Changes in the properties of allosteric and orthosteric GABAB receptor ligands after a continuous, desensitizing agonist pretreatment

It has been estimated that only 15% of the compounds classified as silent G protein-coupled receptor antagonists are indeed devoid of either positive or negative intrinsic efficacy. Considering that 40% of all drugs on the market target G protein-coupled receptors mainly as orthosteric ligands, elucidating their intrinsic properties is becoming increasingly important. While agonism can be demonstrated using appropriately sensitive experimental setups, the detection of inverse agonism can be limited by a low degree of constitutive activity in many assay systems. In this study, changes in ligand behavior upon a lasting pretreatment with γ-aminobutyric acid (GABA), that induced receptor desensitization, were observed, measuring the second messenger cyclic AMP (cAMP) in a GABAB receptor-expressing recombinant cell line. The GABAB receptor partial agonist 2-OH-saclofen lost its ability to inhibit 7β-forskolin-induced cAMP production upon GABA-pretreatment. The “silent” receptor antagonists CGP62349, CGP52432, CGP56999 and SCH50911, on the other hand, stimulated 7β-forskolin-induced cAMP production under these conditions. The inverse agonism of CGP56999 was inhibited by the efficacy-deficient 2-OH-saclofen, proving it was truly mediated through the orthosteric site of the GABAB receptor. Finally, the positive allosteric modulator GS39783, which previously only marginally inhibited cAMP production, suppressed it by 60% both alone and in the presence of the competitive receptor antagonist 2-OH-saclofen, thus GS39783 became an allosteric receptor agonist at desensitized GABAB receptors. These changes likely reflect adaptations in the mechanisms of GABAB receptor function following desensitization and may be important in the elucidation of intrinsic ligand efficacies as well as for the consequences of continuous drug treatment.