Room G05, Royal School of Mines
Abstract: The Hedgehog pathway is one of the most important signalling pathways in tumourigenic processes and vertebrate organogenesis (e.g. limb pattering or organization of the brain). Thereby, the key mediator of the signalling pathway in mammals is the protein Sonic Hedgehog (Shh). N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is crucial for efficient signalling. A recently identified series of dihydrothienopyridines (RUSKIs) was shown to inhibit this essential post-translational modification. In the course of this project we profiled a selection of the literature known RUSKI compounds in cell-based signalling assays and found that the commercially available lead compound (RUSKI-43) exhibits off-target effects, while other members of the series showed no off-target cytotoxicity. Additionally, we used quantitative whole-proteome palmitoylation profiling with a biorthogonal alkyne-palmitate reporter to demonstrate specific inhibition of Hhat in cells. We also performed a comprehensive structure activity relationship study to identify residues crucial for the inhibitory potency of the small molecule inhibitors. In this context we optimised the synthetic strategy of the RUSKI compounds and investigated a conformational isomerism caused by a restricted rotation of the acyl substituents using NMR studies and density functional theory modelling. Our findings decisively contribute to the development of novel Hhat inhibitors as selective chemical probes for the investigation of Shh palmitoylation and its role in cancer development and progression.