Glucagon Receptor Agonists

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Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists Specifically Optimized for Multi-dose Formulations Andreas Evers, Martin Bossart, Stefania Pfeiffer-Marek, Ralf Elvert, Herman A. Schreuder, Michael Kurz, Siegfried Stengelin, Martin Lorenz, Andreas Herling, Anish Ashok Konkar, Ulrike Lukasczyk, Anja Pfenninger, Katrin Lorenz, Torsten Haack, Dieter Kadereit, and Michael Wagner J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00292 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Journal of Medicinal Chemistry

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, † Michael Wagner†*

†

Sanofi-Aventis Deutschland GmbH, R&D, Industriepark Höchst, D-65926 Frankfurt am Main, Germany

ACS Paragon Plus Environment

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Abstract Novel peptidic dual agonists of the glucagon-like 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 & synthesis, experimental activity and solubility profiling, a candidate molecule was identified. Novel SAR points are reported that allowed to fine-tune the desired receptor activity ratio and increased solubility in the presence of antimicrobial preservatives – findings which 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 whereas 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 a major interest in the search for new therapies, which 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, which 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 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 promising approach to enhance anti-diabetic properties and reduce body weight.7–10 A prominent approach is the combination of GLP-1 receptor mediated food intake suppression with glucagonreceptor mediated increase in energy expenditure for synergistic and improved body weight loss. Such dual GLP-1/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 references 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,


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however, 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 co-agonist demonstrated intermediate weight loss but with glucose control comparable to that of the most GLP1 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 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 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 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 1015 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 translational model for the human situation. Further target attributes were (i) selectivity versus the GIP receptor


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and (ii) sufficient solubility for the candidate molecule over a broad pH range (pH 4.5 – pH 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 & 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 presence of a phenolic preservative was achieved by structure-based 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 6-week trial (using once-daily subcutaneous injection) in obese diabetic monkeys, a relevant translational model for the human situation, are described.

Table 1. a) Amino acid sequences of glucagon, exendin-4, peptide 1, native GLP-1, liraglutide and native GIP. Amino acids, which 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 is modified by addition of a C16 fatty acid (palmitic acid) at the ɛ-amino group of lysine using a γ-glutamic acid spacer. 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 (standard error of mean) values and number of measurements (n). a) No glucagon exe ndin-4 1 GLP1(7-36) liraglutide GIP(1-42)

1 H H H H H Y

S G s A A A

Q E Q E E E

G G G G G G

5 T T T T T T

F F F F F F

T T T T T I

S S S S S S

D D D D D D

10 Y L L V V Y

S S S S S S

K K K S S I

Y Q Q Y Y A

L M K(yE-C16) L L M

15 D E D E E D

S E S G G K

R E R Q Q I

R A R A A H

A V A A A Q

20 Q R Q K K(yE-C16) Q

D L D E E D

F F F F F F

V I I I I V

Q E E A A N

25 W W W W W W

30 L L L L L L

M K K V V L

N N N K R A

T G G G G Q

G G R R K

35 P S S GA P S S GA NH2 G GKKND

P P P S NH2 P P P S NH2

W K H N I

T

Q

b) EC50 [pM] No glucagon exendin-4 1 GLP1(7-36) liraglutide GIP(1-42)

GLP-1R

SEM n

43.9 0.4 3.9 0.9 6.4 >2.0·107

2.2 0.0 0.3 0.1 0.7

GCGR SEM n 59 3 3 38 3 3

0.5 >1.0·108 0.7 >1.0·108 >1.0·108 >1.0·108

0.1 0.1

GIPR 180 3 3 3 3 3

SEM n

>1.0·108 2.0·105 1210.0

22719.0 124.0

>1.0·108 >1.0·108 0.4

0.0

3 3 3 3 3 3


5


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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 glucagon and GIP receptor binding. These insights were used as structural basis for optimization of a lead peptide (peptide 2, table 2) towards the desired GLP-1/glucagon/GIP receptor activity ratio. Structural analysis of peptides and binding hypotheses to the GLP-1, glucagon and GIP receptor ECD Molecular recognition of exendin-4 and glucagon at the ECDs of the GLP-1 and glucagon receptor has been described in detail before.13 Figure 1a shows the solution-state NMR ensemble of exendin4 (PDB code 1jrj).30 In exendin-4, the C-terminal 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 threedimensional 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 towards 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 GLP1/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 GLP-135,36 and glucagon37,38 receptor.


