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• W2115944078 abstract "The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes. The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes. Glucagon-like peptide-1 (GLP-1) 2The abbreviations used are: GLP-1, glucagon-like peptide-1; IPGTT, intraperitoneal glucose tolerance test; DPP, dipeptidylpeptidase; PEG, polyethylene glycol; PEGylation, PEGylated, covalent attachment of long chained PEG to a target molecule; PBS, phosphate-buffered saline; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; AUC, area under the curve; PEG-DAPD, PEGylated dual-acting peptide for diabetes; FA, fatty acid. 2The abbreviations used are: GLP-1, glucagon-like peptide-1; IPGTT, intraperitoneal glucose tolerance test; DPP, dipeptidylpeptidase; PEG, polyethylene glycol; PEGylation, PEGylated, covalent attachment of long chained PEG to a target molecule; PBS, phosphate-buffered saline; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; AUC, area under the curve; PEG-DAPD, PEGylated dual-acting peptide for diabetes; FA, fatty acid. and glucagon are members of a family of structurally related peptide hormones, the glucagon/secretin family. GLP-1 and glucagon originate from a common precursor, pre-proglucagon, which upon tissue-specific processing leads to production of GLP-1 in the intestine and glucagon in the pancreas (for review see Ref. 1Mayo K.E. Miller L.J. Bataille D. Dalle S. Goke B. Thorens B. Drucker D.J. Pharmacol. Rev. 2003; 55: 167-194Crossref PubMed Scopus (380) Google Scholar). GLP-1 and glucagon constitute highly homologous peptides, displaying identity at 14 positions (45% identity) and similarity at 3 positions. The receptors for these two peptides are homologous (58% identity) and belong to the family of G-protein-coupled receptors (2Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P.A. Kuijper J.L. Sheppard P.O. Sprecher C.A. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (365) Google Scholar, 3Thorens B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8641-8645Crossref PubMed Scopus (831) Google Scholar). However, despite the high degree of homology between the peptides and the homology between their receptors, GLP-1 and glucagon bind with a high degree of selectivity to their respective receptors (4Hjorth S.A. Adelhorst K. Pedersen B.B. Kirk O. Schwartz T.W. J. Biol. Chem. 1994; 269: 30121-30124Abstract Full Text PDF PubMed Google Scholar). Exendin-4 is a structural homolog of GLP-1 (50% amino acid identity) isolated from saliva of the Gila monster that also acts as a potent agonist of the GLP-1 receptor (5Goke R. Fehmann H.C. Linn T. Schmidt H. Krause M. Eng J. Goke B. J. Biol. Chem. 1993; 268: 19650-19655Abstract Full Text PDF PubMed Google Scholar).Both GLP-1 and glucagon play major, but opposing, roles in overall glucose homeostasis. GLP-1 is synthesized in intestinal endocrine cells and induces glucose-dependent insulin secretion from the pancreas and thereby acts to lower plasma glucose concentrations without the risk of hypoglycemia (6Mojsov S. Kopczynski M.G. Habener J.F. J. Biol. Chem. 1990; 265: 8001-8008Abstract Full Text PDF PubMed Google Scholar, 7Weir G.C. Mojsov S. Hendrick G.K. Habener J.F. Diabetes. 1989; 38: 338-342Crossref PubMed Google Scholar). This activity of GLP-1 has made it attractive as a therapeutic approach for the treatment of type 2 diabetes (for review see Ref. 8Roges O.A. Baron M. Philis-Tsimikas A. Expert. Opin. Investig. Drugs. 2005; 14: 705-727Crossref PubMed Scopus (16) Google Scholar). Glucagon is secreted fromα-cells in pancreatic islets in a glucose-dependent manner. Glucagon stimulates glycogenolysis and gluconeogenesis in the liver, resulting in elevation of plasma glucose. Glucagon has a critical role in maintaining serum glucose concentration, and glucagon receptor antagonists are being pursued as a potential therapeutic approach to inhibit hepatic glucose output (for review see Ref. 9Jiang G. Zhang B.B. Am. J. Physiol. 