From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

Combinatorial expression of GPCR isoforms affects signalling and drug responses.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.

The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease.

Several neurodegenerative diseases associated with protein misfolding (Alzheimer's and Parkinson's disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated posttranslational modifications impact upon protein functions that can exacerbate pathology. Nonenzymatic and irreversible glycation signaling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood. Here we investigated the therapeutic potential of pharmacologically suppressing neuroinflammatory NO signaling during early disease progression of prion-infected mice. Mice were injected daily with an NO synthase (NOS) inhibitor at early disease stages, hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analyzed, and electrophysiological characterization of pyramidal CA1 neurons was performed. Increased neuroinflammatory signaling was observed in mice between 6 and 10 wk postinoculation (w.p.i.) with scrapie prion protein. Their hippocampi were characterized by enhanced nitrergic stress associated with a decline in neuronal function by 9 w.p.i. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg prevented the functional degeneration of hippocampal neurons in prion-diseased mice. We further found that this intervention in diseased mice reduced 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation. Furthermore, L-NAME application led to a reduced expression of the receptor for advanced glycation end-products and the diminished accumulation of hippocampal prion misfolding. Our data suggest that suppressing neuroinflammatory NO signaling slows functional neurodegeneration and reduces nitrergic and glycation-associated cellular stress.

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease.

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.

Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands.

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.

ß-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

(ß-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) ß-arrestin proteins (ß-arrestin1 and ß-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (ß-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of ß-arrestin with GPCRs, and the ß-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based ß-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in ß-arrestin2 that occur rapidly after the receptor-ß-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and ß-arrestins. They further indicate that ß-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of ß-arrestins, which permits their active signalling.

Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors.

FFA1 and FFA4, two G protein-coupled receptors that are activated by long chain fatty acids, play crucial roles in mediating many biological functions in the body. As a result, these fatty acid receptors have gained considerable attention due to their potential to be targeted for the treatment of type-2 diabetes. However, the relative contribution of canonical G protein-mediated signalling versus the effects of agonist-induced phosphorylation and interactions with ß-arrestins have yet to be fully defined. Recently, several reports have highlighted the ability of ß-arrestins and GRKs to interact with and modulate different functions of both FFA1 and FFA4, suggesting that it is indeed important to consider these interactions when studying the roles of FFA1 and FFA4 in both normal physiology and in different disease settings. Here, we discuss what is currently known and show the importance of understanding fully how ß-arrestins and GRKs regulate the function of long chain fatty acid receptors.

Chemogenetics defines receptor-mediated functions of short chain free fatty acids.

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein-coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography-mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P-NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P-binding receptor via liquid chromatography-tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Design of Next-Generation G Protein-Coupled Receptor Drugs: Linking Novel Pharmacology and In Vivo Animal Models.

Despite the fact that G protein-coupled receptors (GPCRs) are the most successful drug targets in history, this supergene family of cell surface receptors has yet to be fully exploited as targets in the treatment of human disease. Here, we present optimism that this may change in the future by reviewing the substantial progress made in the understanding of GPCR molecular pharmacology that has generated an extensive toolbox of ligand types that include orthosteric, allosteric, and bitopic ligands, many of which show signaling bias. We discuss how combining these advances with recently described transgenic, chemical genetic, and optogenetic animal models will provide the framework to allow for the rational design of next-generation GPCR drugs that possess increased therapeutic efficacy and decreased adverse/toxic responses.

Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors.

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and ß-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.