Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 57
Filtrar
1.
Cell ; 184(24): 5886-5901.e22, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34822784

RESUMO

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.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Desenho de Fármacos , Receptor Muscarínico M1/agonistas , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Células CHO , Inibidores da Colinesterase/farmacologia , Cricetulus , Cristalização , Modelos Animais de Doenças , Cães , Donepezila/farmacologia , Eletroencefalografia , Feminino , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Degeneração Neural/complicações , Degeneração Neural/patologia , Primatas , Ratos , Receptor Muscarínico M1/química , Transdução de Sinais , Homologia Estrutural de Proteína
2.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34893539

RESUMO

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.


Assuntos
Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Receptor Muscarínico M1/metabolismo , Animais , Células Cultivadas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Doenças Priônicas/genética , Receptor Muscarínico M1/genética , Transdução de Sinais
3.
J Biol Chem ; 298(3): 101655, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35101446

RESUMO

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.


Assuntos
Receptores Acoplados a Proteínas G , Animais , Humanos , Soros Imunes/farmacologia , Camundongos , Fosforilação , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , beta-Arrestina 2/metabolismo
4.
Nat Chem Biol ; 15(5): 489-498, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992568

RESUMO

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.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Camundongos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética
5.
FASEB J ; 33(4): 5005-5017, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30601679

RESUMO

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.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Isoleucina/química , Leucina/química , Linhagem Celular , Biologia Computacional , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/química , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Isoleucina/genética , Cinética , Leucina/genética , Medições Luminescentes , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fator de Crescimento Transformador alfa/química , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , beta-Arrestinas/química , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
6.
Chem Rev ; 117(1): 67-110, 2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-27299848

RESUMO

G protein-coupled receptors (GPCRs) are historically the most successful family of drug targets. In recent times it has become clear that the pharmacology of these receptors is far more complex than previously imagined. Understanding of the pharmacological regulation of GPCRs now extends beyond simple competitive agonism or antagonism by ligands interacting with the orthosteric binding site of the receptor to incorporate concepts of allosteric agonism, allosteric modulation, signaling bias, constitutive activity, and inverse agonism. Herein, we consider how evolving concepts of GPCR pharmacology have shaped understanding of the complex pharmacology of receptors that recognize and are activated by nonesterified or "free" fatty acids (FFAs). The FFA family of receptors is a recently deorphanized set of GPCRs, the members of which are now receiving substantial interest as novel targets for the treatment of metabolic and inflammatory diseases. Further understanding of the complex pharmacology of these receptors will be critical to unlocking their ultimate therapeutic potential.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Regulação Alostérica , Animais , Corantes Fluorescentes/química , Humanos , Ligantes , Receptores Acoplados a Proteínas G/metabolismo
7.
FASEB J ; 31(5): 2195-2209, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28183801

RESUMO

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.-Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.


Assuntos
Macrófagos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Resistência a Medicamentos/fisiologia , Ácidos Graxos Ômega-3/metabolismo , Camundongos Endogâmicos BALB C , Transdução de Sinais/fisiologia
8.
Mol Pharmacol ; 91(6): 630-641, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28385906

RESUMO

High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date, a single molecule, 4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide (AH-7614), has been described as an FFA4 antagonist; however, its mechanism of antagonism remains unknown. We synthesized AH-7614 and a chemical derivative and demonstrated these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signaling induced by a range of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anticancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, 4-methyl-N-(9H-xanthen-9-yl)benzamide (TUG-1387), will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells toward a more mature, adipocyte-like phenotype.


Assuntos
Compostos de Bifenilo/farmacologia , Fenilpropionatos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Camundongos Endogâmicos C3H , Receptores Acoplados a Proteínas G/antagonistas & inibidores
9.
J Biol Chem ; 291(1): 303-17, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26518871

RESUMO

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.


Assuntos
Aminoácidos/metabolismo , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Butiratos/química , Butiratos/farmacologia , Ácido Butírico/farmacologia , Ésteres/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Receptores de Superfície Celular/química , Tiofenos/química , Tiofenos/farmacologia , Trítio/metabolismo
10.
Handb Exp Pharmacol ; 236: 79-100, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27757763

RESUMO

The free fatty acid (FFA) family of G protein coupled receptors (GPCRs) has generated significant interest for exploiting its members as potential drug targets. However, unravelling the complex pharmacology of this family of receptors has proven challenging. In recent years the use of biosensor technologies capable of assessing biological functions in living cells, and in real time, has greatly enhanced our ability to study GPCR pharmacology and function. These include genetically encoded sensors that change the intensity or wavelength of light emitted from a bioluminescent or fluorescent protein in response to a stimulus, as well as non-genetically encoded sensors able to measure more global cellular changes, such as mass redistribution within a cell. This chapter will examine how these sensors can be used to study GPCRs, and in particular how they are helping uncover the pharmacology of the FFA family of receptors.