2003; 284: E671-E678Crossref PubMed Scopus (575) Google Scholar). A molecule capable of both activation of the GLP-1 receptor and inhibition of the glucagon receptor has potential to be a highly effective and novel approach to control blood glucose in the treatment of type 2 diabetes.Structure-activity studies have been performed to determine the role of individual amino acids within both GLP-1 and glucagon sequences. GLP-1 and glucagon have no defined structure in aqueous solution, but in the presence of micelles, adopt an α-helical structure in the midsection, with flexible N- and C-terminal regions (10Thornton K. Gorenstein D.G. Biochemistry. 1994; 33: 3532-3539Crossref PubMed Scopus (104) Google Scholar, 11Ying J. Ahn J.M. Jacobsen N.E. Brown M.F. Hruby V.J. Biochemistry. 2003; 42: 2825-2835Crossref PubMed Scopus (19) Google Scholar). This suggests that the helical structure is required for binding to their respective receptors. Mutations in the N-terminal region of both peptides result in receptor antagonists, suggesting the importance of the N terminus for receptor activation by both GLP-1 (12Knudsen L.B. Pridal L. Eur. J. Pharmacol. 1996; 318: 429-435Crossref PubMed Scopus (210) Google Scholar, 13Montrose-Rafizadeh C. Yang H. Rodgers B.D. Beday A. Pritchette L.A. Eng J. J. Biol. Chem. 1997; 272: 21201-21206Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and glucagon (14Azizeh B.Y. Ahn J.M. Caspari R. Shenderovich M.D. Trivedi D. Hruby V.J. J. Med. Chem. 1997; 40: 2555-2562Crossref PubMed Scopus (15) Google Scholar, 15Gysin B. Trivedi D. Johnson D.G. Hruby V.J. Biochemistry. 1986; 25: 8278-8284Crossref PubMed Scopus (21) Google Scholar, 16Unson C.G. Andreu D. Gurzenda E.M. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4083-4087Crossref PubMed Scopus (92) Google Scholar, 17Unson C.G. Macdonald D. Merrifield R.B. Arch. Biochem. Biophys. 1993; 300: 747-750Crossref PubMed Scopus (38) Google Scholar, 18Unson C.G. Macdonald D. Ray K. Durrah T.L. Merrifield R.B. J. Biol. Chem. 1991; 266: 2763-2766Abstract Full Text PDF PubMed Google Scholar).In addition to the N-terminal region, many amino acids within the glucagon sequence have been shown to contribute to receptor binding and activation (for review see Ref. 19Hruby V.J. Bittar E.E. Bittar N. Principles of Medical Biology. JAI Press, Greenwich, CT1997: 387-401Google Scholar). For example, mutations at positions 11, 12, 16, 17, and 18 of glucagon appear to negatively affect receptor activation more than binding (20Unson C.G. Wu C.R. Cheung C.P. Merrifield R.B. J. Biol. Chem. 1998; 273: 10308-10312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 21Unson C.G. Wu C.R. Fitzpatrick K.J. Merrifield R.B. J. Biol. Chem. 1994; 269: 12548-12551Abstract Full Text PDF PubMed Google Scholar, 22Unson C.G. Wu C.R. Merrifield R.B. Biochemistry. 1994; 33: 6884-6887Crossref PubMed Scopus (24) Google Scholar, 23Unson C.G. Gurzenda E.M. Merrifield R.B. Peptides. 1989; 10: 1171-1177Crossref PubMed Scopus (110) Google Scholar). Some possible increase in glucagon antagonism may also be provided by changes in amino acids 5, 7, 9, 11, 13, 21, and 29 (24Smith R.A. Sisk R. Lockhart P. Mathewes S. Gilbert T. Walker K. Piggot J. Mol. Pharmacol. 1993; 43: 741-748PubMed Google Scholar).For GLP-1, extensive mutational analysis has been reported. For example, alanine-scanning mutagenesis on GLP-1-(7-36)-amide implicates positions 1, 4, 6, 7, 9, 13, 15, 22, 23, and 26 to be critical for receptor binding (25Adelhorst K. Hedegaard B.B. Knudsen L.B. Kirk O. J. Biol. Chem. 1994; 269: 6275-6278Abstract Full Text PDF PubMed Google Scholar, 26Gallwitz B. Witt M. Paetzold G. Morys-Wortmann C. Zimmermann B. Eckart K. Folsch U.R. Schmidt W.E. Eur. J. Biochem. 