Glucagon Receptor Agonists

Article Cite This: J. Med. Chem. 2018, 61, 5580−5593

pubs.acs.org/jmc

Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists Specifically Optimized for Multidose Formulations Andreas Evers,* Martin Bossart,* Stefania Pfeiffer-Marek, Ralf Elvert, Herman Schreuder, Michael Kurz, Siegfried Stengelin, Martin Lorenz, Andreas Herling, Anish Konkar, Ulrike Lukasczyk, Anja Pfenninger, Katrin Lorenz, Torsten Haack, Dieter Kadereit, and Michael Wagner* Sanofi-Aventis Deutschland GmbH, R&D, Industriepark Höchst, D-65926 Frankfurt am Main, Germany

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ABSTRACT: Novel peptidic dual agonists of the glucagonlike peptide 1 (GLP-1) and glucagon receptor are reported to have enhanced efficacy over pure GLP-1 receptor agonists with regard to treatment of obesity and diabetes. We describe novel exendin-4 based dual agonists designed with an activity ratio favoring the GLP-1 versus the glucagon receptor. As result of an iterative optimization procedure that included molecular modeling, structural biological studies (X-ray, NMR), peptide design and synthesis, experimental activity, and solubility profiling, a candidate molecule was identified. Novel SAR points are reported that allowed us to fine-tune the desired receptor activity ratio and increased solubility in the presence of antimicrobial preservatives, findings that can be of general applicability for any peptide discovery project. The peptide was evaluated in chronic in vivo studies in obese diabetic monkeys as translational model for the human situation and demonstrated favorable blood glucose and body weight lowering effects.

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INTRODUCTION Type 2 diabetes (T2D) has reached pandemic levels and therefore represents a major health burden to modern society. Currently there are more than 400 million patients living worldwide with T2D;1 still many of them are not diagnosed, not treated at all, or not treated in the right way to achieve their target glycemic status. Very often, T2D is coupled with obesity, in fact 80−90% of the patients with T2D are obese.2 Both conditions, diabetes and obesity, have been proven to be risk factors for many associated diseases, such as micro- and macrovascular complications, but also nonalcoholic steatohepatitis (NASH).3 Consequently, there is major interest in the search for new therapies that provide adequate glycemic control in patients with T2D and in addition also lead to a significant body weight reduction in overweight to obese people. There are currently only two classes of medications approved that besides their glucose lowering effect lead to a moderate weight reduction: orally available small molecule sodium−glucose cotransporter 2 inhibitors (SGLT2-I) and injectable peptidic glucagon-like peptide 1 (GLP-1) receptor agonists. For the latter class, novel formulations and dose regimens are currently being explored to further increase efficacy, especially with regards to weight loss.4−6 In recent years, novel unimolecular peptides with activity at multiple target receptors have emerged as a promising approach to enhance antidiabetic properties and reduce body weight.7−10 A prominent approach is the combination of GLP1 receptor mediated food intake suppression with glucagon© 2018 American Chemical Society

receptor mediated increase in energy expenditure for synergistic and improved body weight loss. Such dual GLP1/glucagon receptor agonists have first been described by Day et al.11 and Pocai et al.12 Meanwhile, numerous dual agonists have been reported by other groups, including us, and quite a few of them have progressed into the clinic; see refs 12−18 or recent reviews.7−10 Despite the recent progress of GLP-1/glucagon receptor dual agonists, it is still not clear today what is the optimal ratio between GLP-1 and glucagon receptor activation.19 GLP-1 receptor activation leads to glucose lowering, in conjunction with moderate body weight reduction, whereas an excessive enhancement of the glucagon receptor activity provides more significant weight loss, but at the risk of glucose elevation. In preclinical studies performed in diet-induced obese (DIO) mice, Day et al. showed that a nearly equally balanced coagonist demonstrated intermediate weight loss but with glucose control comparable to that of the most GLP-1 receptor-selective peptide.20 It was concluded that the optimal GLP-1/glucagon activity ratio needs to be carefully evaluated and could be different for different species. These findings suggest additional investigation in more translational animal models, such as obese diabetic monkeys. Besides the pharmacological profile, pharmaceutical properties such as solubility and aggregation properties of peptides Received: February 20, 2018 Published: June 7, 2018 5580

DOI: 10.1021/acs.jmedchem.8b00292 J. Med. Chem. 2018, 61, 5580−5593

Journal of Medicinal Chemistry

Article

Table 1. (a) Amino Acid Sequences of Glucagon, Exendin-4, Peptide 1, Native GLP-1, Liraglutide, and Native GIPa and (b) EC50 Values at the Human GLP-1, Glucagon (GCG), and GIP Receptor as Measured in a cAMP Assay in Receptor Overexpressing HEK-293 Cell Lines with SEM Values and Number of Measurements (n)

a

Amino acids that are identical among glucagon and exendin-4 are colored green, residues unique to glucagon are shown in yellow, residues unique to exendin-4 are colored grey, residues unique to GLP-1 are white, residues unique to GIP are violet, and further modifications are shown in orange. Residue 14 in peptide 1 and residue 20 in liraglutide are modified by addition of a C16 fatty acid (palmitic acid) at the ε-amino group of lysine using a γ-glutamic acid spacer.

Table 2. (a) Peptide Sequencesa and (b) EC50 Valuesb at the Human GLP-1, Glucagon and GIP Receptors with SEM Values and Number of Measurements (n)

a

Color coding of amino acids is identical to Table 1. bMeasured in a cAMP assay in receptor overexpressing HEK-293 cell lines.

alcohol to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual use doses.21 Indeed, aggregation or solubility issues have been reported under certain conditions for several peptides with agonistic activity at the GLP-1, glucagon, or glucose-dependent insulinotropic peptide (GIP) receptor.22−26 Consequently, in order to maximize the probability of success during drug

need to be optimized to ensure clinical use in humans. Peptide drugs that are formulated in a ready-to-use solution for subcutaneous injection are frequently presented to the patient using multiple-dose pen devices. Such drug−device combinations require a low-viscosity solution, long-term chemical and physical stability, and the compatibility of the peptide with aromatic preservatives, such as phenol, m-cresol, or benzyl 5581



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Figure 1. (a) Ensemble of solution-state NMR structure of exendin-4 (PDB code 1jrj). (b) Three-dimensional alignment of the extracellular domain (ECD) of the GLP-1 (GLP-1R, PDB code 3c59), glucagon (GCGR, PDB code 4ers), and GIP receptor (GIPR, PDB code 2qkh) and binding hypothesis of exendin-4 (derived from the NMR structure in solution, PDB code 1jrj) to the GLP-1 receptor ECD. The color coding of peptide amino acids is equivalent to the sequence color coding outlined in Table 1. Residue Trp25 is shown in space fill representation. (c) Magnified view of the peptide binding interface of the receptor ECDs highlighting specific amino acids. According to the binding hypothesis, these amino acids are interacting with Lys27 of exendin-4 and are assumed to represent a spot for modulation of peptide activity and selectivity at different receptors.

