A new molecular mechanism to promote protean agonism at a G protein-coupled receptorl

Abstract

Protean agonists are of great pharmacological interest as their apparent efficacy may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this thesis, the main aim was to investigate whether a dualsteric design principle, i.e. molecular probes carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to generate protean agonism at the M₂ receptor. First, we overcame the methodological limitations: we established two experimental systems, which have either a high level of spontaneous activity (Tris buffer) or a low amount of constitutive activity (Tris NaCl buffer), and we demonstrated that a high buffer osmolarity rather than a high concentration of sodium ions is responsible for receptor inactivation. Then, we identified two new dualsteric protean agonists at the M₂ muscarinic acetylcholine receptors, namely isox-6-naph and OOM-6-naph and we pinpointed three molecular requirements within dualsteric compounds that elicit protean agonism at this receptor subtype: (i) an orthosteric part endowed with an acetylcholine-like efficacy, (ii) a bulky allosteric part to impair the flexibility of the extracellular loop area, and (iii) a flexible linker chain of 6 carbon atoms.<br /> Using radioligand binding and functional assays we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Therefore, given that protean agonism was thought to reside in the intrinsic efficacy of a ligand, these results introduce an unprecedented molecular mechanism to elicit protean agonism.<br /> Finally, assays performed with an allosteric loss-of-affinity mutant confirmed that a functional allosteric site is critical for eliciting protean agonism at the M₂ receptor. Indeed, a single mutation in this binding site converted the behaviour of isox-6-naph and OOM-6-naph from protean to partial agonism.<br /> In conclusion, the findings of this thesis provide new mechanistic insights into the still enigmatic phenomenon of protean agonism and form a rationale for the design of such compounds for a G protein-coupled receptor.<br /> Thus, in addition to the great potential of biased GPCR signaling, improved understanding of protean agonism may provide another level towards a targeted exploitation of the GPCR signaling machinery, which could be relevant to the knowledge-based design of innovative drug candidates.