RESUMO
We present a comprehensive overview of changes in thyroxine (T4) and thyroid stimulating hormone (TSH) serum concentrations after pre-gestational, gestational and/or lactation exposures of rodents to various chemicals that affect the thyroid hormone system. We show that T4 and TSH changes consistent with the idealized view of the hypothalamic-pituitary-thyroid (HPT) feedback loop (T4 decrements accompanied by TSH increases) are observed with only a relatively small set of chemicals. Most substances affect concentrations of various thyroid hormones without increasing TSH. Studies of altered T4 concentrations after gestational exposures are limited to a relatively small set of chemicals in which pesticides, pharmaceuticals and industrial chemicals are under-represented. Our risk-of-bias analysis exposed deficits in T4/TSH analytics as a problem area. By relating patterns of T4 - TSH changes to mode-of-action (MOA) information, we found that chemicals capable of disrupting the HPT feedback frequently affected thyroid hormone synthesis, while substances that produced T4 serum decrements without accompanying TSH increases lacked this ability, but often induced liver enzyme systems responsible for the elimination of TH by glucuronidation. Importantly, a multitude of MOA leads to decrements of serum T4. The current EU approaches for identifying thyroid hormone system-disrupting chemicals, with their reliance on altered TH serum levels as indicators of a hormonal mode of action and thyroid histopathological changes as indicators of adversity, will miss chemicals that produce T4/T3 serum decreases without accompanying TSH increases. This is of concern as it may lead to a disregard for chemicals that produce developmental neurotoxicity by disrupting adequate T4/T3 supply to the brain, but without increasing TSH.
Assuntos
Hormônios Tireóideos , Animais , Feminino , Gravidez , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismo , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tiroxina/sangue , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Disruptores Endócrinos/toxicidade , Tireotropina/sangue , Roedores , Exposição Materna/efeitos adversos , Ratos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismoRESUMO
The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.
Assuntos
Disruptores Endócrinos/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Hormônios Tireóideos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Descoberta de Drogas , Disruptores Endócrinos/química , Humanos , Técnicas In Vitro , InternetRESUMO
A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Compostos Heterocíclicos/química , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Sequência de Aminoácidos , Desenho de Fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Receptores de Glucagon/antagonistas & inibidores , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
This corrects the article DOI: 10.1038/nature22800.
RESUMO
Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica , Ratos , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Glucagon/químicaRESUMO
Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.
Assuntos
Pirazóis/metabolismo , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/química , beta-Alanina/análogos & derivados , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Glucagon/metabolismo , Glucagon/farmacologia , Humanos , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Glucagon/classificação , Receptores de Glucagon/metabolismo , beta-Alanina/química , beta-Alanina/metabolismo , beta-Alanina/farmacologiaRESUMO
G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M(1) muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of approximately 30 ms and approximately 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, approximately 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M(1) receptors undergoing interconversion between monomers and dimers on the timescale of seconds.
Assuntos
Microscopia de Fluorescência/métodos , Pirenzepina/análogos & derivados , Receptor Muscarínico M1/metabolismo , Animais , Benzenossulfonatos/química , Ligação Competitiva , Células CHO , Carbocianinas/química , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Corantes Fluorescentes/química , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Estrutura Molecular , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , Pirenzepina/metabolismo , Pirenzepina/farmacologia , Multimerização Proteica , Ensaio Radioligante , Receptor Muscarínico M1/antagonistas & inibidores , Receptor Muscarínico M1/genética , Fatores de Tempo , TransfecçãoRESUMO
Research into the functions and mechanisms of action of the melanocortin 2 receptor (MC2R) has been severely hampered by difficulties in expressing this gene in heterologous cells. This probably arises because of the need for a cofactor for cell surface expression. Using either the Y1 cell line that expresses endogenous MC2R or the Y6 cell line that expresses this putative expression factor, we have explored the mechanisms of desensitization and internalization after agonist stimulation. Protein kinase A dependence of desensitization has been demonstrated, although internalization is apparently independent of this kinase and dependent on a G protein receptor kinase. Possible underlying reasons for this paradox are discussed.