Development of cobalamin surrogates as probes and carriers through synthetic and chemical biology approaches

Synthetic biology holds the potential to expand the chemical repertoire of natural products through a combinatorial reshuffling of metabolic enzymes. Whilst such an approach has been predicted to escalate the library of modular-constructed antibiotics, such as polyketides, a similar approach for the modification of coenzymes and cofactors should also be considered. This approach could produce compounds that act as powerful inhibitors that interfere with specific cellular processes. To this end we have outlined a method for the modification of cobalamin, vitamin B12, which will lead to the synthesis of a number of analogues. These analogues will be constructed through re-engineering the cobalamin biosynthetic pathway allowing the isolation of stabilised intermediates. These will then be subject to chemical biology approaches whereby cofactors with expanded activity will allow the introduction of new functional groups into the corrin macrocycle.
Initially, the first phase of the project will be to make metal isosteres of cobalamin, whereby the central cobtalt ion will be replaced by either rhodium or iridium. This will be achieved by using genetically engineered strains of E. coli to make hydrogenobyrinic acid a,c-diamide. The alternative metal will then be chelated into this compound non-enzymatically. An electrochemical approach will allow the adenosylation of the metal to generate the adenosylcobyrinic acid equivalent. Enzymatic amidation will allow the transformation into the adenosylcobyric acid counterpart, which can be converted into the final coenzyme form by either a chemical reaction or through biotransformation using a suitable genetically engineered strain.
To complement this work we will also construct a number of cobalamin variants that will be generated either through an enzymatic combinatorial approach or by employing S-adenosylmethionine SAM analogues containing extended functional groups. For instance, by dissecting and shuffling enzymes it is possible to make hydrogenobyrinic acid missing the C5 methyl group or containing a lactone on the C8 position. By using SAM analogues containing alkenyl, alkynyl and keto substituents it will be possible to activate the C5 and C15 positions of the corrin macrocycle. Such modification of the corrin molecule will allow for attachment of a range of chemicals/drugs.
All the cobalamin surrogates will be investigated for their ability to interact with cobalamin binding proteins and to be taken up by a range of cells. Their effect on cobalamin-dependent enzymes will also be examined as will their ability to interact with cobalamin riboswitches. Such a comprehensive study will allow us to determine the potential of these molecules as anti-bacterial agents and biotherapeutics.

The project deals with the advancement of synthetic biology and especially its application in the field of novel biotherapeutics generated by the re-design of an existing, natural biological system. In this instance we are planning the re-design of cobalamin, vitamin B12, the anti-pernicious anaemia factor. Many organisms are dependent upon this nutrient for key metabolic enzymes such as methionine synthase, methylmalonyl CoA mutase and ribonucleotide reductase. The key component of cobalamin is the centrally chelated cobalt ion, which generates a unique cobalt-carbon bond in the major biological forms giving rise to methylcobalamin and adenosylcobalamin. By changing the chemical properties of this organic-metal linkage the properties of the molecule will be greatly disrupted, preventing the cofactor/coenzyme from participating fully in its associated biochemical process and allowing it to act as a highly selective inhibitor. In the first part of the project we will make metal isosteres of cobalamin, where the central cobalt ion will be replaced by other group nine metals such as rhodium and iridium. In terms of cellular molecular recognition the coenzyme forms of these cobalamin surrogates, adenosyl-rhodibalamin and adenosyl-iridibalamin, will be indistinguishable from adenosylcobalamin but the differences in the redox potential of rhodium and iridium will prevent these molecules completing any catalytic turnover.
A second aspect of the project deals with the synthesis of cobalamin variants containing different functional groups on the periphery of the tetrapyrrole framework. Cobalamin has evolved to have a largely inert molecular scaffold, making it difficult to attach drugs or chemicals to the vitamin. The reason to do this is to enable cobalamin can act as a Trojan Horse, to smuggle components into cells by hitching a lift via the body's natural B12 uptake mechanism. Such analogues will be investigated for their toxicity on pathogenic bacteria and cancer cells.

The research described in this application will have a major impact on several areas of science, including synthetic and chemical biology and the development of novel biotherapeutics. It will permit the generation of a library of compounds that are based on a natural biological scaffold. It will also help expand the use of vitamins as carriers for transport into cells.
The research also describes a simple but highly efficient method for the isolation of pathway intermediates, allowing for their further modification and generation of surrogate nutrients. This method will be applicable to a broad range of natural products and should help promote the idea of developing antibiotics based on the molecular blueprint coenzymes and cofactors. With an increase in the interest especially of secondary metabolites such an approach is likely to prove popular with chemical biologists and medicinal chemists alike.
The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. In essence, the project employs the re-design of existing, natural biological systems for useful purposes. The research also has the potential engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will have application in the biomedicine and bioprocessing of pharmaceuticals and nutrient.
The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the biosynthesis of cobalamin. It will demonstrate how cofactor analogues can be used to introduce functional groups into natural products. We will ensure that our findings are widely disseminated, through example short review articles. Furthermore, there is no doubt that the research will be of significance to those devising new strategies against disease and thus we will ensure that our findings are disseminated to those working in drug development.
The Kent and Queen Mary groups are heavily involved in outreach programmes, through interactions with local schools and community groups. Both are member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues.
The skills acquired by those involved in this project include not only a wide range of important biological techniques ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology but also the chance to make a significant contribution towards the development of biotherapeutics. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the new Centre for Molecular Processing within the University of Kent, the research will be brought to the attention of many leading industrial companies.