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A Non-Perturbative Molecular Grafting Strategy for Stable and Potent Therapeutic Peptide Ligands
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  • Kathleen M. Sicinski
    Kathleen M. Sicinski
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
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  • Vittorio Montanari
    Vittorio Montanari
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
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  • Venkata S. Raman
    Venkata S. Raman
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
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  • Jamie R. Doyle
    Jamie R. Doyle
    Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
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  • Benjamin N. Harwood
    Benjamin N. Harwood
    Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
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  • Yi Chi Song
    Yi Chi Song
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
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  • Micaella P. Fagan
    Micaella P. Fagan
    Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
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  • Maribel Rios
    Maribel Rios
    Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
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  • David R. Haines
    David R. Haines
    Department of Chemistry, Wellesley College, Wellesley, Massachusetts 02481, United States
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  • Alan S. Kopin
    Alan S. Kopin
    Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
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  • Martin Beinborn*
    Martin Beinborn
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
    *E.mail: [email protected]
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  • Krishna Kumar*
    Krishna Kumar
    Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    *Email: [email protected]
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    Orcidhttp://orcid.org/0000-0002-0548-5014
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Cite this: ACS Cent. Sci. 2021, 7, 3, 454–466
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https://doi.org/10.1021/acscentsci.0c01237
Published February 15, 2021

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Abstract

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The gut-derived incretin hormone, glucagon-like peptide-1 (GLP1), plays an important physiological role in attenuating post-prandial blood glucose excursions in part by amplifying pancreatic insulin secretion. Native GLP1 is rapidly degraded by the serine protease, dipeptidyl peptidase-4 (DPP4); however, enzyme-resistant analogues of this 30-amino-acid peptide provide an effective therapy for type 2 diabetes (T2D) and can curb obesity via complementary functions in the brain. In addition to its medical relevance, the incretin system provides a fertile arena for exploring how to better separate agonist function at cognate receptors versus susceptibility of peptides to DPP4-induced degradation. We have discovered that novel chemical decorations can make GLP1 and its analogues completely DPP4 resistant while fully preserving GLP1 receptor activity. This strategy is also applicable to other therapeutic ligands, namely, glucose-dependent insulinotropic polypeptide (GIP), glucagon, and glucagon-like peptide-2 (GLP2), targeting the secretin family of receptors. The versatility of the approach offers hundreds of active compounds based on any template that target these receptors. These observations should allow for rapid optimization of pharmacological properties and because the appendages are in a position crucial to receptor stimulation, they proffer the possibility of conferring “biased” signaling and in turn minimizing side effects.

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Synopsis

Diverse N-terminal modifications of various peptide templates render them refractory to protease action and retain full biological activity. These constructs have potential for development of therapeutics for many diseases.

