A Novel Peptide with Similar Pharmacology to Exenatide in Rodents as GLP-1 Receptor Agonist

The prevalence of type-2 diabetes is rapidly increasing. Currently, exenatide is the first medicine which mimics incretin. However, it requires subcutaneous injection twice a day, an inconvenient way for patients. In this study, we identified a novel peptide with similar pharmacology to exenatide in rodents as GLP-1 receptor agonist which consists of 17 amino acids (17P). It promotes Ins-1 cell proliferation and insulin secretion and lowers blood glucose of diabetic rats. 17P was synthetized by solid-phase peptide synthesis. Interactions between GLP-1 receptor and 17P were studied by Bio-layer interferometry. Ins-1 cell proliferation was studied by MTT assay. ELISA was used to study Ins-1 cell insulin secretion. In vivo tests were performed with male Wistar rats. We used high fat diet and STZ injection to induce a type-2 diabetic rats model. Then, those rats were randomized to different test groups, and administered exenatide, 17P and saline water injection, to evaluate different responses. Based on HPLC (high performance liquid chromatography) and MS (mass spectrometry), 17P synthesis was successful. Bio-layer interferometry data showed a close interaction between GLP-1 receptor and 17P. 17P treatment of Ins-1 cells for 24 h could promote cell proliferation and insulin secretion in a dose-dependent manner. Administration of 17P in HF-STZ male Wistar rats demonstrated that 17P could lower the level of blood glucose and stabilize the body weight of the diabetic rats. All 17P treatments were similar to treatment with exenatide. In vitro and in vivo studies demonstrated that 17P could relieve symptoms of type-2 diabetes. Therefore, 17P could be developed as a promising type-2 diabetes therapeutic drug.     

Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Like Agonist,Exendin-4-Fc

International Journal of Peptide Research and Therapeutics - Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes....     

GLP-1及其受体激动剂Exendin-4临床适应症的扩展

GLP-1及其受体激动剂Exendin-4是治疗糖尿病的一种理想药物,是近年来新的研究热点之一。近年发现,该类药物可从多个生理角度发挥功能,揭示其临床适应症可能有进一步的扩展空间。对GLP-1及Exendin-4的各种已知和潜在的临床适应症进行了概述,这些适应症除了各型糖尿病外,还包括肥胖症、神经系统和心脏的疾病以及其他各种潜在适应症。     

An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke

Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC₅₀ value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia.     

A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor

Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r^−/− animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner proteins, and testing of novel therapeutic agents targeting the hGLP-1R.     

Muscimol as an Ionotropic GABA Receptor Agonist

Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol^®) and 4-PIOL.     

Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

Glucagon-like peptide-1 (GLP-1) analogs are approved for treatment of type 2 diabetes and are in clinical trials for disorders including neurodegenerative diseases. GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Given a normally basal circulating level of GLP-1, knowledge of mechanisms regulating GLP-1R signaling, which has diverse functions in extrapancreatic tissues, remains elusive. In this study, we found that the potency of GLP-1, not exendin 4, is specifically enhanced by the endocannabinoid-like lipids oleoylethanolamide (OEA) and 2-oleoylglycerol but not by stearoylethanolamide (SEA) or palmitoylethanolamide. 9.2 μm OEA enhances the potency of GLP-1 in stimulating cAMP production by 10-fold but does not affect its receptor binding affinity. OEA and 2-oleoylglycerol, but not SEA, bind to GLP-1 in a dose-dependent and saturable manner. OEA but not SEA promoted GLP-1(7–36) amide to trypsin inactivation in a dose-dependent and saturable manner. Susceptibility of GLP-1(7–36) amide to trypsin inactivation is increased 40-fold upon binding to OEA but not to SEA. Our findings indicate that OEA binds to GLP-1(7–36) amide and enhances the potency that may result from a conformational change of the peptide. In conclusion, modulating potency of GLP-1 by physiologically regulated endocannabinoid-like lipids allows GLP-1R signaling to be regulated spatiotemporally at a constant basal GLP-1 level.     

A Galanin Receptor Subtype 1 Specific Agonist

The chimeric peptide M617, galanin(1–13)-Gln¹⁴-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K_i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K_i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca²⁺, high Mg²⁺, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.     

