ABSTRACT
Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer's patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.
Subject(s)
Fatty Acids, Nonesterified , Receptors, G-Protein-Coupled , Animals , Humans , Mice , Cell Line , Fatty Acids, Volatile/metabolism , Mice, Transgenic , Phosphorylation , Receptors, G-Protein-Coupled/metabolismABSTRACT
Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M1 acetylcholine muscarinic receptor (M1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Prion Diseases , Prions , Humans , Animals , Mice , Prions/genetics , Neurodegenerative Diseases/genetics , Pathology, Molecular , Proteomics , Prion Diseases/genetics , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Receptor, Muscarinic M1/genetics , Receptor, Muscarinic M1/metabolismABSTRACT
Alzheimer's disease (AD) remains a major cause of morbidity and mortality worldwide, and despite extensive research, only a few drugs are available for management of the disease. One strategy has been to up-regulate cholinergic neurotransmission to improve cognitive function, but this approach has dose-limiting adverse effects. To avoid these adverse effects, new drugs that target specific receptor subtypes of the cholinergic system are needed, and the M1 subtype of muscarinic acetylcholine receptor (M1-mAChR) has been shown to be a good target for this approach. By using several strategies, M1-mAChR ligands have been developed and trialled in preclinical animal models and in human studies, with varying degrees of success. This article reviews the different approaches to targeting the M1-mAChR in AD and discusses the advantages and limitations of these strategies. The factors to consider in targeting the M1-mAChR in AD are also discussed.
ABSTRACT
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.
Subject(s)
Arrestins , Threonine , Arrestins/metabolism , Humans , Ligands , Mutation , Phosphorylation , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Serine/metabolism , Threonine/metabolism , beta-Arrestin 2/metabolismABSTRACT
Urogenital schistosomiasis remains a major public health concern worldwide. In response to egg deposition, the host bladder undergoes gross and molecular morphological changes relevant for disease manifestation. However, limited mechanistic studies to date imply that the molecular mechanisms underlying pathology are not well-defined. We leveraged a mouse model of urogenital schistosomiasis to perform for the first time, proteome profiling of the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to elucidate the protein pathways involved in urogenital schistosomiasis-induced pathology. Purified S. haematobium eggs or control vehicle were microinjected into the bladder walls of mice. Mice were sacrificed seven days post-injection and bladder proteins isolated and processed for proteome profiling using mass spectrometry. We demonstrate that biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism were significantly enriched in S. haematobium infection. S. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Our findings highlight the potential bladder protein indicators for host-parasite interplay and provide new insights into the complex dynamics of pathology and characteristic bladder tissue changes in urogenital schistosomiasis. The findings will be relevant for development of improved interventions for disease control.
Subject(s)
Host-Parasite Interactions/physiology , Schistosoma haematobium/pathogenicity , Schistosomiasis haematobia/physiopathology , Urinary Bladder/parasitology , Animals , Disease Models, Animal , Female , Mice, Inbred BALB C , Ovum , Proteome , Urinary Bladder/metabolism , Urinary Bladder/pathologyABSTRACT
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.
Subject(s)
Prion Diseases/metabolism , Prion Diseases/pathology , Receptor, Muscarinic M1/metabolism , Animals , Cells, Cultured , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Gene Expression Regulation/physiology , Mice , Mice, Knockout , Neurons/metabolism , Prion Diseases/genetics , Receptor, Muscarinic M1/genetics , Signal TransductionABSTRACT
Neurodegenerative diseases remain a major global health challenge, affecting millions of individuals worldwide. Despite significant research efforts, the etiology and pathological mechanisms of most neurodegenerative disorders remain uncertain. This indicates that new disease models and research strategies need to be developed to enhance biomarker discovery and drug development processes. Studies have shown that dysregulation of protein-protein interactions in the brain may be key to the pathophysiological processes in a number of neurodegenerative diseases. Consequently, a deeper understanding of protein-protein interactions in the brain in both healthy and neurodegenerative states may provide greater insights into disease mechanisms and progress, and aid the development of preventive, management and treatment strategies. Efforts to investigate protein-protein interactions in the brain have largely depended on purifying the protein, together with its interacting partner proteins (interactome) and then analyzing these interactions by mass spectrometry. However, factors including abundance and phosphorylation status of the protein of interest and its interactome can lead to identification of false protein-protein interactions. Moreover, studies have shown that combining quantitative proteomics data with data from affinity-purification-mass spectrometry data can reduce these false positives and also provide more insights into protein-protein interactions. Thus, we have developed a protocol for preparing rodent brain tissue for quantitative- and phospho-proteomics analysis, combining in-house and commercially available kits. Data from this protocol will enhance the analysis and interpretation of protein-protein interactions in both physiological and pathological states.
Subject(s)
Proteomics , Rodentia , Animals , Brain , Mass Spectrometry/methods , Protein Processing, Post-Translational , Proteomics/methodsABSTRACT
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.
Subject(s)
Alzheimer Disease/drug therapy , Drug Design , Receptor, Muscarinic M1/agonists , Aged , Aged, 80 and over , Aging/pathology , Alzheimer Disease/complications , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/pathology , Amino Acid Sequence , Animals , Blood Pressure/drug effects , CHO Cells , Cholinesterase Inhibitors/pharmacology , Cricetulus , Crystallization , Disease Models, Animal , Dogs , Donepezil/pharmacology , Electroencephalography , Female , HEK293 Cells , Heart Rate/drug effects , Humans , Male , Mice, Inbred C57BL , Models, Molecular , Molecular Dynamics Simulation , Nerve Degeneration/complications , Nerve Degeneration/pathology , Primates , Rats , Receptor, Muscarinic M1/chemistry , Signal Transduction , Structural Homology, ProteinABSTRACT
The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
Subject(s)
Antimalarials/pharmacology , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Drug Resistance/drug effects , Plasmodium falciparum/drug effects , Protozoan Proteins/antagonists & inhibitors , Animals , Antimalarials/chemistry , Antimalarials/metabolism , Binding Sites , Cyclic GMP-Dependent Protein Kinases/metabolism , Female , Hepatocytes/cytology , Hepatocytes/metabolism , Hepatocytes/parasitology , Humans , Imidazoles/chemistry , Life Cycle Stages/drug effects , Metabolomics , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Proteomics , Protozoan Proteins/genetics , Protozoan Proteins/metabolismABSTRACT
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.
Subject(s)
Receptor, Muscarinic M1/genetics , Receptor, Muscarinic M1/metabolism , Acetylcholinesterase/metabolism , Alzheimer Disease/drug therapy , Animals , Cholinergic Agents/pharmacology , Cholinesterase Inhibitors/metabolism , Cholinesterase Inhibitors/pharmacology , Disease Models, Animal , Drug Design , Female , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BL , PhosphorylationABSTRACT
The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.
Subject(s)
Acetylcholine/metabolism , Muscarinic Agonists/pharmacology , Receptor, Muscarinic M1/metabolism , Receptors, Muscarinic/metabolism , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Animals , Benzimidazoles/pharmacology , Binding Sites/drug effects , CHO Cells , Cell Line , Clinical Trials as Topic , Cricetinae , Cricetulus , Humans , Learning/drug effects , Male , Memory/drug effects , Mice , Mice, Inbred C57BL , Protein Binding/drug effects , Rats , Rats, WistarABSTRACT
Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns' dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181 bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies.