Corrination mitigates peptide aggregation as exemplified for Glucagon.

Pharmaceutical development of glucagon for use in acute hypoglycemia has proved challenging, due in large part to poor solubility, poor stability and aggregate formation. Herein, we describe highly soluble, low aggregating, glucagon conjugates generated through use of the commercially available vitamin B12 precursor dicyanocobinamide ('corrination'), which retain full stimulatory action at the human glucagon receptor. The modified glucagon analogs were tested in a chemical stability assay in 50 mM phosphate buffer and the percentage of original concentration retained was determined after two weeks of incubation at 37° C. Aggregate formation assays were also performed after 48 h of agitation at 37°C using a thioflavin (ThT) fluorescence-based assay. All corrinated compounds retained original concentration to a higher degree than glucagon controls and showed markedly decreased aggregation compared to their respective noncorrinated analogues. Based on the statistically significant increase in chemical stability coupled with the notably decreased tendency to form aggregates, analogues 2 and its corrinated conjugate 5 were used for a functional assay study performed after agitation at 37°C for 24-hr after which agonism was measured at the human glucagon receptor using a cAMP FRET assay. Corrinated 5 exhibited a 6.6-fold increased potency relative to glucagon, which was shown to have a 165-fold reduction in potency. The relative potency of 5 was also improved compared to that of 2 with EC50 values of 5.5 nM and 9.6 nM for 5 and 2, respectively. In conclusion, corrination of peptides mitigates aggregation, presenting a compound with prolonged stability and agonism as demonstrated for glucagon.

A peptide triple agonist of GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors promotes glycemic control and weight loss.

Mechanisms underlying long-term sustained weight loss and glycemic normalization after obesity surgery include changes in gut hormone levels, including glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). We demonstrate that two peptide biased agonists (GEP44 and GEP12) of the GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors (GLP-1R, Y1-R, and Y2-R, respectively) elicit Y1-R antagonist-controlled, GLP-1R-dependent stimulation of insulin secretion in both rat and human pancreatic islets, thus revealing the counteracting effects of Y1-R and GLP-1R agonism. These agonists also promote insulin-independent Y1-R-mediated glucose uptake in muscle tissue ex vivo and more profound reductions in food intake and body weight than liraglutide when administered to diet-induced obese rats. Our findings support a role for Y1-R signaling in glucoregulation and highlight the therapeutic potential of simultaneous receptor targeting to achieve long-term benefits for millions of patients.

Excess pancreatic elastase alters acinar-ß cell communication by impairing the mechano-signaling and the PAR2 pathways.

Type 1 (T1D) or type 2 diabetes (T2D) are caused by a deficit of functional insulin-producing ß cells. Thus, the identification of ß cell trophic agents could allow the development of therapeutic strategies to counteract diabetes. The discovery of SerpinB1, an elastase inhibitor that promotes human ß cell growth, prompted us to hypothesize that pancreatic elastase (PE) regulates ß cell viability. Here, we report that PE is up-regulated in acinar cells and in islets from T2D patients, and negatively impacts ß cell viability. Using high-throughput screening assays, we identified telaprevir as a potent PE inhibitor that can increase human and rodent ß cell viability in vitro and in vivo and improve glucose tolerance in insulin-resistant mice. Phospho-antibody microarrays and single-cell RNA sequencing analysis identified PAR2 and mechano-signaling pathways as potential mediators of PE. Taken together, our work highlights PE as a potential regulator of acinar-ß cell crosstalk that acts to limit ß cell viability, leading to T2D.

Nonpeptidic Z360-Analogs Tagged with Trivalent Radiometals as Anti-CCK2R Cancer Theranostic Agents: A Preclinical Study.

(1) Background: Theranostic approaches in the management of cholecystokinin subtype 2 receptor (CCK2R)-positive tumors include radiolabeled gastrin and CCK motifs. Moving toward antagonist-based CCK2R-radioligands instead, we herein present three analogs of the nonpeptidic CCK2R-antagonist Z360, GAS1/2/3. Each was conjugated to a different chelator (DOTA, NODAGA or DOTAGA) for labeling with medically relevant trivalent radiometals (e.g., Ga-68, In-111, Lu-177) for potential use as anti-CCK2R cancer agents; (2) Methods: The in vitro properties of the thee analogs were compared in stably transfected HEK293-CCK2R cells. Biodistribution profiles were compared in SCID mice bearing twin HEK293-CCK2R and wtHEK293 tumors; (3) Results: The GAS1/2/3 analogs displayed high CCK2R-affinity (lower nM-range). The radioligands were fairly stable in vivo and selectively targeted the HEK293-CCK2R, but not the CCK2R-negative wtHEK293 tumors in mice. Their overall pharmacokinetic profile was found strongly dependent on the radiometal-chelate. Results could be visualized by SPECT/CT for the [111In]In-analogs; (4) Conclusions: The present study highlighted the high impact of the radiometal-chelate on the end-pharmacokinetics of a new series of Z360-based radioligands, revealing candidates with promising properties for clinical translation. It also provided the impetus for the development of a new class of nonpeptidic radioligands for CCK2R-targeted theranostics of human cancer.