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(Figure 1)

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 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 DPPIVmediated 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 ɛ-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 does not only show high activity at the GLP-1 and glucagon receptor, 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 towards the GIP receptor (GLP1/GIP receptor activity ratio in the order < 1:200). As shown in table 2, only three modifications were sufficient to introduce the desired activity profile: (1) Guided by the structural receptor differences close to residue 27 of exendin-4 (see figure 1c), an Aib residue was introduced in position 27 (peptide 3), which modified the GLP-1/glucagon receptor selectivity ratio in favour of GLP-1R activity (activity ratio GLP-1/glucagon receptor = 1:5) and simultaneously improved selectivity towards the GIP receptor (activity ratio GLP-1/GIP receptor = 1:73). (2) Selectivity towards the GIP receptor could be further increased by mutating the glutamate residue in position 21 to leucine (peptide 4: activity ratio GLP-1/GIP receptor = 1:181), however, at the cost of reduced absolute activity at the GLP-1 receptor: whereas peptide 3 activates the GLP-1 receptor with an EC50 value of 1.4 pM, the activity of peptide 4 drops to 6.4 pM. (3) Finally, it was found that modification of the lysine sidechain in position 14 by introduction of a second γ-glutamic acid


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spacer and exchange of the stearic against palmitic acid restored high GLP-1 receptor activity (peptide 5: EC50 = 2.2 pM), while further improving selectivity towards the GIP receptor (EC50 = 2031.7 pM) and providing the desired GLP-1/glucagon receptor activity ratio (1:13).

Table 2. a) EC50 values (measured in pM in a cAMP assay in receptor overexpressing HEK-293 cell lines) of peptide sequences at the human GLP-1, glucagon and GIP receptors. Color coding of amino acids is identical to Table 1. b) EC50 values with SEM (standard error of mean) values and number of measurements (n). a) No 2 3 4 5 6 7 8 9 10 11

1 H Aib Q G H Aib Q G H Aib Q G

5 10 T F T S D L S K Q K(yE-C18) T F T S D L S K Q K(yE-C18) T F T S D L S K Q K(yE-C18)

15 D E Q R D E Q R D E Q R

20 A K A K A K

E F I E F I L F I

25 E W E W E W

30 # L K A G G P S S GA P P P S L Aib A G G P S S G A P P P S L Aib A G G P S S G A P P P S

NH2 NH2 NH2

H Aib Q G T F T S D L

S K Q K(yE-yE-C16) D

E Q R

A K

L F I

E W

L Aib A G G

P S S GA P P P S

NH2

H H H H H

S S S S S S

E E E E E E

A A A A A A

L L L L L L

E E E E E E

L L L L L L

P P P P P P

NH2 NH2 NH2 NH2 NH2 NH2

Aib Aib Aib Aib Aib

Q Q Q Q Q

G G G G G

T T T T T

F F F F F

T T T T T

S S S S S

D D D D D D

L L L L L L

K K K K K K

Q Q Q Q Q Q

K(yE-yE-C16) K(yE-yE-C16) K(yE-yE-C16) K L L

D D D D D D

Q Q Q Q Q Q

R R R R R C(1)

K K K K K K

F F F F F F

I I I I I I

W W W W W W

K L I Aib Aib Aib

A A A A A A

G G G G G G

G G G G G G

S S S S S S

S S S S S S

GA GA GA GA GA GA

P P P P P P

P P P P P P

P P P P P P

S S S S S C(1)