Introduction

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Peptides derived from the processing of the polypeptide preproglucagon define the axis of a synchronized, choreographed system of maintaining glucose homeostasis and regulating other aspects of physiological function. (1) In particular, three distinct peptides, glucagon-like peptide-1 (GLP-1; referred to as GLP1 in this paper), glucagon-like peptide-2 (GLP2), and glucagon are processed from the precursor preproglucagon into mature functioning entities and play distinct roles in glucose metabolism. (2) An additional peptide, produced by K-cells in the GI tract, glucose-dependent insulinotropic polypeptide (GIP), complements this axis. GLP1 is produced in enteroendocrine cells of the gut, and processed to the active form, GLP1(7–36)NH2. Upon food ingestion, active GLP1 is secreted into the bloodstream and initiates multifaceted actions via activation of its cognate GLP1 receptor (GLP1R) expressed in several target tissues that synergistically work to restore normoglycemia. (3) GLP1R stimulation triggers cAMP production and amplification of glucose-induced insulin secretion from pancreatic β-cells resulting in lowered blood glucose levels. This action also results in diminution of glucagon secretion and β-cell proliferation. In parallel, this peptide hormone delays gastric emptying through vagal afferents and suppresses appetite via neurons in the paraventricular nucleus of the hypothalamus. (4) Based in large part on the function of endogenous GLP1, physiologic glycemia is markedly lower after an oral glucose load compared to an equivalent parenteral load—this phenomenological observation has been termed the “incretin” effect. (5) Because insulin secretion is glucose-dependent, this process is self-regulated, and the risk of hypoglycemia remains low. (1)
The efficacy of GLP1 as a treatment for type 2 diabetes (T2D) has long been validated in human subjects, as infusion of exogenous GLP1 to diabetic patients results in near-normalization of blood sugar levels. (6) However, native GLP1 cannot be used as a therapeutic, as it is rapidly inactivated by the ubiquitous serine protease dipeptidyl peptidase-4 (DPP4), which cleaves the N-terminal residues His7-Ala8 from the intact peptide (t1/2 ∼ 90 s) resulting in a shorter inactive fragment, GLP1(9–36)NH2. (7) This observation has motivated development of GLP1 based T2D therapeutics with enhanced protease resistance and delayed renal clearance. The first GLP1 mimetic clinically approved was exenatide, an equipotent reptilian homologue that shows 50% amino acid similarity with human GLP1. (8) Success of glycemic control by exenatide inspired the development of liraglutide, a lipidated form of GLP1. (9) Liraglutide forms a heptamer in solution (12) and also binds the abundant plasma protein albumin resulting in delayed renal clearance. (13) These compounds require twice- and once-daily injections, respectively. Further development has led to a derivative that is attached to the Fc fragment of IgG4 (dulaglutide) (14) and another that uses addition of a lipid to enhance albumin binding along with substitution of aminoisobutyric acid (Aib) at position two (semaglutide) (9,15) rendering these two compounds functional in vivo a week after injection. The future of this class of compounds has been further cemented by new guidance from the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) which recommends GLP1R agonists as the first injectable before insulin for T2D. (16) Small molecule inhibitors of DPP4 have also entered clinical use, (17) although these compounds do not result in weight loss, and the long-term safety profile is still uncertain as there are more than 30 known endogenous substrates of DPP4. (18) Recent efforts to overcome the liability of enzyme catalyzed hydrolysis have seen widespread use of Aib in position 8, but a previous compound (taspoglutide) (19) containing this noncanonical amino acid was withdrawn from Phase III clinical trials due to allergic reactions at the injection site. (20) Taspoglutide contains two Aib residues, at positions 8 and 35, to provide protection from proteases. Given that compounds in the clinic (e.g., semaglutide) contain Aib at position 8, and have good safety profiles, it is unclear whether the drug substance itself or the pharmaceutical formulation is responsible for the adverse reaction in the case of taspoglutide. (21)
Prior attempts at making GLP1 and related peptides more stable to proteolysis have relied on a number of strategies. The use of an α/β peptide scaffold that changes the backbone of the construct, (22) incorporation of fluorinated amino acids at strategic positions, (23) modification of side chains with saccharides, (24) and the use of aminoisobutyric acid (Aib) in position 8 (second from the N-terminus) (25) have been the most fruitful. While these strategies provide proteolytic stability to various degrees, previous attempts at modifying the sacrosanct N-terminal histidine residue, crucial to receptor activation, have led to greatly compromised GLP1 function. (26)
We describe in this report that N-alkyl modifications of several peptide ligands (GLP1, GIP, glucagon, GLP2, and a “triagonist” peptide) of the secretin family of receptors are refractory to DPP4 proteolysis while simultaneously maintaining full activity at respective cognate receptors. Because the strategy is applicable to all developed peptide ligand templates, modulation of activity and retaining stability is possible with N-terminal alkylation. Given the large number of low-molecular-weight aldehydes (estd ∼25,000) commercially available and the ease of installation, the number of derivatives for each peptide template to be useful clinically can easily number in the hundreds providing a blueprint for pharmacokinetic and pharmacodynamic optimization. In addition, because the N-terminus of the peptide ligand interacts deep in the membrane embedded region of the receptor, so as to say in the “belly of the beast”, it provides an avenue to select constructs that may trigger “biased” signaling resulting in fewer side effects. (11,27) We note that Aib residues commonly employed to protect from DPP4 cleavage are used internally within the sequence, and do not confer ability to interact with the hitherto undescribed ligand binding pocket.

