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Final Report Summary - CB-HEDGEHOG (A Chemical Biology Approach to Understand the Release of Sonic Hedgehog)

Cancer is the most important cause of death in the European Union (EU). The most common cancer forms are breast cancer, prostate cancer, bowel cancer and lung cancer. The Hedgehog pathway is one of the most important signalling pathways in tumourigenic processes and known to be involved in all of these four types of tumours that represent 52% of all cancer cases in the EU. Thereby, the key mediator of the signalling pathway in mammals is the protein Sonic Hedgehog (Shh). During its maturation, the morphogen Shh is post-translationally modified in the endoplasmic reticulum (e.g. autocatalytically cleaved and lipidated) and transported to the cell membrane. The subsequent release of the signaling fragment into the surrounding area of the expressing cell results in the formation of Shh multimers that are able to induce genes involved in the development of target cells. Due its emerging impact on cancer development, it is of vital importance to fully understand the mechanism of the signaling pathway in order to design effective therapies. The proposed project was based on highly multidisciplinary approaches that lay at the cutting edge of chemical biology, and permitted the fellow, Dr. Markus Ritzefeld, to gain skills and experience necessary for his future career in the field. All findings have been extensively disseminated through presentations or publications. Furthermore, Dr. Markus Ritzefeld successfully built networks with other scientists (biologists, biochemists, instrumentalists, and chemists) at Imperial College and beyond.
During his Marie Curie IEF the fellow focussed on three different main objectives: The investigation of RUSKI compounds - small molecule inhibitors of the Shh acyltransferase Hhat - to selectively inhibit N-terminal palmitoylation of Shh; The investigation of the effect of Scube 2 on the release of Shh; The preparation of a dually modified recombinant Shh as tool molecule.
During the first objective, the RUSKI inhibitors, the fellow successfully optimised the synthetic strategy and identified stereoisomers of the RUSKI compounds that result from a hindered rotation of the amide bond. The combination of insights from selective NOE experiments with the results from density functional theory (DFT) modelling and non-covalent interaction (NCI) analysis facilitated the synthesis of a RUSKI derivative with a tailored E:Z ratio. Additionally, on- and off-target effects of published RUSKI inhibitors were identified and a comprehensive structure activity relationship study was performed that significantly increased the knowledge regarding these selective Shh pathway inhibitors. Manuscripts regarding these insights have either been published in high impact journals or are currently in submission.
During the second objective, the investigation of the effect of Scube 2 on the Shh release mechanism, preliminary Western blot results after the application of photo cross-linkable fatty acid derivatives and co-immunoprecipitation experiments did not result in the identification of a stable Shh-Scube 2 complex. These findings are in good agreement with the results recently published by by Grobe et al. (Sci. Rep. 2016, 6, 26435.). However, to quantify the release of Sonic Hedgehog and the effect of Scube 2 on this process, a SILAC based proteomics approach was established. The corresponding workflow successfully enabled the quantification of the turnover kinetics of cell surface proteins including Shh. Experiments to quantify the effect of Scube 2 on this process have to be performed in the future.
The third objective, the preparation of a dually modified recombinant Shh, is still under extensive investigation by the fellow. Recently, two collaborations based on the semi-synthetic protein have been stated. To date, a modular route to obtain cholesterylated semi-synthetic Shh proteins has been optimized and the corresponding protein biochemically characterized.
Due to its interdisciplinary nature, the project has contributed to European excellence and competitiveness in many scientific disciplines, such as synthetic chemistry, analytical chemistry, biochemistry, and cell biology. The specific outcomes of this project - the development and progression of novel tool molecules to further elucidate the Shh signalling pathway - are expected to immediately aid researchers in the field and ultimately society as a whole.

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IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE
United Kingdom
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