RESUMO
Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G-protein-coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light-activated positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), a classâ B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca(2+) , cAMP, and insulin responses to glucagon-like peptide-1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small-molecule classâ B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP-1R.
Assuntos
Compostos de Anilina/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Regulação Alostérica/efeitos da radiação , Compostos de Anilina/síntese química , Compostos de Anilina/química , Animais , Compostos Azo/química , Células CHO , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Humanos , Insulina/metabolismo , Isomerismo , Luz , Pirimidinas/química , Pirimidinas/farmacologia , Raios UltravioletaRESUMO
Incretin mimetics are set to become a mainstay of typeâ 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer-biased GLP-1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival.
Assuntos
Incretinas/química , Insulina/metabolismo , Liraglutida/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Transformada , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Secreção de Insulina , Liraglutida/química , Liraglutida/uso terapêutico , Camundongos , Mimetismo Molecular , Dados de Sequência Molecular , Transdução de SinaisRESUMO
The capa peptide family, originally identified in the tobacco hawk moth, Manduca sexta, is now known to be present in many insect families, with increasing publications on capa neuropeptides each year. The physiological actions of capa peptides vary depending on the insect species but capa peptides have key myomodulatory and osmoregulatory functions, depending on insect lifestyle, and life stage. Capa peptide signaling is thus critical for fluid homeostasis and survival, making study of this neuropeptide family attractive for novel routes for insect control. In Dipteran species, including the genetically tractable Drosophila melanogaster, capa peptide action is diuretic; via elevation of nitric oxide, cGMP and calcium in the principal cells of the Malpighian tubules. The identification of the capa receptor (capaR) in several insect species has shown this to be a canonical GPCR. In D. melanogaster, ligand-activated capaR activity occurs in a dose-dependent manner between 10(-6) and 10(-12)M. Lower concentrations of capa peptide do not activate capaR, either in adult or larval Malpighian tubules. Use of transgenic flies in which capaR is knocked-down in only Malpighian tubule principal cells demonstrates that capaR modulates tubule fluid secretion rates and in doing so, sets the organismal response to desiccation. Thus, capa regulates a desiccation-responsive pathway in D. melanogaster, linking its role in osmoregulation and fluid homeostasis to environmental response and survival. The conservation of capa action between some Dipteran species suggests that capa's role in desiccation tolerance may not be confined to D. melanogaster.
Assuntos
Proteínas de Drosophila/metabolismo , Neuropeptídeos/metabolismo , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Drosophila melanogaster , Túbulos de Malpighi/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Photopharmacology describes the use of light to precisely deliver drug activity in space and time. Such approaches promise to improve drug specificity by reducing off-target effects. As a proof-of-concept, we have subjected the fourth generation photoswitchable sulfonylurea JB253 to comprehensive toxicology assessment, including mutagenicity and maximum/repeated tolerated dose studies, as well as in vivo testing in rodents. Here, we show that JB253 is well-tolerated with minimal mutagenicity and can be used to optically-control glucose homeostasis in anesthetized mice following delivery of blue light to the pancreas. These studies provide the first demonstration that photopharmacology may one day be applicable to the light-guided treatment of type 2 diabetes and other metabolic disease states in vivo in humans.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Compostos de Sulfonilureia/administração & dosagem , Animais , Glicemia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Homeostase , Hipoglicemiantes/efeitos adversos , Camundongos , Fármacos Fotossensibilizantes/efeitos adversos , Compostos de Sulfonilureia/efeitos adversos , Resultado do TratamentoRESUMO
The arrangement of ß cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual ß cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the ß cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread ß cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Sistemas Computacionais , Diabetes Mellitus/patologia , Homeostase/efeitos dos fármacos , Homeostase/efeitos da radiação , Humanos , Insulina/metabolismo , Secreção de Insulina , Luz , Lipídeos/toxicidade , Metaboloma/efeitos dos fármacos , Metabolômica , Camundongos , Fenômenos Ópticos , Fenótipo , Especificidade da EspécieRESUMO
Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man.