Results and Discussion

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Peptides are inherently susceptible to proteolytic cleavage, and this deficit has usually been addressed by introduction of unnatural or noncanonical amino acids, by backbone or side chain modification, or by addition of exogenous groups that provide serum stability by binding to plasma components. In the case of GLP1, it has been challenging to decouple enzyme mediated hydrolysis from receptor activation. The ubiquitous serine protease DPP4 removes the N-terminal dipeptide His7Ala8 of GLP1 with a short half-life (t1/2 < 2 min), resulting in a truncated peptide, GLP1(9–37)NH2, with greatly diminished efficacy and potency. The active site of DPP4 has an arrangement of functionality that positions two glutamates for substrate recognition (Glu205 and Glu206), and mutating either or both glutamate(s) to alanine(s) renders the enzyme incompetent at catalysis (Figure 1d). The carboxylate groups from Glu205/Glu206 form electrostatic interactions between the positively charged amino terminus of the substrate and position the labile amide proximal to the catalytic hydroxyl of Ser630 that is primed for nucleophilic attack. Previous studies have focused on amino acid alterations of GLP1 to increase stability toward DPP4, but this strategy has usually come at the expense of GLP1R potency and/or efficacy. The loss in receptor activity underscores the importance of His7 in GLP1. Therefore, instead of altering His7, we first focused our attention on abolishing the N-terminal charge in GLP1 to prevent enzyme recognition.

Figure 1

Figure 1. (a) Cryo-EM structure of the GLP1:GLP1R complex (PDB: 5VAI). (10) GLP1 (ribbon, gold) bound to cognate G protein-coupled receptor, GLP1R (ribbon and surface, gray), with solid gray lines representing approximate locations of the cellular membrane that separate the extracellular domain (ECD) from the transmembrane domain (TMD) of GLP1R. (b) Illustration of receptor amino acids within 4 Å of the N-terminal histidine of GLP1. Residue numbers denote the Wootten nomenclature (11) for class B GPCRs. To note, human GLP1R contains R3105.40, but this PDB structure contains an alanine mutation at this position. The dashed pink circle indicates approximate space where N-terminal decorations may reside. (c) Flattened 2D rendering of the interactions of GLP1R with His7 of GLP1. R299ECL2 forms two hydrogen bonds with the N-terminal histidine (dashed line, gray) and a putative cation−π interaction of the guanidine group of R299ECL2 with the imidazole of His7 of GLP1 (dotted line, maroon). Select neighboring side chains of the receptor are shown at approximate positions relative to GLP1. (d) 2D depiction of DPP4 active site, with a known inhibitor bound (valine–pyrrolidide, blue, PDB: 1N1M). Important electrostatic interactions (dashed lines, gray) occur between the primary amine of the substrate and Glu205 and Glu206 of DPP4 (highlighted yellow). The carbonyl of the first amide bond is anchored by a hydrogen bond to Asn710 (dashed line, gray). The catalytic triad (Ser630, His710, and Asp708) forms a hydrogen bonding network (dashed lines, gray) and is positioned proximal to the pyrrole ring. If the structure bound were a peptide substrate, the labile amide bond would be located close to the canonical nucleophile Ser630.

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Acylation of the N-Terminus of GLP1 Provides DPP4 Protection but Diminishes Receptor Activity

An obvious strategy to overcome GLP1 degradation by DPP4 is removal of the N-terminal charge, crucial to substrate recognition, via acylation, as in 1-GLP1 (Figure 1). Prior reports have established that 1-GLP1 is resistant to DPP4 proteolysis; however, it results in a 60- to 97-fold decrease (28,29) in potency at the receptor. In our hands, 1-GLP1 stimulated GLP1R with an EC50 of 116 pM, a 58-fold loss in potency compared to native GLP1 (EC50 = 2.5 pM; Table 1, Figure S2). This result is consistent with other examples of N-acylation of GLP1, for example, with pyroglutamyl (26) or hexanoyl (28) groups, that show vastly reduced affinity and activation of GLP1R. All acyl groups suffer from this fate, as the receptor has evolved to recognize substrates that are processed via proteolytic cleavage of the precursor to reveal the canonical peptide of correct length, which thus lacks an acyl (amide) bond at the N-terminus. Processing in intestinal L-cells of a larger polypeptide, preproglucagon, first results in production of GLP1(1–37) which is further cleaved to active fragments GLP1(7–37) or GLP1(7–36)NH2. Improperly processed fragments such as GLP1(1–37), GLP1(8–37), and GLP1(6–37) are unable to induce insulin secretion at normal levels. (29) It is only once GLP1 is proteolyzed to display the free N-terminal histidine in GLP1(7–37) or GLP1(7–36)NH2 that native insulin release action is observed. (30)
Table 1. Receptor Activation of Nα-Modified Peptidesa
PeptidebpEC50 ± SEMcEC50 (pM)cnd
    