b) EC50 [pM] No

GLP-1R

SEM n

GCG-R

SEM n

GIP-R

SEM n

2 3 4

2.5 1.4 6.4

0.4 0.1 0.4

3 3 3

1.9 6.7 15.4

0.1 0.5 1.1

3 3 3

39.7 102.0 1160.0

1.9 5.6 84.2

3 3 3

5

2.2

0.3

6

27.6

3.7

6

2031.7

281.9

6

2.2 4.8 5.3 52.5 8.0 >1.0·108

0.4 0.4 0.4 3.7 1.3

6 3 3 3 3 3

4.4 1.6 2.5 42900 3090 >1.0·108

18.1 0.1 0.2 5651 269

6 3 3 3 3 3

224.0 189.0 58.2 177000 114000 >1.0·108

18.1 11.1 3.1 42433 13401

6 3 3 3 3 3

6 7 8 9 10 11

SAR and modeling studies rationalizing selectivity determining features in peptide position 14 and 27. We designed and synthesized variants of peptide 5 to rationalize the effect of the mutations in positions 14 and 27 on receptor activity and selectivity (peptides 6-10). Role of Aib27 for receptor selectivity. The selective GLP-1 receptor agonist exendin-4 has a lysine in position 27, whereas the native hormones glucagon and GIP(1-42) bear hydrophobic residues in this position (Met27 and Leu27, respectively, see table 1). Therefore, it was somewhat surprising ACS Paragon Plus Environment

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that mutating Lys27 in peptide 2 into the hydrophobic residue Aib (peptide 3) increased activity at the GLP-1 receptor and simultaneously reduced activity at the glucagon and GIP receptor. To further investigate the role of residue 27 for receptor selectivity in detail, Aib27 in peptide 5 was backmutated to lysine (peptide 6), leucine (peptide 7) or isoleucine (peptide 8). Indeed, these single-point mutations improved activity at the glucagon and GIP receptors while maintaining or worsening activity at the GLP-1 receptor (see table 2). For example, the mutation Aib27Ile reduces GLP-1 receptor activity by a factor of 2, while improving glucagon activity by a factor of 11 and GIP receptor activity by a factor of 35, respectively (compare peptide 5 versus peptide 8). Inspection of the predicted binding modes (for details, see Experimental section) for peptides 5 and 8 to the GLP1, glucagon and GIP receptor ECDs provides a structural rationale for the selectivity switch of the Aib27Ile mutant (see figure 2). Residue 27 is pointing to a region, where the three receptors bear different residues (GLP-1 receptor: Leu123, glucagon receptor: Ala118 and GIP receptor: His115). In peptide 5, the small sidechain on the Aib27 residue is in ideal distance for an attractive van-derWaals interaction with Leu123 of the GLP-1 receptor. The glucagon receptor, however, bears only a small alanine (Ala118) in this position, which is too distant for an attractive interaction with Aib27 of peptide 5; the GIP receptor has a histidine (His115) in the corresponding position, which is supposedly – due to its planar imidazole ring - restricted in its conformational freedom and, thus, also unable to form an attractive interaction with Aib27 of peptide 5. In contrast, the hydrophobic sidechain of Ile27 in peptide 8 is large enough to establish attractive van-der-Waals interactions with Ala118 in the glucagon receptor and His115 in the GIP receptor, resulting in a significant activity increase at the glucagon and GIP receptor versus peptide 5. At the same time, there seems to be a repulsion of Ile27 in peptide 8 with Leu123 in the GLP-1 receptor, resulting in the slight activity loss of factor 2.

(Figure 2)


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Role of the fatty acid in position 14 for receptor selectivity. Peptides 9 and 10 were synthesized to investigate the effect of the fatty acid side chain in position 14 on receptor activity. Peptide 9 is an analogue of peptide 5, where the lipophilic sidechain attached to Lys14 was simply removed. This mutation results in a considerable activity loss at the GLP-1 receptor (factor 10) and significant activity loss at the glucagon and GIP receptor (factor 1554 and factor 87, respectively). Subsequently, a leucine residue was introduced in position 14. This residue is also present in glucagon, oxyntomodulin and other peptides that show high potency at the GLP-1, glucagon or GIP receptors, e.g.39. This analogue (peptide 10) maintains considerable activity at the GLP-1 receptor (EC50 = 8.0 pM), but also shows significant activity loss at the glucagon and GIP receptor (EC50 = 3090 pM and 114000 pM, respectively, see table 2). It was already observed and discussed in earlier studies (e.g.13,40) that introduction of a fatty acid substituent may provide enhanced activity compared with the natural amino acid, potentially due to stabilization of relevant bioactive conformations, establishment of additional hydrophobic peptide-receptor interactions or by increasing the local peptide concentration in the proximity of membrane-bound receptors due to membrane association.

