[DDC 2018] Modifying Standard Assay for Assessing Allosteric Activity

File Name/Number:
DDC 2018


Since 2004, when the FDA approved the first allosteric G protein-coupled receptor modulator Cinacalcet (Sensipar®), which acts as a calcimimetic by allosteric activation of the calcium-sensing receptor, there has been much interest in allosterism models as a way to maximize drugs receptor selectivity or safety particularly with receptor families where selective compounds have been difficult to identify. Allosteric ligands can potentiate (PAM) or inhibit (NAM) the agonist activity of orthosteric ligands as a dimmer to modulate natural regulation rather than completely inhibit or continuously activate proteins. There are several approaches to identify allosteric effects of test compounds and in this poster we present three positive allosteric modulation models derived from three different types of standard assays. In a M2 muscarinic GTPgammaS membrane assay, we used a fixed concentration of test compound with a full concentration-response curve of a standard agonist, Oxotremorine M to see if the presence of test compound can potentiate the ligand’s potency and efficacy as evidenced by the left-ward shift. In a muscarinic ileum tissue functional assay, we evaluated the effect of a full concentration-range of a test compound with a fixed sub- EC50 concentration (ie. EC20) of the standard agonist Methacholine to see if the test compound can potentiate the Methacholine’s response with enhanced efficacy. In a NMDA receptor binding assay using [3H]-MK-801 as the radioligand, we evaluated the effect of a test compound on [3H]-MK-801 binding in a glycine-deprived assay condition. Indeed, NMDA receptor activation that opens the channels and permits [3H]-MK-801 to bind to the channel is positively modulated by glycine. D-cycloserine, a known partial agonist, binding at glycine site was used as a positive control. Those three examples demonstrate  straightforward strategies to examine positive allosteric modulators using different models.