Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

Abstract

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood.Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M 2 receptors to demonstrate the existence and function of such inactive agonist⅐receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist⅐receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist⅐receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.Specific and coordinated cell-to-cell communication regulates the flow of information between cells, and proper information processing ensures physiological functions of biological systems. G protein-coupled receptors (GPCRs), 8 constituting the largest class of membrane proteins in mammals, are essential mediators of chemically and light-encoded information (1-4). GPCRs sense a great variety of extracellular stimuli, e.g. neurotransmitters and hormones, and subsequently translate this information into an intracellular response via G proteins, ␤-arrestins, and possibly GPCR-interacting proteins (2-5). Because of their abundance and relevance in regulating the majority of (patho-)physiological processes in humans, GPCRs have for a long time represented the most important drug targets being addressed by at least a third of all currently marketed drugs (6, 7).Agonist binding leads to receptor activation, which is followed by intracellular G protein recruitment and subsequent cell signaling. Breakthroughs in GPCR crystallography have led to inactive and active crystal structures of the same receptor protein. Among these are rhodopsin (8 -10) and more recently the ␤ 2 -adrenergic (11-14), M 2 muscarinic (15, 16), and -opioid receptors (17, 18). These structures most likely represent energetically favored, relatively stable inactive and active receptor⅐ligand complexes. Despite the diversity of crystallized receptors, a common...

A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy

“…[5] Such ligands are able to overcome the problem of subtype selectivity for the M 1 subtype by making use of cooperativity in the selective binding and activation of the receptor. [6] To take this concept one step further, dualsteric (or bitopic) ligands are developed herein that covalently connect ah igh-affinity/low-selectivity orthosteric moiety to highly selective allosteric building blocks for concomitant interac- tion with both binding sites.R ecently,o ur groups have reported several sets of dualsteric M 1 and M 2 agonists for which dynamic ligand binding is assumed, leading to partial agonism for the M 1 receptor ( Figure 1) [7] as well as for the M 2 receptor. [8] Based on these findings,d ualsteric ligands hold great potential both as future therapeutics and as pharmacological tools owing to their unprecedented selectivity at Mreceptor subtypes,t heir specificity for signaling pathways ("biased signaling"), their capability for partial agonism, and their potential for studying the process of receptor activation at the molecular level.…”

Novel Analgesic Agents Obtained by Molecular Hybridization of Orthosteric and Allosteric Ligands

“…Disingrini et al, 2006) We found that these hybrid compounds are characterized by a truly bitopic (orthosteric and allosteric) binding mode (hence the term "dualsteric" ligands), (Antony et al, 2009) as well as other distinctive features when compared to their parent compounds, such as receptor subtype selectivity and biased agonism. (Bock et al, 2016;De Min et al, 2017) We also demonstrated that some of these hybrid compounds display interesting pharmacological properties, (Cristofaro et al, 2018;Riefolo et al, 2019) including remarkable analgesic effects in vivo in mice. Noteworthy, one of these compounds, from now on named Iper-8-N ( Figure 1, compound 8b in the reporting article), even combined the potent antinociceptive activity with the absence of relevant cholinergic side effects.…”

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium

“…Atropine either moves out of the M 3 receptor or into the orthosteric site, as is also evident from the on‐rate of atropine which is about 10 times faster than that of tiotropium (Dowling & Charlton, ). Previously, dual binding at both binding sites was described for the M 2 receptor, were one ligand occupied both sites (Bock et al, ). M 2 receptors are not involved in airway smooth muscle contraction, which is solely mediated via M 3 receptors (Roffel et al, ).…”