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Oxford Innovative Organic Synthesis in Cancer Research Doctoral Programme

Final Report Summary - OXIOSCR (Oxford Innovative Organic Synthesis in Cancer Research Doctoral Programme)

PROJECT CONTEXT AND OBJECTIVES
Over the last 50 years, many small molecules isolated from natural sources have been shown to possess anticancer activities. Of all the New Chemical Entities (NCEs) so far approved for oncology more than half are natural products or related molecules – substances that are generally found in, or produced by, living organisms such as plants, fungi or bacteria. Many examples are successful marketed drugs – including vinblastine, etoposide and taxol – which have been shown to act via a diverse range of mechanisms. Despite their potential, the development of promising compounds towards a clinical application is often hindered by poor bioavailability and toxicity, and by the inability to access sufficient quantities of material. The development of sustainable synthetic routes towards these compounds would address many of these issues and help to pave the way for a new generation of natural product-derived anticancer agents.

Oxford Innovative Organic Synthesis in Cancer Research (OxIOSCR), an Innovative Doctoral Programme (IDP) under the Framework Seven Marie Curie Initial Training Network (ITN) programme, had the overarching objective to train 13 Early Stage Researchers (ESRs) – all graduate organic chemists from across Europe – to develop optimal synthetic routes to natural products and analogues with anticancer activities. Cancer cells do not look and behave like normal cells of the body; they may replicate uncontrollably, grow more rapidly, and develop mechanisms to avoid dying; this uncontrolled growth can form a collection of aggressive cancer cells called a tumour. The research and training activities within OxIOSCR were all designed to support initiatives in chemistry and oncology to underpin our understanding and clinical treatment of the diverse set of diseases known collectively as ‘cancer’. These activities were conducted primarily within the Chemistry Research Laboratory, University of Oxford in collaboration with teams from the University’s Department of Oncology and supported by our Associated Partners (APs): (CLEA Technologies, Gensoric GmBH and SUNY at the University of Albany, Institute of Microelectronics of Barcelona (IMB-CNM), LIOS, Novartis Institutes for Biomedical Research, Prous Institute of Biomedical Research (Prous IBR), TU Delft, and Sorso Ltd).

WORK DESCRIPTION AND FINAL RESULTS
The 13 ESRs were grouped into four research teams in which research was focused on four classes of natural products and their analogues associated with proposed activities in cancers: (i) Restoration of programmed cell death (apoptosis): in response to stress or damage, normal cells in the body undergo a regulated process of cell death called apoptosis. In some cancer cells there are raised levels or activities of certain anti-apoptotis proteins (eg Bcl-2 and Bcl-xL), which contributes to an uncontrolled proliferation of cells. The goal of this sub-project (Project 1, WPs 1–3) was for the ESRs to study the synthesis of natural products such as incednine and its analogues, shown to inhibit anti-apoptotis proteins in cancer cells; (ii) Inhibition of proteasome activity: Proteasomes are complex biological molecules, whose function in normal cells is the breakdown of unneeded or damaged proteins, thus helping to control key cellular and regulatory processes such as the cell cycle and apoptosis. The survival of cells in some cancers is promoted by hyperactivation of proteasomes so that the proteins normally in place to regulate their growth and proliferation are degraded. In this sub-project (Project 2, WPs 4–7), the ESRs aimed to synthesise proteasome inhibitors based on so-called heterocyclic substructures including beta-lactones and gamma-lactams; (iii) Microtubule stabilisation: Microtubules are critical structural features of dividing cells, and molecules that bind to and stabilise them can halt their division and replication, forming the basis for the successful application of a number of well-known anticancer drugs. ESRs in this sub-project (Project 3, WPs 8–10) aimed to develop short synthetic routes to taxol and eleutherobin, both known microtubule-stabilising agents, and their analogues; (iv) Activation of caspases: Caspases are enzymes (proteins which can speed up chemical reactions) that are involved in the tight regulation of the apoptotic pathway. They are present in cells in an inactive form but trigger cell death upon activation; the initiation of caspase activation by an anticancer agent could modulate the sensitivity to or resistance against cytotoxic chemotherapies. The goal of this sub-project (Project 4, WPs 11–13) was for the ESRs to develop synthetic pathways to pro-apoptotic caspase activators such as betulinic acid and ferruginol.

