-
PloS One 2019GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic β cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding...
GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic β cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding behavior. Several GPR40 full agonists have been reported to reduce food intake in rodents by regulating gut hormone secretion in addition to their potent glucose-lowering effects; however, detailed mechanisms of feeding suppression are still unknown. In the present study, we characterized T-3601386, a novel compound with potent full agonistic activity for GPR40, by using in vitro Ca2+ mobilization assay in Chinese hamster ovary (CHO) cells expressing FFAR1 and in vivo hormone secretion assay. We also evaluated feeding suppression and weight loss after the administration of T-3601386 and investigated the involvement of the vagal nerve in these effects. T-3601386, but not a partial agonist fasiglifam, increased intracellular Ca2+ levels in CHO cells with low FFAR1 expression, and single dosing of T-3601386 in diet-induced obese (DIO) rats elevated plasma incretin levels, suggesting full agonistic properties of T-3601386 against GPR40. Multiple doses of T-3601386, but not fasiglifam, in DIO rats showed dose-dependent weight loss accompanied by feeding suppression and durable glucagon-like peptide-1 elevation, all of which were completely abolished in Ffar1-/- mice. Immunohistochemical analysis in the nuclei of the solitary tract demonstrated that T-3601386 increased the number of c-Fos positive cells, which also disappeared in Ffar1-/- mice. Surgical vagotomy and drug-induced deafferentation counteracted the feeding suppression and weight loss induced by the administration of T-3601386. These results suggest that T-3601386 exerts incretin release and weight loss in a GPR40-dependent manner, and that afferent vagal nerves are important for the feeding suppression induced by GPR40 full agonism. Our novel findings raise the possibility that GPR40 full agonist can induce periphery-derived weight reduction, which may provide benefits such as less adverse effects in central nervous system compared to centrally-acting anti-obesity drugs.
Topics: Animals; Blood Glucose; CHO Cells; Calcium; Cell Line; Cricetulus; Enteroendocrine Cells; Female; Glucagon-Like Peptide 1; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Male; Mice; Obesity; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Rats, Wistar; Receptors, G-Protein-Coupled; Signal Transduction; Vagus Nerve; Weight Loss
PubMed: 31525244
DOI: 10.1371/journal.pone.0222653 -
Journal of Immunotherapy (Hagerstown,... Jun 2023Toll-like receptor (TLR) 7/8 and 9 agonists stimulate an innate immune response that supports the development of tumor-specific immunity. Previous studies showed that...
Toll-like receptor (TLR) 7/8 and 9 agonists stimulate an innate immune response that supports the development of tumor-specific immunity. Previous studies showed that either agonist individually could cure mice of small tumors and that when used in combination, they could prevent the progression of larger tumors (>300 mm 3 ). To examine whether these agents combined could control metastatic disease, syngeneic mice were challenged with the highly aggressive 66cl4 triple-negative breast tumor cell line. Treatment was not initiated until pulmonary metastases were established, as verified by bioluminescent imaging of luciferase-tagged tumor cells. Results show that combined therapy with TLR7/8 and TLR9 agonists delivered to both primary and metastatic tumor sites significantly reduced tumor burden and extended survival. The inclusion of cyclophosphamide and anti-PD-L1 resulted in optimal tumor control, characterized by a 5-fold increase in the average duration of survival.
Topics: Mice; Animals; Toll-Like Receptor 9; Immunotherapy; Cell Line; Adjuvants, Immunologic; Lung Neoplasms
PubMed: 37103328
DOI: 10.1097/CJI.0000000000000467 -
British Journal of Pharmacology Jan 2019We aimed to identify and develop novel, selective muscarinic M receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease.
BACKGROUND AND PURPOSE
We aimed to identify and develop novel, selective muscarinic M receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease.
EXPERIMENTAL APPROACH
We developed and utilized a novel M receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators.
