Glucagon Receptor Agonists - ACS Publications - American Chemical

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Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP1)/Glucagon Receptor Agonists Andreas Evers,* Torsten Haack, Martin Lorenz, Martin Bossart, Ralf Elvert, Bernd Henkel, Siegfried Stengelin, Michael Kurz, Maike Glien,† Angela Dudda, Katrin Lorenz, Dieter Kadereit, and Michael Wagner* R&D, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst Building G838, D-65926 Frankfurt am Main, Germany S Supporting Information *

ABSTRACT: Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure− activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.

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INTRODUCTION The dramatic rise of the twin epidemics, type 2 diabetes and obesity collectively referred to as “diabesity”, is associated with increased mortality and morbidity worldwide.1 On the basis of this global development, there is clinical need for antidiabetic therapies with accompanied weight reduction. Already a modest reduction in body weight (∼4−5 kg) is an effective means of managing diabesity, resulting in highly beneficial effects on glycemic control as well as reduced morbidity and mortality.2 From the approved antidiabetic therapies, sodium−glucose cotransporter 2 inhibitors (SGLT2-I) and the injectable glucagonlike peptide-1 (GLP-1) receptor agonists are the only classes of agents that are associated with such a modest weight reduction. Physiological effects of the gastrointestinal hormone GLP-1 are improvement of glycemic control as well as a reduction in appetite and food intake. The body weight reduction which can be achieved with synthetic GLP-1 analogs is in the range of 3− 6%. A high dose variant of liraglutide (3 mg) was approved in 2014 for the treatment of obese adults with weight related health conditions. In that target population liraglutide has shown a body weight reduction of 5−6% over 56 weeks in late stage clinical trials.3,4 Beyond the currently approved GLP-1 receptor agonists, the next generation of peptidic agonists could combine the activity of GLP-1 and additional gastrointestinal hormones in one molecule, leading to superior therapeutic benefits. For example, the native gastrointestinal hormone oxyntomodulin (OXM), a 37 amino acid peptide (comprising the entire 29 amino acid © 2017 American Chemical Society

sequence from glucagon plus a C-terminal extension of 8 residues) secreted by intestinal L-cells (together with GLP-1 and PYY) following meal ingestion, is such an endogenous dual agonist for the GLP-1 and glucagon receptor. Its activity is weaker for the GLP-1 and glucagon receptors when compared to the cognate native ligands GLP-1 and glucagon (see Table 1). Glucagon is an insulin counter-regulatory hormone secreted by the α-cells of the pancreas that raises blood glucose levels by stimulating gluconeogenesis and glycogenolysis, thus circumventing a hypoglycemic state. More recent data in rodents and humans reveal that glucagon could have beneficial effects on energy balance, body fat mass, and nutrient intake, although the first observation that glucagon reduces food intake was already made 50 years ago.5 These effects seem to be mediated at least in part by FGF21-dependent pathways, a protein acting at the level of the brain, liver, and adipose tissue to reduce body weight.6,7 In overweight and obese people the endogenous dual GLP-1/glucagon agonist oxyntomodulin was shown to significantly reduce body weight by ∼1.7 kg vs placebo following 3 times daily subcutaneous administration (to compensate for the short half-life of the native peptide) after 4 weeks.8 Furthermore, oxyntomodulin was mechanistically proven to reduce food intake after an ad libitum test meal and increase energy expenditure in humans.9 Therapeutic utility of Received: February 10, 2017 Published: April 27, 2017 4293

DOI: 10.1021/acs.jmedchem.7b00174 J. Med. Chem. 2017, 60, 4293−4303

Journal of Medicinal Chemistry

Article

Table 1. Amino Acid Sequences of Glucagon, Oxyntomodulin (OXM), Exendin-4, Native GLP-1, and Liraglutide and Respective Activities (and SEM Values) at the GLP-1 Receptor and Glucagon Receptor (GCGR) As Measured in a cAMP Assay Overexpressing HEK-293 Cell Linesa

a

In all cases, three replicates were measured. Amino acids, which are identical among glucagon/oxyntomodulin and exendin-4, are colored green. Residues unique to glucagon/oxyntomodulin are shown in yellow. Residues unique to exendin-4 are colored gray. Residues unique to GLP-1 are white, and further modifications are shown in orange. Residue 20 in liraglutide is modified by addition of a C16 fatty acid (palmitic acid) at the εamino group of lysine using a γ-glutamic acid spacer.

enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing of gastric emptying, thereby reducing postprandial glucose absorption, and reduced food intake resulting in a reduction in body weight.22−24 Exenatide BID, a synthetic version of exendin-4, represents the first GLP-1 receptor agonist approved in 2005 (at the time by Amylin/Eli Lilly, now marketed by AstraZeneca) as antidiabetic therapy for the treatment of T2DM. It has a terminal half-life of ∼2.4 h after subcutaneous administration and is applied twice daily (10 μg). In contrast, liraglutide developed by Novo Nordisk is a marketed chemically modified GLP-1 analog. In position 20, a fatty acid is linked to a lysine leading to a half-life of 13 h in humans following subcutaneous injection via oligomerization and reversible albumin binding, suitable for once-daily administration (see Table 1).25,26 Compared to GLP-1, glucagon, and oxyntomodulin, exendin4 has improved physicochemical properties, such as solubility and stability in both solution and under physiological conditions: the Leu21-Ser39 span of exendin-4 forms a compact tertiary fold (termed as tryptophan cage or Trp cage), which is one of the smallest protein folds.27 The Trp cage shields the side chain of Trp25 from solvent exposure and enhances helicity and stability of the peptide by intramolecular interactions (see Figure 1).28 DiMarchi and co-workers have shown that C-terminal addition of the exendin-4 tail, e.g., residues 31−39, to the glucagon sequence resulted in an analogue with significantly improved solubility and chemical stability compared to glucagon itself, probably by shielding hydrophobic and chemically labile surface motifs of the glucagon C-terminus.29 Furthermore, exendin-4 shows an enhanced metabolic stability compared to GLP-1, glucagon, and oxyntomodulin. This improved stability is partially due to the introduction of a Gly residue at position 2, which renders it resistant to DPPIV mediated degradation. In addition, due to the formation of the Trp cage, exendin-4 shows an increased helicity30,31 that might also prevent structural unwinding and enzymatic digestion by other proteases. Indeed, it was shown that the C-terminal exendin-4 extension, which is responsible for the formation of the Trp cage, provides additional metabolic stability and prolonged in vivo potency.14,32 Finally, it was shown that exendin-4 forms specific oligomers in solution33 that increase particle size and might, thus reducing renal clearance. Due to these favorable properties, we considered exendin-4 as an attractive scaffold for the development of exendin-4 analogues with dual activities for the GLP-1 and glucagon receptor. Many of the so far described GLP-1/glucagon agonists are based on the glucagon or oxyntomodulin structure,