glucagon and GIP receptor binding. These insights were used as structural basis for optimization of a lead peptide (peptide 2, Table 2) toward the desired GLP-1/glucagon/GIP receptor activity ratio. Structural Analysis of Peptides and Binding Hypotheses to the GLP-1, Glucagon and GIP Receptor ECDs. Molecular recognition of exendin-4 and glucagon at the ECDs of the GLP-1 and glucagon receptor has been described in detail previously.13 Figure 1a shows the solution-state NMR ensemble of exendin-4 (PDB code 1jrj).30 In exendin-4, the Cterminal sequence stretch (residues 30−39) cages Trp25 (tryptophan cage), which enhances helicity and structural stability of the peptide by intramolecular interactions and thereby provides improved physicochemical and metabolic stability.31 A three-dimensional alignment of the X-ray structures of the GLP-1 (PDB code 3c59),32 glucagon (PDB code 4ers),33 and GIP receptor (PDB code 2qkh)34 ECDs is shown in Figure 1b. Furthermore, Figure 1b depicts the solution-state NMR structure of exendin-4 (PDB code 1jrj) that was superimposed onto exendin(9−39) in the GLP-1 receptor-bound conformation (PDB code 3c59).32 This structural model (shown in Figure 1b) was used as reference for the generation of peptide binding models to guide the design of peptides toward the desired GLP-1/glucagon/GIP receptor activity ratio (for details, see Experimental Section). Figure 1c highlights two positions where the receptors have dissimilar residues. These residues are close to Lys27 of exendin-4, suggesting that the GLP-1/glucagon/GIP receptor activity and selectivity ratio might be specifically optimized by peptide modifications in position 27. The conformations of the ECDs and the exendin-4 binding mode shown in Figure 1 are highly similar to recently published full-length structures of the peptide-bound GLP135,36 and glucagon37,38 receptor. Peptide Design and Optimization. In the search for a new dual GLP-1/glucagon receptor agonist with lower glucagon agonism, peptide 2 was identified, which shows a

development, it is increasingly recognized that physical stability should be assessed as early as possible in a discovery program, to provide robust development candidates with respect to the final drug product and to physical stress conditions that will be encountered in the downstream processes.27−29 In our recently published article, we described identification of a potent dual GLP-1/glucagon receptor agonist based on the exendin-4 structure (peptide 1, Table 1).13 The goal of the present study was to design an exendin-4 based dual GLP-1/ glucagon receptor agonist with an approximately 10−15-fold lower preference for the glucagon versus GLP-1 receptor for pharmacological evaluation with respect to body weight loss and glycemic control in obese diabetic monkeys as a translational model for the human situation. Further target attributes were (i) selectivity versus the GIP receptor and (ii) sufficient solubility for the candidate molecule over a broad pH range (pH 4.5−7.4) in the presence of antimicrobial preservatives. We conducted an iterative optimization procedure that included molecular modeling, structural biological studies (X-ray, NMR), peptide design and synthesis, and experimental activity and solubility profiling under relevant conditions. Novel SAR points were found that represent selectivity switches to fine-tune the desired GLP-1/glucagon/ GIP receptor selectivity ratio. Optimization of solubility in the presence of a phenolic preservative was achieved by structurebased identification of an aggregation hot spot and subsequent introduction of a novel solubility-enhancing motif. Finally, the beneficial effects on body weight and glucose control in a 6week trial (using once-daily subcutaneous injection) in obese diabetic monkeys, a relevant translational model for the human situation, are described.

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RESULTS AND DISCUSSION

In the following, we will present an overview over relevant published structural data (X-ray, NMR) for the recognition of exendin-4 at the extracellular domain (ECD) of the GLP-1 receptor and describe key features with respect to ECD 5582



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activity by a factor of 11 and GIP receptor activity by a factor of 35 (compare peptide 5 versus peptide 8). Inspection of the predicted binding modes (for details, see Experimental Section) for peptides 5 and 8 to the GLP-1, glucagon, and GIP receptor ECDs provides a structural rationale for the selectivity switch of the Aib27Ile mutant (see Figure 2).

slightly higher activity at the glucagon (EC50 = 1.9 pM) than the GLP-1 receptor (EC50 = 2.5 pM), see Table 2. The presence of the 2-aminoisobutyric acid (Aib) residue in position 2 renders the peptide resistant to DPPIV-mediated degradation, and a glutamine residue in position 3 is essential for activity at the glucagon receptor. Among further modifications compared to exendin-4, this peptide is modified in position 14 by addition of a stearic acid at the ε-amino group of a lysine using a γ-glutamic acid spacer. A similar fatty acid modification was shown to have beneficial effects on the activity at the GLP-1 and glucagon receptors in the case of peptide 1 in our previous study.13 However, it was found that peptide 2 not only shows high activity at the GLP-1 and glucagon receptors but also demonstrates considerable activity at the GIP receptor (EC50 = 39.7 pM). Starting from peptide 2, we designed variants aiming to (1) maintain high potency at the GLP-1 receptor, (2) reduce the relative ratio of GLP-1/glucagon receptor activity to a factor of approximately 1:10 to 1:15, and (3) introduce sufficient selectivity toward the GIP receptor (GLP-1/GIP receptor activity ratio in the order 1.0 × 108 (3) >1.0 × 108 (3) 1.5 ± 0.04 (60)

2050 ± 146 (3) >1.0 × 108 (3) >1.0 × 108 (3) >1.0 × 108 (3)

1.8 6.4 0.9 43.9

± ± ± ±

0.3 (3) 0.7 (3) 0.05 (38) 2.2 (59)

GCGR

GIPR

29 ± 2.1 (3) >1.0 × 108 (3) >1.0 × 108 (3) 0.5 ± 0.05 (180)

2032 ± 172 (3) >1.0 × 108 (3) >1.0 × 108 (3) >1.0 × 108 (3)

a Measured in a cAMP assay in receptor overexpressing HEK-293 cell lines stably expressing GLP-1, glucagon or GIP receptor. Values are mean ± SEM. Number of measurements is given in parentheses.

between exendin-4 and peptides 5 and 12, indicating that the amino acid difference in this position (lysine in exendin-4, Aib in peptide 5 and 12) does not alter the conformational preference in this region. The mutation Lys27Aib (see peptide 6 versus peptide 5 in Table 2) was shown to reduce activity at the glucagon (factor 6) and GIP receptor (factor 9) while

maintaining activity at the GLP-1 receptor. These activity differences are obviously caused by differential interactions with the receptors as indicated in Figure 2 and are not due to changes in backbone conformation. (2) The modifications introduced in the C-terminal peptide tail (peptide 5 versus 12) have an effect on the conformation of the tryptophan cage. 5587



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Figure 6. Absolute body weight (top) and relative body weight change (bottom) in obese diabetic cynomolgus monkeys during study period. Values are mean ± SEM, n = 8−10/group. ##P < 0.01 peptide 12 versus vehicle control. Liraglutide was dosed during the dose-ramping period from 10 (day 1−3) to 20 (day 4−6) to 30 μg/kg (day 7−9) and was maintained at 40 μg/kg sc from day 10 to completion of the study. Peptide 12 was dosed during the dose-ramping period from 1.5 (day 1−3) to 3 (day 4−6) to 4.5 (day 7−9) to 6 μg/kg (day 10−17) and was maintained at 8 μg/kg sc from day 18 to completion of the study.

Table 9. Plasma Drug Concentration (ng/mL) on Day 43 after Administration of 8 μg/kg sc of Peptide 12 and 40 μg/kg sc of Liraglutidea hours postdosing peptide

0

1

2

4

8

22

AUC0−22 (h·ng/mL)

12 liraglutide

0.66 87.8 ± 17.2

21.6 ± 2.4 145.0 ± 24.6

28.0 ± 2.9 206.0 ± 36.7

24.3 ± 3.1 279.0 ± 51.0

12.4 ± 2.3 184.0 ± 31.4

0.69 93.7 ± 19.9

254 3580

Values are mean ± SEM, n = 8−10/group.

a

natural glucagon and peptide 12 show a somewhat lower activity at the monkey versus human glucagon receptor (factor 3 for glucagon and factor 4 for peptide 12). Thus, in terms of relative potency (i.e., receptor potency versus the natural hormone), peptide 12 shows a similar GLP-1/glucagon activity preference in humans as in monkeys. Liraglutide and peptide 12 were dosed once daily to ensure sufficient and approximately continuous plasma levels repeatedly over the study duration of 6 weeks. To reduce the well-known gastrointestinal side effects like nausea and vomiting, GLP-1 receptor agonists are dose-ramped in humans.47 We used a similar up-titration protocol, considering the impact on total energy intake as a major indicator for tolerability. Liraglutide was dose-ramped from 10 to 40 μg/kg, and peptide 12 was ramped from 1.5 to 8 μg/kg (see Experimental Section). While vehicle-treated monkeys lost 2.1% ± 0.74% of body weight during the study, liraglutide treatment led to a weight loss of 4.9% ± 1.0% (not significant). The dual GLP-1/glucagon receptor agonist peptide 12 had the highest impact and decreased body weight by 6.9% ± 1.1% (P < 0.01, Figure 6).

Whereas the C-terminal sequence stretch (residues 30−39) of peptide 5 aligns nearly perfectly with exendin-4 (Cα rmsd = 1.1 Å, standard deviation 0.3 Å), peptide 12 shows a larger deviation (Cα rmsd = 3.4 Å, standard deviation 0.3 Å). Notably, these conformational differences in the C-terminal tail do not impact the helical part or the conformational stability of peptide 12. In Vivo Evaluation in Monkeys. The above-described dual GLP-1/glucagon receptor agonist peptide 12 was evaluated in a 6-week multiple dose study in obese diabetic monkeys following once-daily subcutaneous dosing. The marketed selective GLP-1 receptor agonist liraglutide was used as reference compound. Liraglutide carries, as does peptide 12, a fatty acid side chain, leading to a significantly prolonged exposure and duration of action when compared with the natural hormones GLP-1, glucagon, oxyntomodulin, or exendin-4. For pharmacological interpretation, agonist activities of peptide 12, liraglutide, and the natural hormones GLP-1 and glucagon were determined at the monkey and human GLP-1, glucagon, and GIP receptors (Table 8). Potency is rather conserved for the GLP-1 receptors across both species. Both 5588



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Pharmacokinetic analysis on day 43 showed that the 40 μg/ kg daily dose of liraglutide translated into a 14-fold higher exposure than the 8 μg/kg daily dose of peptide 12 as estimated by AUC comparison (see Table 9). The liraglutide AUC in monkeys is somewhat higher than the AUC observed in human clinical trials for liraglutide 3.0 mg, confirming that the up-titration scheme leads to the identification of relevant doses.48 Measurements of the long-term blood glucose marker % HbA1C were performed on day −25 to obtain baseline values and on day +43 following chronic treatment. While HbA1C in the vehicle-treated group slightly increased during the study period (increase from 6.4% ± 0.5% to 6.9% ± 0.5%), liraglutide treatment improved HbA1C (8.3% ± 0.6% at baseline to 7.8% ± 0.6%, not significant). A significant decrease and therefore improvement in HbA1C was observed in monkeys treated with peptide 12, clearly superior to liraglutide (Figure 7, P < 0.001): baseline value of 8.7% ± 0.8%

lower dose compared to the selective GLP-1 receptor agonist liraglutide (8 versus 40 μg/kg, sc) produced higher body weight loss. Body weight reduction and HbA1C improvement was greater despite a 14-fold lower exposure of the dual agonist at the highest dose tested. These findings indicate that the relatively low glucagon versus GLP-1 activity of peptide 12 does not negatively impact the favorable effects on glycemic control of the GLP-1 component. Hence, the relative glucagon receptor activity of a dual agonist might be further increased for additional glucagon-receptor mediated weight loss. We have conducted further detailed monkey studies on a peptide with a higher relative glucagon receptor activity than peptide 12 to address the important question on how much glucagon activation provides beneficial effects in total, which will be published in due time (Elvert et al. 2018, manuscript submitted). The therapeutic potential of dual GLP-1/glucagon receptor agonists will certainly depend on the question in how far the efficacy with respect to body weight reduction exceeds the benefits of marketed GLP-1 receptor agonists without impacting glucoregulatory effects in humans. Several dual GLP-1/glucagon coagonists are undergoing clinical testing,10 and the confirmation of their potential in human proof of concept (POC) studies is urgently awaited. Besides these pharmacological aspects, we have demonstrated how peptide optimization of solubility aspects under relevant conditions of the targeted formulation (e.g., with respect to the application pH range and the presence of antimicrobial preservatives) should be considered as an additional attribute in rational multiparameter optimization. Molecular modeling, experimental structure determination, and SAR analysis were used to introduce a novel solubilityenhancing motif (Pro32-Aib34-Lys35-Lys39) that significantly improved solubility in the presence of phenol as preservative at different pH values while maintaining the favorable receptor activity profile. Overall, peptide 12 with its promising pharmacological profile and well optimized solubility properties was suitable for entry into preclinical development.

Figure 7. HbA1C determination after 43 days of chronic treatment in obese diabetic cynomolgus monkeys compared to baseline according to the dosing scheme as described in Figure 6. Values are mean ± SEM, n = 8−10/group. ***P < 0.001 versus baseline.

declined to 7.1% ± 0.7% on day +43. These findings suggest that the 10−15-fold weaker glucagon versus GLP-1 receptor activity of the dual agonist peptide 12 does not negatively impact GLP-1 receptor mediated glucose control.

