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