scholarly journals Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists

2019 ◽  
Author(s):  
Maria Lucey ◽  
Philip Pickford ◽  
James Minnion ◽  
Jan Ungewiss ◽  
Katja Schoeneberg ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Trafficking ◽  
Appetite Suppression ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
And Diffusion

AbstractObjectiveTo determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy and appetite suppression.MethodsIn vitro signalling responses were measured using biochemical and biosensor assays. GLP-1 receptor trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects and appetite suppression were measured in acute, sub-chronic and chronic settings in mice.ResultsA C-terminally acylated ligand, exendin-phe1-C16, was identified with undetectable β-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator exendin-asp-3-C16 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation.ConclusionsC-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

Glucagon-like Peptide 1 Receptor Agonists, Diabetic Retinopathy and Angiogenesis: The AngioSafe Type 2 Diabetes Study

2019 ◽  
Vol 105 (4) ◽  
pp. e1549-e1560 ◽  
Author(s):  
Bénédicte Gaborit ◽  
Jean-Baptiste Julla ◽  
Samaher Besbes ◽  
Matthieu Proust ◽  
Clara Vincentelli ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Diabetic Retinopathy ◽  
Fundus Photography ◽  
Endothelial Cell Proliferation ◽  
Receptor Agonists ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Abstract Aims Recent trials provide conflicting results on the association between glucagon-like peptide 1 receptor agonists (GLP-1RA) and diabetic retinopathy (DR). The aim of the AngioSafe type 2 diabetes (T2D) study was to determine the role of GLP-1RA in angiogenesis using clinical and preclinical models. Methods We performed two studies in humans. In study 1, we investigated the effect of GLP-1RA exposure from T2D diagnosis on the severity of DR, as diagnosed with retinal imaging (fundus photography). In study 2, a randomized 4-week trial, we assessed the effect of liraglutide on circulating hematopoietic progenitor cells (HPCs), and angio-miRNAs. We then studied the experimental effect of Exendin-4, on key steps of angiogenesis: in vitro on human endothelial cell proliferation, survival and three-dimensional vascular morphogenesis; and in vivo on ischemia-induced neovascularization of the retina in mice. Results In the cohort of 3154 T2D patients, 10% displayed severe DR. In multivariate analysis, sex, disease duration, glycated hemoglobin (HbA1c), micro- and macroangiopathy, insulin therapy and hypertension remained strongly associated with severe DR, while no association was found with GLP-1RA exposure (o 1.139 [0.800–1.622], P = .47). We further showed no effect of liraglutide on HPCs, and angio-miRNAs. In vitro, we demonstrated that exendin-4 had no effect on proliferation and survival of human endothelial cells, no effect on total length and number of capillaries. Finally, in vivo, we showed that exendin-4 did not exert any negative effect on retinal neovascularization. Conclusions The AngioSafe T2D studies provide experimental and clinical data confirming no effect of GLP-1RA on angiogenesis and no association between GLP-1 exposure and severe DR.

scholarly journals Förster Resonance Energy Transfer-Based Stability Assessment of PLGA Nanoparticles in Vitro and in Vivo

2019 ◽  
Vol 2 (3) ◽  
pp. 1131-1140 ◽  
Author(s):  
Edyta Swider ◽  
Sanish Maharjan ◽  
Karlijne Houkes ◽  
Nicolaas Koen van Riessen ◽  
Carl Figdor ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Plga Nanoparticles ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Stability Assessment ◽  
Förster Resonance

scholarly journals G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection

2013 ◽  
Vol 51 (1) ◽  
pp. 191-202 ◽  
Author(s):  
Patricia M Lenhart ◽  
Stefan Broselid ◽  
Cordelia J Barrick ◽  
L M Fredrik Leeb-Lundberg ◽  
Kathleen M Caron
Keyword(s):  
G Protein ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cardiac Cells ◽  
Receptor Activity ◽  
Hek293 Cells ◽  
G Protein Coupled

Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30–RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.

Oral intake of slowly digestible α-glucan, isomaltodextrin, stimulates glucagon-like peptide-1 secretion in the small intestine of rats

2019 ◽  
Vol 123 (6) ◽  
pp. 619-626
Author(s):  
Yoshihiko Komuro ◽  
Takashi Kondo ◽  
Shingo Hino ◽  
Tatsuya Morita ◽  
Naomichi Nishimura
Keyword(s):  
Small Intestine ◽  
Oral Delivery ◽  
Oral Intake ◽  
Membrane Vesicles ◽  
Rat Small Intestine ◽  
Maize Starch ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

AbstractTo investigate whether oral intake of highly branched α-glucan isomaltodextrin (IMD) could stimulate ileal glucagon-like peptide-1 (GLP-1) secretion, we examined (1) the digestibility of IMD, (2) the digestion and absorption rates of IMD, in rat small intestine and (3) portal GLP-1 concentration in rats given IMD. In Expt 1, ileorectostomised rats were given a 3 % IMD diet for 10 d. Separately, a 16-h in vitro digestion of IMD, using porcine pancreatic α-amylase and brush-border membrane vesicles from rat small intestine, was conducted. In Expt 2, upon 24-h fasting, rats were given any of glucose, IMD and high-amylose maize starch (HAMS) (1 g/kg of body weight). In Expt 3, caecectomised rats were given 0·2 % neomycin sulphate and a 5 % IMD diet for 10 d. The in vivo and in vitro digestibility of IMD was 70–80 %. The fraction of IMD digested in vitro for the first 120 min was 67 % of that in maize starch. The AUC for 0–120 min of plasma glucose concentration was significantly lower in HAMS group and tended to be lower in IMD group than in the glucose group. Finally, we also observed that, when compared with control rats, glucose of IMD significantly stimulated and improved the concentration of portal active GLP-1 in antibiotic-administered, caecectomised rats. We concluded that IMD was slowly digested and the resulting glucose stimulated GLP-1 secretion in rat small intestine. Oral delivery of slowly released IMD glucose to the small intestine probably exerts important, yet unknown, physiological effects on the recipient.

scholarly journals Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1218-1230 ◽  
Author(s):  
Alicja J. Copik ◽  
M. Scott Webb ◽  
Aaron L. Miller ◽  
Yongxin Wang ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Glucocorticoid Receptor ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Function ◽  
Tata Binding Protein

Abstract The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

Sign in / Sign up

Export Citation Format

Share Document