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CONCLUSION Several dual GLP-1/glucagon receptor agonists have entered clinical trials for the treatment of obese patients with T2D and are assessed to show improved therapeutic benefits versus sole GLP-1 receptor agonists. The key question to be still answered is the optimal ratio of relative GLP-1/glucagon receptor activities to optimize beneficial weight loss efficacy without negatively elevating blood glucose due to excessive glucagon pharmacology. Day and colleagues recommended that the relative ratio of GLP-1 and glucagon dual agonism needs to be carefully selected for each species. It was shown in mice that a rather GLP-1 receptor biased agonistic ratio leads to weight loss and glucose lowering. On the other hand, extensive enhancement of the relative glucagon receptor agonistic potency yielded greater weight loss but at the expense of considerable glucose elevation.20 The goal of the present study was the design of a dual agonist candidate with a 10−15-fold higher GLP-1 receptor activity compared to the glucagon receptor activity for chronic pharmacological evaluation with respect to weight loss and glucose control in monkeys, a relevant translational model for the human situation. In this study, daily administration of the dual GLP-1/glucagon receptor agonist peptide 12 at a 5-fold

EXPERIMENTAL SECTION

Homology Modeling of Peptides and Generation of Peptide Binding Hypotheses at the Receptors (ECD). ECDs of the GLP-1 receptor (PDB code 3c59), glucagon receptor (GCGR, PDB code 4ers), and GIP receptor (PDB code 2qkh) were structurally aligned. The NMR structure of exendin-4 (PDB code 1jrj) was then structurally superimposed to receptor-bound exendin(9−39) (PDB code 3c59), followed by energy minimization of the receptor−peptide complex. Structural models of peptides were generated by homology modeling, using the predicted binding mode of exendin-4 as structural template. For this purpose, a customized routine that allows us to consider unnatural and modified amino acids was used that has been implemented by Schrodinger. Subsequently, the peptides were minimized in the rigid GLP-1 receptor extracellular domain structure using MacroModel.49 Molecular graphics were generated using PyMol.50 Synthesis of Peptides. Solid phase synthesis was carried out on Rink-resin (e.g., from Agilent Technologies with a loading of 0.38 mmol/g, 75−150 μm). The Fmoc-synthesis strategy was applied with HBTU/DIPEA activation. For analogs with fatty acid side chains, in position 14 N-α-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3methylbutyl-N-ε-Fmoc-L-lysine (Fmoc-Lys(ivDde)-OH) and in position 1 Nα-Boc-N(im)-trityl-L-histidine (Boc-His(Trt)-OH) were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin using hydrazine in DMF according to literature.51 Hereafter, the respective lipophilic side chain was coupled 5589



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(total binding) and presence (nonspecific binding) of 1 μM unlabeled exendin-4, respectively. Each condition was measured in triplicate, and reported values are mean IC50 (nM) from n = 3 independent experiments. Solubility Testing. Prior to the solubility measurement of a peptide batch, its content was determined via UPLC-MS (% purity) and ion chromatography (both cations and anions). For solubility testing the target concentration was 10 mg of pure compound per milliliter. Therefore solutions from solid samples were prepared in a buffer system with a concentration of 10 mg/mL compound based on the previously determined content. The sample was gently agitated for 2 h, and UPLC-UV was performed from the supernatant, which was obtained by centrifugation (15 min at 3000 relative centrifugal force, rcf) and consecutive 1:10 dilution with the respective buffer. Solubility buffer system were (1) acetate buffer, pH 4.5, 100 mM sodium acetate trihydrate, or (2) phosphate buffer, pH 7.4, 100 mM sodium hydrogen phosphate, either with or without 0.5% phenol. The solubility was then determined by comparison of the UV peak area observed at a 2 μL injection of the diluted sample with UV areas of the respective peptide in DMSO (1.2 mg/mL based on the previously determined content) in a standard curve obtained from various injection volumes ranging from 0.2 μL to 2 μL. NMR. Peptides 5 and 12 gave rise to NMR spectra of high quality using a solution of ca. 6 mg/mL peptide in 70% 100 mM phosphate buffer, pH 5.0, H2O/D2O (9:1), and 30% trifluoroethanol (TFE) at 313 K. Based on the analysis of several 2D-spectra including DQFCOSY, TOCSY, NOESY, 1H,15N-HSQC, and 1H−13C-HSQC a complete assignment of 1H chemical shifts was achieved (see Tables S2 and S4). Distance constraints were obtained from NOESY spectra (mixing time was 200 ms) recorded in a mixture of 350 μL of H2O/ D2O, 50 mM phosphate buffer, pH 5.0, and 150 μL of TFE at 313 K. NOEs among protons of side chains have been obtained from NOESY spectra recorded in a mixture of 350 μL of D2O, 50 mM phosphate buffer, pH 5.0, and 150 μL of TFE at 313 K (mixing time was 200 ms). The 3D-structures of peptides 5 and 12 have been determined by restrained molecular dynamics calculations and energy minimization. Molecular dynamics (MD) simulations and interactive modeling were performed using the software package SYBYL, version 2.1.1. All energy calculations were based on the Tripos force field. For energy minimizations, the Powell method was used. The analysis of the NMR-derived distance restraints was carried out with SPL (Sybyl Programming Language) scripts. For peptide 5, the experimental data set used as input for the MD calculations included 402 distance constraints including 48 intraresidual distances, 134 sequential distances, 153 medium distances (2−4 amino acids apart), and 67 long-range distances (>4 amino acids apart). Upper and lower distance limits were set to plus and minus 10% of the calculated (experimental) distances, respectively. For nondiastereotopically assigned methylene protons and methyl groups, 0.9 and 1.0 Å, respectively were added to the upper bounds as pseudo-atom corrections. The set of utilized distance constraints is summarized in Table S2. Dihedral angle constraints and hydrogen bond constraints have not been used. A homology model derived from the NMR structure of exendin-4 (PDB code 1jrj) was used as starting structure. The NOE derived distance constraints were applied with a force constant of 41.9 kJ/mol·Å2. The MD simulation was performed in a water box using 6727 explicit water molecules. In the production run at 300 K, conformers were sampled every 50 ps for a duration of 1000 ps yielding a total of 20 structures. The obtained structures were energy-minimized (1000 steps). The 10 structures with the lowest constraint violation were used for further analyses. The average constraint violation is 0.11 Å. Only 6 constraints are violated by more than 0.6 Å, the largest violation is 0.73 Å. Considering only residues 7−37 the rmsd over all backbone atoms (N, NH, Cα, Hα, C′, O) is 0.56 Å with a standard deviation of 0.14 Å. For the same residues (residue 14 has been excluded), the rmsd over all heavy atoms is 0.80 Å with a standard deviation of 0.12 Å. The 3D-structure of peptide 12 was determined in the same way. For peptide 12, the experimental data set used as input for the MD calculations included 361 distance constraints including 55 intra residual distances, 114 sequential

