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

在所描述的项目中，将研究G蛋白偶联受体(GPCRs)超家族中的一个子家族的受体，即FrizzledGPCRs类(F类)。这个亚家族由10个Frizzled型(FZD)和Smoothened型(SMO)组成，其中的前一个是这里感兴趣的。主要的重点将集中在发现和表征新的小分子配体，使用在电子和体外的方法相结合的方法。四个FZD同源簇(FZD4、FZD5、FZD6和FZD7)的代表成员将成为与大分子文库对接的目标，以寻找这些受体的新的小分子配体。在进一步的过程中，从这些筛选中选择的潜在配体将在基于细胞的体外分析中表征，包括亲和力、受体激活和效应蛋白的招募以及FZD信号通路的诱导。此外，还将评估配体对不同FZD的选择性。基于这些结果，结构-活性分析可以深入了解蛋白质-配体相互作用与这些配体的亲和力和作用之间的联系。得到的新型配体将用于FZD及其信号转导的进一步研究。深入了解配体对FZD信号的合理调节对于药物开发以及对FZD的一般理解都有很大的影响。此外，在描述的项目中，将进一步研究杂乱(DVL)和异源三聚体G蛋白与FZD的招募机制和相互作用。在计算机技术中，分子动力学等方法将被用来阐明哪种受体构象导致哪种效应蛋白的募集。首先，模拟将在带有小分子配体的复合体中进行，而这可能会在项目的后期扩展到包括Wingless/Int-1(WNT)家族的内源配体。由此产生的假说将在基于细胞的体外试验中进行评估和确认。该项目这一部分的结果将有助于理解导致不同效应蛋白招募的FZD构象是如何稳定的，以及如何解释和调节DVL和G蛋白之间的招募偏见。

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.