Crystal structure of GLP-1 receptor extracellular domain (ECD). For experimental validation of the predicted peptide binding mode crystallization of peptide 5 in complex with the GLP-1 receptor ECD was attempted. These activities did not produce crystals, presumably due to conformationally flexible regions in the peptide. NMR structure determination (see below) indicated that in particular the N-terminal residues of the peptide (residues 1-8) and the fatty acid substituent in position 14 had high structural flexibility. Guided by analysis of the NMR structure and receptorpeptide modeling hypothesis, we used the following strategy to design peptide 11 as a stable variant of peptide 5 for subsequent crystallization trials: (i) Elimination of residues 1 to 8. The corresponding variant of exendin-4, exendin(9-39), was successfully crystallized in the presence of the GLP-1 receptor ECD.32 This N-terminal truncation was shown to lead to a competitive antagonist (exendin(9-39)), displacing both GLP-1 and exendin-4 from receptor binding.41 (ii)


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Replacement of the fatty-acid substituted lysine residue in position 14 by a leucine residue, a strategy that has also been applied to obtain a crystal structure for the semaglutide backbone with the GLP-1 receptor ECD.42 (iii) Peptide cyclisation by introduction of an intramolecular disulfide bond between residues 18 and 39 (see table 2).With peptide 11, crystals of the complex with the GLP-1 receptor ECD were obtained that were suitable for X-ray diffraction studies, resulting in a 2.7 Å crystal structure of the complex (PDB code 6gb1) as shown in figure 3a. The region of the Nterminal truncation and the position of the disulfide bridge are indicated. As expected, the resulting complex structure is highly similar to the GLP-1 receptor-exendin(9-39) complex (PDB code 3c59, see three-dimensional alignment in figure S2a). Whereas in the crystal structure of the GLP-1 receptor-exendin(9-39) complex (PDB code 3c59) the C-terminus (residues 36-39) is disordered, these residues are well ordered in our structure, due to the tethering of the C-terminus to the center of the helix by the engineered Cys18-Cys39 disulfide link (see figure S2). Surprisingly, there are only two direct hydrogen bonds between the peptide (p) and the receptor (r) between Asp15p – Leu32r and Aib27p – Arg121r (not shown). As is shown in figure 3b, at the “top” of the bound peptide, a hydrogen bonding network is present involving the side chains of Glu68r, Arg121r and Ser32p, the main chain carbonyl oxygens of Phe66r, Asp67r, Leu26p and Aib27p as well as three bound water molecules.

However, the majority of the contacts are non-polar van der Waals

interactions, for example between the residues Phe22, Ile23, Trp25, Leu26, Aib27 and Trp25 of the peptide and hydrophobic regions of the receptor. This crystal structure is very similar to the ECD-peptide binding mode of the recently published full-length structure of the GLP-1 receptor in complex with the exendin-4 analogue exendin-P5 (PDB code 6b3j)43 (see figure S2b). Structural comparison furthermore revealed that the crystal structure of peptide 11 is nearly identical to the predicted binding mode of peptide 5 (see figure S3). In summary, these data indicate that our structure-based molecular modeling approach leads to realistic and relevant receptor-peptide binding modes.

(Figure 3)


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Receptor binding. To further validate the relevance of the binding mode of peptide 11, characterization of receptor binding at the GLP-1 receptor was investigated for peptide 11 and the non-truncated analogue peptide 10 in a radioligand binding assay using [125I]GLP-1 as tracer. Peptide 10 shows an EC50 value of 8.0 pM in the functional cAMP assay at the GLP-1 receptor (see table 2). Due to truncation of residues 1-8, peptide 11 does not reveal agonistic activity in the functional cAMP assay (table 2). The binding data demonstrate that peptide 11 (IC50 = 1132.3 nM) is binding in a specific and competitive manner to the GLP-1 receptor. Its binding affinity is, however, significantly (43-fold) lower compared to peptide 10 (IC50 = 26.0 nM) (see table 3). According to published data, the corresponding truncation results in a lower affinity drop in the case of exendin-4 (leading to the antagonist exendin(9-39)),44,45 but a similar or even higher affinity drop in the case of GLP-1 or exendin-4/GLP-1 chimeric peptides.45 These data demonstrate that the Nterminal peptide segment – which is interacting with the transmembrane domain of the receptor - is not only relevant for receptor activation, but may also have significant contributions to receptor affinity.

