Unlocking the potential of engineered C-C bond forming enzymes for biocatalysis

The future success of the global pharmaceutical and chemical industries is dependent on the successful development of efficient, selective and sustainable ways of making new organic molecules with useful properties. Historically, such methods have centered on the use of synthetic organic chemistry to fuse and tailor simple chemical building blocks into a vast array of complex molecular architectures, which may in-turn be used as the basis for amongst other things new drugs, crop protection agents, or materials. Despite the undoubted success of synthetic chemistry, problems exist, including the over-reliance on certain types of reactions that have led to an excessive representation of molecules with predominantly flat, 2D architectures, which are of limited value as drugs. In contrast to chemical catalysts, biological catalysts, termed enzymes, are able to perform challenging chemical reactions that can rapidly build complex 3D chemical structures with multiple bonds under precise stereochemical control. In addition, enzymes can perform such reactions under ambient conditions and without any requirement for environmentally damaging reagents. For these reasons there is significant interest in developing biocatalytic routes to current and future pharmaceuticals and similarly important molecules. A carbon-carbon bond forming reaction known as the Diels-Alder reaction is an effective means of building complex 3D molecules in a single step. However, a limitation of this reaction is that to achieve high yields, stereoselectivity and regioselectivity, the electronic properties of the reactants need to be complementary and often harsh reaction conditions are required. This research project builds on our exciting recent discovery of a naturally evolved co-factor independent enzyme that catalyzes the Diels-Alder reaction at room temperature and on substrates which cannot be transformed using conventional synthetic organic chemistry. This discovery opens up the possibility of using this enzyme to generate a whole new series of complex molecules that could form the basis of new drugs or similarly important chemical compounds. During this project we will establish the practical and theoretical limits of the reactions that this and other related Diels-Alderases can catalyze, we will also rationally re-engineer these enzyme to purposefully change their function to allow access to an even greater variety of products. We will partner these engineered biocatalysts with auxiliary enzymes which catalyze further ring forming reactions to develop routes to industrially useful molecules. This project is a strategically important partnership between the University of Bristol and the pharmaceutical company AstraZeneca, and we will work together to develop natural and engineered enzymes and deploy them to generate a vast array of new 3D molecules that can be used as the basis for new drugs to treat a diverse array of human diseases.

The Diels-Alder reaction, a [4+2] cycloaddition of a conjugated diene to a dienophile, is widely recognised as one of the cornerstone synthetic organic reactions of the 20th century. It is commonly employed in the synthesis of bioactive natural products and in the rapid construction of sp3 rich cyclic and polycyclic compounds. This reaction opened the way to the synthesis of numerous important pharmaceuticals including the anti-viral agents Tamiflu and Peramivir, the anti-fungal Tolciclate, and opiates including Morphine and the smoking cessation aid Varenicline. It has also been widely used in the preparation of vitamins, steroid hormones, agrochemicals (Isopyrazam, Bixafen, Cycocel), and numerous fragrance and flavour compounds. Despite its unquestionable usefulness, the versatility of this reaction is limited by its distinctive steric and electronic requirements, along with the harsh reaction conditions which often must be employed. By contrast, the development of protein catalysts for this reaction remains a major goal, as access to enzymes capable of catalyzing Diels-Alder reactions under ambient conditions and in the absence of co-factors, would enable new, green routes to a wide variety of valuable chemical building blocks, natural products and lead scaffolds. In this academic-industrial LINK project, which builds on substantive collaborative research undertaken by the academic PI and Co-Is in the identification and characterisation of natural Diels-Alderases and associated ring forming enzymes, the researchers will:(i) Answer pressing, unresolved fundamental questions, regarding the enzymology of naturally evolved Diels-Alderases, (ii) undertake structure and simulation-guided reengineering and directed evolution of these enzymes; and (iii) deploy this portfolio of biocatalysts, in combination with auxiliary enzymes, to enable the preparation of chemical building blocks, pharmaceutical lead scaffolds and bioactive natural products.

This project will deliver (i) detailed molecular insight into a poorly understood and hitherto unexploited group of naturally evolved biocatalysts, (ii) a suite of engineered variants of these enzymes with modified substrate selectivities and catalytic properties, and (iii) a portfolio of new bioactive molecules and lead compounds for further investigation and exploitation. As such we consider this research to have potential broad ranging intellectual, economic and potential clinical impact. The outlined research programme will contribute substantially to the UK's global leadership in the areas of biocatalysts and drug discovery, and aligns well with current BBSRC strategic priorities and cross-council initiatives. The applicants are committed to ensuring that the outputs from this research impact upon policy-makers, funding bodies, academic institutions, and industry by providing clear evidence of the value of strategically aligned fundamental and applied interdisciplinary research.

This is a collaborative LINK project with the multi-national pharmaceutical company AstraZeneca (AZ), with whom we will work in partnership to realise the impacts of this research. This will be through the generation of IP and licensing agreements centered on the enzymes and small molecules that will be generated. The project will establish a strategically important partnership between UK academia and a multi-national pharmaceutical company in an application area that is of major economic significance to the UK. The outcomes of this project are of direct strategic relevance to AZ's future research strategy and will deliver significant industrial and commercial impact. Should any of the new compounds isolated during the course of this study prove to be useful drug leads we will work with AZ to deliver the potentially significant resulting medical and industrial impact. A key output of this work will also be the establishment of general methods to advance our fundamental understanding of enzyme catalysis and our ability to manipulate naturally evolved enzymes to produce new chemical compounds. These discoveries will impinge on the emerging field of synthetic biology, and have the potential to transform industrial processes and practices specifically in the areas of biotechnology and pharmaceutical development. We will maintain an open dialogue with key industrial stakeholders and policy makers during the lifetime of the project, such that they are informed and in a position of readiness to act appropriately as outputs emerge.

The outlined programme will offer those involved (PDRAs, students, etc.) experience and training at the chemistry-biology interface, providing them with a range of technical and intellectual skills required to succeed in careers in academia, industry or the third sector. The research outputs from this program will be reported to the wider scientific community through publication in leading peer-reviewed journals, presentation at national and international conferences and at meetings with industrial and academic collaborators. Training will be given to those involved in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of widely transferable skills. Further, significant opportunities exist for the presentation of research findings by all those involved to a more general audience through public engagement activities organised by the University of Bristol and AZ (school talks, science festivals, etc.). Such activities will ensure that as broad an audience as is feasible will be informed of our ongoing research.