Innovative Functional Methods Useful for New Cellular Target Identification and for Receptor Ligand Characterization

G-protein coupled receptors (GPCRs) are a large family of receptor proteins that play important roles in many physiological and pathological conditions. In drug discovery, they are one of the most important drug targets. In particular, adenosine receptors, classified as A1, A2A, A2B and A3 (A1AR, A2AAR, A2BAR, and A3AR) subtypes, have been actively studied as potential therapeutic targets in several disorders such as cardiovascular and CNS pathologies. The aim of the present work is the development of innovative and non-radioactive screening systems in order to study a large number of adenosine P1 receptor ligands. On the other hand, since new assays could be useful to contribute to receptor deorphanization, the task work has been focused to characterize the putative guanosine receptor and the novel dual uracil nucleotide/cysteinyl-leukotriene receptor, GPR17. In particular, functional cAMP and DELFIA GTP-Eu binding assays were used. The cAMP immuno-competitive assay, performed in homogeneous time resolved fluorescence (HTRF), was optimized and validated using as reference agonists NECA at A2AAR and A2BAR, and Cl-IBMECA at A3AR. Hence, after this assay validation, the biological profile of a series of 8-substituted 9-ethyladenines was assessed at A2AAR. Results showed that all compounds behave as A2AAR antagonists. On the other hand, an innovative luminescence cAMP assay, that uses a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety, was developed for ARs. Two new improved plasmids containing the gene for the modified form of firefly luciferase were optimized and validated at ARs in order to verify their efficacy at Gαs and Gαi systems. In particular, the experiments were carried out by transiently expressing these new vectors at CHO cell line stably transfected with A2A and A3 ARs. Results showed that both plasmids can be used at ARs and since this assay demonstrated to be robust and reproducible, it was used as new tool to investigate the novel ''dual'' GPR17. Furthermore, the better evaluation of the ''dual'' nature of the human GPR17 could be carried out in 1321N1 cells since they do not constitutively express nucleotide and cysteinyl-leukotriene receptors. In particular, different transient transfection methods such as calcium phosphate, lipofectamine, fugene, arrest-InTM and microporation, have been investigated. Experiments were performed using also in parallel GFP as reporter gene and results analyzed by RT-PCR suggested that microporator as well as Arrest-In are both the best method to promote an enriched hgpr17 delivery into 1321N1 cell line. Moreover, DELFIA GTP-Eu binding assay was carried out using CHO-A3 membranes. The standardization of DELFIA GTP-Eu assay is a long process, in which the determination of the specific receptor buffer stimulation was the first crucial step investigated. The assay validation was carried out using know ligands and subsequently, a new series of 2-alkynyl-N6-methyl-5'-N-methylcarboxamidoAdo derivatives were screened and results suggested that they are full agonists at A3AR. Subsequently, the assay potentialities were extended at native tissue. In particular, rat brain membranes were prepared from Male Wistar rat and used to investigate putative guanosine receptor by testing known and novel ligands. Results obtained suggested the presence of a G-protein coupled receptor able to be activated by guanosine and some guanosine derivatives. The AGC is a family of kinase that is composed of 60 different enzymes including PKC isoforms, PKB/AKT, S6K and their upstream kinase, the phosphoinositide-dependent protein kinase 1 (PDK1). In particular, located in the small loop of the kinase domain is present a pocket called PIF-binding pocket that is involved in the regulation of the intrinsic activity of these kinases. The molecular mechanism of allosteric activation of PDK1 by small molecular weight prodrugs and their effects in C2C12 cells were evaluated. In particular experiments were carried out incubating C2C12 with PS423 and PS0403, small chemical entities synthesized by the PhosphoSites research group, and after cell insulin stimulation the PDK1 pathway was investigated. Results obtained suggested that these compounds were able to bind the PIF-binding pocket inhibiting the S6K phosphorylation without affecting the PKB activity. On the other hand, the in cell western was optimized at C2C12, as new assay to screen and to evaluate in a throughput way the effects of several PS-compounds on the S6K phosphorylation by PDK1. Results demonstrated that the in cell western is a robust, reproducible and easy to use assay to screen directly in the cell several compounds in the same experiments.