Molecular modelling studies on Frizzled receptors
G protein-coupled receptors (GPCRs) mediate effects of many endogenous and exogenous substances such as small molecules, peptides, lipids, ions, and odorants. According to homology, GPCRs were grouped into Classes A, B, C, F (Frizzleds), adhesion receptors, and other 7 transmembrane (TM) spanning receptors. Frizzleds (FZDs) regulate some processes during embryonic development, stem cell regulation, and adult tissue homeostasis.
Deregulation of FZDs leads to pathogenesis, including, but not limited to, cancer and neurologic disorders; thus, making them attractive drug targets. In mammals, there are 10 Frizzleds (FZD1–10), which are activated by the WNT family of lipoglycoproteins through interaction with the extracellular cysteine-rich domain (CRD) of FZD.
Our group has shown that FZD6 dynamically dimerizes in an agonist-dependent fashion and that the dimer interface of FZD6 is formed by the transmembrane α-helices four and five. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of signaling.
We plan to expand our understanding of molecular mechanisms of FZD activation and signal initiation by analysis of membrane localization, dimerization status and constitutive activity of mutants of FZD6. Mutation of R416 enhances the receptor’s ability to negatively impact on the WNT/b-catenin pathway, prevents dimerization and increases receptor internalization and turnover. R416 is located at the lower end of the TM6 mediating interaction with TM7 thereby providing a potential lock mechanism that could explain the mutations apparent constitutive activity. Furthermore, R416 is conserved in all human FZDs except for FZD4 and FZD9. This mutation has also been found in many forms of cancer, including uterine and bladder.
We are planning to carry on the molecular dynamics (in general all-atom but we may also use coarse grained and other accelerated methods for larger systems) simulations of FZDs but potentially also of other proteins communicating with FZDs, e.g., DVL, heterotrimeric G proteins. The work is also planned to use the SMO and the new FZD4 crystal structures as templates to model other FZD paralogues. These models will be used for in silico screening of large databases of small molecules. The ligands with the lowest binding energies and highest docking scores will later be validated using all-atom molecular dynamics simulations. In order to target FZDs pharmacologically, we need to define the binding modes of the ligands in the orthosteric binding pocket of the receptor.
The information will be used to mutate residues that form essential interactions (e.g., hydrogen bonds) with ligands. Subsequently, these FZDs mutants will be tested for their cell membrane expression and used in functional studies that will aid in defining the ligand-protein interaction, ligand-receptor selectivity and guide further hit optimization.
We have already several high-impact papers in the pipeline, in which we have used SNIC resources. These include a Nature Communications paper published in 2019 and a Nature Chemical Biology paper currently under review.