GLP1R
GLP111.6 ± 0.12.53
1-GLP1e10.0 ± 0.2115.93
2-GLP111.5 ± 0.12.83
3-GLP111.2 ± 0.16.73
4-GLP111.4 ± 0.36.93
5-GLP111.4 ± 0.073.83
6-GLP112.0 ± 0.21.12
7-GLP111.3 ± 0.26.53
8-GLP110.8 ± 0.217.62
9-GLP111.7 ± 0.42.83
10-GLP111.4 ± 0.024.43
11-GLP111.6 ± 0.74.73
12-GLP111.6 ± 0.12.72
13-GLP111.3 ± 0.25.32
14-GLP110.6 ± 0.125.32
15-GLP111.4 ± 0.13.82
16-GLP111.0 ± 0.039.62
17-GLP110.4 ± 0.0541.63
18-GLP111.1 ± 0.068.44
19-GLP17.5 ± 0.13.3 × 1042
Liraglutide11.6 ± 0.23.23
2-Liraglutide11.4 ± 0.034.03
Exenatide11.6 ± 0.022.63
2-Exenatide11.6 ± 0.062.93
2-Triagonist(Ala2)11.26.91
GIP6.921.2 × 1051
Glucagon9.314901
    
GIPR
GIP12.2 ± 0.040.63
1-GIP10.3 ± 0.254.62
2-GIP12.6 ± 0.020.33
Glucagon8.078.6 × 1031
GLP18.305.0 × 1031
2-Triagonist(Ala2)11.53.31
    
GCGR
Glucagon11.5 ± 0.043.45
2-Glucagon11.6 ± 0.12.35
7-Glucagon9.49 ± 0.13252
GLP16.186.6 × 1051
GIP6.305.1 × 1051
2-Triagonist(Ala2)11.44.51
    
GLP2R
GLP210.7 ± 0.1213
2-GLP211.2 ± 0.173
a

Potency of synthesized peptides using HEK293 cells expressing GLP1R, GIPR, GCGR, or GLP2R and luciferase reporter system. Results are separated by target receptors.

b

See Figure 2 for peptide structures.

c

EC50 is the concentration of peptide required for half-maximal activity of the targeted receptor. pEC50 = −log(EC50) ± standard error of the mean (SEM) of independent experiments where applicable.

d

Number of independent experiments that were run in triplicate or quadruplicate.

e

Peptides were incubated at 37 °C overnight before stimulating transfected cells.

