In silico prediction of small molecule ligands for class Frizzled GPCRs and investigation of the structural basis of FZD signalling

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In the described project receptors from a subfamily of the superfamily of G protein-coupled receptors (GPCRs), the class Frizzled (class F) GPCRs, will be investigated. This subfamily consists of ten Frizzled (FZD) paralogues and Smoothened (SMO), of which the prior are of interest here. The main focus will be directed towards the discovery and characterisation of novel small molecule ligands by using a combined approach of in silico and in vitro methods. Representative members of the four FZD homology clusters (FZD4, FZD5, FZD6 and FZD7) will be targeted in docking screens with large molecular libraries to find novel small molecule ligands for these receptors. In the further process, the potential ligands that were selected from these screens will be characterised in cell-based in vitro assays regarding the affinity, receptor activation and recruitment of effector proteins as well as induction of FZD signalling pathways. Further, the selectivity of the ligands regarding different FZDs will be evaluated. Based on these results, structure-activity analyses can be pursued to gain insights into the connection between protein-ligand interactions and the affinity as well as effect of these ligands. The resulting novel ligands will be used for further investigations of FZDs and their signalling. Insights into the rational modulation of FZD signalling by ligands can be of great impact for drug development as well as a general understanding of FZDs. Furthermore, the mechanisms of recruitment and the interactions of Dishevelled (DVL) and heterotrimeric G proteins with FZDs will be investigated further in the described project. In silico methods such as Molecular Dynamics simulations will be employed to elucidate which receptor conformations lead to the recruitment of which effector proteins. First, the simulations will be conducted in complex with small molecule ligands, while this might be expanded to including the endogenous ligands of the Wingless/Int-1 (WNT) family at a later point of the project. Hypotheses resulting from this will be evaluated and confirmed in cell-based in vitro assays. Results from this part of the project will contribute to the understanding how FZD conformations leading to recruitment of different effector proteins are stabilised and how a recruitment bias between DVL and G proteins can be explained and modulated.