Article

Innovative functional cAMP assay for studying G protein-coupled receptors: application to the pharmacological characterization of GPR17.

Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, 62032, Camerino, Italy.
Purinergic Signalling (Impact Factor: 3.51). 07/2011; 7(4):463-8. DOI: 10.1007/s11302-011-9245-8
Source: PubMed

ABSTRACT In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC(50) or IC(50) values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [(35)S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G(αi).

Full-text

Available from: Ajiroghene Thomas, Jun 10, 2015
0 Followers
 · 
145 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The anticancer activity of isofuranodiene, extracted from Smyrnium olusatrum, was evaluated in human breast adenocarcinomas MDA-MB 231 and BT 474, and Caucasian prostate adenocarcinoma PC 3 cell lines by MTS assay. MTS assay showed a dose-dependent growth inhibition in the tumor cell lines after isofuranodiene treatment. The best antiproliferative activity of the isofuranodiene was found on PC 3 cells with an IC50 value of 29 M, which was slightly less than the inhibition against the two breast adenocarcinoma cell lines with IC50 values of 59 and 55 M on MDA-MB 231 and BT 474, respectively. Hoechst 33258 assay was performed in order to study the growth inhibition mechanism in prostate cancer cell line; the results indicate that isofuranodiene induces apoptosis. Overall, the understudy compound has a good anticancer activity especially towards the PC 3. On the contrary, it is less active on Chinese hamster ovary cells (CHO) and human embryonic kidney (HEK 293) appearing as a good candidate as a potential natural anticancer drug with low side effects.
    The Scientific World Journal 05/2014; 2014(Article ID 264829). DOI:10.1155/2014/264829 · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.
    ACS Medicinal Chemistry Letters 04/2014; 5(4):326-330. DOI:10.1021/ml400399f · 3.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.
    International Journal of Molecular Sciences 04/2014; 15(4):6252-64. DOI:10.3390/ijms15046252 · 2.34 Impact Factor