We show here that removal of charge by alkylation (for example, with a trifluoroethyl group) is a viable strategy. Since this modification does not introduce an acyl (amide) bond at the N-terminus, it is tolerated by the receptor. A second approach is to increase steric bulk at the N-terminus so that the binding of the free amine in the enzyme active site is compromised. Surprisingly, the receptor is again accommodating of a large variety of such bulky groups introduced as N-alkyl tethers.
We then investigated alternative strategies that are capable of altering the pKa of the N-terminal amine. The cryo-EM structure of a GLP1-GLP1R complex (PDB: 5VAI) (10) provides a basis to inform which His7 modifications on the ligand could be accommodated within the membrane embedded region of the receptor (Figure 1a). Within the binding pocket of GLP1R, there are receptor side chains that make key noncovalent interactions with His7 of GLP1 (Figures 1b, S1A). Notably, R299ECL2 is involved in a π–cation interaction with the aromatic imidazole ring of His7, as well as W3065.36 and I3095.39 that form hydrogen bonds, but the primary N-terminal amine of GLP1 in this structure is devoid of contacts with the receptor (Figure 1c). The only residue within 4 Å of the free amine is V2373.40, and this hydrophobic residue is not proximal enough to GLP1 to contribute toward intermolecular interactions between receptor and ligand. Although this amine is not perceived to be interacting with GLP1R, removal of the primary amine results in a 15-fold loss in potency illustrating the importance of the amino terminus. (31) Not only has the free positive amino terminus been determined to be crucial, but the methyl imidazole side chain of His7 has also been recalcitrant to modification efforts. For example, substitution of His7 with Ala, Arg, Lys, or Tyr results in losses in both potency (174- to 1280-fold) and efficacy (12–66% of native peptide). (32) Further interrogation with six analogues that differ in the number and position of nitrogens in the methyl imidazole side chain revealed potency losses from 2.1-fold (H2.2-GLP1(8–36), Figure S1B) to 475-fold (H3.1-GLP1(8–36), Figure S1B) without a significant improvement in DPP4 resistance. Further, a His7Tyr modification leads both to decrease in receptor affinity (10-fold) and EC50 values of stimulatory activity (13-fold). (28) These observations serve as a foundation to make chemical modifications at this location to prevent DPP4 proteolysis while simultaneously retaining receptor efficacy and potency.
We hypothesized that modification of the amino terminus via alkylation through the agency of a trifluoroethyl group would afford an equipotent and equiefficacious GLP1 analogue that is impervious to DPP4 catalyzed inactivation. We envisioned that proteolytic stability would result from the electron withdrawing character and steric bulk of the 2,2,2-trifluoroethyl group. Alkylation of the α-amine of glycine with the trifluoroethyl moiety reduces the pKa of the ammonium species (33) from 9.6 to 5.3, and we envisioned that such a modification would result in removal of charge and simultaneously eliminate the acyl functionality at the N-terminus that GLP1R recognizes as an improperly processed ligand.

N-Trifluoroethyl Modification of GLP1 and of Related Receptor Agonists Result in Maintenance of Receptor Activity

We assessed the potency and efficacy of 2-GLP1 (Figure 2) at GLP1R by transfecting HEK293 cells with three cDNA constructs that encode for the target G protein-coupled receptor, GLP1R, CRE6x-luciferase reporter, and β-galactosidase to determine transfection efficiency. After 24 h, 2-GLP1 was titrated and the amount of luciferase produced after 4–6 h was used to calculate EC50 values, that were found to be comparable to native GLP1 (Table 1). This was a surprising and remarkable discovery given that the free amino terminus of GLP1 has long been considered crucial for receptor binding and activation. (28) Additionally, 2-GLP1 was entirely refractory to DPP4 catalyzed hydrolysis (vide infra). To the best of our knowledge, this is the first instance that an N-terminal modification of GLP1 has resulted in an equipotent and equally efficacious GLP1R agonist.

Figure 2

Figure 2. Library of N-terminally modified peptides. (a) Alignment of GLP1 and related peptides with positions and numbering above each residue (gray). GLP1 starts with amino acid 7 based on established convention. (3) Blue residues are homologous to GLP1, and residues highlighted yellow are conserved between all peptides. Liraglutide and triagonist contain a lysine (K, maroon) modified with a γ-glutamic acid spacer and by palmitoylation (right). Semaglutide contains a modified lysine (K, orange) with two oliogoethylene glycol (OEG) spacers, γ-glutamic acid, and octadecanedioic acid (right). “X” denotes the noncanonical amino acid, aminoisobutyric acid (Aib, bottom right). (b) Native amino acid sequences are modified with N-terminal chemical modifications 1–19 resulting in a library of peptides, nominally “R-Peptide” where “R” is the number referencing the N-terminus modification and “Peptide” indicates the template sequence as in (a). Semaglutide and triagonist peptides were also assembled with Aib2Ala mutation denoted as R-semaglutide(Ala2) and R-triagonist(Ala2), respectively.

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GLP1R agonists currently used clinically (liraglutide, exenatide, and semaglutide) operate by similar modes of binding and activation; we therefore evaluated the effect of N-terminal alkylation on these peptide ligands. For these GLP1R agonists, the potency and efficacy toward the receptor were not altered. The potency of 2-exenatide (2.9 pM) was essentially unchanged from that of exenatide (Figure 2, Table 1). A similar trend was seen for 2-liraglutide and 2-semaglutide(Ala2) (Figure 2, Table 1, Figure S3). The fact that this same N-terminal modification is useful in multiple GLP1R agonists confirms the robustness of this chemical grafting method and fueled our investigation to probe for stability against DPP4 action.