natural gastrointestinal hormones like GLP-1, glucagon, or oxyntomodulin is limited by its rapid degradation by serum proteases, predominantly dipeptidyl peptidase IV (DPPIV) but also other enzymes such as neutral endopeptidase (NEP), plasma kallikrein, or plasmin.10 Therefore, such native peptides have a very short half-life of a few minutes following intravenous administration. One strategy to stabilize peptides against DPPIV cleavage is to exchange amino acid sequences which are prone to enzymatic degradation. Another approach for peptide stabilization is chemical modification, e.g., by conjugation to a fatty acid side chain or cholesterol derivatives for reversible binding to plasma albumin or to large hydrophilic polymers such as polyethylene glycol (PEG) to reduce renal clearance. Such dual GLP-1/glucagon receptor agonists that are additionally stabilized by chemical modifications have been described, for example, by Pocai et al.,11,12 Flatt et al.,13−16 Day et al.,17 and most recently by Henderson et al. (MedImmune).18 For example, Lys(yE-C16) 10Glu12Glu17Arg20Ala24Glu27Ala28Gly29Gly30-glucagon (MEDI0382)17 is a once-daily administered glucagon analog with dual GLP-1 and glucagon receptor activity, which is modified with a fatty acid side chain in position 10. It is currently evaluated in a phase 1/2 study in patients with type 2 diabetes mellitus (T2DM). Poly(oxy-1,2ethanediyl), α-hydro-ω-methoxy-, 38,39-diether with L-histidyl2-methylalanyl-L-glutaminylglycyl-L-threonyl-L-phenylalanyl-Lthreonyl-L-seryl-L-α-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-Lleucyl-L-α-aspartyl-L-seryl-L-lysyl-L-lysyl-L-alanyl-L-glutaminyl-Lα-glutamyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-Lleucyl-L-leucyl-L-asparaginyl-2-methylalanylglycyl-L-arginyl-L-asparaginyl-L-arginyl-L-asparaginyl- L-asparaginyl- L-isoleucyl-Lalanyl-S-(1-(3-((3-hydroxypropyl)amino)-3-oxopropyl)-2,5dioxo-3-pyrrolidinyl)-L-cysteinyl-S-(1-(3-((3-hydroxypropyl)amino)-3-oxopropyl)-2,5-dioxo-3-pyrrolidinyl)-L-cysteinamide (pegapamodutide)18,19 from Transition Therapeutics, which is a PEGylated dual agonist for once-weekly dosing, is another dual agonist that has been evaluated in phase 2 studies in patients with T2DM. An extensive review that includes dual GLP-1/glucagon agonists in discovery, preclinical validation, and first clinical examination has recently been published by Tschöp et al.20 Exendin-4 is a 39 amino acid peptide that is produced by the salivary glands of the Gila monster (Heloderma suspectrum).21 Exendin-4 is an activator of the GLP-1 receptor; however it does not activate the glucagon receptor (see Table 1). Clinical and nonclinical studies have shown that exendin-4 has several beneficial antidiabetic properties including an increase in β cell mass and markers of β cell function, a glucose dependent 4294



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agonist. Additional chemical modifications resulted in a further improved metabolic stability. Structural analysis of the most promising peptide by NMR spectroscopy revealed that (in agreement with the design rationales) the resulting peptide indeed shows an exendin-like fold. This peptide was then pharmacologically assessed in rodent models of diabetes and obesity.

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RESULTS AND DISCUSSION Structural Analysis of Peptides and Binding Hypotheses to the GLP-1 and Glucagon Receptor. The GLP-1 and glucagon receptors both belong to the class B G-proteincoupled receptors (GPCRs), sharing 47% sequence identity and adopting a similar fold. At the time of peptide design and synthesis, X-ray structures were available only for the extracellular domain (ECD) of the GLP-1 receptor (PDB codes 3iol, 3c59, 3c5t).30,31 Therefore, a structural hypothesis for the ECD of the glucagon receptor was generated by homology modeling. Consistent with a recently published crystal structure (PDB code 4ers),34 a high structural similarity to the ECD of the GLP-1 receptor is observed (Figure 1). Structural data for the peptides were obtained from solutionstate NMR of exendin-4 (PDB code 1jrj)28 and X-ray data of glucagon (PDB code 1gcn)35 or exendin(9−39) in the receptor-bound conformation (PDB code 3c59).30 Binding models of exendin-4 and glucagon to their respective receptors were generated after 3D alignment of the receptor structures and subsequent structure-based alignments of the peptides to receptor-bound exendin(9−39), followed by energy minimization of receptor−peptide complexes. The resulting 3D alignments were then analyzed. Although exendin-4 and glucagon share only 13 identical residues, they show similar helical conformations in the receptor-bound state and establish several similar key interactions to their target receptors (Figure 1). Interestingly, several differences were found in the structural regions of the GLP-1 receptor and glucagon receptor that interact with residues 15−21 from exendin-4 and glucagon, respectively. The C-terminal exendin-4 tail, which is responsible for the formation of the Trp cage, does not establish relevant interactions with the receptors, suggesting that this motif does not significantly impact activation of the glucagon receptor. In contrast, it is well-known that the interaction of the N-terminal

Figure 1. 3D structures of exendin-4 (derived from a NMR structure in solution, PDB code 1jrj), glucagon (derived from an X-ray structure, PDB code 1gcn), and binding hypotheses for these peptides to the extracellular domain (ECD) of the GLP-1 receptor (PDB code 3c59) and glucagon receptor (PDB code 4ers). The color coding of peptide residues is equivalent to the sequence color coding outlined in Table 1. Residue Trp25, which is exposed to the solvent in glucagon and shielded from the solvent by the Trp cage motif in exendin-4, is shown in spacefill representation.

resulting in a considerable challenge to optimize and tune the structural and physicochemical properties. Although glucagon and oxyntomodulin already show considerable activity at the GLP-1 and glucagon receptor, exendin-4 is a highly selective GLP-1 receptor agonist (see Table 1). Here, we report our efforts to identify such novel dual agonistic peptides, aiming to introduce significant activity at the glucagon receptor into exendin-4 while preserving its favorable structure-stabilizing and solubility-enhancing fold. Relevant pharmacophoric features for dual activity were identified by sequence and structure-based comparison of exendin-4 and glucagon and their interactions with the extracellular domains of the respective receptors (see Table 1 and Figure 1). On the basis of this analysis, sequence stretches from glucagon were systematically introduced into exendin-4 to obtain a potent dual

Table 2. EC50 Values (plus SEM Values, Measured in pM in a cAMP Assay in Overexpressing HEK-293 Cell Lines) of Peptide Sequences Derived from Grafting Glucagon/Oxyntomodulin Sequence Stretches into Exendin-4a

a

In all cases, three replicates were measured. 4295



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Table 3. Amino Acid Sequences of Glucagon and GlucagonCex, a Glucagon Variant That Carries the C-Terminal Exendin4(30−39) Sequence Stretch, and Peptide 7a

a

EC50 values are taken from ref 29. Absolute EC50 values for glucagon differ from measurements of this study, presumably due to different assay settings.

One of the most relevant requirements for dual activity at the GLP-1 and glucagon receptor is the presence of Ser2 and Gln3. Reverse mutation of one of these residues into exendin-4 (i.e., Ser2Gly or Gln3Glu) results in a significant activity loss at the glucagon receptor (compare peptides 11 and 12 vs peptides 6 and 7). Activity data for peptide 13 in comparison to peptides 6 and 7 reveal that in addition residues 15−21 are important for activity at the glucagon receptor. In summary, the combination of amino acids from the exendin-4 and glucagon sequence as realized in peptide 7 results in a peptide with a balanced GLP-1/glucagon in vitro activity ratio of 1:1. The in vitro activity data in Table 2 show that adjustment for another activity ratio toward the one or other receptor might be achieved by specific amino acid exchange. This is further supported by the in vitro activity data that have been reported by Chabenne et al.:29 in their study, glucagon shows an EC50 value of 3.74 nM at the GLP-1 receptor and of 0.06 nM at the glucagon receptor, respectively, which translates into a GLP-1R/glucagon activity ratio of 1:62. Addition of the exendin-4(30−39) sequence stretch to glucagon, resulting in glucagonCex (see Table 3), switches the GLP-1R/glucagon selectivity ratio from 1:62 to 1:14.29 This peptide still differs in seven positions from peptide 7, indicating that a further stepwise adjustment toward the GLP-1/glucagon activity ratio of 1:1 can be achieved by systematic mutation of the glucagon-originating residues into the corresponding exendin-4 variants. While peptide 7 shows potent dual activity at both the GLP1 and glucagon receptors, it is thought to be cleared by renal filtration and/or enzymatic digestion, e.g., by DPPIV.12 We used the following strategies in order to improve metabolic stability: (1) modification of the serine residue in position 2 to avoid DPPIV-mediated degradation and (2) addition of a palmitic acid at the ε-amino group of a lysine residue using a γglutamic acid spacer, a strategy that has been successfully proven in the case of liraglutide.37,38 On the basis of the peptide-receptor binding models in Figure 1, several positions were identified where the modified Lys residue could be introduced without perturbing receptor binding interactions (Figure 2). While the position of the fatty acid substituent in peptides 14 vs 15 and 16 are distant in the sequence positions (14 vs 39 and 40), they are spatially similar. In all cases, the modified peptides show even enhanced activation at both receptors compared to the nonacylated peptides (Table 4). This observation was also described by DiMarchi et al. for glucagon analogues.39 Experimental data based on different biophysical and structural characterization techniques suggest that in these particular cases, lipidation enhances receptor activity by stabilizing relevant bioactive conformations. Further studies addressing other peptide targets also demonstrated that lipid conjugation can increase the functional activity of peptides.40−42 However, the molecular basis for the activity

part of the peptides with the transmembrane domains of the receptors is essential for receptor activity. However, at the time of design and synthesis, structural data about the full-length sequences or the transmembrane domain of the receptors were not available. In the meantime, an X-ray structure of the transmembrane domain and a structural model for full-length glucagon receptor have been published.36 A peptide binding hypothesis in this full-length model of the glucagon receptor is provided below. In summary, structure- and sequence-based analysis suggests that transfer of N-terminal and “central” sequence stretches of glucagon into exendin-4 might result in exendin-4 analogues with dual GLP-1/glucagon receptor activity. Peptide Design and Optimization. As outlined above, peptide sequences were designed by sequence- and structurebased analysis. Following synthesis of these peptides, their potency on the GLP-1 and glucagon receptors was tested in a cAMP assay in receptor overexpressing HEK-293 cells. In the first optimization cycle (peptides 1−5), sequence stretches from positions 15−33 of the exendin-4 sequence were replaced by the corresponding glucagon/oxyntomodulin stretches (Table 2). Potency at the GLP-1 receptor was only affected to a low extent for most analogues, while activity at the glucagon receptor could not be significantly increased. At least, peptide 2 (containing residues 15−21 from glucagon) showed a very modest activity (EC50 = 565 nM) at the glucagon receptor and was used as a starting point for further optimization. It was found that introduction of solely Ser2 and Gln3 from the glucagon sequence into exendin-4 resulted in a peptide (peptide 6) that maintained high potency at the GLP-1R (EC50 = 0.7 pM) and showed one-digit nanomolar activity at the glucagon receptor (EC50 = 1.12 nM). Finally, combination of sequence motifs of peptides 2 and 6 resulted in peptide 7, which shows potent dual activity at the GLP-1 and glucagon receptor (EC50(GLP-1R) = 22.2 pM, EC50(GCGR) = 26.4 pM). In order to identify the importance of specific mutations for relative activity at both receptors, several variants of peptide 7 were synthesized and tested (see Table 2, peptides 8−13). According to our structural analysis (Figure 1), the C-terminal tail of exendin-4 that cages residue Trp25 does not show significant interactions with the GLP-1 or glucagon receptor. Therefore, modifications in this region were not expected to have a significant impact on receptor in vitro activities. Peptides 8−10 reveal that the C-terminal tail derived from exendin-4 can be modified or shortened, resulting in similar or even higher in vitro activity at both receptors. Since this Trp-cage motif is known to significantly enhance solubility and stability,29 this motif was retained for further peptide optimization, and therefore, peptide 7 was chosen as starting point for additional modifications (see below). 4296



