Apelin-36 Modulates Blood Glucose and Body Weight Independently of Canonical APJ Receptor Signaling.

Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.

A novel glucagon-like peptide 1/glucagon receptor dual agonist improves steatohepatitis and liver regeneration in mice.

Because nonalcoholic steatohepatitis (NASH) is associated with impaired liver regeneration, we investigated the effects of G49, a dual glucagon-like peptide-1/glucagon receptor agonist, on NASH and hepatic regeneration. C57Bl/6 mice fed chow or a methionine and choline-deficient (MCD) diet for 1 week were divided into 4 groups: control (chow diet), MCD diet, chow diet plus G49, and M+G49 (MCD diet plus G49). Mice fed a high-fat diet (HFD) for 10 weeks were divided into groups: HFD and H+G49 (HFD plus G49). Following 2 (MCD groups) or 3 (HFD groups) weeks of treatment with G49, partial hepatectomy (PH) was performed, and all mice were maintained on the same treatment schedule for 2 additional weeks. Analysis of liver function, hepatic regeneration, and comprehensive genomic and metabolic profiling were conducted. NASH was ameliorated in the M+G49 group, manifested by reduced inflammation, steatosis, oxidative stress, and apoptosis and increased mitochondrial biogenesis. G49 treatment was also associated with replenishment of intrahepatic glucose due to enhanced gluconeogenesis and reduced glucose use through the pentose phosphate cycle and oxidative metabolism. Following PH, G49 treatment increased survival, restored the cytokine-mediated priming phase, and enhanced the proliferative capacity and hepatic regeneration ratio in mice on the MCD diet. NASH markers remained decreased in M+G49 mice after PH, and glucose use was shifted to the pentose phosphate cycle and oxidative metabolism. G49 administered immediately after PH was also effective at alleviating the pathological changes induced by the MCD diet. Benefits in terms of liver regeneration were also found in mice fed HFD and treated with G49. CONCLUSION: Dual-acting glucagon-like peptide-1/glucagon receptor agonists such as G49 represent a novel therapeutic approach for patients with NASH and particularly those requiring PH. (Hepatology 2017;65:950-968).

Cell-permeable cyclic peptides from synthetic libraries inspired by natural products.

Drug design efforts are turning to a new generation of therapeutic targets, such as protein-protein interactions (PPIs), that had previously been considered "undruggable" by typical small molecules. There is an emerging view that accessing these targets will require molecules that are larger and more complex than typical small molecule drugs. Here, we present a methodology for the discovery of geometrically diverse, membrane permeable cyclic peptide scaffolds based on the synthesis and permeability screening of a combinatorial library, followed by deconvolution of membrane-permeable scaffolds to identify cyclic peptides with good to excellent passive cell permeabilities. We use a combination of experimental and computational approaches to investigate structure-permeability relationships in one of these scaffolds, and uncover structural and conformational factors that govern passive membrane diffusion in a related set of cyclic peptide diastereomers. Further, we investigate the dependency of permeability on side-chain identity of one of these scaffolds through single-point diversifications to show the adaptability of these scaffolds toward development of permeability-biased libraries suitable for bioactivity screens. Overall, our results demonstrate that many novel, cell permeable scaffolds exist beyond those found in extant natural products, and that such scaffolds can be rapidly identified using a combination of synthesis and deconvolution which can, in principle, be applied to any type of macrocyclic template.

Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the apelin receptor.

BACKGROUND: Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. METHODS: [Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100µM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and ß-arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. RESULTS: In both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting ß-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and ß-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the ß-arrestin assay but was more potent in the cAMP assay. CONCLUSIONS: Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b.

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.

LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor.

Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.

Potent and selective agonists of human melanocortin receptor 5: cyclic analogues of alpha-melanocyte-stimulating hormone.