The alpha-7 nicotinic acetylcholine receptor agonist GTS-21 engages the glucagon-like peptide-1 incretin hormone axis to lower levels of blood glucose in db/db mice.

AIM: To establish if alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a blood glucose-lowering action in db/db mice, and to test if this action requires coordinate α7nAChR and GLP-1 receptor (GLP-1R) stimulation by GTS-21 and endogenous GLP-1, respectively. MATERIALS AND METHODS: Blood glucose levels were measured during an oral glucose tolerance test (OGTT) using db/db mice administered intraperitoneal GTS-21. Plasma GLP-1, peptide tyrosine tyrosine 1-36 (PYY1-36), glucose-dependent insulinotropic peptide (GIP), glucagon, and insulin levels were measured by ELISA. A GLP-1R-mediated action of GTS-21 that is secondary to α7nAChR stimulation was evaluated using α7nAChR and GLP-1R knockout (KO) mice, or by co-administration of GTS-21 with the dipeptidyl peptidase-4 inhibitor, sitagliptin, or the GLP-1R antagonist, exendin (9-39). Insulin sensitivity was assessed in an insulin tolerance test. RESULTS: Single or multiple dose GTS-21 (0.5-8.0 mg/kg) acted in a dose-dependent manner to lower levels of blood glucose in the OGTT using 10-14 week-old male and female db/db mice. This action of GTS-21 was reproduced by the α7nAChR agonist, PNU-282987, was enhanced by sitagliptin, was counteracted by exendin (9-39), and was absent in α7nAChR and GLP-1R KO mice. Plasma GLP-1, PYY1-36, GIP, glucagon, and insulin levels increased in response to GTS-21, but insulin sensitivity, body weight, and food intake were unchanged. CONCLUSIONS: α7nAChR agonists improve oral glucose tolerance in db/db mice. This action is contingent to coordinate α7nAChR and GLP-1R stimulation. Thus α7nAChR agonists administered in combination with sitagliptin might serve as a new treatment for type 2 diabetes.

Intra-islet glucagon confers ß-cell glucose competence for first-phase insulin secretion and favors GLP-1R stimulation by exogenous glucagon.

We report that intra-islet glucagon secreted from α-cells signals through ß-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9-39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on ß-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a "spillover" effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the ß-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9-39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test that is diagnostic of type 2 diabetes in the clinical setting.

Synthesis, in vitro biological investigation, and molecular dynamics simulations of thiazolopyrimidine based compounds as corticotrophin releasing factor receptor-1 antagonists.

Corticotrophin releasing factor receptor-1 (CRFR1) is a potential target for treatment of depression and anxiety through modifying stress response. A series of new thiazolo[4,5-d]pyrimidine derivatives were designed, prepared and biologically evaluated as potential CRFR1 antagonists. Four compounds produced more than fifty percent inhibition in the [125I]-Tyr0-sauvagine specific binding assay. Assessment of binding affinities revealed that compound (3-(2,4-dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-one) 8c was the best candidate with highest binding affinity (Ki = 32.1 nM). Further evaluation showed the ability of compound 8c to inhibit CRF induced cAMP accumulation in a dose response manner. Docking and molecular dynamics simulations were used to investigate potential binding modes of synthesized compounds as well as the stability of 8c-CRFR1 complex. These studies suggest similar allosteric binding of 8c compared to that of the co-crystalized ligand CP-376395 in 4K5Y pdb file.

Cyclic AMP-dependent activation of ERK via GLP-1 receptor signalling requires the neuroendocrine cell-specific guanine nucleotide exchanger NCS-RapGEF2.

Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signalling initiated by the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), via the Gs -protein coupled receptor (Gs PCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in Neuroscreen-1 (NS-1) neuroendocrine cells. We examined the expression of other Family B Gs PCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist vasoactive intestinal polypeptide, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably as a result of insufficient receptor protein expression. Vasoactive intestinal polypeptide and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK-dependent (blocked by U0126), indicating its use of the cAMP→RapGEF2→ERK neuritogenic signalling pathway previously identified for PACAP/PAC1 signalling in NS-1 cells. NCS-RapGEF2 is expressed in the rodent insulinoma cell lines MIN6 and INS-1, as well as in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after RapGEF2-shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after 1 hour of exposure to exendin-4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signalling targets. We propose that cAMP signalling initiated by glucagon-like peptide 1 (GLP-1) in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS-RapGEF2, Epac and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation.

Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4.

Corrination is the conjugation of a corrin ring containing molecule, such as vitamin B12 (B12) or B12 biosynthetic precursor dicyanocobinamide (Cbi), to small molecules, peptides, or proteins with the goal of modifying pharmacology. Recently, a corrinated GLP-1R agonist (GLP-1RA) exendin-4 (Ex4) has been shown in vivo to have reduced penetration into the central nervous system relative to Ex4 alone, producing a glucoregulatory GLP-1RA devoid of anorexia and emesis. The study herein was designed to optimize the lead conjugate for GLP-1R agonism and binding. Two specific conjugation sites were introduced in Ex4, while also utilizing various linkers, so that it was possible to identify Cbi conjugates of Ex4 that exhibit improved binding and agonist activity at the GLP-1R. An optimized conjugate (22), comparable with Ex4, was successfully screened and subsequently assayed for insulin secretion in rat islets and in vivo in shrews for glucoregulatory and emetic behavior, relative to Ex4.

Design and Evaluation of Peptide Dual-Agonists of GLP-1 and NPY2 Receptors for Glucoregulation and Weight Loss with Mitigated Nausea and Emesis.

There is a critical unmet need for therapeutics to treat the epidemic of comorbidities associated with obesity and type 2 diabetes, ideally devoid of nausea/emesis. This study developed monomeric peptide agonists of glucagon-like peptide 1 receptor (GLP-1R) and neuropeptide Y2 receptor (Y2-R) based on exendin-4 (Ex-4) and PYY3-36. A novel peptide, GEP44, was obtained via in vitro receptor screens, insulin secretion in islets, stability assays, and in vivo rat and shrew studies of glucoregulation, weight loss, nausea, and emesis. GEP44 in lean and diet-induced obese rats produced greater reduction in body weight compared to Ex-4 without triggering nausea associated behavior. Studies in the shrew demonstrated a near absence of emesis for GEP44 in contrast to Ex-4. Collectively, these data demonstrate that targeting GLP-1R and Y2-R with chimeric single peptides offers a route to new glucoregulatory treatments that are well-tolerated and have improved weight loss when compared directly to Ex-4.

Therapeutic potential of α7 nicotinic acetylcholine receptor agonists to combat obesity, diabetes, and inflammation.

The cholinergic anti-inflammatory reflex (CAIR) represents an important homeostatic regulatory mechanism for sensing and controlling the body's response to inflammatory stimuli. Vagovagal reflexes are an integral component of CAIR whose anti-inflammatory effects are mediated by acetylcholine (ACh) acting at α7 nicotinic acetylcholine receptors (α7nAChR) located on cells of the immune system. Recently, it is appreciated that CAIR and α7nAChR also participate in the control of metabolic homeostasis. This has led to the understanding that defective vagovagal reflex circuitry underlying CAIR might explain the coexistence of obesity, diabetes, and inflammation in the metabolic syndrome. Thus, there is renewed interest in the α7nAChR that mediates CAIR, particularly from the standpoint of therapeutics. Of special note is the recent finding that α7nAChR agonist GTS-21 acts at L-cells of the distal intestine to stimulate the release of two glucoregulatory and anorexigenic hormones: glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). Furthermore, α7nAChR agonist PNU 282987 exerts trophic factor-like actions to support pancreatic ß-cell survival under conditions of stress resembling diabetes. This review provides an overview of α7nAChR function as it pertains to CAIR, vagovagal reflexes, and metabolic homeostasis. We also consider the possible usefulness of α7nAChR agonists for treatment of obesity, diabetes, and inflammation.

FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells.

Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors H188 and YC3.60 so that it is possible to monitor the kinetics with which alterations of [cAMP] or [Ca2+] occur in monolayers or suspensions of living cells exposed to GPCR agonists. This protocol uses HEK293 cell lines doubly transfected with a FRET biosensor and a recombinant GPCR of interest (e.g., glucagon receptors, CCK2 receptors, or NPY2R receptors). The protocol allows for rapid screening of small molecule GPCR agonists and antagonists, and it is also useful for discovery of synthetic mono-, dual-, and tri- agonist peptides with GPCR activating properties.

Corrination of a GLP-1 Receptor Agonist for Glycemic Control without Emesis.

