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Figure legends for Supplementary Figures

Caspase-8 dependent apoptosis induced by IR

IR-induced changes in cell surface expression of death receptors

Response of H460 and A549 cells to IR

Combination of SAHA with IR in H460 and A549 cells using different treatment schedules

The application of biocatalysis in organic synthesis is rapidly gaining popularity amongst chemists. Compared to traditional synthetic methodologies biocatalysis offers a number of advantages in terms of enhanced selectivity (chemo-, regio-, stereo-), reduced environmental impact and lower cost of starting materials. Together these advantages can contribute to more sustainable manufacturing processes across a wide range of industries ranging from pharmaceuticals to biofuels. The biocatalytic toolbox has expanded significantly in the past five years and given the current rate of development of new engineered biocatalysts it is likely that the number of available biocatalysts will double in the next few years. This textbook gives a comprehensive overview of the current biocatalytic toolbox and also establishes new guidelines or rules for “biocatalytic retrosynthesis”. Retrosynthesis is a well known and commonly used technique whereby organic chemists start with the structure of their target molecule and generate potential starting materials and intermediates through a series of retrosynthetic disconnections. These disconnections are then used to devise a forward synthesis, in this case using biocatalytic transformations in some of the key steps. Target molecules are disconnected with consideration for applying biocatalysts, as well as chemical reagents and chemocatalysts, in the forward synthesis direction. Using this textbook, students will be able to place biocatalysis within the context of other synthetic transformations that they have learned earlier in their studies. This additional awareness of biocatalysis will equip students for the modern world of organic synthesis where biocatalysts play an increasingly important role. In addition to guidelines for identifying where biocatalysts can be applied in organic synthesis, this textbook also provides examples of current applications of biocatalysis using worked examples and case studies. Tutorials enable the reader to practice disconnecting target molecules to find the ‘hidden’ biocatalytic reactions which can be applied in the synthetic direction. The book contains a complete description of the current biocatalyst classes that are available for use and also suggests areas where new enzymes are likely to be developed in the next few years. This textbook is an essential resource for lecturers and students studying synthetic organic chemistry. It also serves as a handy reference for practicing chemists who wish to embed biocatalysis into their synthetic toolbox.

Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers and leukemias and has been shown to be a major mediator of resistance to chemo- and radio-therapies and to cell death induced by immune effector cells. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby regulates induction of apoptosis and necroptosis - two major cell death mechanisms. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP. Molecular modelling identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using protein-protein interaction assays identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP’s interaction with the TRAIL-R2 DISC and display nanomolar activity in NSCLC cell-based assays in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of standard-of-care chemotherapeutics, ionizing radiation and targeted anti-cancer agents. FLIP overexpression and CRISPR-mediated deletion of procaspase-8 and FADD abrogated the effects of these novel inhibitors consistent with their expected mechanism-of-action. Recent studies have also demonstrated that lead FLIP inhibitors target Treg cells, which are key immuno-suppressive, tumor-promoting immune cells. Lead molecules have also demonstrated in vivo efficacy in NSCLC cancer models both as a single agent and in combination with a multivalent TRAIL agonist. The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of several cancers, including solid tumors and haematological malignancies, either as monotherapy or in combination with other agents. Acknowledgements: This work was supported by a Seeding Drug Discovery award from the Wellcome Trust. Citation Format: Catherine A. Higgins, Jennifer Fox, Joanna Majkut, Greti E. Fiedler, Jamie Roberts, Luke Humphreys, Ray J. Boffey, Trevor R. Perrior, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 382.

International audience; A syn-enantioselective aldol reaction has been developed using Brønsted acid catalysis based on H(8)-BINOL-derived phosphoric acids. This method affords an efficient synthesis of various beta-hydroxy ketones, some of which could not be synthesized using enamine organocatalysis.

Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, Mentha spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)synthesis of (1R,2S,5R)-(-)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an "ene"-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.

The phosphoric acid (R)-POS is an efficient catalyst which allows formation of a number of β-hydroxy ketones with moderate to good diastereo- and enantioselectivity.