ESRs in Project 1 have achieved new catalytic chemistry relevant to the incednine structure and one ESR completed a total synthesis of aruncin B (a Bcl-2 inhibitor) that also necessitated a revision of the published structure for this natural product. The chemistry was developed such that 15 analogues of the structure were also prepared to enable testing for biological activity with cancer cell lines. Within Project 2, ESRs achieved new reactions that gave precursors to beta-lactones of interest, and effected reactions using both metal-based reagents and electrochemistry to provide advanced intermediates towards the target compounds. One ESR worked on the total synthesis of the natural product inthomycin C and was one step away from its elaboration to oxazolomycin B, an important bioactive natural product and potential proteasome inhibitor. Research within Project 3 focused on streamlined methods for building the complex chemical frameworks of eleutherobin and taxol with both ring systems of their chemical structures completed by two ESRs. Following exposure of these intermediates to electrochemical and biocatalytic oxidations, a number of derivatives and analogues of taxol and eleutherobin were generated to enable testing in biological assays against cancer cell lines. Efforts within Project 4 resulted in novel catalytic chemistry leading to the core structures of estrone, salvinorin A, and betulinic acid, and both electrochemical and photochemical methodology have been explored.

A key objective of OxIOSCR was to screen the compounds developed by the ESRs in biological assays. These assays were performed in collaboration with Daniel Ebner at the Target Discovery Institute (TDI) and with Valentine Macaulay, Frances Willenbrock and Christopher Towers at the pre-clinical validation lab, Department of Oncology. Compounds were first tested in cell viability assays and any with promising activities against cancer cells were then put forward for more screening assays according to their proposed mode of action: proteasome, caspase, and apoptosis assays. Cell cycle analysis was carried out to determine the effects of some test compounds (particularly those with potential microtubule-stabilising activities) on the cell cycle. To complement and compare with the experimental biological assay results our AP, Prous IBR, performed computational algorithmic predictions on the compounds. Additionally, results derived from the initial viability studies at the TDI were analysed in collaboration with Francesca Buffa’s Computational Biology and Integrative Genomics group, Department of Oncology. In parallel, an intensive training programme was implemented that exposed the researchers to the experience and knowledge of leading experts from academia and industry in specialised strategies and technologies in synthetic chemistry, including computational aspects. An integral part of the programme was for the ESRs to incorporate new synthetic techniques (biocatalysis, electrosynthesis, and flow chemistry) within their work. Activities within WPs 14–16 led to the delivery of courses (workshops) in biocatalysis and sustainable chemistry, electrosynthesis, and flow chemistry, respectively. ESRs also spent two months in the laboratories of the APs in order to acquire additional skills, extend their research, explore how skills and techniques are applied to particular industrial concerns, and gain practical industry experience.

EXPECTED FINAL RESULTS AND POTENTIAL IMPACT
202 natural product-inspired compounds (in three sets, or ‘libraries’) prepared by the ESRs were assessed for anticancer activity, each library of compounds becoming more focused and natural product-like as the synthetic chemistry advanced. Preliminary results derived from the proteasome assays on the first two compound libraries identified two compounds as inhibitory in lung cancer cells, one of which was also inhibitory in multiple myeloma cells. Two compounds were shown to induce caspase 3/7 activity. Cell viability studies on the third compound library indicated nine compounds with promising cytotoxicity in lung cancer and breast cancer cells. Cell cycle and proteasome studies are continuing to assess the compounds according to their proposed mode of action. A full methodological paper is planned for once the results have been collected from the assays on the third compound library. At the close of the project, the compound libraries will exist as a collection of structures with experimental and predicted activities associated for subsequent analysis and follow-on research.

OxIOSCR has resulted in creative and innovative synthetic strategies for complex molecule synthesis utilising efficient, combined applications of traditional chemical techniques with new techniques of biocatalysis, electrochemistry and flow chemistry with the emergence of new compound libraries mapped against cancer-relevant biochemical pathways. OxIOSCR provided a comprehensive training programme for this new generation of chemists, instilling an open-minded approach to synthesis, exposing them to less widely known techniques, and helping them to develop a clear vision of where innovative research techniques can make a significant impact both in an academic and industrial setting. The research has resulted in academic journal articles, research lectures and posters complemented by outreach and public engagement activities to describe the research and training programme. ESRs have emerged from OxIOSCR with a wide range of highly desirable skills having the potential to select from a range of career paths including university research, industry research, evaluating and consulting roles: at the time of writing this report (July 2017) five students (four of which have submitted their thesis) have either started or secured new research new academic or industrial postdoctoral jobs in the Europe or the US. We have demonstrated a model example of doctoral programme to produce highly trained and motivated researchers, and perceive that this will inspire others to form multinational networks to develop multidisplinary research training programmes. A doctoral programme established at the Department of Chemistry, Synthesis for Biology and Medicine – Centre for Doctoral Training (SBM-CDT), was initially inspired by the OxIOSCR programme and modelled elements of their training to the IDP training programme to establish a successful academic-industrial initiative; a lasting legacy of the IDP model.

CONTACT DETAILS
Project coordinator: Professor Jeremy Robertson
Chemistry Research Laboratory, Department of Chemistry, University of Oxford
Email: jeremy.robertson@chem.ox.ac.uk
Telephone: +44 (0)1865 275660
Website: http://oxioscr.chem.ox.ac.uk