Assuntos
Técnicas Biossensoriais/métodos , Ácidos Graxos não Esterificados/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Arrestina/análise , Humanos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Transdução de Sinais
11.
Mol Pharmacol ; 89(5): 505-20, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26873857

RESUMO

It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.


Assuntos
Membrana Celular/metabolismo , Sistema de Sinalização das MAP Quinases , Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Serina/metabolismo , Treonina/metabolismo , Substituição de Aminoácidos , Animais , Arrestinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
12.
J Biol Chem ; 289(29): 20345-58, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24860101

RESUMO

The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Substituição de Aminoácidos , Arrestinas/metabolismo , Sítios de Ligação , Ácidos Graxos não Esterificados/química , Células HEK293 , Humanos , Ligação de Hidrogênio , Ligantes , Metilaminas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Propionatos/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Termodinâmica , Ácido alfa-Linolênico/metabolismo , beta-Arrestinas
13.
J Biol Chem ; 289(26): 18451-65, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24817122

RESUMO

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr(347), Thr(349), Ser(350), Ser(357), and Ser(360)) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu(341), Asp(348), and Asp(355) located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.


Assuntos
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arrestinas/química , Arrestinas/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Receptores Acoplados a Proteínas G/genética
14.
J Biol Chem ; 289(23): 16032-45, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24742673

RESUMO

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 ß-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.


Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Dieta Hiperlipídica , Indometacina/farmacologia , Resistência à Insulina , Obesidade/prevenção & controle , Animais , Linhagem Celular , Ácidos Graxos não Esterificados/sangue , Teste de Tolerância a Glucose , Glicerol/sangue , Mediadores da Inflamação/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxilipinas/sangue , Reação em Cadeia da Polimerase em Tempo Real
15.
Br J Nutr ; 113(11): 1677-88, 2015 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-25916176

RESUMO

Various foods are associated with effects against metabolic diseases such as insulin resistance and type 2 diabetes; however, their mechanisms of action are mostly unclear. Fatty acids may contribute by acting as precursors of signalling molecules or by direct activity on receptors. The medium- and long-chain NEFA receptor FFA1 (free fatty acid receptor 1, previously known as GPR40) has been linked to enhancement of glucose-stimulated insulin secretion, whereas FFA4 (free fatty acid receptor 4, previously known as GPR120) has been associated with insulin-sensitising and anti-inflammatory effects, and both receptors are reported to protect pancreatic islets and promote secretion of appetite and glucose-regulating hormones. Hypothesising that FFA1 and FFA4 mediate therapeutic effects of dietary components, we screened a broad selection of NEFA on FFA1 and FFA4 and characterised active compounds in concentration-response curves. Of the screened compounds, pinolenic acid, a constituent of pine nut oil, was identified as a relatively potent and efficacious dual FFA1/FFA4 agonist, and its suitability for further studies was confirmed by additional in vitro characterisation. Pine nut oil and free and esterified pure pinolenic acid were tested in an acute glucose tolerance test in mice. Pine nut oil showed a moderately but significantly improved glucose tolerance compared with maize oil. Pure pinolenic acid or ethyl ester gave robust and highly significant improvements of glucose tolerance. In conclusion, the present results indicate that pinolenic acid is a comparatively potent and efficacious dual FFA1/FFA4 agonist that exerts antidiabetic effects in an acute mouse model. The compound thus deserves attention as a potential active dietary ingredient to prevent or counteract metabolic diseases.


Assuntos
Gorduras na Dieta/farmacologia , Ácidos Linolênicos/farmacologia , Síndrome Metabólica/prevenção & controle , Receptores Acoplados a Proteínas G/genética , Animais , Diabetes Mellitus Tipo 2/prevenção & controle , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nozes/química , Pinus , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo
16.
Mol Pharmacol ; 86(2): 200-10, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24870406

RESUMO

Analysis of the roles of the short chain fatty acid receptor, free fatty acid 3 receptor (FFA3), has been severely limited by the low potency of its endogenous ligands, the crossover of function of these on the closely related free fatty acid 2 receptor, and a dearth of FFA3-selective synthetic ligands. From a series of hexahydroquinolone-3-carboxamides, we demonstrate that 4-(furan-2-yl)-2-methyl-5-oxo-N-(o-tolyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxamide is a selective and moderately potent positive allosteric modular (PAM)-agonist of the FFA3 receptor. Modest chemical variations within this series resulted in compounds completely lacking activity, acting as FFA3 PAMs, or appearing to act as FFA3-negative allosteric modulators. However, the pharmacology of this series was further complicated in that certain analogs displaying overall antagonism of FFA3 function actually appeared to generate their effects via a combined positive allosteric binding cooperativity and negative allosteric effect on orthosteric ligand maximal signaling response. These studies show that various PAM-agonist and allosteric modulators of FFA3 can be identified and characterized. However, within the current chemical series, considerable care must be taken to define the pharmacological characteristics of specific compounds before useful predictions of their activity and their use in defining specific roles of FFA3 in either in vitro and in vivo settings can be made.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Humanos , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores
17.
Mol Pharmacol ; 85(1): 91-104, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24113750

RESUMO

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.


