Katie Leach

Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties.

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M4 muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M4 mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist–receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [35S]GTPγS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3β phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M4 mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [3H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M4 mAChR knockout mice, validating the M4 mAChR as a key target of action of this novel allosteric ligand.

New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD (designer receptor exclusively activated by a designer drug).

The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated “designer receptors exclusively activated by a designer drug” (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298s cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.

Crystal structures of the M1 and M4 muscarinic acetylcholine receptors

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M4 mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M1/M4 mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M4 mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile93 and Lys95 as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe186 in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp432 in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr439 and Tyr443, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.

Impact of Clinically Relevant Mutations on the Pharmacoregulation and Signaling Bias of the Calcium-Sensing Receptor by Positive and Negative Allosteric Modulators

Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca(2+) mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G(670)R, P(748)R, and L(773)R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V(817)I, L(773)R, and E(767)K) altered the cooperativity between the allosteric modulator and Ca(2+)(o). These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

Impact of species variability and ‘probe‐dependence’ on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

BACKGROUND AND PURPOSE We recently characterized LY2033298 as a novel allosteric modulator and agonist at M4 muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M4 mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.

Towards a structural understanding of allosteric drugs at the human calcium-sensing receptor

Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an “enriched” picture of structure-function requirements for interaction between Ca2+o and allosteric modulators within the CaSRs 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca2+o-binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators with distinct and potentially pathway-biased pharmacological effects.

Engendering biased signalling from the calcium-sensing receptor for the pharmacotherapy of diverse disorders

The human calcium‐sensing receptor (CaSR) is widely expressed in the body, where its activity is regulated by multiple orthosteric and endogenous allosteric ligands. Each ligand stabilizes a unique subset of conformational states, which enables the CaSR to couple to distinct intracellular signalling pathways depending on the extracellular milieu in which it is bathed. Differential signalling arising from distinct receptor conformations favoured by each ligand is referred to as biased signalling. The outcome of CaSR activation also depends on the cell type in which it is expressed. Thus, the same ligand may activate diverse pathways in distinct cell types. Given that the CaSR is implicated in numerous physiological and pathophysiological processes, it is an ideal target for biased ligands that could be rationally designed to selectively regulate desired signalling pathways in preferred cell types.

Calcium-sensing receptor-dependent activation of CREB phosphorylation in HEK293 cells and human parathyroid cells

In addition to its acute effects on hormone secretion, epithelial transport, and shape change, the calcium-sensing receptor (CaSR) modulates the expression of genes that control cell survival, proliferation, and differentiation as well as the synthesis of peptide hormones and enzymes. In the present study, we investigated the impacts of a CaSR agonist and several CaSR modulators on phosphorylation of transcription factor CREB residue Ser(133) in CaSR-expressing HEK293 (HEK-CaSR) cells and human adenomatous parathyroid cells. Elevated Ca(2+)o concentration had no effect on CREB phosphorylation (p-CREB) in control HEK293 cells but stimulated p-CREB in both HEK-CaSR cells and human parathyroid cells. In addition, p-CREB was stimulated by the positive modulator cinacalcet and inhibited by the negative modulator NPS 2143 in both CaSR-expressing cell types. Two positive modulators that bind in the receptors Venus Fly Trap domain, l-phenylalanine and S-methylglutathione, had no effect on p-CREB in HEK-CaSR cells, demonstrating the existence of pronounced signaling bias. Analysis of the signaling pathways using specific inhibitors demonstrated that phosphoinositide-specific phospholipase C and conventional protein kinase C isoforms make major contributions to Ca(2+)o-induced p-CREB in both cell-types, suggesting key roles for Gq/11. In addition, in parathyroid cells but not HEK-CaSR cells, activation of p-CREB was dependent on Gi/o, demonstrating the existence of cell type-specific signaling.

Murine GPRC6A mediates cellular responses to L-amino acids, but not osteocalcin variants

Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that require GPRC6A may be indirect, rather than via direct activation of the receptor.