Assembly-line biosynthesis of polyethers that selectively kill cancer stem cells

The motivation for this research arises from a wholly unexpected collision between two previously-unlinked fields of biological science, between the way in which a certain large group of natural antibiotics called polyethers are produced in Nature; and the problem of how to stop cancers which have initially responded to therapy, from coming back in a resistant form and killing the patient. Polyethers are antibiotics, whose clinical use has been restricted by their relative toxicity and by the difficulty of synthesising them or modifying them chemically, but which have already been recently discovered to be highly effective against drug-resistant malarial parasites, a major global health threat. There was therefore already great interest in developing new biological ways of synthesising libraries of such molecules to test as the starting point for potentially improved drugs of lower toxicity. Here in Cambridge, with previous BBSRC support, we have been the first to define the genes and enzymes involved in constructing polyethers. To build up such complex small molecules from the simple building blocks inside bacterial cells requires multiple steps, each one catalysed by an enzyme. Some of these are physically tethered together into massive multienzyme complexes, the most complex biological catalysts so far discovered on the planet, but all are orchestrated to provide a smooth cascade or chain of reactions so that nothing is wasted and typically a single end-product is made. Meanwhile, the latest explanation for the return of cancers is that a small proportion of the tumour consists of so-called cancer stem cells (CSCs) which are more resistant to therapy and which remain behind, to seed the regrowth of the tumour in virulent form. If this is true, it is argued, then what is needed is a drug to specifically kill CSCs, to combine with existing drugs that kill non-CSC cancer cells. Obviously normal stem cells are precious and damaging them gives serious side-effects. Accordingly, in a sophisticated cell-based biological screen, biologists at MIT and Harvard have sifted a large library of chemical compounds (16,000) to see if any would kill the CSCs but not normal stem cells. It turned out that this was a relatively rare property, only four compounds (all natural products) passed the test, and two of these (including the very best, salinomycin,) were - to general surprise - polyethers. We aim in this project to take the polyether construction rules we have learned and apply them to the salinomycin pathway, to define that and related pathways and initiate biochemical engineering of these pathways to generate altered versions of salinomycin that might be even more specific for CSCs and might serve as leads in cancer drug development, with a major impact on human quality of life. We intend to do this in a partnership with Biotica, an established biotech company spun out of the University of Cambridge. Their role will be to evaluate the results, to test any compounds that we make, and (if the research is sufficiently promising) to take the project forward as a discovery program and hopefully into commercial development.

The biosynthesis of complex antibiotic polyketides such as polyethers by multienzyme pathways in actinomycete soil bacteria represents one of the best studied examples of assembly-line enzymology, in which multiple enzyme activities are orchestrated to produce a specific chemical and stereochemical outcome in the final product. The starting point for this project is an unexpected link that has been recently made between polyethers and the study of cancer stem cells (CSCs), tumour cells resistant to conventional therapy, which later seed tumour regrowth. In previous work, funded by BBSRC, we have successfully defined the roles of most of the genes and enzymes involved in polyether biosynthesis. Meanwhile, 16,000 chemical compounds have been screened to identify any that selectively kill CSCs but not normal stem cells. This selective activity was found to be a relatively rare property, shared by only four compounds (all natural products), two of which (including the very best, salinomycin,) were - to general surprise - polyethers. The polyether construction rules we have learned will be applied to the salinomycin pathway. We will clone the genes for the pathway, define the mechanisms of chain growth, tailoring and release in this and related pathways (for nigericin and lasalocid), using specific blocked mutants to accumulate and identify intermediates. We will study the specificity and detailed mechanism of novel enzymes in the pathway; and initiate biochemical engineering of these pathways, using where possible industrially-improved overproducing strains of the polyether-producing organisms to increase productivity. Any altered versions of polyethers will be tested by our industrial partner, Biotica Technology, for their effects on CSCs and other cancer cells. Such compounds might help delineate the mechanism of the effect of polyethers on these cells, and serve as additional leads in cancer drug development.

Who will benefit? The chief and direct beneficiaries of this research over the timescale of the next 5-10+ years are in the private commercial sector, especially in the pharmaceutical industry and in biotechnology. The most obvious and proximal beneficiary, over the next 2-5 years, and given that this is an Industrial Partnership Award application, will be the partner organisation Biotica (www.biotica.com), a Cambridge-based University spin-out company I co-founded in 1996 and whose mission is to commercialise drugs based on engineered polyketide natural products. The size of the potential clinical and commercial impact is tremendous. Both Wyeth (Toricel) and Novartis (Afinitor) have launched polyketide drugs in the last 18 months for advanced and refractory cancer, and both are expected to become blockbusters at their peak (sales > $1 Billion/annum). However, the failure rate in clinical trials is extremely high and the timescales for such success may be 10-15 years from the completion of the scientific work. These particular direct beneficiaries also hold the key to an important long-term indirect benefit for the next generation of scientists being trained in the UK. If opinion-formers in the pharmaceutical industry do not see high levels of scientific creativity and cutting edge technologies being developed and maintained in the UK's universities and research institutes, the trend for such companies to relocate their activities elsewhere will be reinforced, a long-term threat to skilled scientific employment in the UK, which should concern us all. Other beneficiaries are the general public: this project promotes a knowledge-based platform for producing next generation pharmaceuticals and related products, using clean and sustainable fermentation technology. This has many environmental and societal benefits over alternative synthetic routes to such products, which rely on petrochemicals. How will they benefit? For this project, Biotica and Cambridge University will sign a Collaboration Agreement which will specify precisely the responsibilities of the parties, and how the outputs of the project will be managed for maximal impact. I would expect Biotica, in addition to their binding themselves to contribute over 10% of the gross budget by paying £40,000 in cash, to also offer some or all of the following 'in-kind' contributions to strengthen the link between the partners: advice on accessing patent literature; technical advice on specialised fermentation; and screening at their cost of any compounds generated by the project for their activities in specified assays and indications. We will ensure regular technical briefing of company staff on the progress of the project. The Collaboration Agreement will also regulate the proper management of foreground IP, and will specify the financial terms. There will be safeguards for future academic use of the IP and for publication of the results as soon as important IP has been protected by patent applications; as well as for the ability of the University to offer the outputs to alternative industry beneficiaries if Biotica decides not to take the project forward. Full attention to this early step is easily the most vital part of the impact plan. Finally the PDRA and the (part-time) technician who will be recruited on this project will develop key interdisciplinary skills which will be extremely valuable to future industrial employers. What will be done to ensure that they benefit? As PI I will take active responsibility for identification and protection of IP generated in this project, working closely with Cambridge Enterprise, in order to maximise its exploitation. I will also work closely with the university press office in Cambridge to ensure our work is disseminated when appropriate by press release, to accompany a significant publication in a major international journal.