1994; 225: 1151-1156Crossref PubMed Scopus (87) Google Scholar). On the other hand, combinatorial alanine substitutions at positions 8, 11, 12, and 16 of GLP-1 have a minimal effect on GLP-1 receptor binding and activation (27Xiao Q. Giguere J. Parisien M. Jeng W. St-Pierre S.A. Brubaker P.L. Wheeler M.B. Biochemistry. 2001; 40: 2860-2869Crossref PubMed Scopus (92) Google Scholar). In vivo, GLP-1 is rapidly degraded by dipeptidylpeptidase IV (DPP-IV), a protease responsible for cleaving peptides containing proline or alanine residues in the penultimate N-terminal position, resulting in the inactive GLP-1-(9-36)-amide metabolite (28Mentlein R. Gallwitz B. Schmidt W.E. Eur. J. Biochem. 1993; 214: 829-835Crossref PubMed Scopus (1028) Google Scholar). Removal of the DPP-IV site by mutagenesis greatly improves plasma stability (29Ritzel U. Leonhardt U. Ottleben M. Ruhmann A. Eckart K. Spiess J. Ramadori G. J. Endocrinol. 1998; 159: 93-102Crossref PubMed Scopus (62) Google Scholar). Exendin-4 has a prolonged half-life in vivo relative to GLP-1 likely due to the lack of the DPP-IV site (5Goke R. Fehmann H.C. Linn T. Schmidt H. Krause M. Eng J. Goke B. J. Biol. Chem. 1993; 268: 19650-19655Abstract Full Text PDF PubMed Google Scholar).The similarity between GLP-1 and glucagon, and between their respective receptors, raises the possibility of producing a hybrid peptide that can bind to both receptors. Indeed, Hjorth et al. (4Hjorth S.A. Adelhorst K. Pedersen B.B. Kirk O. Schwartz T.W. J. Biol. Chem. 1994; 269: 30121-30124Abstract Full Text PDF PubMed Google Scholar) generated a chimeric peptide consisting of the N-terminal part of the glucagon molecule joined to the C-terminal part of the GLP-1 molecule that displayed high affinity for both receptors. However, the design of a peptide that binds both receptors and exhibits agonist activity on the GLP-1 receptor but antagonist activity on the glucagon receptor is more challenging.A therapeutic drug based upon a hybrid peptide with dual GLP-1 agonist and glucagon antagonist action would still have to overcome the very short half-life (<5 min) inherent to its parent peptides, which makes them unsuitable for the treatment of type 2 diabetes. PEGylation, the covalent attachment of long chained polyethylene glycol (PEG) to a target molecule, has been successfully employed to increase the circulation half-life of proteins, allowing once-a-week dosing (for review see Ref. 30Harris J.M. Chess R.B. Nat. Rev. Drug. Discov. 2003; 2: 214-221Crossref PubMed Scopus (2681) Google Scholar). PEGylation protects the protein from protease digestion and keeps the protein out of the kidney filtrate. However, the high molecular weight PEGs required for significant improvement of plasma half-life tend to have a negative effect on protein activity, due to steric hindrance. Given the small size of GLP-1 and glucagon peptides, and the fact that functionally important amino acids are spread throughout the peptide sequences, it is a further challenge to generate a PEGylated peptide that retains dual GLP-1 agonist and glucagon antagonist activities.In the current study, we have engineered hybrid glucagon/GLP-1 peptides with targeted mutations that result in the identification of a peptide with both GLP-1 agonist and glucagon antagonist activity. We believe this is the first example of a single chimeric peptide that possesses opposing agonist and antagonist effects on related receptors. To enhance the duration of action, we have site-specifically modified the hybrid peptides with high molecular weight PEGs and succeeded in identifying a PEGylated peptide retaining both GLP-1 agonist and glucagon antagonist activities.EXPERIMENTAL PROCEDURESPeptide Synthesis and PEGylation—Peptides were supplied by Sigma Genosys (The Woodlands, TX) or SynPep (Dublin, CA). The peptides were