Glucagon-like peptide-1 receptor (GLP-1R) agonists used to treat type 2 diabetes mellitus often produce nausea, vomiting, and in some patients, undesired anorexia. Notably, these behavioral effects are caused by direct central GLP-1R activation. Herein, we describe the creation of a GLP-1R agonist conjugate with modified brain penetrance that enhances GLP-1R-mediated glycemic control without inducing vomiting. Covalent attachment of the GLP-1R agonist exendin-4 (Ex4) to dicyanocobinamide (Cbi), a corrin ring containing precursor of vitamin B12, produces a "corrinated" Ex4 construct (Cbi-Ex4). Data collected in the musk shrew (Suncus murinus), an emetic mammal, reveal beneficial effects of Cbi-Ex4 relative to Ex4, as evidenced by improvements in glycemic responses in glucose tolerance tests and a profound reduction of emetic events. Our findings highlight the potential for clinical use of Cbi-Ex4 for millions of patients seeking improved glycemic control without common side effects (e.g., emesis) characteristic of current GLP-1 therapeutics.

[99mTc]Tc-DGA1, a Promising CCK2R-Antagonist-Based Tracer for Tumor Diagnosis with Single-Photon Emission Computed Tomography.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

"A-kinase" regulator runs amok to provide a paradigm shift in cAMP signaling.

The activity of the archetypal protein kinase A (PKA) is typically thought of in regards to the catalytic subunit, which is inhibited by the regulatory subunits in the absence of cAMP. However, it is now reported that one of the regulatory subunit isoforms (PKA-RIα) takes on a function of its own upon binding to cAMP, acting independently of this canonical cAMP signaling mechanism. PKA-RIα instead binds to and stimulates the catalytic activity of a guanine nucleotide exchange factor (P-REX1) that itself promotes Rac1 GTPase activation. This newly discovered function of PKA-RIα adds an additional layer of complexity to our understanding of cAMP signal transduction.

Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP.

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the ß-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

Restoration of Glucose-Stimulated Cdc42-Pak1 Activation and Insulin Secretion by a Selective Epac Activator in Type 2 Diabetic Human Islets.

Glucose metabolism stimulates cell division control protein 42 homolog (Cdc42)-p21-activated kinase (Pak1) activity and initiates filamentous actin (F-actin) cytoskeleton remodeling in pancreatic ß-cells so that cytoplasmic secretory granules can translocate to the plasma membrane where insulin exocytosis occurs. Since glucose metabolism also generates cAMP in ß-cells, the cross talk of cAMP signaling with Cdc42-Pak1 activation might be of fundamental importance to glucose-stimulated insulin secretion (GSIS). Previously, the type-2 isoform of cAMP-regulated guanine nucleotide exchange factor 2 (Epac2) was established to mediate a potentiation of GSIS by cAMP-elevating agents. Here we report that nondiabetic human islets and INS-1 832/13 ß-cells treated with the selective Epac activator 8-pCPT-2'-O-Me-cAMP-AM exhibited Cdc42-Pak1 activation at 1 mmol/L glucose and that the magnitude of this effect was equivalent to that which was measured during stimulation with 20 mmol/L glucose in the absence of 8-pCPT-2'-O-Me-cAMP-AM. Conversely, the cAMP antagonist Rp-8-Br-cAMPS-pAB prevented glucose-stimulated Cdc42-Pak1 activation, thereby blocking GSIS while also increasing cellular F-actin content. Although islets from donors with type 2 diabetes had profound defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion, these defects were rescued by the Epac activator so that GSIS was restored. Collectively, these findings indicate an unexpected role for cAMP as a permissive or direct metabolic coupling factor in support of GSIS that is Epac2 and Cdc42-Pak1 regulated.

Publisher Correction: Chimeric peptide EP45 as a dual agonist at GLP-1 and NPY2R receptors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Chimeric peptide EP45 as a dual agonist at GLP-1 and NPY2R receptors.

We report the design and target validation of chimeric peptide EP45, a novel 45 amino acid monomeric dual agonist peptide that contains amino acid sequence motifs present within the blood glucose-lowering agent exendin-4 (Ex-4) and the appetite-suppressing agent PYY(3-36). In a new high-throughput FRET assay that provides real-time kinetic information concerning levels of cAMP in living cells, EP45 recapitulates the action of Ex-4 to stimulate cAMP production via the glucagon-like peptide-1 receptor (GLP-1R), while also recapitulating the action of PYY(3-36) to inhibit cAMP production via the neuropeptide Y2 receptor (NPY2R). EP45 fails to activate glucagon or GIP receptors, whereas for cells that co-express NPY2R and adenosine A2B receptors, EP45 acts in an NPY2R-mediated manner to suppress stimulatory effects of adenosine on cAMP production. Collectively, such findings are remarkable in that they suggest a new strategy in which the co-existing metabolic disorders of type 2 diabetes and obesity will be treatable using a single peptide such as EP45 that lowers levels of blood glucose by virtue of its GLP-1R-mediated effect, while simultaneously suppressing appetite by virtue of its NPY2R-mediated effect.