to the liberated amino group using preactivated N-hydroxysuccinimide ester derivatives (e.g., Stea(γOSu)(αOtBu)Glu) or the side chain was built up stepwise using Fmoc-protected building blocks. The peptide was cleaved from the resin with King’s cocktail.52 The crude product was purified via preparative HPLC on a Waters column (e.g., XBridge, BEH130, Prep C18, 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). Purity and chemical identity of the product were assessed by UPLC and LC-MS (for details see Table S5) and confirmed to have ≥95% purity for all key compounds. In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Potency. Agonism of peptides for the three receptors was determined by functional assays measuring cAMP response of HEK293 cell lines stably expressing human or monkey GLP-1, glucagon, or GIP receptor. cAMP content of cells was determined using a kit from Cisbio Corp. (cat. no. 62AM4PEC) based on HTRF (homogenous time resolved fluorescence). For preparation, cells were split into T175 culture flasks and grown overnight to near confluence in medium (DMEM/10% FBS). Medium was then removed, and cells were washed with PBS lacking calcium and magnesium, followed by proteinase treatment with accutase (Sigma-Aldrich cat. no. A6964). Detached cells were washed and resuspended in assay buffer (1× HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX), and cellular density was determined. They were then diluted to 400 000 cells/mL, and 25 μL aliquots were dispensed into the wells of 96-well plates. For measurement, 25 μL of test compound in assay buffer was added to the wells, followed by incubation for 30 min at room temperature. After addition of HTRF reagents diluted in lysis buffer (kit components), the plates were incubated for 1 h, followed by measurement of the fluorescence ratio at 665/620 nm. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC50). Reported values are mean (SEM) EC50 (pM) from n ≥ 3 independent experiments. Crystallization and Structure Determination. A 6-fold excess of peptide 11 was dissolved in a solution of 12 mg/mL GLP-1 receptor-ECD in 10 mM Tris buffer, pH 7.5, 100 mM sodium sulfate, and 2% glycerol. This protein solution (100 nL) and 100 nL of reservoir solution were equilibrated against reservoir solution consisting of 10 mM CoCl2, 9.4% (v/v) 1,6-hexanediol, and 100 mM sodium acetate, pH 4.8. Crystals appeared after about 1 week. A crystal was picked up with a nylon loop that just had been dipped in glycerol and flash frozen in liquid nitrogen. X-ray diffraction data were collected at beamline PX-II of the Swiss Light Source (SLS) in Villigen, Switzerland, and processed with XDS53 and scaled with Aimless54 as implemented in AutoPROC.55 The crystal was of space group P43212 and contained one peptide−GLP-1 receptor ECD complex in the asymmetric unit. The unit cell dimensions of this crystal are as follows: a = b = 55.6 Å and c = 138.8. The crystal diffracted to 2.73 Å resolution, and the Rmean was 8.1%. The structure was solved by molecular replacement with Phaser,56 using a single monomer from PDB entry 3c59 as a starting model. Clear density was present for the bound peptide (see Figure S1). Model building was done with Coot,57 and refinement was done with Buster.58 The final was 20.6% and the free R-factor was 26.3%. Final data collection and refinement statistics are given in Table S6. Radioligand Binding Assay. The radioligand [125I]GLP-1 was purchased from PerkinElmer, USA. Cell membranes from HEK-293 cells expressing recombinant human GLP-1 receptors were used in the binding assays as follows. PVT-WGA SPA beads (0.125 mg/well, cat. no. RPNQ0001, PerkinElmer) coated with HEK-293 cells membranes (1 μg/well of protein) were incubated for 60 min at room temperature in HEPES buffer (20 mM HEPES, 1 mM EDTA, 150 mM NaCl, 5 mM MgCl2, and 1 mM CaCl2 (pH 7.4), with protease inhibitors and 0.1% BSA) with radiolabeled peptide (0.1 nM) in the absence and presence of a range of concentrations of unlabeled peptide. After incubation, the 96-well assay plate (cat. no. 3604, Corning), was read in a Wallac Microbeta plate reader (PerkinElmer) after a 180 min delay to measure cell membraneassociated radioactivity. Specific binding was calculated as the difference between the amount of [125I]GLP-1 bound in the absence 5590



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Statistical Analysis. All in vivo data are presented as means ± SEM. A one-way analysis of variance for factor treatment or two-way analysis including factor time were used followed by Dunnett’s test for multiple comparisons versus the vehicle or high-fat diet control group and Newman−Keuls test for comparison of the dual agonist versus the reference liraglutide. Statistical significance was considered with P < 0.05. All analyses were performed using SAS (version 9.2) under HP-UX via interface software EverStat V6.0-alpha5.

