Biosynthesis of five-membered heterocyclic rings

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry

Abstract

Many of todays drugs that we rely on for treatment of cancer, bacterial infection, immune disorders and viral infections are either natural products or are derived from natural products. Natural products remain, even today, a source of drugs and diagnostic molecules. In contrast to man made chemicals natural products are complex in terms of shape and composition. This structural novelty is in part the reason that they work in specific ways, (less side effects). In general, these natural products are made in bacteria rather than in humans or animals. Humans have evolved to ditch much of their complex chemistry. We need vitamins in food, because we cannot make them; rather rely on bacteria or plants to make them and we eat them. Bacteria have an amazing repetoire of complex chemistry that organic chemists can only dream of. In making new man made materials, a key challenge is often to identify plausible new scaffolds or skeletons. Molecules which are easy to draw are hard to make. Yet new scaffolds and motifs (known as chemical diversity) is at the heart of drug discovery. We are going to study the bacterial enzyme that makes heterocyclic amino acids. These five membered rings are a common motif in biologically active compounds but there does not exist any good way of making them in natural products by synthetic chemistry. The use of bacterial enzymes to accomplish chemical tasks is very well known, washing powder being the best known example but they are widespread in the food industry and increasingly the organic chemistry lab. By working out how the enzyme which makes five membered rings works, we will gain control of the enzyme. By doing this we will be able to make novel materials and we believe completely new biologically active compounds. What is more these enzymes work in water at room temperature without producing noxious waste materials.

Technical Summary

We propose to investigate the mechanism of the formation of heterocycles by enzyme. These five membered rings are critical components of many important biologically active molecules. We have made significant progress in determining the chemical mechanism, which is the first step to harnessing the enzyme. We intend to pursue the mechanism by novel labeling strategies including stable isotope labeling of substrates. We will also probe the basis of recognition using NMR approaches to identify the crucial residues involved in binding substrate to the enzyme. This is important because in the long term we would wish to replace amino acids by non peptide like groups. We have shown that NMR appears to detect intermediates that form during the reaction. The oxidation state of the rings is important, since even this subtle modification controls activity and stability of the compounds. We have shown that air can be sued to replace in part the enzyme mechanism. We intend to develop further the chemical route but also to study the enzymatic route. There are important and puzzling differences between enzymes that catalyse this reaction. The ability to control the stereocentres of amino acids is also central the activity of these compounds. The mechanism by which residues adjacent to thiazolines are epimerised is unknown. We will determine whether it is spontaneous or enzyme catalysed. We have developed an approach using a combination of peptide synthesis and protein ligation that will allow us incorporate non natural amino acids. This will not only give us exquisite control of the biochemical experiments but lead to more interesting chemical scaffolds in the future.

Planned Impact

Who might benefit from this research? In terms of economic impact the main beneficiaries will be the UK Industrial
Biotechnology sector. We see the technology as enabling new biologically active molecules. The adoption of new 'greener'
biotransformation processes will enable the difficult transformations required for the production of fine chemicals and
pharmaceuticals. The pharmaceutical industry in the UK generates a positive trade balance, and innovation in these
sectors is critical for the success of the UK as a whole. Society will benefit by the production of new pharmaceutical lead
molecules.
How might they benefit from this research? The Industrial Biotechnology sector will benefit from adopting new but de-risked
technology. Understanding this very useful flexible enzyme will give rise to whole new materials we test for bioactivity in a
number of disease targeted screens. These compounds can then be developed for commercial application and can be
licenced or co-developed with industry. There is an urgent need for a diverse arrays of complex molecules to refill the
pharmaceutical drug discovery pipelines. Our approach will produce materials that can be modified easily and thus tuned to
a particular application. New materials with unusual properties will also be produced as part of this work and these may
provide new products or ideas for new products. The production of heterocycles is difficult to achieve synthetically and
often gives low yields despite the use of large quantities of reagents. The use of efficient biotransformation enzymes will
reduce the use of chemical reagents, solvents, energy and waste products.

Publications

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BrĂ¡s NF (2016) The Catalytic Mechanism of the Marine-Derived Macrocyclase PatGmac. in Chemistry (Weinheim an der Bergstrasse, Germany)

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Houssen WE (2014) An efficient method for the in vitro production of azol(in)e-based cyclic peptides. in Angewandte Chemie (International ed. in English)

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Jaspars M (2014) The origins of cyanobactin chemistry and biology in Chem. Commun.

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Koehnke J (2014) The structural biology of patellamide biosynthesis. in Current opinion in structural biology

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Oueis E (2016) Enzymatic Macrocyclization of 1,2,3-Triazole Peptide Mimetics. in Angewandte Chemie (International ed. in English)

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Oueis E (2017) Bypassing the proline/thiazoline requirement of the macrocyclase PatG. in Chemical communications (Cambridge, England)

 
Description We have engineered enzymes that can make entirely new molecules. The molecules are hydrids of peptides and organic molecules. These molecules have value as drugs and diagnostics. We have formuated an entirely new process for making complex macrocycles. Along the way there are many key enabling findings. These include the characterisation of the kinetics of the enzymes that control modification of peptides including macrocyclisation. We have also presented findings on how the enzymes in the pathway control the timing of the formation of modifications. These findings were crucial in moving to reach the key finding, the making of a new molecule.
Exploitation Route We are going to try form a company. The company is called GyreOx.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have been able to solve the structure of a novel protein that may be involved in the epimerisation of amino acids. This is important as controlled epimerisation is chemically very useful in the pharmaceutical industry. Our work has inspired new efforts in peptide drug discovery. We have shown that it is possible to combine organic chemistry to make highly diverse and modifiable macrocycles. This has changed how people think about peptides in medicine.
First Year Of Impact 2014
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title An engineered heterocyclase 
Description The engineering of an enzyme that can generate azoline heterocycles from dipeptides containing XC, XS or XT by attaching the precursor peptide leader to the N terminus of the native enzyme thus activating it permanently. 
IP Reference GB1419650.5 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact Start-up company Ripptide formed in Sept 2015, which will formally in-license the IP in July 2016.
 
Company Name Ripptide Pharma 
Description Ripptide Pharma has developed a chemoenzymatic process for the efficient production of macrocycles and cyclic peptides, addressing entirely novel areas of chemical space for drug discovery and development. Macrocycles are new therapeutics in areas only currently accessible through biologics, such as protein-protein interactions. Unlike biologics they can be administered orally and access therapeutic targets within the cell. Our technology is available to partners to discover or optimise macrocycles in their areas of therapeutic interest Ripptide is advancing its in-house drug discovery/development activities in unmet areas of inflammation, autoimmune disease and cancer. 
Year Established 2015 
Impact None yet, we will in-license IP formally in July 2016
Website http://www.ripptide-pharma.com
 
Description Build a medicine 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Led to further invitations to have a stand at European Researcher's night and to present to schools liaison officers

NA
Year(s) Of Engagement Activity 2014
URL http://www.facebook.com/marinebiodiscoverycentre