LC-MS Analysis of DPP4 Stability for GLP1R Agonists

After having determined that N-trifluoroethyl histidine modified GLP1 is accommodated by GLP1R, we investigated if the change in the N-terminal pKa influenced DPP4 proteolysis. (33) Native GLP1 or 2-GLP1 was incubated with and without DPP4 at 37 °C and the reaction mixture analyzed by ESI LC-MS after 16 h. In the presence of DPP4, native GLP1 was cleaved resulting in a distinct shift in retention time, from 22.2 to 23.3 min, with an accompanying reduction in mass by 208.1 Da indicating removal of the His7Ala8 dipeptide from the N-terminus. The product of GLP1 incubated with DPP4 eluted at the same time as the control GLP1(9–37)NH2, further confirming that the product resulted from dipeptide cleavage (Figure 3a). When 2-GLP1 was incubated with DPP4 overnight, no change in retention time or mass was observed, illustrating that the N-trifluoroethyl alkylation of GLP1 prevented DPP4 hydrolysis likely due to both decrease in pKa and addition of steric bulk on the N-terminal amine. This modification abolishes the interaction between the amino terminus of 2-GLP1 and Glu205 and Glu206 in the DPP4 active site, rendering it refractory to enzyme recognition and hydrolysis. We also explored whether the modified peptides are inhibitors of the enzyme or are just plainly not recognized as substrates. Steady-state kinetics with a known inhibitor (linagliptin) and substrate (GlyPro-pNA, Figure S4) reveal that these compounds are neither inhibitors (no reduction in Km) nor bind to the enzyme (no change in Vmax). These data suggest that either removal of charge or addition of bulk at the N-terminus of GLP1 abrogates recognition by DPP4, the frontline protease.

Figure 3

Figure 3. (a) LC-MS/MS total ion chromatogram depicting the stability of GLP1 (maroon) and 2-GLP1 (pink) with (shaded) and without (nonshaded) DPP4. GLP1 incubated with DPP4 shows the same retention time as control GLP1(9–36) (gray) indicating excision of dipeptide His7Ala8 to give cleaved, c, peptide. 2-GLP1 incubated with DPP4 exhibits no change in retention time or mass. (b) LC-MS/MS total ion chromatogram depicting the stability of exenatide (purple) and 2-exenatide (light purple) with (shaded) and without DPP4 (nonshaded). Exenatide incubated with DPP4 results in a mixture of cleaved, c, and native (unreacted), n, exenatide. The retention time of cleaved exenatide, c, is the same as control exenatide(3–39) (gray). 2-Exenatide incubated with DPP4 is unreactive with no change in retention time or mass. (c) LC-MS/MS total ion chromatogram depicting the stability of liraglutide (navy) and 2-liraglutide (light blue) with (shaded) and without DPP4 (nonshaded). Liraglutide incubated with DPP4 results in a mixture of cleaved, c, and native (unreacted), n, liraglutide. 2-Liraglutide incubated with DPP4 does not undergo reaction with unchanged retention time and mass.

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Exenatide is more resistant to DPP4 than GLP1; however, it is still susceptible to enzymatic degradation as judged by LC-MS analysis. After overnight incubation with DPP4, exenatide was partially cleaved resulting in a mixture containing an additional product (tR = 6.1 min). The elution profile and identified mass (−194.1 g/mol) corresponded to the peptide fragment exenatide(3–39) (Figure 3b). When 2-exenatide was subjected to DPP4 incubation, no cleavage products were observed. Our results can be explained with the same rationale as 2-GLP1, where recognition of the substrate and cleavage are prevented by removal of charge and addition of steric bulk at the N-terminus.
Liraglutide contains the same N-terminal motif as GLP1, and in the absence of serum albumin, it is susceptible to proteolysis. When we incubated liraglutide with DPP4 overnight, we saw MS signatures of two unique compounds by LC-MS (Figure 3c). The first compound eluted at the same time (tR = 9.3 min) and had the same MW as liraglutide. The second compound eluted later and had a mass that was 208.1 Da lower than liraglutide, suggestive of DPP4 removal of the HisAla dipeptide. When the N-terminally modified peptide 2-liraglutide was incubated with DPP4 under similar conditions, no change in mass or retention time was observed indicating the failure of DPP4 to process and inactivate substrate.