characterized by high-performance liquid chromatography and mass spectrometry and were >90% pure (data not shown). Peptides were site-specifically PEGylated with 22- or 43-kDa PEG-maleimide purchased from Nektar Therapeutics (San Carlos, CA) and purified to >95% purity (data not shown).RINm5F Cell Culture—RINm5F cells were maintained in RPMI 1640 medium containing 5% fetal bovine serum (JRH Biosciences) and 1% antibiotic-antimycotic solution (Invitrogen) at 37 °C in a humidified 5% CO2 incubator.Preparation of RINm5F Cell Membranes—RINm5F cells were washed with PBS, scraped in ice-cold HES buffer (20 mm Hepes, 1 mm EDTA, 250 mm sucrose buffer containing protease inhibitors), and homogenized. Unbroken cells and nuclei were removed by centrifugation at 500 × g for 5 min at 4 °C. The supernatant was centrifuged at 40,000 × g for 20 min. Membranes were resuspended in HES.Preparation of Plasma Membranes from Rat Liver—Male Sprague-Dawley rats were sacrificed, and livers were removed and placed into ice-cold TES buffer (20 mm Tris, pH 7.5, 1 mm EDTA, 255 mm sucrose containing protease inhibitors). The tissue was minced in 5 volumes of ice-cold TES, and a slurry was prepared using a Polytron. The slurry was further homogenized using a handheld Dounce homogenizer. The homogenate was passed through several layers of cheesecloth and spun at 25,000 × g for 10 min. The pellets were resuspended in TES, and plasma membranes were isolated from the interface of 1.2 m sucrose and TES cushion centrifugation at 180,000 × g. The resulting membranes were washed with TES using a 15-min centrifugation at 200,000 × g, resuspended in TES again, and purified on a second sucrose cushion at 200,000 × g. The final pellet was resuspended in TES.Competitive Binding of Peptide to the GLP-1 and Glucagon Receptors—GF/C filtration plates (Millipore, Bedford, MA) were blocked with 0.3% or 0.1% polyethyleneimine for RINm5F membranes and rat liver plasma membranes, respectively. Filter plates were washed twice with binding buffer consisting of 20 mm Tris, 2 mm EDTA, pH 7.5, 1 mg/ml bovine serum albumin, and 1 mg/ml bacitracin. 5 μg of RINm5F cell plasma membranes or 3-5 μg of rat liver plasma membranes diluted in binding buffer were combined with 0.05 μCi of 125I-labeled GLP-1 or 125I-labeled glucagon and unlabeled test peptide at the indicated concentrations. Following a 1-h incubation at room temperature, the plates were washed three times with ice-cold PBS containing 1 mg/ml bovine serum albumin. The plates were dried, scintillant was added to each well, and plates were counted using a Wallac Microbeta counter.Measurement of Peptide Signaling through GLP-1 Receptor—1.5 × 105 RINm5F cells per well were seeded in 96-well plates and grown overnight. The cells were then washed twice with PBS and then incubated with peptide in Hepes-PBS containing 1% bovine serum albumin and 100 μm isobutylmethylxanthine for 15 min at 37 °C. The cells were lysed, and intracellular cAMP was determined using the cAMP Scintillation Proximity Assay direct screening assay system (Amersham Biosciences).Measurement of Peptide Signaling through Glucagon Receptor—Hepatocytes were isolated according to a modified procedure of Berry (31Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-520Crossref PubMed Scopus (3607) Google Scholar). Freshly isolated rat hepatocytes were plated in 96-well plates at 7.5 × 104 cells/well in Hepes-bicarbonate-PBS containing 1% bovine serum albumin and 100 μm isobutylmethylxanthine. Following equilibration at 37 °C in a 5% CO2/95% O2 environment for 10 min, peptide was added for an additional 15 min. The cells were lysed, and intracellular cAMP was determined as described above. The ability of the hybrid peptide to inhibit glucagon activity was measured as follows: Following equilibration at 37 °C in a 5% CO2/95%O2 environment for 