distances, 155 medium distances (2−4 amino acids apart), and 37 long-range distances (>4 amino acids apart). The set of utilized distance constraints is summarized in Table S4. The MD simulation was performed in a water box using 5760 explicit water molecules. In the production run at 300 K, conformers were sampled every 50 ps for a duration of 1000 ps yielding a total of 20 structures. The 10 structures with the lowest constraint violation were used for further analyses. The average constraint violation is 0.15 Å. Nineteen constraints are violated by more than 0.6 Å; the largest violation is 0.84 Å. Considering only residues 7−37, the rmsd over all backbone atoms (N, NH, Cα, Hα, C′, O) is 0.65 Å with a standard deviation of 0.19 Å. For the same residues (residue 14 has been excluded), the rmsd over all heavy atoms is 0.98 Å with a standard deviation of 0.24 Å. DIO Monkey Study. The monkey study was performed at Kunming Biomed International (KBI, Yunnan Province, China). KBI adheres to the guidelines for the care and use of animals for scientific purposes established by Chinese National Advisory Committee for Laboratory Animal Research (NACLAR) as well as the safety and quality assurance guidelines documented in the Guideline for Experiments Document of Kunming Biomed International (KBI-01GE v2.0). These guidelines set out the responsibilities of all parties involved in the care and use of animals for scientific purposes in accordance with widely accepted scientific, ethical, and legal principles. The guidelines stipulating the proposed use of animals for scientific purposes must be evaluated by an Institutional Animal Care and Use Committee (IACUC) in compliance with the guidelines. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of KBI prior to start of the experimental phase. The cynomolgus monkey (Macaca fascicularis) was selected as the test species. Up to fifty-one (n = 51) monkeys were trained in order to identify 30 monkeys (n = 8−10 per group) that were used in chronic treatment phase and fulfill metabolic criteria: weighing at least 8−16 kg, age of 12−20 years, fasting glucose >110 mg/dL, and fasting insulin of >70 μU/mL. Monkeys were individually housed in species- and size-appropriate metabolic stainless steel caging. A time controlled lighting system was used (lights on 7:00 am to 7:00 pm) to provide a regular 12-h light/12-h dark diurnal cycle. Monkeys were provided three meals per day with ad libitum access to water. The total offered amount of daily energy was about 680 kcal. Animals were stratified by randomized block stratification into 3 homogeneous groups according body weight, fasted plasma glucose, and %HbA1C. During the entire dosing period food and water intake was monitored daily, and body weight was measured every 3−4 days. Two monkeys within the vehicle group were found to be undergoing weight loss despite normal (high) food intake throughout the study. Both were considered advanced diabeticinsulin requiring and were excluded from final data analysis. Therefore baseline mean values for the vehicle treated monkeys were lower compared to treatment groups. Liraglutide was purchased directly from Novo Nordisk distributors in Beijing and Kunming and stored at 4 °C in accordance with manufacturer requirements. Victoza pens contain liraglutide at a concentration of 6 mg/mL. The solution was diluted to the desired concentrations of 100, 200, 400 μg/mL using a PBS vehicle solution. Liraglutide was dosed during the dose-ramping period from 10 (day 1−3) to 20 (day 4−6) to 30 μg/kg (day 7−9) and was maintained at 40 μg/kg sc (in 0.1 mL/kg of formulation) from day 10 to completion of the study. Peptide 12 was diluted with a PBS vehicle volume adjusted for the specific mass of the peptide. It was dosed during the dose-ramping period from 1.5 (day 1−3) to 3 (day 4−6) to 4.5 (day 7−9) to 6 μg/kg (day 10−17) and was maintained at 8 μg/kg sc from day 18 to completion of the study. Pharmacokinetics. Blood samples for PK analysis were collected on day 43 directly before and 1, 2, 4, 8, and 22 h postdosing. The samples were directly transferred into potassium ethylene diamine tetraacetic acid (EDTA-2K) tubes. Plasma was separated by centrifugation at 2500 × g for 10 min at 4 °C. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC-MS) for the dual agonist and by ELISA for liraglutide.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00292. 1 H-chemical shifts of peptides 5 and 12, distance constraints used for the MD simulation obtained from NOESY spectra, analytical data with RP-HPLC retention times and molecular masses of the synthesized peptides, crystallographic data collection and refinement statistics, omit map of the bound peptide 11, threedimensional alignment of X-ray structure of GLP-1 (ECD) receptor-bound peptide 11, receptor-bound modeling hypothesis of peptide 5, and NMR structure of peptide 5 in solution, three-dimensional alignment of X-ray structure of GLP-1 (ECD) receptor-bound peptide 11 and ECD, X-ray structure of GLP-1 (ECD) receptor-bound exendin(9−39), as well as three-dimensional alignment of X-ray structure of GLP-1 (ECD) receptor-bound peptide 11 and cryo-EM GLP-1 (fulllength) receptor-bound exendin-P5. (PDF) Accession Codes

Atomic coordinates for NMR structure of peptides 5 and 12 in solution can be accessed using PDB codes 6GDZ and 6GE2. Coordinates of the crystal structure of GLP-1 receptor extracellular domain (ECD) with peptide 11 can be assessed using PDB code 6GB1. The authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Authors

*A.E. Phone: +49 305 12636. E-mail: Andreas.Evers@sanofi. com. *M.B. Phone: +49 305 16655. E-mail: Martin.Bossart@sanofi. com. *M.W. Phone: +49 305 46875. E-mail: Michael.Wagner@ sanofi.com. ORCID

Andreas Evers: 0000-0003-4643-1941 Herman Schreuder: 0000-0003-2249-2782 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank R. Loder, R. Micciche, S. Peukert, S. Rauch, A. Sadikovic, K. Schlitt, C. Schneider, M. Schnierer, and L. Wäß for peptide synthesis; M. Schaffrath, S. Kohlitz, R. Hennig, K. Jung, M. Schnierer, and T. Zeisberg for purification of peptides; M. Jung, J. Ströbele, and N. Jacoby for peptide characterization (purity, solublity, stability); R. Noll, S. Apel, and T. Harth for in vitro potency characterization; A. Liesum for crystallizing the complex, J. Diez for the data collection, and P. Loenze for help with data processing; T. Weiss for program 5591



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(12) Pocai, A.; Carrington, P. E.; Adams, J. R.; Wright, M.; Eiermann, G.; Zhu, L.; Du, X.; Petrov, A.; Lassman, M. E.; Jiang, G.; et al. Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice. Diabetes 2009, 58 (10), 2258−2266. (13) Evers, A.; Haack, T.; Lorenz, M.; Bossart, M.; Elvert, R.; Henkel, B.; Stengelin, S.; Kurz, M.; Glien, M.; Dudda, A.; et al. Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/ Glucagon Receptor Agonists. J. Med. Chem. 2017, 60 (10), 4293− 4303. (14) Henderson, S. J.; Konkar, A.; Hornigold, D. C.; Trevaskis, J. L.; Jackson, R.; Fritsch Fredin, M.; Jansson-Löfmark, R.; Naylor, J.; Rossi, A.; Bednarek, M. A.; et al. Robust Anti-Obesity and Metabolic Effects of a Dual GLP-1/Glucagon Receptor Peptide Agonist in Rodents and Non-Human Primates. Diabetes, Obes. Metab. 2016, 18 (12), 1176− 1190. (15) Lynch, A. M.; Pathak, N.; Pathak, V.; O’Harte, F. P. M.; Flatt, P. R.; Irwin, N.; Gault, V. A. A Novel DPP IV-Resistant C-Terminally Extended Glucagon Analogue Exhibits Weight-Lowering and Diabetes-Protective Effects in High-Fat-Fed Mice Mediated through Glucagon and GLP-1 Receptor Activation. Diabetologia 2014, 57 (9), 1927−1936. (16) O’Harte, F. P. M.; Ng, M. T.; Lynch, A. M.; Conlon, J. M.; Flatt, P. R. Dogfish Glucagon Analogues Counter Hyperglycaemia and Enhance Both Insulin Secretion and Action in Diet-Induced Obese Diabetic Mice. Diabetes, Obes. Metab. 2016, 18 (10), 1013− 1024. (17) O’Harte, F. P. M.; Ng, M. T.; Lynch, A. M.; Conlon, J. M.; Flatt, P. R. Novel Dual Agonist Peptide Analogues Derived from Dogfish Glucagon Show Promising in Vitro Insulin Releasing Actions and Antihyperglycaemic Activity in Mice. Mol. Cell. Endocrinol. 2016, 431, 133−144. (18) Transition Therapeutics Inc. http://www. transitiontherapeutics.com/technology/tt401.php (accessed Mar 21, 2017). (19) Soni, H. Peptide-Based GLP-1/Glucagon Co-Agonists: A Double-Edged Sword to Combat Diabesity. Med. Med. Hypotheses 2016, 95, 5−9. (20) Day, J. W.; Gelfanov, V.; Smiley, D.; Carrington, P. E.; Eiermann, G.; Chicchi, G.; Erion, M. D.; Gidda, J.; Thornberry, N. A.; Tschöp, M. H.; et al. Optimization of Co-Agonism at GLP-1 and Glucagon Receptors to Safely Maximize Weight Reduction in DIORodents. Biopolymers 2012, 98 (5), 443−450. (21) General Chapters: ⟨51⟩ ANTIMICROBIAL EFFECTIVENESS TESTING http://www.pharmacopeia.cn/v29240/ usp29nf24s0_c51.html (accessed Jan 23, 2018). (22) De Jong, K. L.; Incledon, B.; Yip, C. M.; DeFelippis, M. R. Amyloid Fibrils of Glucagon Characterized by High-Resolution Atomic Force Microscopy. Biophys. J. 2006, 91 (5), 1905−1914. (23) Heljo, P.; Ross, A.; Zarraga, I. E.; Pappenberger, A.; Mahler, H.C. Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations. Pharm. Res. 2015, 32 (10), 3201−3212. (24) Pedersen, J. S. The Nature of Amyloid-like Glucagon Fibrils. J. Diabetes Sci. Technol. 2010, 4 (6), 1357−1367. (25) Pedersen, J. S.; Dikov, D.; Otzen, D. E. N- and C-Terminal Hydrophobic Patches Are Involved in Fibrillation of Glucagon. Biochemistry 2006, 45 (48), 14503−14512. (26) Pedersen, J. S.; Dikov, D.; Flink, J. L.; Hjuler, H. A.; Christiansen, G.; Otzen, D. E. The Changing Face of Glucagon Fibrillation: Structural Polymorphism and Conformational Imprinting. J. Mol. Biol. 2006, 355 (3), 501−523. (27) Bak, A.; Dai, W. Peptide Developability at the Discovery-toDevelopment Interface−Current State and Future Opportunities. AAPS J. 2015, 17 (4), 777−779. (28) Bak, A.; Leung, D.; Barrett, S. E.; Forster, S.; Minnihan, E. C.; Leithead, A. W.; Cunningham, J.; Toussaint, N.; Crocker, L. S. Physicochemical and Formulation Developability Assessment for Therapeutic Peptide Delivery−a Primer. AAPS J. 2015, 17 (1), 144− 155.

management and organizational support; B. Zhang, T. Wang, P. Higgins, L. Yang, F. Du, N. Lood, and staff at KBI for performing the monkey in vivo study; F. Levai and K. Schröter for providing us the kinetic profiles of compounds used from monkey plasma samples; and G. Tiwari for reading the manuscript and providing valuable comments.

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ABBREVIATIONS USED Aib, 2-aminoisobutyric acid; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle’s medium; DPPIV, dipeptidyl peptidase IV; ECD, extracellular domain; FBS, fetal bovine serum; GCGR, glucagon receptor; GIP, glucosedependent insulinotropic peptide; HbA1C, hemoglobin A1C; HBSS, Hank’s balanced salt solution; HBTU, O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HP-UX, Hewlett-Packard Unix; HTRF, homogenous time resolved fluorescence; IBMX, isobutylmethylxanthine; NASH, nonalcoholic steatohepatitis; OXM, oxyntomodulin; PVT, polyvinyltoluene; SEM, standard error of the mean; SGLT2-I, sodium−glucose cotransporter 2 inhibitor; SPA, scintillation proximity assay; SPL, Sybyl programming language; TFE, trifluorethanol; WGA, wheat germ agglutinin

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REFERENCES

(1) World Health Organization. Media Centre. Diabetes (Fact sheet no. 312) Available at http://www.who.int/mediacentre/factsheets/ fs312/en/ [accessed 23 Jan 2018]. (2) 2014 Diabetes Report Card - diabetesreportcard2014.pdf [Internet], Available at http://www.cdc.gov/diabetes/pdfs/library/ diabetesreportcard2014.pdf [accessed 23 Jan 2018]. (3) Nonalcoholic Steatohepatitis: Risk Factors and Diagnosis Available at http://www.medscape.com/viewarticle/731163 [accessed 23 Jan 2018]. (4) Lee, C. Y. Glucagon-Like Peptide-1 Formulation − the Present and Future Development in Diabetes Treatment. Basic Clin. Pharmacol. Toxicol. 2016, 118 (3), 173−180. (5) McBrayer, D. N.; Tal-Gan, Y. Recent Advances in GLP-1 Receptor Agonists for Use in Diabetes Mellitus. Drug Dev. Res. 2017, 78 (6), 292−299. (6) Uccellatore, A.; Genovese, S.; Dicembrini, I.; Mannucci, E.; Ceriello, A. Comparison Review of Short-Acting and Long-Acting Glucagon-like Peptide-1 Receptor Agonists. Diabetes Ther. 2015, 6 (3), 239−256. (7) Finan, B.; Clemmensen, C.; Mü l ler, T. D. Emerging Opportunities for the Treatment of Metabolic Diseases: Glucagonlike Peptide-1 Based Multi-Agonists. Mol. Cell. Endocrinol. 2015, 418, 42−54. (8) Knerr, P. J.; Finan, B.; Gelfanov, V.; Perez-Tilve, D.; Tschöp, M. H.; DiMarchi, R. D. Optimization of Peptide-Based Polyagonists for Treatment of Diabetes and Obesity. Bioorg. Med. Chem. 2018, 26 (10), 2873−2881. (9) Sánchez-Garrido, M. A.; Brandt, S. J.; Clemmensen, C.; Müller, T. D.; DiMarchi, R. D.; Tschöp, M. H. GLP-1/Glucagon Receptor Co-Agonism for Treatment of Obesity. Diabetologia 2017, 60 (10), 1851−1861. (10) Tschöp, M. H.; Finan, B.; Clemmensen, C.; Gelfanov, V.; Perez-Tilve, D.; Müller, T. D.; DiMarchi, R. D. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity. Cell Metab. 2016, 24 (1), 51−62. (11) Day, J. W.; Ottaway, N.; Patterson, J. T.; Gelfanov, V.; Smiley, D.; Gidda, J.; Findeisen, H.; Bruemmer, D.; Drucker, D. J.; Chaudhary, N.; et al. A New Glucagon and GLP-1 Co-Agonist Eliminates Obesity in Rodents. Nat. Chem. Biol. 2009, 5 (10), 749− 757. 5592



Article

(29) Fosgerau, K.; Hoffmann, T. Peptide Therapeutics: Current Status and Future Directions. Drug Discovery Today 2015, 20 (1), 122−128. (30) Neidigh, J. W.; Fesinmeyer, R. M.; Prickett, K. S.; Andersen, N. H. Exendin-4 and Glucagon-like-Peptide-1: NMR Structural Comparisons in the Solution and Micelle-Associated States †. Biochemistry 2001, 40 (44), 13188−13200. (31) Chabenne, J. R.; DiMarchi, M. A.; Gelfanov, V. M.; DiMarchi, R. D. Optimization of the Native Glucagon Sequence for Medicinal Purposes. J. Diabetes Sci. Technol. 2010, 4 (6), 1322−1331. (32) Runge, S.; Thøgersen, H.; Madsen, K.; Lau, J.; Rudolph, R. Crystal Structure of the Ligand-Bound Glucagon-like Peptide-1 Receptor Extracellular Domain. J. Biol. Chem. 2008, 283 (17), 11340−11347. (33) Koth, C. M.; Murray, J. M.; Mukund, S.; Madjidi, A.; Minn, A.; Clarke, H. J.; Wong, T.; Chiang, V.; Luis, E.; Estevez, A.; et al. Molecular Basis for Negative Regulation of the Glucagon Receptor. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (36), 14393−14398. (34) Parthier, C.; Kleinschmidt, M.; Neumann, P.; Rudolph, R.; Manhart, S.; Schlenzig, D.; Fanghänel, J.; Rahfeld, J.-U.; Demuth, H.U.; Stubbs, M. T. Crystal Structure of the Incretin-Bound Extracellular Domain of a G Protein-Coupled Receptor. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (35), 13942−13947. (35) Jazayeri, A.; Rappas, M.; Brown, A. J. H.; Kean, J.; Errey, J. C.; Robertson, N. J.; Fiez-Vandal, C.; Andrews, S. P.; Congreve, M.; Bortolato, A.; et al. Crystal Structure of the GLP-1 Receptor Bound to a Peptide Agonist. Nature 2017, 546 (7657), 254−258. (36) Zhang, Y.; Sun, B.; Feng, D.; Hu, H.; Chu, M.; Qu, Q.; Tarrasch, J. T.; Li, S.; Sun Kobilka, T.; Kobilka, B. K.; et al. Cryo-EM Structure of the Activated GLP-1 Receptor in Complex with a G Protein. Nature 2017, 546 (7657), 248−253. (37) Zhang, H.; Qiao, A.; Yang, D.; Yang, L.; Dai, A.; de Graaf, C.; Reedtz-Runge, S.; Dharmarajan, V.; Zhang, H.; Han, G. W.; et al. Structure of the Full-Length Glucagon Class B G-Protein-Coupled Receptor. Nature 2017, 546 (7657), 259−264. (38) Zhang, H.; Qiao, A.; Yang, L.; Van Eps, N.; Frederiksen, K. S.; Yang, D.; Dai, A.; Cai, X.; Zhang, H.; Yi, C.; et al. Structure of the Glucagon Receptor in Complex with a Glucagon Analogue. Nature 2018, 553 (7686), 106−110. (39) Finan, B.; Yang, B.; Ottaway, N.; Smiley, D. L.; Ma, T.; Clemmensen, C.; Chabenne, J.; Zhang, L.; Habegger, K. M.; Fischer, K.; et al. A Rationally Designed Monomeric Peptide Triagonist Corrects Obesity and Diabetes in Rodents. Nat. Med. 2015, 21 (1), 27−36. (40) Ward, B. P.; Ottaway, N. L.; Perez-Tilve, D.; Ma, D.; Gelfanov, V. M.; Tschöp, M. H.; DiMarchi, R. D. Peptide Lipidation Stabilizes Structure to Enhance Biological Function. Mol. Metab. 2013, 2 (4), 468−479. (41) López de Maturana, R.; Willshaw, A.; Kuntzsch, A.; Rudolph, R.; Donnelly, D. The Isolated N-Terminal Domain of the Glucagonlike Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1. J. Biol. Chem. 2003, 278 (12), 10195− 10200. (42) Lau, J.; Bloch, P.; Schäffer, L.; Pettersson, I.; Spetzler, J.; Kofoed, J.; Madsen, K.; Knudsen, L. B.; McGuire, J.; Steensgaard, D. B.; et al. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J. Med. Chem. 2015, 58 (18), 7370− 7380. (43) Liang, Y.-L.; Khoshouei, M.; Glukhova, A.; Furness, S. G. B.; Zhao, P.; Clydesdale, L.; Koole, C.; Truong, T. T.; Thal, D. M.; Lei, S.; et al. Phase-Plate Cryo-EM Structure of a Biased Agonist-Bound Human GLP-1 Receptor-Gs Complex. Nature 2018, 555 (7694), 121−125. (44) Göke, R.; Fehmann, H. C.; Linn, T.; Schmidt, H.; Krause, M.; Eng, J.; Göke, B. Exendin-4 Is a High Potency Agonist and Truncated Exendin-(9−39)-Amide an Antagonist at the Glucagon-like Peptide 1(7−36)-Amide Receptor of Insulin-Secreting Beta-Cells. J. Biol. Chem. 1993, 268 (26), 19650−19655.

(45) Montrose-Rafizadeh, C.; Yang, H.; Rodgers, B. D.; Beday, A.; Pritchette, L. A.; Eng, J. High Potency Antagonists of the Pancreatic Glucagon-like Peptide-1 Receptor. J. Biol. Chem. 1997, 272 (34), 21201−21206. (46) Zhang, Y.; Roy, S.; Jones, L. S.; Krishnan, S.; Kerwin, B. A.; Chang, B. S.; Manning, M. C.; Randolph, T. W.; Carpenter, J. F. Mechanism for Benzyl Alcohol-Induced Aggregation of Recombinant Human Interleukin-1 Receptor Antagonist in Aqueous Solution. J. Pharm. Sci. 2004, 93 (12), 3076−3089. (47) Prasad-Reddy, L.; Isaacs, D. A Clinical Review of GLP-1 Receptor Agonists: Efficacy and Safety in Diabetes and Beyond. Drugs Context 2015, 4, 1. (48) Overgaard, R. V.; Petri, K. C.; Jacobsen, L. V.; Jensen, C. B. Liraglutide 3.0 Mg for Weight Management: A Population Pharmacokinetic Analysis. Clin. Pharmacokinet. 2016, 55 (11), 1413−1422. (49) MacroModel; Schrö dinger https://www.schrodinger.com/ macromodel (accessed Jan 31, 2018). (50) PyMOL; Schrödinger https://www.schrodinger.com/pymol (accessed Jan 31, 2018). (51) Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C. An Appraisal of New Variants of Dde Amine Protecting Group for Solid Phase Peptide Synthesis. Tetrahedron Lett. 1998, 39 (12), 1603−1606. (52) King, D. S.; Fields, C. G.; Fields, G. B. A Cleavage Method Which Minimizes Side Reactions Following Fmoc Solid Phase Peptide Synthesis. Int. J. Pept. Protein Res. 1990, 36 (3), 255−266. (53) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (2), 125−132. (54) Evans, P. Scaling and Assessment of Data Quality. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62 (1), 72−82. (55) Vonrhein, C.; Flensburg, C.; Keller, P.; Sharff, A.; Smart, O.; Paciorek, W.; Womack, T.; Bricogne, G. Data Processing and Analysis with the AutoPROC Toolbox. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67 (4), 293−302. (56) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J. Appl. Crystallogr. 2007, 40 (4), 658−674. (57) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (4), 486−501. (58) Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.; Paciorek, W.; Roversi, P.; Sharff, A.; Smart, O. S.; Vonrhein, C.; Womack, T. O. Buster 2011; Global Phasing Ltd: Cambridge, United Kingdom, 2017.

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