KEY RESULTS
Using this paradigm, we identified a series of M receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M receptors with activity conserved across species. SPP1 displays high functional selectivity for M receptors over native M and M receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M receptors.
CONCLUSIONS AND IMPLICATIONS
We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M receptors in physiology and disease.
Topics: Animals; CHO Cells; Cells, Cultured; Cricetulus; Male; Mice; Mice, Inbred C57BL; Molecular Structure; Osteopontin; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Spiro Compounds; Structure-Activity Relationship; Xenopus
PubMed: 30276808
DOI: 10.1111/bph.14510 -
JAMA Network Open May 2024
Topics: Humans; Diabetes Mellitus, Type 2; Glucagon-Like Peptide-1 Receptor; Obesity; Male; Female; Middle Aged; Hypoglycemic Agents; Aged; Adult
PubMed: 38787563
DOI: 10.1001/jamanetworkopen.2024.13172 -
The Journal of Physiology Nov 2016The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is...
KEY POINTS
The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium-free environment and this sensitisation is very long lasting following agonist removal. We show that agonist-induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist-induced sensitisation may provide an explanation for sensitisation following long-term exposure to harmful irritants and pollutants, particularly in the airways.
ABSTRACT
The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non-covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N-terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non-covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1.
Topics: Animals; Calcium; Cells, Cultured; Cymenes; Female; Humans; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Monoterpenes; Neurons; Signal Transduction; Transient Receptor Potential Channels
PubMed: 27307078
DOI: 10.1113/JP272237 -
British Journal of Pharmacology Jul 2019Receptor internalisation is by nature kinetic. Application of a standard equilibrium dose response model to describe the properties of a ligand inducing internalisation,...
BACKGROUND AND PURPOSE
Receptor internalisation is by nature kinetic. Application of a standard equilibrium dose response model to describe the properties of a ligand inducing internalisation, while commonly used, are therefore problematic. Here, we propose two quantitative approaches to address this issue-(a) a model-free method and (b) a kinetic modelling approach-and systematically evaluate the performance of these methods against traditional equilibrium methods to characterise the internalisation profiles of cannabinoid CB receptor agonists.
EXPERIMENTAL APPROACH
Kinetic internalisation assays were conducted using a concentration series of six CB receptor ligands. Internalisation rate analysis and snapshot equilibrium analysis were performed. A model-free method was developed based on the mean residence time of internalisation. A kinetic internalisation model was developed under the quasi-steady state assumption.
KEY RESULTS
Rates of receptor internalisation depended on both agonist and concentration. Agonist potencies from snapshot equilibrium analysis increased with stimulation time, and there was no single time point at which internalisation profiles could infer agonist properties in a comparative manner. The model-free method yielded a time-invariant measure of potency/efficacy for internalisation. The kinetic model adequately described the internalisation of CB receptors over time and provided robust estimates of both potency and efficacy.
CONCLUSION AND IMPLICATIONS
Applying equilibrium analysis to a non-equilibrium pathway cannot provide a reliable estimate of agonist potency. Both the model-free and kinetic modelling approaches characterised the internalisation profiles of CB receptor agonists. The kinetic model provides additional advantages as a method to capture changes in receptor number during other functional assays.
Topics: Cannabinoid Receptor Agonists; Cells, Cultured; Humans; Kinetics; Models, Biological; Receptor, Cannabinoid, CB1
PubMed: 30945265
DOI: 10.1111/bph.14684 -
Scientific Reports Sep 2020Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The...
Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model (OM) of pharmacological agonism is a useful means for achieving this goal. Allosteric ligands bind to receptors at sites that are distinct from those of endogenous agonists that interact with the orthosteric domain on the receptor. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.