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Figure 3. NMR structure of peptide 14 in solution. For structural clarity, only heavy atoms are depicted. (a) Superposition of 10 structures derived from a 2 ns restrained MD simulation “in water”. All backbone atoms of residues 9−37 were used for fitting. (b) Individual structure with the lowest constraint violation compared to the NMR structure of exendin-4 (blue, PDB code 1jrj). The secondary structure is shown in ribbon presentation, the palmitic acids side chain of peptide 14 is shown as spacefill.

Figure 2. Modeling hypotheses of D-Ser2 variants containing an additional lysine residue, which is modified by addition of a palmitic acid at the ε-amino group using a γ-glutamic acid spacer in positions expected not to disturb receptor binding/activation. Full amino acid sequences and EC50 values (in pM) of the corresponding peptides are shown in Table 3.

superposition of 10 representative snapshots from a 2 ns restrained MD simulation. Though a certain degree of structural flexibility is observed in the N-terminal region (residues 1−8), the upper part of the helix (i.e., residues 9−28) and in particular the Trp cage motif show very high structural stability. A very high degree of flexibility is observed for the palmitic acid substituted at position 14. No NOEs were observed between the fatty acid side chain and any other amino acid of the peptide. Overall, peptide 14 displays high structural similarity to exendin-4 (Figure 3b), although both peptides differ in 10 positions from each other. Of particular note, the Trp cage, which is a hydrophobic cluster surrounding Trp25, shows a highly similar conformation to exendin-4 and a similar low degree of structural fluctuation. Binding Hypothesis of Peptide 14 to Full-Length Glucagon Receptor. The X-ray crystal structure of the seven transmembrane (TM) domain of the human glucagon receptor, together with a full-length homology model of the glucagon bound-receptor complex, has been reported.27 We used this structure prediction to model glucagon receptor binding of peptide 14, including the interactions with the TM region of the receptor (Figure 4). Importantly, several key interactions that have been reported for glucagon, and/or related (such as GLP-1) receptors, are also observed in our model including (1) a salt bridge between the peptidic N-terminal amino group of His1 and the carboxy side chain of Asp195 of the receptor, (2) a second salt bridge between Asp9 of the peptide and Arg378 of

increase is not clear due to lack of experimental evidence. It was hypothesized that additional hydrophobic interactions between the lipid side chain and hydrophobic regions of the target receptor might be responsible.42 Another theory that is potentially relevant for the GLP-1 and glucagon receptor is that lipidation fosters membrane association and thereby increases the local peptide concentration in the proximity of membrane-bound receptors.43,44 In the present context, the fatty acid side chain might simultaneously establish hydrophobic interactions with the receptor(s) and the surrounding membrane. Inspection of the binding hypothesis of peptide 14 to the full-length glucagon receptor indicates that this might be possible (see Figure 4). However, for a final conclusion about the molecular mechanism, further experimental data are required, for example, experimental (crystal) structural data of a lipidated peptide in complex with the full-length GLP-1 or glucagon receptor. NMR Structure of Peptide 14 in Solution. The 3D structure of peptide 14 was determined by restrained molecular dynamics (MD) calculations and energy minimization, imposing distance constraints obtained from NOESY spectra (Table S2 in Supporting Information). In total, 455 distance constraints were obtained from analysis of the NOESY spectra and used for the MD simulation (Table S3). Figure 3a shows a

Table 4. Full Amino Acid Sequences and EC50 Values (plus SEM Values) of D-Ser2 Variants of Peptide 7 Containing an Additional Lysine Residue, Which Is Modified by Addition of a Palmitic Acid at the ε-Amino Group Using a γ-Glutamic Acid Spacer in Positions Expected Not To Disturb Receptor Binding/Activationa

a

In all cases, three replicates were measured. 4297



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carries (as peptide 14) 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 respective receptor activation (measured in a cAMP assay in overexpressing HEK-293 cell lines) in mice was the following: peptide 14 activated the murine GLP-1 receptor with an EC50 of 2.3 pM and the glucagon receptor with an EC50 of 25 pM. In comparison, liraglutide activated murine GLP-1 receptor with an EC50 of 4.4 pM (see Table 6). Table 6. EC50 Values (Measured in pM in a cAMP Assay in Overexpressing HEK-293 Cell Lines) of Peptides at the Murine GLP-1 and Glucagon Receptorsa EC50 (pM)

Figure 4. (a) Predicted binding mode of peptide 14 (color) to the fulllength homology model of the glucagon receptor (orange), shown in ribbon representation.36 The palmitic acid side chain of peptide 14 is shown in spacefill representation. (b) Key interactions of the Nterminal domain of the peptide with specific regions in the transmembrane domain of the receptor are indicated.

a

the receptor, and (3) hydrogen bonding between the amide side chain of Gln3 and the hydroxyl side chain of Tyr149 of the receptor. According to the binding model, the palmitic acid side chain projects into the solvent space. As shown above, our NMR studies reveal a high structural flexibility of this side chain in solution. It is unclear whether this flexibility is maintained in the receptor-bound state or if hydrophobic interactions with the receptor or the membrane are established. In Vivo Studies. The in vivo assessment of above-described peptidic dual GLP-1/glucagon receptor agonists comprised evaluation of the pharmacokinetic (PK) properties as well as pharmacodynamic characterization in diabetic and obese mice. Subcutaneous administration of peptide 14 (1 mg/kg) in healthy mice showed a long half-life of 3.2 h (similar to liraglutide but with a lower exposure), whereas peptide 15 showed a somewhat shorter half-life. Nevertheless, exposure for peptide 15 was higher than for peptide 14. Table 5 summarizes the pharmacokinetic properties of peptides 14 and 15 in comparison to liraglutide in healthy mice dosed at 1 mg/kg subcutaneously. The pharmacological activity of the dual GLP-1/glucagon agonist peptide 14 was assessed in a variety of chronic mouse studies, among those studies in db/db mice as well as studies in diet induced obese (DIO) mice. In all studies the selective GLP-1 receptor agonist liraglutide was used as reference compound. As mentioned in the Introduction, liraglutide

dose (mg/kg)

Cmax (ng/mL)

Tmax (h)

AUC (ng h/mL)

T1/2 (h)

14 15 liraglutide

1 1 1

1930 3450 7700

1 2 4

11000 17000 80300

3.2 2.2 3.6

mGLP-1R

SEM

mGCGR

SEM

2.3 4.4 0.5 0.9 43.5

0.5 1.0 0.2 0.1 3.2

25.0 >10 000 000 >10 000 000 >10 000 000 1.3

10.1

0.1

In all cases, three replicates were measured.

The antiobese activity of peptide 14 and liraglutide was tested in diet induced obese (DIO) mice. Liraglutide as well as peptide 14 were dosed at 50 μg/kg twice daily to ensure sufficient and approximately continuous 24 h plasma levels repeatedly over 33 days. For both peptides a significant decrease in body weight was observed compared to baseline values as well as to high fat diet (HFD) controls (Table 7 and Table 7. Overall Body Weight and Body Fat Change (%) versus Baseline Values Prior Start of Treatment over a Treatment Period of 33 Daysa compd

overall weight change

body fat change

control standard diet control high-fat diet 14 liraglutide

+1.5% (±0.9%) +0.2% (±0.7%) −29.1% (±2.2%)*,# −13.6% (±2.2%)*,#

+6.4% (±3.5%) +2.5% (±1.4%) −57.3% (±3.8%)*,# −31.2% (±2.5%)*,#

a

Female C57BL/6N mice are fed with standard diet (SD control), with high fat diet (HFD control), and HFD plus treatment with peptide 14 and liraglutide. Both control groups were treated with vehicle. All mice were treated twice daily (b.i.d.). Data are shown as the mean ± SEM, n = 8 per group: (∗) p < 0.001 vs baseline and (#) vs HFD controls, one-way ANOVA with Dunnett’s post hoc test.

Figure 5). A significant decrease of 29.1% (±2.2%, p < 0.001) in relative body weight loss for peptide 14 and 13.6% (±2.2%, p < 0.001) for liraglutide compared to baseline values prior to start of treatment was observed. This strong reduction in body weight coincided with a marked reduction in body fat mass, as determined by NMR spectroscopy before the start of treatment and after 33 days. While the high fat diet control group maintained body fat content over the study period, treatment with peptide 14 led to a significant reduction of 57.3% (±3.8%) and liraglutide of 31.2% (±2.5%); see Table 7. As discussed earlier, body weight reduction for GLP-1/glucagon agonists is caused by inhibition of food intake but also increased energy expenditure (details of such studies will be published elsewhere in due time45).

Table 5. Pharmacokinetic Parameters for Peptides 14, 15 and Liraglutide after sc Administration to Micea compd

compd 14 liraglutide exendin-4 GLP-1 glucagon

a

Data represent average data calculated from mean plasma concentrations at different time points. The respective raw data including SD values can be found in Table S4. 4298



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dual GLP-1 and glucagon receptor agonists represent a very promising therapeutic principle for the chronic treatment of diabesity. On the basis of the exceptional physiochemical properties, in vivo stability, and prolonged pharmacokinetic profile of the selective GLP-1 receptor agonist exendin-4, we have re-engineered the exendin-4 sequence to (i) include glucagon receptor agonistic activity while (ii) maintaining the potent GLP-1 receptor activity and (iii) achieving sufficient in vivo stability for a therapeutic evaluation in preclinical models of diabetes and obesity. We have described a series of novel peptides with dual activity on these GLP-1 and glucagon receptors that were discovered by rational design, utilizing sequence and structural information from X-ray and NMR structures as well as molecular models for peptide optimization and structure− activity rationalization. On the basis of this strategy, structural elements of glucagon/oxyntomodulin were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in a hybrid peptide (7) with potent and balanced dual GLP-1/ glucagon receptor activity. New structure−activity relationships demonstrate where specific mutations of exendin-4-originating residues into the corresponding glucagon variants or vice versa can be introduced to switch the GLP-1/glucagon receptor activity ratio toward the one or other receptor. Importantly, the innovative challenge for such a dual coagonist design is to identify the optimal balance of the relative GLP-1/glucagon receptor activity ratio. GLP-1 receptor activation could lead to glucose lowering, in conjunction with moderate body weight reduction, whereas an additional enhancement of the glucagon receptor activity provides more significant weight loss, however, at the risk of glucose elevation. This concept is in line with findings from Day et al. that the GLP-1/glucagon activity ratio needs to be carefully evaluated and could be different for different species.46 Introduction of a D-serine in position 2 of peptide 7 and a lysine residue in position 14 modified by addition of a palmitic acid at the ε-amino group using a γ-glutamic acid spacer provided the dual GLP-1/glucagon receptor agonist peptide 14. Although this peptide differs in 10 (out of 39) residues from exendin-4, the structural fold of both peptides is (in agreement with our design rationales) highly similar, as demonstrated by NMR structure determination in solution. In particular, the stability- and solubility-enhancing Trp cage motif was structurally maintained. Peptide 14 showed significant glucose lowering effects associated with body weight reduction in murine models of diabetes and obesity. The observed weight loss in DIO mice was significantly greater compared to the selective GLP-1 receptor agonist liraglutide, while glycaemic control was similar to liraglutide when assessed in diabetic db/db mice. Overall, these beneficial preclinical data seen with peptide 14 support the hypothesis that dual GLP-1/glucagon receptor agonists have the potential to lead to significantly higher body weight reduction (due to reduced food intake and increased energy expenditure) compared to pure GLP-1 receptor agonists but similar glucose control. Due to lipidation, the pharmacokinetic properties of peptide 14 are comparable to liraglutide, showing that the half-life in comparison to exendin-4 could be improved pointing toward a suitability for once-daily administration in humans. The therapeutic potential of dual GLP-1/glucagon receptor agonists as antidiabetic therapy will certainly depend on the question of how far the efficacy, especially with respect to body

Figure 5. Relative body weight change (%) versus baseline values on day 0 prior to start of treatment. Female diet-induced obese (DIO) C57BL/6N mice are fed with standard diet (SD control), with high fat diet (HFD control) and HFD plus treatment with peptide 14 and liraglutide. Both control groups were treated with vehicle. All mice were treated twice daily with 50 μg/kg (b.i.d.). An oral glucose tolerance test (oGTT) was performed on day 21 of chronic treatment (data not shown), with an overnight starvation prior to glucose load. Relative weight loss is shown as mean values ± SEM, n = 8 per group. Black bar indicates statistically different body weight change from day 1 to day 33 of treatment for both peptides: (∗) p < 0.001 vs baseline and (#) vs HFD controls, one-way ANOVA with Dunnett’s post hoc test.

The antidiabetic activity of peptide 14 was studied in diabetic db/db mice. Liraglutide and peptide 14 were administered twice daily at 50 μg/kg over 32 days. Compared to the diabetic control group at day 32, treatment with peptide 14 prohibited worsening of the long-term glucose marker HbA1c and led to a significantly lower value of approximately 1.5%, very similar to liraglutide. Although the dual acting peptide 14 stimulates the glucagon receptor, no evidence for an increase in HbA1c indicating an elevation of blood glucose compared to liraglutide was seen (Figure 6). Chronic activation of the glucagon receptor to a certain amount does not seem to limit the glucose lowering potential of a dual GLP-1/glucagon coagonist.

Figure 6. HbA1c (%), start vs end of study after 32 days. Female diabetic (db/db) mice were treated with vehicle or peptide 14 or liraglutide with 50 μg/kg twice daily. Data are shown as mean values ± SEM, n = 8 per group: (∗) p < 0.001 vs baseline and (#) vs db/db controls, one-way ANOVA with Dunnett’s post hoc test.

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CONCLUSION On the basis of preclinical and exploratory human data for the native gut hormone oxyntomodulin, the combined activation of the GLP-1 and glucagon receptors could simultaneously improve blood glucose and reduce body weight. If the short half-life of this native peptide could be significantly improved, 4299



Article

350 μL of H2O/D2O (9:1), 50 mM phosphate buffer, pH 5.0, and 150 μL of trifluoroethanol at 310 K (mixing time was 200 ms) and from a NOESY spectrum recorded in a mixture of 350 μL of D2O, 50 mM phosphate buffer, pH 5.0, and 150 μL of trifluoroethanol at 310 K (mixing time was 200 ms). The 455 constraints included 83 intraresidual distances, 155 sequential distances, 147 medium distances (2−4 amino acids apart), and 70 long-range distances (>4 amino acids apart). The NOE between Trp25-H1 and H7 has been used as reference integral (2.9 Å). Upper and lower distance limits were set to ±10% of the calculated (experimental) distances, respectively. For nondiastereotopically assigned methylene protons and methyl groups, 0.9 and 1.0 Å were added to the upper bounds as pseudo atom corrections, respectively. The set of utilized distance constraints is summarized in Table S3. 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−1 Å−2. The MD simulation was performed in a water box using 7133 explicit water molecules. In the production run at 300 K, conformers were sampled every 100 ps for a duration of 2000 ps yielding a total of 20 structures. The obtained structures were energy-minimized (2000 steps). The 10 structures with the lowest constraint violation were used for further analyses. The average constraint violation is 0.10 Å. Only 9 constraints are violated by more than 0.6 Å; the largest violation is 0.87 Å. Considering only residues 9−37, the rmsd over all backbone atoms (N, NH, Cα, Hα, C′, O) is 0.698 Å with a standard deviation of 0.148 Å. For the same residues (residue 14 has been excluded) the rmsd over all heavy atoms is 1.068 Å with a standard deviation of 0.157 Å. Pharmacokinetics in Mice. Female C57/Bl6 mice were dosed 1 mg/kg subcutaneously (sc). The mice were sacrificed, and blood samples were collected after 0.083, 0.25, 1, 2, 4, 8, 16, and 24 h after application. Plasma samples were analyzed after protein precipitation via liquid chromatography−mass spectrometry (LC−MS/MS). Pharmacokinetic parameters and half-life were calculated using WinonLin, version 5.2.1 (noncompartment model). Pharmacodynamics. Acute and Subchronic Effects on Body Weight in Female Diet-Induced Obese (DIO) C57BL/6NCrl Mice. All mouse related experimental procedures were conducted in accordance with the German Animal Protection Law, as well as according to international animal welfare legislation and rules. Female C57BL/6N mice (Charles River) were housed in groups with n = 8 mice per cage in a specific pathogen-free barrier facility on a 12 h light/dark cycle with free access to water and high-fat diet. Mice were prefed for 18 weeks on high-fat diet (Ssniff adjusted to Teklad diet, TD.97366) and reached a plateau in body weight before start of the study. Mice were stratified at an age of approximately 24 weeks to treatment groups (n = 8) so that each group had similar mean body weight (HFD controls, 43.7 ± 0.93g; peptide 14, 43.9 ± 0.4 g; liraglutide, 43.5 ± 0.5 g). Individual values for body fat content varied between 16.9 and 21.3 g. An aged-matched group with ad libitum access to standard chow (Ssniff E15769-04 EF) was included as standard control group (SD controls: 24.4 ± 0.3 g). Before the experiment, mice were subcutaneously (sc) injected with vehicle solution (PBS, Gibco) and weighed for 3 days for acclimation. All animals were treated twice daily sc with an application volume of 5 mL/kg in the morning at the beginning of the light phase (12 h lights on) and in the evening prior lights off for 33 days with vehicle or compound, respectively, to ensure bioavailability of compound levels. Body weight was recorded daily. Two days before start of treatment and on day 32, total fat mass was measured by nuclear magnetic resonance (NMR) using a Bruker minispec (Ettlingen, Germany). Statistical analyses were performed with Everstat 6.0 by one-way ANOVA, followed by Dunnetts post hoc test for body weight and body fat compared to baseline values as well as to HFD controls. Differences were considered statistically significant at the p < 0.05 level. Chronic Treatment Effects on HbA1c in Female LeptinReceptor Deficient Diabetic db/db Mice. Female BKS.Cg-m +/+ Leprdb/J (db/db) and BKS.Cg-m +/+ Leprdb/+ (lean control) mice

weight reduction, exceeds the benefits of marketed GLP-1 receptor agonists without impacting glucoregulatory effects. Currently, there are a number of dual GLP-1/glucagon coagonists undergoing clinical testing,20 and the confirmation of their potential in human POC studies is urgently awaited.

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EXPERIMENTAL SECTION

Molecular Modeling. For binding mode generation of peptide 14 in full-length homology model of the glucagon receptor, the full-length glucagon receptor homology model in complex with glucagon36 was used as starting point. Peptide 14 was aligned to the 3D structure of receptor-bound glucagon. Subsequently, a conformational search of the N-terminal peptide residues 1−9 was performed in the presence of the glucagon receptor using Schrodinger’s loop refinement routine.47 The top-ranked solutions were minimized in the presence of the glucagon receptor, allowing all peptide and receptor atoms to move. After minimization, the different binding poses were analyzed and we selected the pose that showed the best agreement with available mutagenesis data. Synthesis of Peptides. Solid phase synthesis was carried out on Rink resin with a loading of 0.38 mmol/g, 75−150 μm from Agilent Technologies. The Fmoc synthesis strategy was applied with HBTU/ DIPEA activation. The peptide was cleaved from the resin with King’s cocktail.48 The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18, 5 μm) using an acetonitrile/water gradient (both buffers with 0.1% TFA). For analogs with fatty acid side chain Fmoc-Lys(ivDde)-OH and in position 1, Boc-His(Boc)-OH was used in the solid phase synthesis protocol. The ivDde group was cleaved from the peptide on resin using hydrazine in DMF according to literature.49 Hereafter Palm(γOSu)(αOtBu)Glu was coupled to the liberated amino group. Peptides were purified by RP-HPLC on a C18-column in acetonitrile/TFA, and purity and identity of the product were established by UPLC and LC−MS (for details see Table S1). Chemical identity and purity of the peptides were assessed by LC−MS and confirmed to have ≥95% purity for all key compounds. In Vitro Cellular Assays for GLP-1 Receptor and Glucagon Receptor Efficacy. Agonism of compounds for the two receptors was determined by functional assays measuring cAMP response of HEK293 cell lines stably expressing human or murine GLP-1 or glucagon receptor. cAMP content of cells was determined using a kit from Cisbio Corp. (catalog no. 62AM4PEC) based on HTRF (homogeneous 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 catalog 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). By default, three replicates were measured. Data are provided as the mean ± SEM. NMR. The 3D structure of peptide 14 has 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. The experimental data set used as input for the MD calculations included 455 distance constraints obtained from NOESY spectra (Table S2) recorded in a mixture of 4300



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were obtained from Charles River Laboratories, Germany, at an age of 9−10 weeks. The animals were housed in groups of n = 8 mice per cage in a specific pathogen-free barrier facility on a 12 h light/dark cycle with free access to water and rodent standard chow (see above). After 1 week of acclimatization, blood samples were drawn from the tail without anesthesia and HbA1c level (method, hemolysate, Cobas6000 c501, Roche Diagnostics, Germany) was determined. HbA1c is a glycosylated form of hemoglobin whose level reflects the average level of glucose to which the erythrocyte has been exposed during its lifetime. In mice, HbA1c is a relevant biomarker for the average blood glucose level during the preceding 4 weeks (erythrocyte life span in mouse ∼47 days). Db/db mice were stratified to treatment groups (n = 8) so that each group had similar baseline blood glucose and HbA1c levels (blood glucose, 20.7 ± 1.0 mmol/L (db/db controls), 20.5 ± 0.8 mmol/L (peptide 14), and 20.8 ± 0.6 mmol/L (liraglutide); HbA1c, 5.3 ± 0.2% (db/db controls), 5.3 ± 0.2% (peptide 14), and 5.3 ± 0.1% (liraglutide). An aged-matched lean and healthy littermate group was included as lean control (7.2 ± 0.1 mmol/L blood glucose and 3.8 ± 0.03% for HbA1c). For chronic effect on HbA1c, all animals were treated twice daily sc for 32 days. At the end of the study, blood samples (tail, no anesthesia) were analyzed for HbA1c. Statistical analyses were performed with Everstat 6.0 by repeated measures by one-way ANOVA and Dunnetts post hoc analyses. Differences versus baseline and vehicle-treated db/db control mice were considered statistically significant at the p < 0.05 level.

