NN1213 - A Potent, Long-Acting, and Selective Analog of Human Amylin.

Amylin, a member of the calcitonin family, acts via amylin receptors in the hindbrain and hypothalamus to suppress appetite. Native ligands of these receptors are peptides with short half-lives. Conjugating fatty acids to these peptides can increase their half-lives. The long-acting human amylin analog, NN1213, was generated from structure-activity efforts optimizing solubility, stability, receptor affinity, and selectivity, as well as in vivo potency and clearance. In both rats and dogs, a single dose of NN1213 reduced appetite in a dose-dependent manner and with a long duration of action. Consistent with the effect on appetite, studies in obese rats demonstrated that daily NN1213 dosing resulted in a dose-dependent reduction in body weight over a 21-day period. Magnetic resonance imaging indicated that this was primarily driven by loss of fat mass. Based on these data, NN1213 could be considered an attractive option for weight management in the clinical setting.

Development of Zalfermin, a Long-Acting Proteolytically Stabilized FGF21 Analog.

Here, we describe the development of the FGF21 analog zalfermin (NNC0194-0499, 15), intended for once-weekly sc dosing. Protein engineering was needed to address inherent druggability issues of the natural FGF21 hormone. Thus, deamidation of Asp121 was solved by mutation to glutamine, and oxidation of Met168 was solved by mutation to leucine. N-terminal region degradation by dipeptidyl peptidase IV was prevented by alanine residue elongation. To prevent inactivating metabolism by fibroblast activation protein and carboxypeptidase-like activity in the C-terminal region, and to achieve t1/2 extension (53 h in cynomolgus monkeys), we introduced a C18 fatty diacid at the penultimate position 180. The fatty diacid binds albumin in a reversible manner, such that the free fraction of zalfermin potently activates the FGF-receptor complex and retains receptor selectivity compared with FGF21, providing strong efficacy on body weight loss in diet-induced obese mice. Zalfermin is currently being clinically evaluated for the treatment of metabolic dysfunction-associated steatohepatitis.

Discovery of a potent GIPR peptide antagonist that is effective in rodent and human systems.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) is one of the two major incretin factors that regulate metabolic homeostasis. Genetic ablation of its receptor (GIPR) in mice confers protection against diet-induced obesity (DIO), while GIPR neutralizing antibodies produce additive weight reduction when combined with GLP-1R agonists in preclinical models and clinical trials. Conversely, GIPR agonists have been shown to promote weight loss in rodents, while dual GLP-1R/GIPR agonists have proven superior to GLP-1R monoagonists for weight reduction in clinical trials. We sought to develop a long-acting, specific GIPR peptide antagonist as a tool compound suitable for investigating GIPR pharmacology in both rodent and human systems. METHODS: We report a structure-activity relationship of GIPR peptide antagonists based on the human and mouse GIP sequences with fatty acid-based protraction. We assessed these compounds in vitro, in vivo in DIO mice, and ex vivo in islets from human donors. RESULTS: We report the discovery of a GIP(5-31) palmitoylated analogue, [Nα-Ac, L14, R18, E21] hGIP(5-31)-K11 (γE-C16), which potently inhibits in vitro GIP-mediated cAMP generation at both the hGIPR and mGIPR. In vivo, this peptide effectively blocks GIP-mediated reductions in glycemia in response to exogenous and endogenous GIP and displays a circulating pharmacokinetic profile amenable for once-daily dosing in rodents. Co-administration with the GLP-1R agonist semaglutide and this GIPR peptide antagonist potentiates weight loss compared to semaglutide alone. Finally, this antagonist inhibits GIP- but not GLP-1-stimulated insulin secretion in intact human islets. CONCLUSIONS: Our work demonstrates the discovery of a potent, specific, and long-acting GIPR peptide antagonist that effectively blocks GIP action in vitro, ex vivo in human islets, and in vivo in mice while producing additive weight-loss when combined with a GLP-1R agonist in DIO mice.

Next generation GLP-1/GIP/glucagon triple agonists normalize body weight in obese mice.

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.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Development of Cagrilintide, a Long-Acting Amylin Analogue.

A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide.

