necessary to express the individual genes and to demonstrate concerted expression of Pat cluster genes.
2. The necessary biological and analytical assays to identify and characterise the products of E.coli engineered with Pat cluster genes were defined and demonstrated.
3. Unnatural amino acids were successfully introduced to modify the PatE peptide using both an intein based method and modified tRNA/amber codon approach.
4. Successful heterocyclization of the target was demonstrated in vivo.
5. Genes encoding additional enzyme activities (prenylase, thiazoline oxidase) able to modify the structure and potential efficacy of PatE were cloned and expressed in E.coli and the gene products demonstrated to be active. These targets were revised from the original plan which included the isolation of genes encoding Pat transporter proteins. These were considered unnecessary since the PatE protein did not prove to have antimicrobial activity towards E.coli.
Exploitation Route The project team established a strong basis of synergy from which to continue this work and were accordingly successful in securing a follow on 3 year IB Catalyst award which will complete the establishment of the scalable heterocycles containing cyclic peptide production system as well as demonstrating the potential to extensively diversify the target compounds. In addition the feasibility study will be expanded to include the development of scalable production systems based on additional native gene clusters for ribosomal peptides, introducing novel enzyme activities to achieve diversification of these targets also. The objective is to obtain compound libraries from multiple natural compounds of high pharmaceutical relevance that can be screened by end users for activity towards targets of interest and which can then be subsequently produced using the production platform engineered in a suitable microbe. We will include additional factors in the future study to address and overcome gene expression difficulties such as codon revision and alternate promoter/RBS evaluation, in the form of combinatorial libraries. We will also concertedly express target genes/enzymes in Pichia pastoris as well as E.coli. During the follow-on project we will establish relationships with the pharma companies who have already expressed significant interest in these targets with a view to sharing data at a suitable point and beginning to solicit input on potential prioritisation of targets or enzymatic activities for product modification or derivatisation. We have already established a collaboration agreement between the original partners to facilitate commercial exploitation. A spin-out company will be formed to embody specific aspects of the biosynthetic technology.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The study allowed a very valuable assessment of the more readily achievable as well as the more challenging steps in establishing a recombinant bacterial system to overproduce and diversify mature patellamide-like products using recombinant E.coli. Clear potential of the recombinant E.coli system to produce PatE using a scalable in vivo system was shown for future application, provided that fairly typical molecular biology hurdles can be overcome. Individual key enzymes of PatE maturation were shown to behave very differently when expressed in E.coli. For example, while heterocylase activity upon PatE could clearly be demonstrated in vivo, expression of other targets did not yield soluble enzyme. The study provided very valuable data on the availability of alternate patellamide processing enzymes, such as oxidases and prenylase activities to potentially impact activity and bioavailability of resulting products. It is notable that the new C-prenylases discovered were a direct result from the collaboration established with Prof Sivonen group and Dr Wael Houssen during his 3 months placement funded by SULSA. The study also established a variety of adaptable expression vectors that can be used as a platform for future reconfiguration of individual gene expression, choice and location of regulatory regions, substituted codon bias or introduction of novel patellamide core sequences. The market potential for the patellamide products remains very significant with an ongoing high level of pharma company interest in these anti-tumour compounds. The project team established a strong basis of synergy from which to continue this work and were accordingly successful in securing a follow on 3 year IB Catalyst award which will complete the establishment of the scalable heterocycles containing cyclic peptide production system as well as demonstrating the potential to extensively diversify the target compounds. In addition the feasibility study will be expanded to include the development of scalable production systems based on additional native gene clusters for ribosomal peptides, introducing novel enzyme activities to achieve diversification of these targets also. The objective is to obtain compound libraries from multiple natural compounds of high pharmaceutical relevance that can be screened by end users for activity towards targets of interest and which can then be subsequently produced using the production platform engineered in a suitable microbe. We will include additional factors in the future study to address and overcome gene expression difficulties such as codon revision and alternate promoter/RBS evaluation, in the form of combinatorial libraries. We will also concertedly express target genes/enzymes in Pichia pastoris as well as E.coli. During the follow-on project we will establish relationships with the pharma companies who have already expressed significant interest in these targets with a view to sharing data at a suitable point and beginning to solicit input on potential prioritisation of targets or enzymatic activities for product modification or derivatisation. We have already established a collaboration agreement between the original partners to facilitate commercial exploitation. A spin-out company will be formed to embody specific aspects of the biosynthetic technology.
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Follow on Fund
Amount £145,781 (GBP)
Funding ID BB/M013669/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description IB Catalyst
Amount £1,600,000 (GBP)
Funding ID 48487-341231 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 09/2015 
End 08/2018
 
Description IBIOIC Exemplar
Amount £142,000 (GBP)
Funding ID NA 
Organisation Industrial Biotechnology Innovation Centre 
Sector Academic/University
Country Unknown
Start 08/2015 
End 07/2016
 
Description Scottis Enterprise High Growth Spinout Programme
Amount £455,000 (GBP)
Funding ID NA 
Organisation Scottish Enterprise 
Sector Public
Country United Kingdom
Start 04/2015 
End 06/2016