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ABBREVIATIONS USED ANOVA, analysis of variance; GCGR, glucagon receptor; DPPIV, dipeptidyl peptidase IV; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle medium; ECD, extracellular domain; FBS, fetal bovine serum; HbA1c, hemoglobin A1c; HBSS, Hanks’ balanced salt solution; HBTU, O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa fl u o r o p h o s p h a t e ; H E P ES , 4 - ( 2 - h y d r o x y e t h y l ) - 1 piperazineethanesulfonic acid; HFD, high-fat diet; HTRF, homogeneous time resolved fluorescence; IBMX, isobutylmethylxanthine; NEP, neutral endopeptidase; OXM, oxyntomodulin; POC, proof of concept; PYY, peptide tyrosine tyrosine; RPC, reversed phase chromatography; SGLT2-I, sodium−glucose co-transporter 2 inhibitor; SPL, Sybyl programming language; TM, transmembrane

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REFERENCES

(1) Farag, Y. M. K.; Gaballa, M. R. Diabesity: An Overview of a Rising Epidemic. Nephrol., Dial., Transplant. 2011, 26 (1), 28−35. (2) Ross, S. A.; Dzida, G.; Vora, J.; Khunti, K.; Kaiser, M.; Ligthelm, R. J. Impact of Weight Gain on Outcomes in Type 2 Diabetes. Curr. Med. Res. Opin. 2011, 27 (7), 1431−1438. (3) Wadden, T. A.; Hollander, P.; Klein, S.; Niswender, K.; Woo, V.; Hale, P. M.; Aronne, L. NN8022−1923 Investigators. Weight Maintenance and Additional Weight Loss with Liraglutide after Low-Calorie-Diet-Induced Weight Loss: The SCALE Maintenance Randomized Study. Int. J. Obes. 2013, 37 (11), 1443−1451. (4) Pi-Sunyer, X.; Astrup, A.; Fujioka, K.; Greenway, F.; Halpern, A.; Krempf, M.; Lau, D. C. W.; le Roux, C. W.; Violante Ortiz, R.; Jensen, C. B.; Wilding, J. P. H. SCALE Obesity and Prediabetes NN8022− 1839 Study Group. A Randomized, Controlled Trial of 3.0 Mg of Liraglutide in Weight Management. N. Engl. J. Med. 2015, 373 (1), 11−22. (5) Schulman, J. L.; Carleton, J. L.; Whitney, G.; Whitehorn, J. C. Effect of Glucagon on Food Intake and Body Weight in Man. J. Appl. Physiol. 1957, 11 (3), 419−421. (6) Habegger, K. M.; Heppner, K. M.; Geary, N.; Bartness, T. J.; DiMarchi, R.; Tschöp, M. H. The Metabolic Actions of Glucagon Revisited. Nat. Rev. Endocrinol. 2010, 6 (12), 689−697. (7) Habegger, K. M.; Stemmer, K.; Cheng, C.; Müller, T. D.; Heppner, K. M.; Ottaway, N.; Holland, J.; Hembree, J. L.; Smiley, D.; Gelfanov, V.; Krishna, R.; Arafat, A. M.; Konkar, A.; Belli, S.; Kapps, M.; Woods, S. C.; Hofmann, S. M.; D’Alessio, D.; Pfluger, P. T.; PerezTilve, D.; Seeley, R. J.; Konishi, M.; Itoh, N.; Kharitonenkov, A.; Spranger, J.; DiMarchi, R. D.; Tschöp, M. H. Fibroblast Growth Factor 21 Mediates Specific Glucagon Actions. Diabetes 2013, 62 (5), 1453− 1463. (8) Wynne, K.; Park, A. J.; Small, C. J.; Patterson, M.; Ellis, S. M.; Murphy, K. G.; Wren, A. M.; Frost, G. S.; Meeran, K.; Ghatei, M. A.; Bloom, S. R. Subcutaneous Oxyntomodulin Reduces Body Weight in Overweight and Obese Subjects: A Double-Blind, Randomized, Controlled Trial. Diabetes 2005, 54 (8), 2390−2395. (9) Wynne, K.; Park, A. J.; Small, C. J.; Meeran, K.; Ghatei, M. A.; Frost, G. S.; Bloom, S. R. Oxyntomodulin Increases Energy Expenditure in Addition to Decreasing Energy Intake in Overweight and Obese Humans: A Randomised Controlled Trial. Int. J. Obes. 2006, 30 (12), 1729−1736. (10) Deacon, C. F.; Nauck, M. A.; Toft-Nielsen, M.; Pridal, L.; Willms, B.; Holst, J. J. Both Subcutaneously and Intravenously Administered Glucagon-like Peptide I Are Rapidly Degraded from the NH2-Terminus in Type II Diabetic Patients and in Healthy Subjects. Diabetes 1995, 44 (9), 1126−1131. (11) Pocai, A.; Carrington, P. E.; Adams, J. R.; Wright, M.; Eiermann, G.; Zhu, L.; Du, X.; Petrov, A.; Lassman, M. E.; Jiang, G.; Liu, F.; Miller, C.; Tota, L. M.; Zhou, G.; Zhang, X.; Sountis, M. M.; Santoprete, A.; Capito’, E.; Chicchi, G. G.; Thornberry, N.; Bianchi, E.; Pessi, A.; Marsh, D. J.; SinhaRoy, R. Glucagon-Like Peptide 1/

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00174. Analytical data with RP-HPLC retention times and molecular masses of the synthesized peptides, 1H chemical shifts of peptide 14, distance constraints used for the MD simulation obtained from NOESY spectra, and tabular results of pharmacokinetic data not shown in the manuscript (PDF) Accession Codes

Atomic coordinates for peptide 14 can be accessed using PDB code 5NIQ. The authors will release the atomic coordinates and experimental data upon article publication.

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Article

AUTHOR INFORMATION

Corresponding Authors

*A.E.: phone, +49 305 12636; e-mail, Andreas.Evers@sanofi. com. *M.W.: phone, +49 305 46875; e-mail, Michael.Wagner@ sanofi.com. ORCID

Andreas Evers: 0000-0003-4643-1941 Present Address †

M.G.: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, D-88397 Biberach an der Riss, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Ray C. Stevens and Chris de Graaf for providing us the coordinates of their full-length homology model of the glucagon receptor. We thank Melissa Besenius and Garima Tiwari for reading the manuscript and providing valuable comments. 4301



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Glucagon Receptor Agonists - ACS Publications - American Chemical

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