Differential receptor selectivity of the FGF15/FGF19 orthologues determines distinct metabolic activities in db/db mice.

Fibroblast growth factors (FGF) 19, 21 and 23 are characterized by being endocrinely secreted and require co-receptor α-klotho or ß-klotho (BKL) for binding and activation of the FGF receptors (FGFR). FGF15 is the rodent orthologue of human FGF19, but the two proteins share only 52% amino acid identity. Despite the physiological role of FGF21 and FGF19 being quite different, both lower blood glucose (BG) when administered to diabetic mice. The present study was designed to clarify why two human proteins with distinct physiological functions both lower BG in db/db mice and if the mouse orthologue FGF15 has similar effect to FGF19 and FGF21. Recombinant human FGF19, -21 and a mouse FGF15 variant (C110S) were expressed and purified from Escherichia coli While rhFGF19 (recombinant human fibroblast growth factor 19) and rhFGF21 (recombinant human fibroblast growth factor) bound FGFRs in complex with both human and mouse BKL, rmFGF15CS (recombinant mouse fibroblast growth factor 15 C110S) only bound the FGFRs when combined with mouse BKL. Recombinant hFGF21 and rhFGF19, but not rmFGF15CS, increased glucose uptake in mouse adipocytes, while rhFGF19 and rmFGF15CS potently decreased Cyp7a1 expression in rat hepatocytes. The lack of effect of rmFGF15CS on glucose uptake in adipocytes was associated with rmFGF15CS's inability to signal through the FGFR1c/mouse BKL complex. In db/db mice, only rhFGF19 and rhFGF21 decreased BG while rmFGF15CS and rhFGF19, but not rhFGF21, increased total cholesterol. These data demonstrate receptor- and species-specific differential activity of FGF15 and FGF19 which should be taken into consideration when FGF19 is used as a substitute for FGF15.

Molecular elements in FGF19 and FGF21 defining KLB/FGFR activity and specificity.

OBJECTIVE: To signal, FGF19 and FGF21 require co-receptor ßKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. METHODS: We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. RESULTS: C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. CONCLUSIONS: We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.

Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography.

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR). The crystal structure of the human calcitonin receptor ectodomain (CTR ECD) in complex with a truncated analogue of salmon calcitonin ([BrPhe(22)]sCT(8-32)) has been determined to 2.1-Å resolution. Parallel analysis of a series of peptide ligands showed that the rank order of binding of the CTR ECD is identical to the rank order of binding of the full-length CTR, confirming the structural integrity and relevance of the isolated CTR ECD. The structure of the CTR ECD is similar to other Class B GPCRs and the ligand binding site is similar to the binding site of the homologous receptors for the calcitonin gene-related peptide (CGRP) and adrenomedulin (AM) recently published (Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Mol. Cell 58, 1040-1052). Interestingly the receptor-bound structure of the ligand [BrPhe(22)]sCT(8-32) differs from the receptor-bound structure of the homologous ligands CGRP and AM. They all adopt an extended conformation followed by a C-terminal ß turn, however, [BrPhe(22)]sCT(8-32) adopts a type II turn (Gly(28)-Thr(31)), whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.

Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes.

Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.

Increased expression of toll-like receptor 4 and inflammatory cytokines, interleukin-6 in particular, in islets from a mouse model of obesity and type 2 diabetes.

Toll-like receptor 4 (TLR4) has received much attention in the recent years due to its role in development of insulin resistance in type 2 diabetes mellitus. Its expression is elevated in fat and muscle from insulin-resistant mice. Several cells of the pancreatic islets, including ß-cells and resident macrophages, express TLR4. Our hypothesis is that expression of TLR4 and downstream signalling molecules in islets increases during progression of type 2 diabetes, thereby contributing to ß-cell damage. We investigated the hypothesis in the db/db mouse. Islets from male db/db (4, 8 and 15 weeks old) and control db/+ (4 and 15 weeks old) mice were examined for mRNA expression of TLR4 and selected cytokines using qPCR. In addition, cytokine secretion from islets was quantified. TLR4 is expressed in islets from lean and obese mice, displaying a 7.4-fold higher level in 15 weeks old db/db relative to age-matched control (p < 0.01). During progression of clinical type 2 diabetes manifested by hyperglycaemia, TLR4 expression increases 5.6-fold in islets from 15 weeks compared with 4 weeks old db/db mice (p < 0.01). Furthermore, both protein and mRNA levels of all cytokines examined increased. In particular, expression of IL-6 increased with 37 fold. Expression of TLR4 in db/db mouse islets increased in parallel with hyperglycaemia. A similar increase in expression and secretion of TNFα, IL-1 and IL-6 was observed. Our results demonstrate that, in addition to its contribution to insulin resistance, TLR4 might also play a role in ß-cell dysfunction in type 2 diabetes.

