Binding and Dynamics of Dualsteric Agonists for M2R in Glioblastoma

Glioblastoma (GBM), the most aggressive brain tumour, still today presents poorly effective treatments. In fact, pharmacological therapies failed to impact patient survival. Therefore, the identification of new drugs capable of counteracting malignancy remains a promising challenge in Glioblastoma treatment. To this aim, we investigated the role of naphthalimide derivatives in inhibiting GBM cell proliferation and survival. Interestingly, the dualsteric agonist Iper-8-Naphthalimide (N8), binding both allosteric and orthosteric binding sites of M2R, can induce “biased agonism” downstream receptor activation and promote a strong effect upon low-dose treatment. Recently, thanks to the addition of an amine group in the allosteric region, we have obtained an autofluorescent N8, named fluo-N8. It seems very useful to better understand the ligands' functional properties and the interaction with M2R. Moreover, fluo-N8 can allow to follow the fate of naphthalimide derivatives once internalized into tumour cells. In the present work, we analysed drug-receptor interactions from a pharmacological, biochemical and bioinformatic perspective, to expand our knowledge about M2R activation mechanisms. Bioinformatic analysis has shown aminoacidic residues involved in drug-receptor docking, while binding experiments have allowed to identify the binding parameters to confirm the pharmacological properties of the two dualsteric agonists. Over-expressing M2R-flag in combination with fluo-N8, it was possible to evaluate the receptor-ligand interaction and their dynamics within cellular compartments, observing the translocation of M2R from cytoplasm to the plasma membrane after agonist stimulation and its return in the cytoplasm in vesicle-like structures, after a long time of stimulation or in the presence of high doses of ligands. Interestingly, fluo-N8 was internalised in the cells and appears at least in part co-localize with mitochondria. Ongoing experiments will allow us to understand if the cytotoxic effects mediated by N8 are exclusively dependent on the M2R activation or if the molecule can have its independent effects once internalized.