GLP-1 Receptor Agonist Inhibited the Activation of RIPK1 for Alleviation the Neuronal Death and Neuroinflammation in APP/PS1 Mice

International Journal of Peptide Research and Therapeutics - Alzheimer’s disease (AD) is characterized by neuronal necroptosis and neuroinflammation, retardation of these pathological...     

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Proteomic Analysis of INS-1 Rat Insulinoma Cells: ER Stress Effects and the Protective Role of Exenatide,a GLP-1 Receptor Agonist

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.     

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161^Bright mucosal-associated invariant T (MAIT) and CD56^Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.     

PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the in trans presence of the PCSK9 prosegment would interfere with PCSK9''s activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular in vitro co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln₁₅₂ of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.     

The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons

Glucagon-like peptide-1 (GLP-1) is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM), an allosteric positive modulator of GABA_A receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC) amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABA_A current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM) plus diazepam (1 μM), only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABA_A receptors in the CA3 pyramidal neurons.     

Development of a Membrane-anchored Chemerin Receptor Agonist as a Novel Modulator of Allergic Airway Inflammation and Neuropathic Pain

The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gα_i, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149–157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21–157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.     

Identification and Characterization of ZEL-H16 as a Novel Agonist of the Histamine H(3) Receptor

The histamine H3 receptor (H3R) has been recognized as a promising target for the treatment of various central and peripheral nervous system diseases. In this study, a non-imidazole compound, ZEL-H16, was identified as a novel histamine H3 receptor agonist. ZEL-H16 was found to bind to human H3R with a Ki value of approximately 2.07 nM and 4.36 nM to rat H3R. Further characterization indicated that ZEL-H16 behaved as a partial agonist on the inhibition of forskolin-stimulated cAMP accumulation (the efficacy was 60% of that of histamine) and activation of ERK1/2 signaling (the efficacy was 50% of that of histamine) at H3 receptors, but acted as a full agonist just like histamin in the guinea-pig ileum contraction assay. These effects were blocked by pertussis toxin and H3 receptor specific antagonist thioperamide. ZEL-H16 showed no agonist or antagonist activities at the cloned human histamine H1, H2, and H4 receptors and other biogenic amine GPCRs in the CRE-driven reporter assay. Furthermore, our present data demonstrated that treatment of ZEL-H16 resulted in intensive H3 receptor internalization and delayed recycling to the cell surface as compared to that of control with treatment of histamine. Thus, ZEL-H16 is a novel and potent nonimidazole agonist of H3R, which might serve as a pharmacological tool for future investigations or as possible therapeutic agent of H3R.     

A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide,GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA²]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA²]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA²]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA²]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA²]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA²]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA²]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.     

6-Cyclopropylpurines as Novel Potent Analogs of Cytokinins

6-Alkynyl-, trans-6-alkenyl-, trans-6-cyclopropyl-and 6-alkylpurines structurally related to the cytokinin 6-benzylaminopurine (BAP) have been synthesized and examined with a radish cotyledon assay as plant growth stimulators. The growth stimulation obtained with the 6-alkylpurines trans-cyclopropylpurines was very close to that obtained with BAP, and the trans-styrylpurines were somewhat less effective. The fact that the conformationally locked cyclopropanes exhibit growth-stimulating effects comparable to the flexible 6-alkylpurines and to BAP, supports the hypothesis that the orientation of the NH-CH₂ bond in “the active conformation” of BAP is close to anti, which means that the torsion angle C(6)-N(6)-CH₂-C is approximately 180 degrees.     

GFP荧光标记GLP-1受体细胞系的建立

为建立胰高血糖素样肽1受体(glucagon-like peptide 1 receptor,GLP-1R)药物筛选模型,真核表达载体pCMV6/GFP/GLP-1R构建好后,转染至U2OS细胞,转染后的细胞经G418筛选获得单克隆细胞株。该细胞株经Western-blot和GLP-1类似物检测,结果表明GLP-1R在该细胞株高表达并对GLP-1类似物有着高灵敏度与特异性的反应,可以用于GLP-1受体激动剂的筛选。