Assuntos
Antialérgicos/farmacologia , Mastócitos/efeitos dos fármacos , Ácido Oxâmico/análogos & derivados , Fenantrolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Linhagem Celular , Simulação por Computador , Cricetinae , Cricetulus , Humanos , Mastócitos/fisiologia , Simulação de Acoplamento Molecular , Mutação , Ácido Oxâmico/farmacologia , Polimorfismo de Nucleotídeo Único , Ratos , Receptores Acoplados a Proteínas G/genética
18.
J Biol Chem ; 288(24): 17296-312, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23589301

RESUMO

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.


Assuntos
Butiratos/farmacologia , Ciclopropanos/farmacologia , Receptores de Superfície Celular/agonistas , Tiazóis/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Benzenoacetamidas/farmacologia , Sítios de Ligação , Ciclopropanos/química , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Lipólise/efeitos dos fármacos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Tiazóis/química
19.
Br J Nutr ; 111 Suppl 1: S3-7, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24382058

RESUMO

It is becoming evident that nutrients and metabolic intermediates derived from such nutrients regulate cellular function by activating a number of cell-surface G-protein coupled receptors (GPCRs). Until now, members of the GPCR family have largely been considered as the molecular targets that communicate cellular signals initiated by hormones and neurotransmitters. Recently, based on tissue expression patterns of these receptors and the concept that they may elicit the production of a range of appetite- and hunger-regulating peptides, such nutrient sensing GPCRs are attracting considerable attention due to their potential to modulate satiety, improve glucose homeostasis and supress the production of various pro-inflammatory mediators. Despite the developing interests in these nutrients sensing GPCR both as sensors of nutritional status, and targets for limiting the development of metabolic diseases, major challenges remain to exploit their potential for therapeutic purposes. Mostly, this is due to limited characterisation and validation of these receptors because of paucity of selective and high-potency/affinity pharmacological agents to define the detailed function and regulation of these receptors. However, ongoing clinical trials of agonists of free fatty acid receptor 1 suggest that this receptor and other receptors for free fatty acids may provide a successful strategy for controlling hyperglycaemia and providing novel approaches to treat diabetes. Receptors responsive to free fatty acid have been of particular interest, and some aspects of these are considered herein.


Assuntos
Diabetes Mellitus/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Hiperglicemia/metabolismo , Estado Nutricional , Receptores Acoplados a Proteínas G/metabolismo , Regulação do Apetite , Glicemia/metabolismo , Diabetes Mellitus/terapia , Humanos , Hiperglicemia/terapia
20.
Mol Pharmacol ; 84(5): 710-25, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23979972

RESUMO

TUG-891 [3-(4-((4-fluoro-4'-methyl-[1,1'-biphenyl]-2-yl)methoxy)phenyl)propanoic acid] was recently described as a potent and selective agonist for the long chain free fatty acid (LCFA) receptor 4 (FFA4; previously G protein-coupled receptor 120, or GPR120). Herein, we have used TUG-891 to further define the function of FFA4 and used this compound in proof of principle studies to indicate the therapeutic potential of this receptor. TUG-891 displayed similar signaling properties to the LCFA α-linolenic acid at human FFA4 across various assay end points, including stimulation of Ca²âº mobilization, ß-arrestin-1 and ß-arrestin-2 recruitment, and extracellular signal-regulated kinase phosphorylation. Activation of human FFA4 by TUG-891 also resulted in rapid phosphorylation and internalization of the receptor. While these latter events were associated with desensitization of the FFA4 signaling response, removal of TUG-891 allowed both rapid recycling of FFA4 back to the cell surface and resensitization of the FFA4 Ca²âº signaling response. TUG-891 was also a potent agonist of mouse FFA4, but it showed only limited selectivity over mouse FFA1, complicating its use in vivo in this species. Pharmacologic dissection of responses to TUG-891 in model murine cell systems indicated that activation of FFA4 was able to mimic many potentially beneficial therapeutic properties previously reported for LCFAs, including stimulating glucagon-like peptide-1 secretion from enteroendocrine cells, enhancing glucose uptake in 3T3-L1 adipocytes, and inhibiting release of proinflammatory mediators from RAW264.7 macrophages, which suggests promise for FFA4 as a therapeutic target for type 2 diabetes and obesity. Together, these results demonstrate both potential but also significant challenges that still need to be overcome to therapeutically target FFA4.


Assuntos
Compostos de Bifenilo/farmacologia , Fenilpropionatos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Células 3T3-L1 , Animais , Arrestinas/fisiologia , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Glucose/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , beta-Arrestina 1 , beta-Arrestina 2 , beta-Arrestinas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA