An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins.

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells.

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the ß1 adrenergic receptor and the ß2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

Half-Life Extending Modifications of Peptide YY3-36 Direct Receptor-Mediated Internalization.

Peptide YY3-36 (PYY3-36) is an endogenous ligand of the neuropeptide Y2 receptor (Y2R), on which it acts to reduce food intake. Chemically modified PYY3-36 analogues with extended half-lives are potential therapeutics for the treatment of obesity. Here we show that the common half-life extending strategies PEGylation and lipidation not only control PYY3-36's pharmacokinetics but also affect central aspects of its pharmacodynamics. PEGylation of PYY3-36 inhibited endocytosis by increasing receptor dissociation rates (koff), which reduced arrestin-3 (Arr3) activity. This is the first link between Arr3 recruitment and Y2R residence time. C16-lipidation of PYY3-36 had a negligible impact on Y2R signaling, binding, and endocytosis. In contrast, C18acid-lipidation minimized endocytosis, which indicated a decreased internalization through non-arrestin-related mechanisms. We propose a temporal model that connects the properties and position of the half-life extender with receptor Gi versus Arr3 signaling bias. We believe that this will be important for future design of peptide therapeutics.

Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins.

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases.

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane curvature was essential for enrichment in raft-like liquid-ordered phases; enrichment was driven by relief of lateral pressure upon anchor insertion and most likely affects the localization of lipidated proteins in general.

Neoglycolipids for Prolonging the Effects of Peptides: Self-Assembling Glucagon-like Peptide 1 Analogues with Albumin Binding Properties and Potent in Vivo Efficacy.

Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides.

GUB06-046, a novel secretin/glucagon-like peptide 1 co-agonist, decreases food intake, improves glycemic control, and preserves beta cell mass in diabetic mice.

Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight-lowering effects of bariatric surgery involve stimulated secretion of appetite-regulating gut hormones, including glucagon-like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux-en-Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux-en-Y gastric bypass. We therefore developed novel secretin-based peptide co-agonists and identified a lead compound, GUB06-046, that exhibited potent agonism of both the secretin receptor and glucagon-like peptide 1 receptor. Semi-acute administration of GUB06-046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06-046 to diabetic db/db mice for 8 weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta-cell mass after GUB06-046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06-046 on appetite regulation, glucose homeostasis, and beta-cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake.

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti-diabetic and anti-obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC-MS. Native NMU was found to be rapidly cleaved at the C-terminus between Arg(24) and Asn(25) , followed by cleavage between Arg(16) and Gly(17) . Lipidated NMU analogs were generated using solid-phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293-based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor-interacting C-terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose-dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity.

Microwave heating in solid-phase peptide synthesis.

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form ß-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of ß-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).

Membrane curvature sensing by amphipathic helices: a single liposome study using α-synuclein and annexin B12.

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.

Effect of residual water and microwave heating on the half-life of the reagents and reactive intermediates in peptide synthesis.

Precise microwave heating has changed the way many small molecules are being synthesized and, currently, the field of solid-phase peptide synthesis is undergoing dramatic changes owing to the use of microwave heating. To fully reap the benefits of precise microwave heating for the formation of amide bonds in peptide synthesis, it is important to understand the kinetics of formation and break-down of activated esters and their N-acylation of the nascent peptide chain at elevated temperatures. Herein, we present systematic studies of, first, the rate of formation of activated esters by NMR spectroscopy and, second, their N-acylation during peptide synthesis. A study of the amount of residual water in the solvents revealed a significant effect on electrophilic reagents and intermediates. This observation was expanded into a general study of microwave heating in peptide synthesis.

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2-helix variants of PYY3-36.

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions.

Glyco-scan: varying glycosylation in the sequence of the peptide hormone PYY3-36 and its effect on receptor selectivity.

The increasing prevalence of obesity worldwide calls for safe and highly efficacious satiety drugs. PYY3-36 has been implicated in food intake regulation, and novel peptide analogues with high Y2 receptor-subtype selectivity and potency have potential as drugs for the treatment of obesity. It has been hypothesized that PYY3-36 associates with the plasma membrane prior to receptor activation such that the amphipathic alpha-helix of PYY3-36 possibly guides the C-terminal pentapeptide into the correct conformation for receptor activation. Ala-scans are used routinely to study the effect of individual amino acids in a given peptide sequence. Here we report the glyco-scan of the peptide hormone PYY3-36, in which hydroxyl side-chain functionalities were glycosylated; in addition new glycosylation sites were introduced. An array of novel PYY3-36 analogues with a glycan positioned in the water-membrane interface or in the N terminal were screened for Y-receptor affinity and selectivity as well as metabolic stability. Interestingly, in contrast to the Y1 and Y4 receptors, the Y2 receptor readily accommodated glycosylations. Especially glycosylations in the alpha-helical region of PYY3-36 were favorable both in terms of Y-receptor selectivity and endopeptidase resistance. We thus report several PYY3-36 analogues with enhanced Y-receptor selectivity. Our results can be used in the design of novel PYY analogues for the treatment of obesity. The glyco-scan concept, as systematically demonstrated here, has the potential for a wider applicability.

Automated 'X-Y' robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains.

Precise microwave heating has emerged as a valuable method to aid solid-phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the beta-amyloid 1-42 peptide. The instrument is built around a valve-free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an 'X-Y' robotic microwave-assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve-free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficult peptide sequences were achieved.