Table 3. IC50 values with SEM (standard error of mean) values and number of experiments (n) of binding experiments measured in nM based on [125I]GLP-1 displacement studies in GLP-1 receptor overexpressing HEK-293 cell lines.

peptide IC50 [nM]

SEM

n

10

26.0

6.2

3

11

1132.3

104.2

3

Solubility characterization and optimization of peptide 5. In order to provide development candidates that fulfill not only the desired criteria with respect to target activity, it is essential to ACS Paragon Plus Environment

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assess several requirements of the final drug product during compound optimization. Peptide formulations that are intended for subcutaneous application in a multiple-dose pen device require the presence of antimicrobial preservatives, such as m-cresol, phenol or benzyl alcohol, to prevent the growth of microbes and bacteria that may be introduced from repeatedly withdrawing individual use doses.21 Since peptide 5 showed the desired activity profile at the GLP-1, glucagon and GIP receptors, its solubility was tested at a concentration of 10 mg/mL in different buffers at pH 4.5 and pH 7.4 (see table 4), either in the absence or presence of 0.5% phenol as antimicrobial preservative. A good solubility (≥10 mg/mL) was obtained in both buffers in the absence of phenol. However, solubility significantly decreased at pH 4.5 in the presence of phenol down to 1.2 mg/mL. This solubility behavior is considered as potential issue, since it might restrict the final peptide formulation to non-acidic pH ranges. In addition, the downstream process of drug substance manufacturing runs at acidic pH at high peptide concentrations. The sensitivity to the presence of aromatic preservatives indicates a certain aggregation propensity at lower pH values.

Table 4. Solubility profile of peptide 5 in different buffers, at pH 4.5 and pH 7.4. Solubility was measured at a peptide concentration of 10 mg/mL in the absence or presence of 0.5% phenol as antimicrobial preservative. solubility peptide buffer

pH

preservative [mg/mL]

5

acetate

4.5

None

≥10

5

acetate

4.5

0.5% phenol

1.2

5

phosphate 7.4

None

≥10

5

phosphate 7.4

0.5% phenol

≥10

Preservative-induced peptide aggregation can lead to immunogenic and toxic side-effects in patients. The aggregation event may be induced by weak binding of the preservative to the peptide and favor ACS Paragon Plus Environment

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an increase in the level of partially unfolded, aggregation-competent species, e.g.46. Furthermore, addition of preservatives can increase the attractive forces between peptide oligomers, leading to aggregate formation.23 Thus, a strategy for optimizing preservative-induced aggregation of a peptide might be (1) stabilization of the native conformation at potential preservative binding sites and/or (2) introduction of modifications that reduce the binding tendency for preservatives. To eliminate preservative-induced solubility issues for peptide 5, a search for potential aggregation hot spots was performed by identifying clusters of hydrophobic residues on the peptide surface that might represent interaction sites for phenolic molecules. As shown in figure 4a (red color), a region around Trp25 was identified. This aggregation spot includes residues Ala19, Leu21, Phe22, Ile23, Trp25, Leu26, Aib27, Ala28, Gly29, Gly30, Pro31, Gly34, Ala35 and Pro36-38. Several residues in this hydrophobic spot are conserved among incretin peptides (e.g. Phe22, Trp25, Leu26 – see table 1) and establish important interactions with their respective receptors. Replacement of these amino acids by polar residues would result in significant activity decrease. According to our binding hypothesis (see figure 4a), the C-terminal peptide tail, ranging from residue 30 to 39, points away from the receptor. This sequence stretch forms a stable fold around Trp25 (the tryptophan cage), and was shown to be essential for favorable physico-chemical properties of exendin-4 analogues,31 but data from internal studies and literature demonstrated that it does not significantly impact activity at the receptors, e.g.13,31. We were therefore looking for modifications of the C-terminal sequence, which would (1) preserve the structural fold of the tryptophan cage, (2) alter the polarity environment in this region, while (3) maintaining the activity profile at the GLP-1, glucagon and GIP receptors. Optimization activities resulted in peptide 12, where the following mutations have been introduced into peptide 5 (figure 4b and table 5): Ser32Pro, Gly34Aib, Ala35Lys and Ser39Lys. Pro32 and Aib34 are both more conformationally constrained than the corresponding amino acids of peptide 5 (Ser32 and Gly34). The lysine residues in position 35 and 39 increase charge and reduce hydrophobicity in this region, which is otherwise composed of mainly neutral and hydrophobic amino acids.


