Methods to measure g-protein-coupled receptor activity for the identification of inverse agonists

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).