Agonisti inversi del GPR21: un approccio binario in silico ed in vitro per l'identificazione di nuovi composti antidiabetici

Diabetes is a relevant pathology both for the epidemiological and mortality-related aspects. Type two diabetes (T2D), the most widespread, is a multifaceted disease linked to an impairment of the insulin signaling pathway that can occur at different levels. Although being available several pharmacological treatments almost half of the patients do not reach therapeutic goals. The abundance of GPCRs class members, their recognized predisposition to be pharmacological targets and their proven presence in both islets of Langerhans and enteroendocrine cells has rendered these receptors subject of numerous pharmacological studies. Among the GPCRs one has started to attract particular attention due to its role in the onset and maintenance of insulin resistance states: GPR21. GPR21 silencing or blockage has been correlated to an improved glucose tolerance, reduced macrophages chemotaxis, decreased release of pro-inflammatory cytokines and augmented glucose uptake in hepatic cells. The aim of the work was to define a tridimensional model of GPR21 to better understand the binding pocket involved in the docking of inverse agonists and then screen them to identify compounds potentially more affine than the lead, GRA2. The more promising analogues were then tested alongside GRA2 on HepG2 cells defining their activity in reducing GPR21 detrimental action on the insulin signaling pathway using insulin resistance models. From the in silico work seems to emerge not only the presence of a side pocket with not-negligible affinity values for the compounds analyzed but two spatial levels of interactions in the orthosteric pocket, with higher affinity values observed in the deeper one. Testing in vitro two promising analogues, it was observed that GPR21 expression levels increased in a hyperglycemia model and, although the insulin cascade was not affected by prolonged exposure to high glucose medium or high insulin and high glucose, all the compounds tested presented a booster role regarding the phosphorylation of GSK3β and so the insulin pathway activation, when GPR21 was overexpressed. The inhibitory action of the compounds on GPR21 was also observed quantifying the calcium release, marker of GPR21 pathway activation, reduction following their administration. A better understanding of GPR21 binding pocket could lead to a more efficient screening for inverse agonists based on the effective interactions occurring, useful because until now only one ligand is present in literature and it presents low potency values. The overexpression of GPR21 in hyperglycemia models could potentially render the search for new inverse agonists a relevant tool in treating pre-diabetic and diabetic patients.