RESUMO
Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of ß-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and ß-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and ß-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a ß-arrestin dependency and genetic ablation of ß-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of ß-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.
Assuntos
Fenótipo , Receptores dos Hormônios Gastrointestinais , beta-Arrestinas , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Camundongos , Humanos , beta-Arrestinas/metabolismo , Variação Genética , beta-Arrestina 2/metabolismo , beta-Arrestina 2/genética , Transdução de Sinais , Polipeptídeo Inibidor Gástrico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Obesidade/metabolismo , Obesidade/genética , Masculino , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genéticaRESUMO
OBJECTIVE: Pharmacological strategies that engage multiple mechanisms-of-action have demonstrated synergistic benefits for metabolic disease in preclinical models. One approach, concurrent activation of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon (Gcg) receptors (i.e. triagonism), combines the anorectic and insulinotropic activities of GLP-1 and GIP with the energy expenditure effect of glucagon. While the efficacy of triagonism in preclinical models is known, the relative contribution of GcgR activation remains unassessed. This work aims to addresses that central question. METHODS: Herein, we detail the design of unimolecular peptide triagonists with an empirically optimized receptor potency ratio. These optimized peptide triagonists employ a protraction strategy permitting once-weekly human dosing. Additionally, we assess the effects of these peptides on weight-reduction, food intake, glucose control, and energy expenditure in an established DIO mouse model compared to clinically relevant GLP-1R agonists (e.g. semaglutide) and dual GLP-1R/GIPR agonists (e.g. tirzepatide). RESULTS: Optimized triagonists normalize body weight in DIO mice and enhance energy expenditure in a manner superior to that of GLP-1R mono-agonists and GLP-1R/GIPR co-agonists. CONCLUSIONS: These pre-clinical data suggest unimolecular poly-pharmacology as an effective means to target multiple mechanisms contributing to obesity and further implicate GcgR activation as the differentiating factor between incretin receptor mono- or dual-agonists and triagonists.
Assuntos
Polipeptídeo Inibidor Gástrico , Glucagon , Animais , Peso Corporal , Polipeptídeo Inibidor Gástrico/metabolismo , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Camundongos , Camundongos Obesos , Peptídeos/farmacologia , Receptores de Glucagon/metabolismoRESUMO
There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H+-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.
RESUMO
Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.
Assuntos
Cisteína/química , Polímeros/síntese química , Proglucagon/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , Cisteína/análogos & derivados , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Maleimidas/química , Fenômenos de Química Orgânica , Peptídeos/síntese química , Peptídeos/química , Polímeros/químicaRESUMO
Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.
Assuntos
Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/farmacologia , Receptores dos Hormônios Gastrointestinais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Sistema Nervoso Central/metabolismo , Dieta Hiperlipídica , Polipeptídeo Inibidor Gástrico/química , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/metabolismo , Obesidade/patologia , Obesidade/prevenção & controle , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores dos Hormônios Gastrointestinais/deficiência , Receptores dos Hormônios Gastrointestinais/genéticaRESUMO
Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential to establish an in vivo correlation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Barreira Hematoencefálica/citologia , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transportador 1 de Peptídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores/genética , Simportadores/metabolismoRESUMO
The continued global growth in the prevalence of obesity coupled with the limited number of efficacious and safe treatment options elevates the importance of innovative pharmaceutical approaches. Combinatorial strategies that harness the metabolic benefits of multiple hormonal mechanisms have emerged at the preclinical and more recently clinical stages of drug development. A priority has been anti-obesity unimolecular peptides that function as balanced, high potency poly-agonists at two or all the cellular receptors for the endocrine hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon. This report reviews recent progress in this area, with emphasis on what the initial clinical results demonstrate and what remains to be addressed.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico/agonistas , Glucagon/metabolismo , Obesidade/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores de Glucagon/agonistas , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Desenho de Fármacos , Glucagon/química , Humanos , Obesidade/metabolismo , Obesidade/patologia , Fragmentos de Peptídeos/química , Relação Estrutura-AtividadeRESUMO
The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.