N-Trifluoroethyl Modification Is Accommodated by Most Secretin Family Receptors

Three peptide ligands—glucagon, GIP, and GLP2—fall within the same secretin family as GLP1 and contain five conserved amino acids: Gly4, Thr6, Phe22, Trp31, and Leu32 (Figure 2). Each peptide elicits various physiological responses from increasing blood glucose to stimulating intestinal growth. Although the first three N-terminal amino acids are dissimilar, all peptides are cleaved by DPP4. We sought to ascertain if fluoroalkylation would be compatible with the cognate receptors for glucagon, GIP, and GLP2 secretin peptides.
Glucose-dependent insulinotrophic polypeptide (GIP), like GLP1, is a gut hormone, and functionally complements the latter in accounting for the incretin effect after food ingestion. (34) GIP works through a different class B receptor (GIPR) (35,36) and has overlapping (amplification of insulin release from pancreatic β-cells) as well as distinct (modulation of fat cell metabolism) functions compared to GLP1. (7,5) In the diabetic state, the function of GIP is greatly diminished, (37) possibly as a result of GIPR down-regulation. (38,39) However, there is now increasing evidence that this defect is reversible once chronic hyperglycemia is alleviated, (40) e.g., after treatment with GLP1 analogues. There are some sequence similarities between GLP1 and GIP, but the N-terminus is occupied by a Tyr instead of His and this change provides specificity at GIPR. We synthesized an α-amino-modified tyrosine to incorporate into the full-length peptides yielding 2-GIP. Again, utilizing a luciferase-based assay, we transfected HEK293 cells with cDNAs encoding for the receptor of interest, GIPR, CRE6x-luciferase reporter, and a β-galactosidase control. (23) The day following transfection, cells were agonized with native GIP or 2-GIP and production of luciferase was quantified. 2-GIP showed similar potency as native GIP at the cognate receptor, GIPR (EC50 = 0.6 pM and 0.3 pM, respectively, Table 1). Similar to GLP1, this small molecular modification resulted in a GIP analogue with complete proteolytic stability without compromising receptor stimulatory activity (vide infra). The minimal change in potency and efficacy of 2-GIP cements the view that this molecular grafting method by alkylation is suitable for use at multiple receptors of the secretin family.
Glucagon originates from pancreatic islets and, in contrast to the incretins, elevates glycemia by inducing hepatic gluconeogenesis and mobilization of glucose into blood. However, during balanced pharmacological co-stimulation of GLP1R, GIPR, and glucagon receptors with low concentrations of corresponding agonists, glucagon further amplifies the anorectic effect of the incretins by enhancing energy expenditure via receptors on adipocytes and in the brain, thereby further promoting a loss in body weight. (1,41) The glycemic liability of glucagon disappears in combination drug regimens with GLP1 and GIP mimetics where the latter are dominant in reducing blood glucose. A recently engineered GLP1/GIP/glucagon triple agonist was highly effective in treating diabetes and obesity in mice. (42) Akin to GIP and GLP1, glucagon is also a substrate for DPP4 albeit suffers hydrolysis at a slower rate (t1/2 = 5–6 min). (43,44) Glucagon possesses the same N-terminal HisXaa motif as GLP1 and the N-terminal histidine was previously found to be essential for receptor activation. (45) We synthesized glucagon with the N-trifluoroethyl modification, 2-glucagon, and tested its ability to bind and activate its cognate glucagon receptor (GCGR). Consistent with our findings above, the addition of the N-terminal modification did not influence potency—2-glucagon was slightly more potent than native glucagon, with an EC50 of 2.3 pM compared to 3.4 pM of native glucagon in cellular assays (Table 1).
Glucagon-like peptide-2 (GLP2) is an agonist of another secretin family receptor, GLP2R. The 33-residue peptide is cosecreted with GLP1 and is a mediator of mucosal proliferation and enhances the activity of several absorptive enzymes resulting in optimal intestinal uptake of nutrients. (46,47) We synthesized an N-terminally modified GLP2 analogue to determine if this trifluoroethyl decoration would also be useful at its cognate receptor GLP2R. After synthesizing 2-GLP2, cells overexpressing GLP2R were treated with 2-GLP2 or native GLP2. We discovered that the fluorinated derivative 2-GLP2 was more potent than native GLP2 with EC50 of 7 pM compared to 21 pM of native GLP2 and was completely refractory to DPP4 catalyzed proteolysis. This was the most impressive improvement in potency of a secretin peptide that we observed. Taken together, we have shown that multiple peptides within the secretin family tolerate or are actually functionally enhanced by the small N-trifluoroethyl modification.
Because variations in potency was observed for alkylated secretin peptides at their cognate receptors, we then modified a peptide known to activate three of the secretin receptors (GLP1R, GIPR, and GCGR). Previous work by Finan et al. noticed a similarity in amino acid sequences between GLP1, GIP, and glucagon and generated a single peptide constructed with a synergistic effect on body weight reduction. (42) This peptide, named “triagonist”, contains Aib at position two to prevent DPP4 cleavage. Our goal was to eliminate Aib and utilize our novel N-terminal modification to retain receptor stimulatory activity while simultaneously preserving DPP4 resistance. We synthesized a N-trifluoroethyl triagonist with alanine in position 2, 2-triagonist(Ala2). Fluoroalkylation of the triagonist resulted in potencies in the single picomolar range, remarkably close to the respective native peptide ligands at their cognate receptors (Table 1, Figure S5).
In order to examine the scope and limitation of receptors that are able to recognize ligands modified through this strategy, we focused on the δ-opioid receptor and its native ligand, MetEnk, which can also be inactivated by DPP4. (48,49) In order to test if the trifluoroethyl modification protects the native peptide from hydrolysis and retain activity at the receptor, the activity of 2-MetEnk was tested at the native δ-receptor. This small modification abolished activity of the native peptide completely, most likely due to the importance of the primary amine for receptor activation (Figure S6) and thus circumscribing the scope of such alterations to the secretin family of receptors and their ligands.