Topics: Allosteric Regulation; Animals; Drug Synergism; Humans; Kinetics; Ligands; Protein Binding; Receptors, G-Protein-Coupled
PubMed: 32879329
DOI: 10.1038/s41598-020-71228-y -
Molecular Diagnosis & Therapy Jul 2022G protein coupled receptors (GPCRs) are a superfamily of transmembrane-spanning receptors that are activated by multiple endogenous ligands and are the most common... (Review)
Review
G protein coupled receptors (GPCRs) are a superfamily of transmembrane-spanning receptors that are activated by multiple endogenous ligands and are the most common target for agonist or antagonist therapeutics across a broad spectrum of diseases. Initial characterization within the superfamily suggested that a receptor activated a single intracellular pathway, depending on the G protein to which it coupled. However, it has become apparent that a given receptor can activate multiple different pathways, some being therapeutically desirable, while others are neutral or promote deleterious signaling. The activation of pathways that limit effectiveness of a primary pathway or promote unwanted signals has led to abandonment of some GPCRs as drug targets. However, it is now recognized that the conformation of the receptor in its ligand-bound state can be altered by the structure of the agonist or antagonist to achieve pathway selectivity, a property termed biased signaling. Biased ligands could dramatically expand the number of novel drugs acting at GPCRs for new indications. However, the field struggles with the complexity and uncertainty of these structure-functions relationships. In this review we define the theoretical underpinnings of the biased effect, discuss the methods for measuring bias, and the pitfalls that can lead to incorrect assignments of bias. Using the recent elucidation of a β-adrenergic receptor agonist that is biased in favor of Gs coupling over β-arrestin binding, we provide an example of how large libraries of compounds that are impartial to preconceived notions of agonist binding can be utilized to discover pathway-specific agonists. In this case, an agonist that lacks tachyphylaxis for the treatment of obstructive lung diseases was uncovered, with a structure that was distinctly different from other agonists. We show how biased characteristics were ascertained analytically, and how molecular modeling and simulations provide a structural basis for a restricted signaling repertoire.
Topics: Drug Development; Humans; Ligands; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 35595932
DOI: 10.1007/s40291-022-00592-4 -
Journal of Medicinal Chemistry Jan 2022NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here,...
NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of ()-3-(5-furanyl)carboxamido-2-aminopropanoic acid analogues - as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogues display highly variable potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound is identified as a potent partial agonist at GluN1/2C (EC = 0.074 μM) with an agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B, and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents.
Topics: Animals; Carrier Proteins; Excitatory Amino Acid Agonists; Glycine; Humans; Membrane Proteins; Models, Molecular; Nerve Tissue Proteins; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship; Xenopus; Xenopus laevis
PubMed: 34918931
DOI: 10.1021/acs.jmedchem.1c01810 -
Toxicology in Vitro : An International... Dec 2020Environmental exposures often occur in complex mixtures and at low concentrations. Generalized concentration addition (GCA) is a method used to estimate the joint effect...
Environmental exposures often occur in complex mixtures and at low concentrations. Generalized concentration addition (GCA) is a method used to estimate the joint effect of receptor ligands that vary in efficacy. GCA models have been successfully applied to mixtures of aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor gamma (PPARγ) ligands, each of which can be modeled as a receptor with a single binding site. Here, we evaluated whether GCA could be applied to homodimer nuclear receptors, which have two binding sites, to predict the combined effect of full glucocorticoid receptor (GR) agonists with partial agonists. We measured transcriptional activation of GR using a cell-based bioassay. Individual concentration-response curves for dexamethasone (full agonist), prednisolone (full agonist), and medroxyprogesterone 17-acetate (partial agonist) were generated and applied in three additivity models, GCA, effect summation (ES), and relative potency factor (RPF), to generate response surfaces. GCA and RPF yielded adequate predictions of the experimental data for two full agonists. However, GCA fit experimental data significantly better than ES and RPF for all other binary mixtures. This work extends the application of GCA to homodimer nuclear receptors and improves prediction accuracy of mixture effects of GR agonists.
Topics: Biological Assay; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Ligands; Models, Theoretical; Receptors, Glucocorticoid
PubMed: 32858110
DOI: 10.1016/j.tiv.2020.104975