The physiological role of melanocortin receptor 5 (MC5R) in humans is not clear despite its broad presence in various peripheral sites and in the brain, cortex, and cerebellum. To differentiate between functions of this receptor and those of the other melanocortin receptors (hMC1,3,4R), peptides with improved receptor subtype selectivity are needed. The endogenous ligands, melanocortins, and their various synthetic analogues are not particularly selective for hMC5R. In this study, cyclic peptides derived from MTII, Ac-Nle-cyclo(Asp-His6-D-Phe7-Arg8-Trp-Lys)-NH2 (a pan-agonist at the melanocortin receptors) were prepared and tested in binding and functional assays on CHO cells expressing hMC1b,3-5R. The analogues included in their structures sterically constrained hydrophobic amino acids in positions 6 (His) and 8 (Arg), and the D-4,4'-biphenyl residue in position 7 (D-Phe). Several of the new compounds were selective potent agonists at hMC5R. They are exemplified by peptide 29, Ac-Nle-cyclo(Asp-Oic6-D-4,4'-Bip7-Pip8-Trp-Lys)-NH2 (Oic=octahydroindole-2-COOH; 4,4'-Bip=4,4'-biphenylalanine; Pip=pipecolic acid) of IC50=0.95 nM and EC50=0.99 nM at hMC5R and selectivity for this receptor with respect to the other melanocortin receptors greater than 5000-fold.

Potent and selective agonists of alpha-melanotropin (alphaMSH) action at human melanocortin receptor 5; linear analogs of alpha-melanotropin.

Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.

The hypothalamic neuropeptide melanin-concentrating hormone acts in the nucleus accumbens to modulate feeding behavior and forced-swim performance.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide with a prominent role in feeding and energy homeostasis. The rodent MCH receptor (MCH1R) is highly expressed in the nucleus accumbens shell (AcSh), a region that is important in the regulation of appetitive behavior. Here we establish a role for MCH and MCH1R in mediating a hypothalamic-limbic circuit that regulates feeding and related behaviors. Direct delivery of an MCH1R receptor antagonist to the AcSh blocked feeding and produced an antidepressant-like effect in the forced swim test, whereas intra-AcSh injection of MCH had the opposite effect. Expression studies demonstrated that MCH1R is present in both the enkephalin- and dynorphin-positive medium spiny neurons of the AcSh. Biochemical analysis in AcSh explants showed that MCH signaling blocks dopamine-induced phosphorylation of the AMPA glutamate receptor subunit GluR1 at Ser845. Finally, food deprivation, but not other stressors, stimulated cAMP response element-binding protein-dependent pathways selectively in MCH neurons of the hypothalamus, suggesting that these neurons are responsive to a specific set of physiologically relevant conditions. This work identifies a novel hypothalamic-AcSh circuit that influences appetitive behavior and mediates the antidepressant activity of MCH1R antagonists.

Passive Membrane Permeability in Cyclic Peptomer Scaffolds Is Robust to Extensive Variation in Side Chain Functionality and Backbone Geometry.

Synthetic and natural cyclic peptides provide a testing ground for studying membrane permeability in nontraditional drug scaffolds. Cyclic peptomers, which incorporate peptide and N-alkylglycine (peptoid) residues, combine the stereochemical and geometric complexity of peptides with the functional group diversity accessible to peptoids. We synthesized cyclic peptomer libraries by split-pool techniques, separately permuting side chain and backbone geometry, and analyzed their membrane permeabilities using the parallel artificial membrane permeability assay. Nearly half of the side chain permutations had permeability coefficients (Papp) > 1 × 10-6 cm/s. Some backbone geometries enhanced permeability due to their ability to form more stable intramolecular hydrogen bond networks compared with other scaffolds. These observations suggest that hexameric cyclic peptomers can have good passive permeability even in the context of extensive side chain and backbone variation, and that high permeability can generally be achieved within a relatively wide lipophilicity range.

Antiobesity effect of a melanin-concentrating hormone 1 receptor antagonist in diet-induced obese mice.

Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus, which plays an important role in regulating energy balance. To elucidate the physiological role of MCH in obesity development, the present study examined the effect of a selective MCH1 receptor (MCH1R) antagonist in the diet-induced obesity mouse model. The MCH1R antagonist has high affinity and selectivity for MCH-1R and potently inhibits intracerebroventricularly injected MCH-induced food intake in Sprague Dawley rats. Chronic intracerebroventricular infusion of the MCH1R antagonist (7.5 microg/d) completely suppressed body weight gain in diet-induced obese mice during the treatment periods and significantly decreased cumulative food intake, by 14%. Carcass analysis showed that the MCH1R antagonist resulted in a selective decrease of body fat in the diet-induced obese mice. In addition, the MCH1R antagonist ameliorated the obesity-related hypercholesterolemia, hyperinsulinemia, hyperglycemia, and hyperleptinemia. These results indicate that MCH has a major role in the development of diet-induced obesity in mice and that a MCH1R antagonist might be a useful candidate as an antiobesity agent.

Going Out on a Limb: Delineating The Effects of ß-Branching, N-Methylation, and Side Chain Size on the Passive Permeability, Solubility, and Flexibility of Sanguinamide A Analogues.

It is well established that intramolecular hydrogen bonding and N-methylation play important roles in the passive permeability of cyclic peptides, but other structural features have been explored less intensively. Recent studies on the oral bioavailability of the cyclic heptapeptide sanguinamide A have raised the question of whether steric occlusion of polar groups via ß-branching is an effective, yet untapped, tool in cyclic peptide permeability optimization. We report the structures of 17 sanguinamide A analogues designed to test the relative contributions of ß-branching, N-methylation, and side chain size to passive membrane permeability and aqueous solubility. We demonstrate that ß-branching has little effect on permeability compared to the effects of aliphatic carbon count and N-methylation of exposed NH groups. We highlight a new N-methylated analogue of sanguinamide A with a Leu substitution at position 2 that exhibits solvent-dependent flexibility and improved permeability over that of the natural product.

Chronic MCH-1 receptor modulation alters appetite, body weight and adiposity in rats.

Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 microg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 microg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 microg/day), Compound B (30 or 48 microg/day) or neuropeptide Y (24 microg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%, P<0.001), caloric efficiency and body weight gain (+38%, P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (-16%, P<0.001), body weight gain (-35%, P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Potency optimization of Huwentoxin-IV on hNav1.7: a neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena.

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNav1.7 (IC50 determined herein as 17 ± 2 nM). Nav1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure-function study into Nav1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu(1), Glu(4), Phe(6) and Tyr(33) were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNav1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNav1.5. Peptide 47 (Gly(1), Gly(4), Trp(33)-HwTx) demonstrated the largest potency increase on hNav1.7 (IC50 0.4 ± 0.1 nM).

MCH receptor peptide agonists and antagonists.

Melanin-concentrating hormone (MCH) is an important neuropeptide hormone involved in multiple physiological processes. Peptide derivatives of MCH have been developed as tools to aid research including potent radioligands, receptor selective agonists, and potent antagonists. These tools have been used to further understand the role of MCH in physiology, primarily in rodents. However, the tools could also help elucidate the role for MCHR1 and MCHR2 in mediating MCH signaling in higher species.

Analogs of alpha-melanocyte stimulating hormone with high agonist potency and selectivity at human melanocortin receptor 1b: the role of Trp(9) in molecular recognition.

alpha-Melanocyte stimulating hormone (alphaMSH), Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), is an endogenous agonist for the melanocortin receptor 1 (MC1R), the receptor found in the skin, several types of immune cells, and other peripheral sites. Three-dimensional models of complexes of this receptor with alphaMSH and its synthetic analog NDP-alphaMSH, Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), have been previously proposed. In those models, the 6-9 segment of the ligand was considered essential for the ligand-receptor interactions. In this study, we probed the role of Trp(9) of NDP-alphaMSH in interactions with hMC1bR. Analogs of NDP-alphaMSH with various amino acids in place of Trp(9) were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4, and 5 (hMC1b,3-5R). Several new compounds displayed high agonist potency at hMC1bR (EC(50) = 0.5-5 nM) and receptor subtype selectivity greater than 2000-fold versus hMC3-5R. The Trp(9) residue of NDP-alphaMSH was determined to be not essential for molecular recognition at hMC1bR.