14

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(Figure 4)

Table 5. Amino acid sequences and EC50 values (measured in pM in a cAMP assay in receptor overexpressing HEK-293 cell lines) of peptides 5 and 12. Amino acids that are part of the predicted aggregation hot spot, and represent a potential interaction site for phenol, are colored red. Residues, which were introduced into peptide 12 to increase conformational stability or polarity are colored blue or green, respectively. a) No 5 12

1 5 10 15 # H Aib Q G T F T S D L S K Q K(yE-yE-C16) D E Q R A K L F I H Aib Q G T F T S D L S K Q K(yE-yE-C16) D E Q R A K L F I

25 E W E W

L Aib L Aib

A A

30 35 GG P S S G A P P P S NH2 G G P P S Aib K P P P K NH2

b) EC50 [pM ] No

GLP-1R

5 12

2.2 1.8

SEM n 0.3 0.1

6 3

GCG-R

SEM n

GIP-R

3.7 2.1

2031.7 2032.0

27.6 29.0

6 3

SEM n 281.9 172.1

6 3

In vitro testing of peptide 12 demonstrates that – in agreement with our design rationales - the activity profile at the GLP-1, glucagon and GIP receptors is nearly identical to peptide 5 (see table 5). Furthermore, peptide 12 showed this time high solubility (≥ 10 mg/mL) at pH 4.5 and pH 7.4 in absence and – as desired - in presence of phenol (see table 6).

Table 6. Solubility profile of peptide 12 in different buffers, at pH 4.5 and pH 7.4. Solubility was measured at a peptide concentration of 10 mg/mL in the absence or presence of 0.5% phenol as antimicrobial preservative. solubility peptide buffer

pH

preservative

[mg/mL]

12

acetate

4.5

None

≥10

12

acetate

4.5

0.5% phenol

≥10

12

phosphate 7.4

none

≥10


15


12

phosphate 7.4

0.5% phenol

Page 16 of 46

≥10

NMR structure of peptides 5 and 12 in solution. We performed NMR studies to investigate the structural preferences of peptides 5 and 12 in solution. 3D-structures were determined by restrained molecular dynamics (MD) simulations and energy minimization, imposing distance constraints obtained from NOESY spectra (table S1-S4). In total, 402 distance constraints were obtained from analysis of the NOESY spectra for peptide 5 (table S2) and 360 distance constraints for peptide 12 (table S4) and used for the MD simulation. Overall, peptides 5 (PDB code 6gdz) and 12 (PDB code 6ge2) show high structural similarity to exendin-4 (figure 5a and 5b), although both peptides differ in 10 or 14 positions from exendin-4 (see table 7). Of particular note and in full agreement with our design rationales, the solution NMR structure of peptide 5 reveals a high similarity to our receptor-bound modeling hypothesis (figure 4) and receptor-bound conformation of peptide 11, which was obtained by X-ray diffraction studies (figure 2). A three-dimensional alignment of these peptides onto the receptor-bound conformation of peptide 11 is provided in figure S2. Our 3D-modeling hypothesis (figure 4) suggests that the C-terminal modifications introduced into positions 32, 34, 35 and 39 of peptide 5 have only minor effects on the structural preferences of the tryptophan cage, which seemed to be confirmed by the fact that the activity profile at the target receptors remained nearly identical. Figure 5b shows a superposition of NMR conformers of peptide 5 (10 conformers), peptide 12 (10 conformers) and exendin-4 (PDB code 1jrj, 36 conformers). For all peptides, a certain degree of structural flexibility is observed in the N-terminal region (residues 110), whereas the upper part of the helix and – in particular - the tryptophan cage motif show very high structural stability. A very high degree of flexibility is observed for the fatty acid side-chain in position 14 for peptide 5 and 12. No NOEs were observed between the fatty acid side chain and any other amino acid of the peptide.


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Two further observations and conclusions can be derived from the structural alignment shown in figure 5c: (1) Backbone conformation around residue 27 is nearly identical 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. 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.

