Publications by authors named "Kilian W Conde-Frieboes"

11 Publications

  • Page 1 of 1

Chemical Synthesis of Phosphorylated Insulin-like Growth Factor Binding Protein 2.

J Am Chem Soc 2021 Apr 2;143(14):5336-5342. Epub 2021 Apr 2.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.

Chemical protein synthesis is a powerful avenue for accessing homogeneously modified proteins. While a significant number of small modified proteins bearing native post-translational modifications and non-natural modifications have been generated to date, access to larger targets has proved challenging. Herein, we describe the use of two ligation manifolds, namely, diselenide-selenoester ligation and native chemical ligation, to assemble a 31.5 kDa phosphorylated insulin-like growth factor binding protein (IGFBP-2) that comprises 290 amino acid residues, a phosphoserine post-translational modification, and nine disulfide bonds.
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http://dx.doi.org/10.1021/jacs.1c02280DOI Listing
April 2021

prediction of pseudo-allergenic response via MRGPRX2.

J Immunotoxicol 2021 Dec;18(1):30-36

Global Research, Novo Nordisk A/S, Maaloev, Denmark.

In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect. Histological analysis of skin samples revealed that the injection site reactions were concurrent with mast cell degranulation, resulting in histamine release. Mast cell activation can be mediated by MRGPRX2, a GPCR that induces a pseudo-allergenic immune response. The present study demonstrates that a commercially-available cell-based MRGPRX2 assay reliably identifies compounds that induce histamine release or localized edema in human and rodent skin samples. screening was subsequently implemented using the MRGPRX2 assay as a substitute for postmortem injection site evaluation, thus achieving a significant reduction in animal use. Thus, in cases where injection site reactions are encountered during screening, to enable faster screening during the early drug discovery process, an MRGPRX2 assay can be used as an efficient, more ethical tool with human translational value for the development of safer pharmacotherapies for patients.
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http://dx.doi.org/10.1080/1547691X.2021.1877375DOI Listing
December 2021

UCP1-independent glucose-lowering effect of leptin in type 1 diabetes: only in conditions of hypoleptinemia.

Am J Physiol Endocrinol Metab 2020 01 19;318(1):E72-E86. Epub 2019 Nov 19.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.
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http://dx.doi.org/10.1152/ajpendo.00253.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985793PMC
January 2020

Diselenide-selenoester ligation for chemical protein synthesis.

Nat Protoc 2019 07 21;14(7):2229-2257. Epub 2019 Jun 21.

School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia.

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at β-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.
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http://dx.doi.org/10.1038/s41596-019-0180-4DOI Listing
July 2019

Sweet Taste Receptor Activation in the Gut Is of Limited Importance for Glucose-Stimulated GLP-1 and GIP Secretion.

Nutrients 2017 Apr 22;9(4). Epub 2017 Apr 22.

NNF Center for Basic Metabolic Research and Department of Biomedical Sciences, Panum Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej, DK-2200, Copenhagen N, Denmark.

Glucose stimulates the secretion of the incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is debated whether the sweet taste receptor (STR) triggers this secretion. We investigated the role of STR activation for glucose-stimulated incretin secretion from an isolated perfused rat small intestine and whether selective STR activation by artificial sweeteners stimulates secretion. Intra-luminal administration of the STR agonists, acesulfame K (3.85% ), but not sucralose (1.25% ) and stevioside (2.5% ), stimulated GLP-1 secretion (acesulfame K: 31 ± 3 pmol/L vs. 21 ± 2 pmol/L, < 0.05, = 6). In contrast, intra-arterial administration of sucralose (10 mM) and stevioside (10 mM), but not acesulfame K, stimulated GLP-1 secretion (sucralose: 51 ± 6 pmol/L vs. 34 ± 4 pmol/L, < 0.05; stevioside: 54 ± 6 pmol/L vs. 32 ± 2 pmol/L, < 0.05, = 6), while 0.1 mM and 1 mM sucralose did not affect the secretion. Luminal glucose (20% ) doubled GLP-1 and GIP secretion, but basolateral STR inhibition by gurmarin (2.5 µg/mL) or the inhibition of the transient receptor potential cation channel 5 (TRPM5) by triphenylphosphine oxide (TPPO) (100 µM) did not attenuate the responses. In conclusion, STR activation does not drive GIP/GLP-1 secretion itself, nor does it have a role for glucose-stimulated GLP-1 or GIP secretion.
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http://dx.doi.org/10.3390/nu9040418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409757PMC
April 2017

Analytic framework for peptidomics applied to large-scale neuropeptide identification.

Nat Commun 2016 May 4;7:11436. Epub 2016 May 4.

Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.
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http://dx.doi.org/10.1038/ncomms11436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857386PMC
May 2016

Melanocortin agonists stimulate lipolysis in human adipose tissue explants but not in adipocytes.

BMC Res Notes 2015 Oct 12;8:559. Epub 2015 Oct 12.

Diabetes and Obesity Biology, Novo Nordisk A/S, 2760, Maaloev, Denmark.

Background: The central melanocortin system is broadly involved in the regulation of mammalian nutrient utilization. However, the function of melanocortin receptors (MCRs) expressed directly in peripheral metabolic tissues is still unclear. The objective of this study was to investigate the lipolytic capacity of MC1-5R in differentiated adipocytes versus intact white adipose tissue.

Results: Non-selective MCR agonist α-MSH, MC5R-selective agonist PG-901 and MC4R-selective agonist LY2112688 significantly stimulated lipolysis in intact white adipose tissue, whereas stimulation of MCRs in differentiated adipocytes failed to do so. The lipolytic response of MC5R was decreased in intact human white adipose tissue when co-treating with β-adrenergic antagonist propranolol, suggesting that the effect may be dependent on neuronal innervation via noradrenalin release.

Conclusion: When developing an anti-obesity therapeutic drug with selective MC4R/MC5R properties, effects on lipolysis in white adipose tissue may be physiologically relevant.
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http://dx.doi.org/10.1186/s13104-015-1539-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604100PMC
October 2015

Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1.

J Biol Chem 2012 Nov 25;287(48):40493-501. Epub 2012 Sep 25.

Novo Nordisk A/S, 2760 Måløv, Denmark.

Background: Cyclotides are useful scaffolds to stabilize bioactive peptides.

Results: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist.

Conclusion: The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds.

Significance: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity. Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23-28) had a K(i) of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC(50) of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23-28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.
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http://dx.doi.org/10.1074/jbc.M112.395442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504764PMC
November 2012

Handling a tricycle: orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.

Peptides 2012 Sep 3;37(1):144-9. Epub 2012 Jul 3.

Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48 h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.
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http://dx.doi.org/10.1016/j.peptides.2012.06.016DOI Listing
September 2012

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved.

Mol Cell Endocrinol 2011 Jul 17;341(1-2):9-17. Epub 2011 May 17.

Diabetes Biology and Pharmacology, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark.

The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphate activated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by α-MSH, NDP-α-MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes.
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http://dx.doi.org/10.1016/j.mce.2011.03.010DOI Listing
July 2011

Fmoc solid-phase synthesis of C-terminal peptide thioesters by formation of a backbone pyroglutamyl imide moiety.

Angew Chem Int Ed Engl 2009 ;48(40):7411-4

IGM, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.

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http://dx.doi.org/10.1002/anie.200903710DOI Listing
December 2009