Peptide hormone isoforms: N-terminally branched PYY3-36 isoforms give improved lipid and fat-cell metabolism in diet-induced obese mice.

The prevalence of obesity is increasing with an alarming rate worldwide and there is a need for efficacious satiety drugs. PYY3-36 has been shown to play a role in hypothalamic appetite regulation and novel analogs targeting the Y2 receptor have potential as drugs for the treatment of obesity. We have designed a series of novel PYY3-36 isoforms, by first adding the dipeptide Ile-Lys N-terminal to the N(α) of Ser-13 in PYY13-36 and then anchoring the N-terminal segment, e.g. PYY3-12, to the new Lys N(ε)-amine. We hypothesized that such modifications would alter the folding of PYY, due to changes in the turn motif, which could change the binding mode to the Y receptor sub-types and possibly also alter metabolic stability. In structure-affinity/activity relationship experiments, one series of PYY isoforms displayed equipotency towards the Y receptors. However, an increased Y2 receptor potency for the second series of PYY isoforms resulted in enhanced Y receptor selectivity compared to PYY3-36. Additionally, acute as well as chronic mice studies showed body-weight-lowering effects for one of the PYY isoforms, which was also reflected in a reduction of circulating leptin levels. Interestingly, while the stability and pharmacokinetic profile of PYY3-36 and the N-terminally modified PYY3-36 analogue were identical, only mice treated with the branched analogue showed marked increases in adiponectin levels as well as reductions in non-esterified free fatty acids and triglycerides.

Remotely Triggered Liquefaction of Hydrogel Materials.

Adaptable behavior such as triggered disintegration affords a broad scope and utility for (bio)materials in diverse applications in materials science and engineering. The impact of such materials continues to grow due to the increased importance of environmental considerations as well as the increased use of implants in medical practices. However, examples of such materials are still few. In this work, we engineer triggered liquefaction of hydrogel biomaterials in response to internal, localized heating, mediated by near-infrared light as external stimulus. This adaptable behavior is engineered into the readily available physical hydrogels based on poly(vinyl alcohol), using gold nanoparticles or an organic photothermal dye as heat generators. Upon laser light irradiation, engineered biomaterials underwent liquefaction within seconds. Pulsed laser light irradiation afforded controlled, on-demand release of the incorporated cargo, successful for small molecules as well as proteins (enzymes) in their biofunctional form.

How Membrane Geometry Regulates Protein Sorting Independently of Mean Curvature.

Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism.

Modifying the conserved C-terminal tyrosine of the peptide hormone PYY3-36 to improve Y2 receptor selectivity.

The Y2 selective PYY derived peptide PYY3-36 was recently shown to play a role in appetite regulation. Novel PYY3-36 analogs with high selectivity for the Y2 receptor could be potential drug candidates for the treatment of obesity. The C-terminal pentapeptide segment of PYY3-36 is believed to bind to the Y receptors. Tyr-36 is highly conserved across species and only few successful modifications of Tyr-36 have been documented. PYY3-36 analogs were prepared using solid-phase peptide chemistry and tested for binding to the Y1, Y2 and Y4 receptor subtypes by radioligand displacement assay. The Y2 receptor agonists with the best affinity and selectivity were further investigated for activity towards the Y1 and Y2 receptor subtypes. Unexpectedly, modifications of Tyr-36 were well-tolerated, and the analogs of PYY3-36 in which the Tyr-36 hydroxyl group was substituted with a halogen or an amino group were particularly well tolerated and yielded an improved selectivity and approximately equipotent affinity to the Y2 receptor. These modifications could be used to design new potential drug candidates for the treatment of obesity.

Adrenomedullin and glucagon-like peptide-1 have additive effects on food intake in mice.

Adrenomedullin (ADM) is a vasoactive peptide expressed in several peripheral organs and known primarily for its beneficial vasoactive effects. However, ADM is also known to inhibit insulin secretion, and central administration of ADM has been shown to elicit anorexigenic effects. Here, we investigated if peripheral co-administration of ADM and glucagon-like peptide 1 (GLP-1) could subdue the hypoglycaemic effects of ADM while enhancing its anorectic properties. The effects of mono- and combination therapy of ADM and GLP-1 on appetite regulation and glucose homeostasis were assessed acutely in male NMRI mice for 12 h, while effects on glucose homeostasis were assessed by oral glucose tolerance tests (OGTT). While the monotherapy with GLP-1 and ADM resulted in modest anorexigenic effects, co-administration of the two peptides led to a marked additive reduction in food intake. Moreover, while OGTT-evoked blood glucose-excursions were significantly increased by ADM monotherapy, co-administration of ADM with a lower dose of GLP-1 normalized glucose excursions. In conclusion, we demonstrate additive anorectic effects of ADM and GLP-1, and that GLP-1 co-administration prevents ADM-induced impairment of glucose tolerance, suggesting that ADM could be potential anti-obesity target when combined with GLP-1 agonist therapy.

Chemical Strategies for Half-Life Extension of Biopharmaceuticals: Lipidation and Its Alternatives.

Strategies for half-life extension are often required in the design of new biopharmaceuticals. This Viewpoint focuses on chemical moieties that convey protraction by albumin binding or by self-assembly to form larger structures, with GLP-1 and insulin as examples.