(Figure 5)

Table 7. Sequences of exendin-4, peptide 5 and 12. Amino acid differences to exendin-4 are indicated by different colors. No Exendin-4 5 12

1 5 H G E G T F T S D H Aib Q G T F T S D H Aib Q G T F T S D

10 L S K Q M L S K Q K(yE-yE-C16) L S K Q K(yE-yE-C16)

15 20 E E E A V R D E Q R A K D E Q R A K

L F I L F I L F I

25 E W E W E W

30 L K N G G P S S G L Aib A G G P S S G L Aib A G G P P S Aib

35 A P P P S NH2 A P P P S NH2 K P P P K NH2


17


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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 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, 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 GLP1/glucagon activity preference in humans as in monkeys.

Table 8. EC50 values (measured in pM in a cAMP assay in receptor overexpressing HEK-293 cell lines stably expressing GLP-1, glucagon or GIP receptor) of peptides at the monkey or human receptors. Values are mean ± SEM. Number of measurements is given in parentheses. EC50 monkey [pM]

EC50 human [pM]

peptide GLP-1R

GCGR

GIPR

GLP-1R

GCGR

GIPR

12

2.0±0.2 (3)

129±9.0 (3)

2050±146 (3)

1.8±0.3 (3)

29±2.1 (3)

2032±172 (3)

liraglutide

4.1±0.4 (3)

>1.0·10 (3)

>1.0·10 (3)

6.4±0.7 (3)

>1.0·10 (3)

>1.0·10 (3)

GLP-1

1.1±0.04 (35)

>1.0·108 (3)

>1.0·108 (3)

0.9±0.05 (38)

>1.0·108 (3)

>1.0·108 (3)

glucagon

30.8±1.3 (42) 1.5±0.04 (60)

>1.0·108 (3)

43.9±2.2 (59) 0.5±0.05 (180)

8

8

8

8

>1.0·108 (3)

Liraglutide as well as peptide 12 were dosed once daily to assure sufficient and approximately continuous plasma levels repeatedly over the study duration of 6 weeks. To reduce the well-known gastro-intestinal 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 ACS Paragon Plus Environment

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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 Methods). 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 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. 19 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-01-GE 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 fiftyone (n = 51) monkeys were trained in order to identify 30 monkeys (n = 8-10 per group) that were


27


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used in chronic treatment phase and fulfil 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-hour light/12-hour 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 homogenous groups according body weight, fasted plasma glucose and %HbA1C. During the entire dosing period food and water intake was monitored daily, 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 diabetic-insulin 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 s.c. (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 s.c. 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 post-dosing. The samples were directly transferred into potassium ethylene diamine tetra-acetic acid (EDTA-2K) tubes. Plasma was separated by centrifugation at 2,500xg 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. Statistical analysis. All in vivo data are presented as means ± SEM. A oneway analysis of variance for factor treatment or two-way analysis including factor time were used


28

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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 a P < 0.05. All analyses were performed using SAS (version 9.2) under HP-UX via interface software EverStat V6.0-alpha5.


29


Page 30 of 46

Associated content. Supporting information. 1H-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; three-dimensional alignment of x-ray structure of GLP-1 (ECD) receptor-bound peptide 11 (grey), receptor-bound modeling hypothesis of peptide 5 (magenta) 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 (full-length) receptor-bound exendin-P5. PDB ID 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. Corresponding Author Information. *Andreas Evers: Phone: +49 305 12636. E-mail: [email protected].

*Martin

[email protected].

*Michael

Bossart: Wagner:

Phone: Phone:

+49 +49

305

16655.

E-mail:

305

46875.

E-mail:

[email protected]. 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, glucose-dependent 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


30

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the mean; SGLT2-I, sodium–glucose cotransporter 2 inhibitor; SPA, Scintillation proximity assay; SPL, Sybyl Programming Language; TFE, Trifluorethanol; WGA, Wheat Germ Agglutinin . Acknowledgements. 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 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; G. Tiwari for reading the manuscript and providing valuable comments.


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World Health Organization. Media Centre. Diabetes (Fact sheet no. 312.) [Cited on : 23 Jan 2018] Available at: http://www.who.int/mediacentre/factsheets/fs312/en/.