10 min, 10 μm peptide was added to the cells followed immediately by either 2 or 10 nm glucagon for 15 min. The cells were lysed, and cAMP was determined.Measurement of Glucose Release from Rat Hepatocytes—Hepatocytes were added into a flat bottom 96-well plate (2 × 105/100 μl/well) and preincubated in a 37 °C incubator with constant shaking and under 95% O2/5% CO2 flow for 10 min. Hepatocytes were incubated for another 30 min after addition of glucagon with or without peptide. Cells were then lysed with 15% perchloric acid and plates were spun. The supernatant was neutralized with 1 m Tris-HCl (pH 8.0):2.5 n KOH (45:55) and spun again. The resulting supernatant was analyzed for glucose with hexokinase and glucose-6-phosphate dehydrogenase and the A340 read on a fMAX plate reader (Molecular Devices, Sunnyvale, CA). Glucose output was calculated in the following manner: after subtracting the amount of glucose produced in the unstimulated hepatocytes from each data point, the percent inhibition was calculated.Insulin Release from Perifused Rat Islets—The bi-phasic response of insulin release stimulated by peptides was tested in perifused rat islets. Fifty islets were loaded in a perifusion chamber and perifused with HEPES-Krebs-Ringer bicarbonate buffer containing 3 mm glucose at 37 °C. After 60 min, islets were exposed to buffer containing 8 mm glucose with or without peptide (50 nm) and perifused for another 30 min. Fractions of perifusate were collected at 1- or 5-min intervals for insulin determination. Insulin was measured using an enzyme-linked immunosorbent assay kit (Alpco Diagnostics, Windham, NH).Measurement of Peptide Stability in Rat Plasma—Fresh rat plasma was collected from male Wistar rats. The peptides were diluted in 100% rat plasma to 10-fold desired final concentration and incubated at 37 °C for 0, 3, 6, 18, or 24 h. Samples were then added to RINm5F cells, and cAMP production was measured after a 15-min exposure as outlined above. Percent peptide remaining was determined by comparing cAMP production at a given time point to cAMP production in a freshly diluted sample (zero time).Intraperitoneal Glucose Tolerance Test—Male Wistar rats (220-250 g) were purchased from Harlan (Indianapolis, IN). All procedures were approved by the Bayer Animal Care and Use Committee, and all experiments were performed in accordance with relevant guidelines and regulations. Male Wistar rats were either fasted overnight and then given peptide or vehicle by subcutaneous injection or they were given peptide or vehicle first and then fasted overnight, depending on the time interval between dosing and when the IPGTT was to be performed. At the appropriate time after dosing, the fasting blood glucose level was measured from tail-tip blood using a Glucometer (Bayer HealthCare, Mishawaka, IN), and the animals were given 2 g/kg glucose by intraperitoneal injection. Blood glucose was measured again after 15, 30, and 60 min. The area under the glucose curve (AUC) was calculated using the trapezoidal method, and the effect of the peptide on the AUC was expressed as a percentage of the AUC for the vehicle-treated group.Statistical Analysis—In vitro results are means ± S.E. for the number of experiments (n) indicated in the figure legends. In vitro assay statistics were calculated by t test using Graphpad Prizm (GraphPad Software, Inc., San Diego, CA). In vivo data are expressed ± S.E. Statistical analyses were performed using InStat (GraphPad). Treatment effects were analyzed by analysis of variance with post hoc analysis using Tukey-Kramer Multiple Comparisons Test (parametric methods) or the Kruskal-Wallis Test (non-parametric methods) when necessary. Differences are considered significant at p values of <0.05.RESULTSTo engineer a long acting dual GLP-1 agonist and glucagon antagonist peptide, a three-stage