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells.

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Neuronal responses to transient hypoglycaemia in the dorsal vagal complex of the rat brainstem.

Several regions of the mammalian brain contain glucosensing neurones. In vivo studies have suggested that those located in the hypothalamus and lower brainstem are involved in glucoprivic feeding and homeostatic control of blood glucose. We have identified and characterized hypoglycaemia-sensitive neurones in the dorsal vagal complex of the brainstem using in situ hybridization, single-cell RT-PCR and whole-cell patch-clamp recordings from rat brainstem slices. Approximately 80% of neurones did not respond to hypoglycaemia (changing artificial cerebrospinal fluid (ACSF) glucose from 10 mM to 0 mM) within 5 min (non-responsive: NR). Another 10% depolarized within 155+/-31 s (mean+/-s.e.m.) of glucose removal (glucose-inhibited: GI), and the remaining neurones hyperpolarized within 53+/-7 s (glucose-excited: GE). The hyperpolarization was reversed by the KATP channel blocker tolbutamide. Single-cell RT-PCR revealed that GI and GE, but not NR, cells expressed glucokinase (GLK). In contrast, SUR1, a KATP channel subunit, was expressed in GE and some NR cells. In situ hybridization with biotin-labelled riboprobes in the dorsal vagal complex revealed ubiquitous expression of SUR1, and widespread, but sparse, expression of GLK. Identification of astrocytes using a GFAP (glial fibrillary acidic protein) antibody showed that GLK and GFAP were not colocalized. In summary, we have demonstrated that GI and GE neurones exist in the brainstem and that GLK is essential for their function. It seems likely that GE neurones work in a way analogous to pancreatic beta-cells in that they require both GLK and KATP channels.

Kir6.2-dependent high-affinity repaglinide binding to beta-cell K(ATP) channels.

1. The beta-cell K(ATP) channel is composed of two types of subunit - the inward rectifier K(+) channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2DeltaN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2DeltaN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37 degrees C. 2. Repaglinide was found to bind with low affinity (K(D)=59+/-16 nM) to SUR1 alone, but with high affinity (increased approximately 150-fold) when SUR1 was co-expressed with Kir6.2 (K(D)=0.42+/-0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3. Repaglinide bound with low affinity (K(D)=51+/-23 nM) to SUR1 co-expressed with Kir6.2DeltaN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. 4. In whole-cell patch-clamp experiments inhibition of Kir6.2DeltaN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.

Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.

Repaglinide and nateglinide represent a new class of insulin secretagogues, structurally unrelated to sulphonylureas, that were developed for the treatment of type 2 diabetes. The inhibitory effect of these drugs was investigated on recombinant wild-type and mutant Kir6.2/SUR1 channels expressed in HEK293 cells. Nateglinide and repaglinide dose-dependently inhibited whole-cell Kir6.2/SUR1 currents with half-maximal inhibitory concentration (IC(50)) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K(+) channel. In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC(50) = 23 nmol/l). Radioligand binding studies revealed a single high-affinity binding site for [(3)H]repaglinide on membranes prepared from HEK293 cells expressing wild-type (equilibrium dissociation constant [K(D)] = 0.40 nmol/l) or mutant (K(D) = 0.31 nmol/l) Kir6.2/SUR1 channels. Nateglinide and tolbutamide displaced [(3)H]repaglinide binding to wild-type channels with IC(50) values of 0.7 and 26 micro mol/l, respectively, but produced <10% displacement of [(3)H]repaglinide bound to mutant channels. This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. These results are discussed in terms of a conformational analysis of the drug molecules.