(2)

2014 Diabetes Report Card - diabetesreportcard2014.pdf [Internet]. [Cited on : 23 Jan 2018]. Available at: http://www.cdc.gov/diabetes/pdfs/library/diabetesreportcard2014.pdf.

(3)

Nonalcoholic Steatohepatitis: Risk Factors and Diagnosis [Cited on : 23 Jan 2018] Available at: http://www.medscape.com/viewarticle/731163.

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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.

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Finan, B.; Clemmensen, C.; Müller, T. D. Emerging Opportunities for the Treatment of Metabolic Diseases: Glucagon-like Peptide-1 Based Multi-Agonists. Mol. Cell. Endocrinol. 2015, 418 Pt 1, 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.


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(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. (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 WeightLowering 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 DietInduced 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).


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(19) Soni, H. Peptide-Based GLP-1/Glucagon Co-Agonists: A Double-Edged Sword to Combat Diabesity. 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 DIO-Rodents. Biopolymers 2012, 98 (5), 443–450. (21) General

Chapters:

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 (Mosc.) 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-to-Development 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.


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(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 Glucagon-like 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 Glucagonlike 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.


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(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. DOI: 10.7573/dic.212283. (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 | Schrödinger 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. D Biol. Crystallogr. 2010, 66 (Pt 2), 125–132. (54) Evans, P. Scaling and Assessment of Data Quality. Acta Crystallogr. D Biol. Crystallogr. 2006, 62 (Pt 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. 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.


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Figure of Contents Graphic 956x462mm (96 x 96 DPI)



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 colour coding of peptide amino acids is equivalent to the sequence colour coding outlined in Table 1. Residue Trp25 is shown in spacefill representation. c) Zoom-in 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. 621x263mm (96 x 96 DPI)


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Figure 2. Predicted binding hypothesis for peptides 5 and 8 at the extracellular domain of the GLP-1 (PDB code 3c59), glucagon (GCG, PDB code 4ers) and GIP (PDB code 2qkh) receptors. Both peptides have identical sequences except for residue 27. Introduction of Aib27 in peptide 5 and Ile27 in peptide 8 cause different activity profiles at the receptors, supposedly due to differential interactions with Leu123 (GLP-1 receptor), Ala118 (glucagon receptor) and His115 (GIP receptor). 835x495mm (96 x 96 DPI)



Figure 3. Crystal structure of peptide 11 in complex with the GLP-1 receptor extracellular domain (PDB code 6gb1). Peptide residues are colored according to the scheme of table 1. a) Overall structure. The GLP1 receptor ECD is shown as a white surface, the bound peptide 11 as a stick model. b) Close-up of a hydrogen bonding network at the “top” of the peptide. This hydrogen bonding network involves the side chains of the receptor residues Glu68 and Arg121, and Ser32 of the peptide, the main chain carbonyl oxygens of the receptor residues Phe66 and Asp67 and of the peptide residues Leu26 and Aib27 as well as three bound water molecules. 1143x807mm (96 x 96 DPI)


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Figure 4. Predicted binding hypothesis of a) peptide 5 and b) peptide 12 at the GLP-1 (PDB code 3c59), glucagon (PDB code 4ers) or GIP (PDB code 2qkh) receptor ECDs. Amino acids that are part of the predicted aggregation hot spot, and represent a potential interaction site for phenol, are colored red. Residues, which were introduced into peptide 12 to increase conformational stability or polarity are colored blue or green, respectively. 1123x719mm (96 x 96 DPI)



Figure 5. a) NMR ensemble structures of peptide 5 (PDB code 6gdz), peptide 12 (PDB code 6ge2) and exendin-4 (PDB code 1jrj) in solution. For structural clarity, only heavy atoms are depicted. Side-chain atoms of residue 14 (palmitic acid linked to the ɛ-amino group of lysine using two γ-glutamic acid spacers) of peptides 5 and 12 are indicated in gray. b) 3D-alignment of NMR structures of peptide 5, peptide 12 and exendin-4. c) Zoom-in view on 3D-alignment of tryptophan cage of peptide 5 and 12. 1119x1187mm (96 x 96 DPI)


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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 s.c. 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 s.c. from day 18 to completion of the study. 1240x967mm (96 x 96 DPI)



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. 1207x566mm (96 x 96 DPI)


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Glucagon Receptor Agonists

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