mutagenesis strategy was employed. In the first stage, glucagon, GLP-1, and the related GLP-1 agonist, exendin-4, were combined to generate a series of chimeric peptides with the goal of identifying peptides with high affinity toward GLP-1 and glucagon receptors. In stage two, mutations known to antagonize the glucagon receptor but not known to antagonize the GLP-1 receptor were selectively introduced into the GLP-1/glucagon binding-optimized chimeric peptide identified in stage one, leading to the identification of a peptide with potential to act as both a GLP-1 agonist and glucagon antagonist. The GLP-1 agonist and glucagon antagonist activities of the stage two selected peptide was then tested in secondary cell assays to examine its ability to promote pancreatic islet insulin secretion and inhibit glucagon-induced hepatocyte glucose output. Finally, in stage three, cysteine and lysine mutagenesis procedures were performed to identify a position suitable for attachment of a high molecular weight PEG to prolong duration of action in vivo.Stage 1: Optimization of Dual Binding Affinities to GLP-1 and Glucagon Receptors—As previously reported, both glucagon and GLP-1 displayed high affinity (IC50) and potency (EC50) for their own receptors but no significant cross-reactivity for the other receptor (Table 2). To identify novel peptides capable of binding to both receptors, 15 peptides (A1-A15) were synthesized (Table 1). Peptides A1 to A6 were designed to progressively introduce GLP-1-specific amino acids into the glucagon sequence from the C terminus. The N-terminal 18 amino acids of glucagon are critical for high affinity binding to its receptor, because C-terminal replacements with GLP-1 sequence beyond this point greatly reduce glucagon binding affinity (Table 2). The IC50 of A4-A6 at the glucagon receptor were at least 10-fold higher than that of peptides A1-A3. Exchange of only the three C-terminal amino acids of glucagon with the corresponding C-terminal GLP-1 amino acids, while only causing <3-fold decrease in glucagon receptor affinity, reduced glucagon agonist activity by >100-fold, as exhibited by peptide A1. Therefore, the precise amino acid sequence of the C-terminal portion of glucagon appears to be required for activation but not binding to the glucagon receptor. In contrast, binding to the GLP-1 receptor is much more tolerant to insertion of glucagon amino acids into the GLP-1 sequence. Even peptide A1, with 12 amino acid differences from GLP-1 in the N-terminal 26 positions, displayed relatively high affinity (IC50 = 6 nm) and potency (EC50 = 21.7 nm) for the GLP-1 receptor. Successive introduction of more GLP-1 amino acids into glucagon further enhanced activity at the GLP-1 receptor culminating in peptide A6, which has four amino acid differences from GLP-1 but almost an identical IC50 and EC50 as GLP-1. Peptide A3, a chimera sequence of glucagon-(1-19) and GLP-1-(20-30) possesses the most desired in vitro profile of a potent GLP-1 agonist (EC50 = 4.9 nm) and a strong glucagon receptor binder (IC50 = 7.7 nm). Moreover, peptide A3 is a weak glucagon agonist (EC50 = 361 nm) and could potentially act as a glucagon antagonist. However, consistent glucagon antagonism could not be demonstrated with peptide A3 (data not shown) perhaps because it can act as a full agonist of the glucagon receptor at sufficiently high concentrations.TABLE 2Hybrid peptide GLP-1/glucagon receptor binding and activity GLP-1 and glucagon receptor binding (IC50) and activation (EC50) to receptors on RINm5F cells or rat liver membranes, respectively. Data are the mean ± S.E. of at least two experiments.PeptideGLP-1 receptorGlucagon receptorIC50EC50IC50EC50Maximumnmnmnmnm%Glucagon>1 μm2.3 ± 0.34.1 ± 1.2100GLP-10.4 ± 0.11.9 ± 0.5>1 μmExendin-40.40.8 ± 0.2A16.0 ± 1.721.7 ± 7.16.6 ± 1.2505 ± 142103.1 ± 6.3A23.5 ± 0.414.4 ± 4.08.4 ± 1.3567 ± 12996.6 ± 7.4A30.9 ± 0.24.9 ± 1.07.7 ± 1.6361 ± 12492.9 ± 4.0A40.18 ± 0.0710.5 ± 6.884 ± 14365100A52.1 ± 1.215.1 ± 0.7193 ± 963021 ± 95499.7 ± 9.5A60.5 ± 0.12.0 ± 0.6243 ± 71938 ± 200106.5 ± 8.6A712.3 ± 3.239 ± 1598 ± 775 ± 2380.8 ± 8.4A81.9 ± 0.75.6 ± 1.89.7 ± 0.312.8 ± 5.380.2 ± 3.8A90.2 ± 0.19.2 ± 4.65310880.3A101.40.8>1 μmA110.4 ± 0.16.6 ± 3.3>1 μm>1 μm49.6 ± 16.1A120.88.8 ± 6.921 ± 131389.4A130.6 ± 0.46.1 ± 5.5105 ± 89392.3A140.2 ± 0.021.6 ± 0.4452 ± 2381458 ± 34097.9 ± 16.7A150.27 ± 0.041.9 ± 0.3216 ± 48543 ± 17778.4 ± 9.2AN141 ± 12>1 μm101 ± 9>10 μm10.5 ± 10.5AN299 ± 4.7>1 μm264 ± 101>10 μm0.6AN328 ± 1191 ± 1913.1 ± 6.3143 ± 2581.3 ± 6.2AN464 ± 25132 ± 19372 ± 180>10 μm6.8AN55.9 ± 3.640 ± 17116 ± 25321 ± 10246.8 ± 17.7AN61.5 ± 0.712.9 ± 3.328.8 ± 6.3468 ± 18272.1 ± 11.1AN74.5 ± 2.311.6 ± 0.631 ± 1024194.7AN80.6 ± 0.12.5 ± 0.516.4 ± 7.8166 ± 80103.1 ± 18.0AN910.2 ± 2.653 ± 1442 ± 34>10 μm0.0AN103.0 ± 0.713.3 ± 4.969 ± 16713 ± 14043.6 ± 4.3AN119.7 ± 0.2>1 μm43 ± 26>10 μm0.0AN1211.4 ± 4.3>1 μm26 ± 20>10 μm0.0AN13>10 μm>1 μmNTaNT, not testedNTAN1461 ± 29463 ± 197591 ± 176NTNTAN152.7 ± 1.631 ± 22136 ± 100705 ± 44075.5 ± 16.0AN1617.5 ± 6.3>1 μm163 ± 47837 ± 65658.2 ± 18.3AN1740 ± 19171799 ± 245>10 μm2.7 ± 3.9AN18>1 μm1429 ± 364NTNTa NT, not tested Open table in a new tab TABLE 1Hybrid peptide sequences Peptide sequences are represented as follows: Normal font = glucagon amino acids, shaded normal font = GLP-1 amino acids, shaded italic font = exendin-4 amino acids, bold font = mutated amino acids. The non-natural amino acid norleucine is denoted as X. C-terminal amide is symbolized by asterisk. Open table in a new tab Similar substitution of glucagon with exendin-4 amino acids (A7-A11) showed comparable results at the GLP-1 receptor, suggesting GLP-1- or exendin-4-specific amino acids in the N-terminal portion are not required for potent activities at the GLP-1 receptor. Unlike the GLP-1 amino acids, substitution of exendin-4 amino acids into glucagon had a more profound effect on glucagon receptor binding. Only peptide A8 displayed high affinity and potency at the glucagon receptor. Introduction of a combination of GLP-1 and exendin-4 amino acids into peptides (A12-A15) showed a similar trend and again did not produce any peptide with a better profile with respect to GLP-1 agonist activity and lack of glucagon agonist activity when compared with peptide A3. Thus, peptide A3 was selected as the lead peptide from this stage and the template sequence for the next stage of mutagenesis.Stage 2: Optimization of GLP-1 Agonism and Glucagon Antagonism—To generate a peptide mutant that retains GLP-1 agonism but blocks glucagon from activating its receptor (i.e. glucagon antagonism) 18 peptides were designed that introduced known glucagon antagonist mutations into the template peptide A3 (Table 1). Mutations at positions 11, 12, and 16, which have been shown to affect glucagon receptor activation much more than glucagon receptor binding (21Unson C.G. Wu C.R. Fitzpatrick K.J. Merrifield R.B. J. Biol. Chem. 1994; 269: 12548-12551Abstract Full Text PDF PubMed Google Scholar, 32Unson C.G. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 454-458Crossref PubMed Scopus (49) Google Scholar), had a modest effect on GLP-1 receptor binding and activation as demonstrated by peptides AN5-AN10 and AN15 (Table 2). On the other hand, mutations in the N-terminal region, including residues 1, 3-5, and 9, which lead to strong glucagon antagonism (14Azizeh B.Y. Ahn J.M. Caspari R. Shenderovich M.D. Trivedi D. Hruby V.J. J. Med. Chem. 1997; 40: 2555-2562Crossref PubMed Scopus (15) Google Scholar, 15Gysin B. Trivedi D. Johnson D.G. Hruby V.J. Biochemistry. 1986; 25: 8278-8284Crossref PubMed Scopus (21) Google Scholar, 16Unson C.G. Andreu D" @default.
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