Before the concept of constitutive or intrinsic activity of the biological systems, which was formulated about thirty years ago, it was thought that agonist compounds were the only drugs capable of activating physiological responses, while antagonists were the ones capable of blocking them. However, this basic classification of drugs in pharmacology started to change only at the end of the eighties, when bioactive ligands, with negative efficacy, were developed. The G-protein-coupled receptors (GPCR) were promptly selected as one of the most useful types of pharmacological targets to study this inverse efficacy. This family of receptors is responsible for the signaling and control of many physiological processes, from the peripheral nervous system to the central. Therefore, the GPCR have become the most studied family of receptors in drug discovery. It has been estimated that around a third of the drugs actually used act via the GPCR, nevertheless there are still many orphan GPCR encoded by the human genome. During the last decade, reports and patents have described new methods to detect GPCR inverse agonist compounds. The detection of the G-protein constitutive activity and the quantification of the positive or negative efficacies induced by agonists or inverse agonists respectively has been studied by analyzing the binding of the nonhydrolyzable GTP analog, [(³⁵S]GTPγS. The present chapter describes an optimized method to detect GPCR inverse agonist ligands such as cannabinoid compounds, in both membrane homogenates and tissue sections (autoradiography).
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.
PLoS ONE 09/2015; 10(9):e0138463. DOI:10.1371/journal.pone.0138463 · 3.23 Impact Factor
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