Deorphanization of Adhesion GPCRs overexpressed in Glioblastoma

G-protein-coupled receptors (GPCRs) represent, nowadays, one of the most productive source of drug targets. Some GPCRs members are, however, poorly studied and hence cannot be targeted for therapies yet. Among these, there are more than 90 GPCRs still orphan, i.e. with no described endogenous ligand and no clearly defined function. The orphan receptors CELSR2 and Frizzled4 (Fz4), have recently drawn the attention of the scientific community in virtue of their involvement in cancer progression. CELSR2 is an adhesion GPCR (class B GPCRs) overexpressed in glioblastoma. Until now, CELSR2 endogenous ligands have not yet been identified. Fz4 belongs to class F GPCRs and it is activated by the lipoproteins WNTs and Norrin. Misregulation of Fz4 activity is involved in tumor proliferation and cancer stem cell genesis in many types of malignancies, such as glioblastoma, colorectal and breast cancer. To date, the existence of low-molecular-weight organic molecules binding to and modulating Fz4 has not been reported. Thus, the identification of ligands/modulators of CELSR2 and Fz4 activity could pave the way for new therapeutic strategies to treat cancer. In this thesis, I attempted to identify CELSR2 and Fz4 ligands by using the “Pharmacological chaperone readout”, an innovative screening platform (PC-platform) that identifies ligands in virtue of their potency in affecting the tridimensional structure and the intracellular localization of a protein target. The first chapter of the thesis describes the results obtained in the attempt of identifying new ligands of Fz4 receptor. To achieve this aim, a cell line expressing a Fz4 mutant, Fz4-L501fsX533, was generated. This frameshift mutation is responsible in vivo for the occurrence of the Familial Exudative Vitreoretinopathy (FEVR), a pathology of the retina. The resulting mutant, here referred to as Fz4-FEVR, aggregates intracellularly in the Endoplasmic Reticulum (ER) without reaching the Plasma Membrane (PM) of the cell where, in contrast, the wt receptor localizes at steady state. To identify Fz4 wt modulators, a library of organic molecules has been screened for pharmacological chaperones of Fz4-FEVR, i.e. for molecules able to rescue the folding and correct localization of Fz4-FEVR at PM. Using such read-out, the organic molecule FzM1 has been thus identified as Fz4-FEVR pharmacological chaperone. The pharmacological chaperone FzM1 acts as allosteric inhibitor of the Fz4-wt receptor, binding directly to the wt receptor and inhibiting the signalling pathway Fz4 is involved in. I also performed a structure-activity relationship (SAR) analysis using FzM1 as lead to identify the first allosteric agonist of Fz4, FzM1.8. As discussed in the second chapter of this thesis, a specific PC-platform was developed for the identification of ligands of CELSR2. To achieve this aim, a misfolded version of CELSR2 was generated and then a library of metabolites was screened looking for compounds binding to the receptor and correcting its folding. Zebrafish eggs were used as biomass to obtain a library of metabolites which were screened for pharmacological chaperone efficiency. The outcome of the screening identified three compounds acting as modulators of CELSR2: cholesterol, PGE2 and β-carotene. They do not target the orthosteric binding site of CELSR2 but they are located in an hydrophobic region of the receptor at the interface between the TM-bundle and the lipid bilayer. The potential ability of these molecules to allosterically modulate CELSR2 function could have important implications both in physiological and pathological cell conditions