N-Trifluoroethyl Alkylation Confers Superior DPP4 Stability

We have established that altering the pKa of the N-terminal amine of GLP1 contributes to increased DPP4 stability, as judged by LC-MS. We then investigated how EC50 is modulated upon incubation with DPP4, which provides a more sensitive analysis of the remaining full-length peptide. We incubated 2-GLP1 and GLP1 with DPP4 at 37 °C overnight. The peptides were then added directly to HEK293 cells overexpressing GLP1R. The receptor was stimulated for 4–6 h and production of luciferase was quantified and concentration response curves with and without DPP4 incubation were plotted. When GLP1 was incubated with DPP4 overnight, there was a shift in potency (EC50) from 3 pM to 2.8 nM corresponding to a massive 800-fold shift due to proteolysis (Figure 4a and Table 2). A significant difference in potency was also observed after incubation of liraglutide with DPP4 shifting an average of 33-fold from an EC50 of 8.6 pM to 283 pM (Figure 4b, Table 2). Only a minimal loss (∼1.7-fold) in potency of exenatide was observed after DPP4 incubation from 3.3 to 5.6 pM (Table 2).

Figure 4

Figure 4. Representative concentration–response curves of unmodified peptides (GLP1, liraglutide, GIP, and glucagon; a–d) or N-trifluoroethyl analogues (2-GLP1, 2-liraglutide, 2-GIP, and 2-glucagon; e–h) incubated overnight with DPP4 or vehicle prior to diluting into microtiter plates containing HEK293 cells overly expressing receptors (GLP1R, GIPR, or GCGR) and reporter CRE6x-luciferase. Luciferase production corresponds directly to activation of cognate GPCR via a cAMP dependent pathway, normalized to 100% maximal activity, and resultant fold-loss in EC50 upon DPP4 incubation is listed when applicable. Error bars represent SEM for three independent experiments (n = 3).

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Table 2. Stability towards DPP4 Proteolysis of Nα-Modified Peptidesa
 –DPP4+DPP4 
PeptidebpEC50 ± SEMcEC50 (pM)cndpEC50 ± SEMcEC50 (pM)cndFold-shift (↓)e
        
GLP1R
GLP111.6 ± 0.23.558.83 ± 0.32.8 × 1035800.0
1-GLP19.98 ± 0.299.139.93 ± 0.2106.431.1
2-GLP111.5 ± 0.13.5311.3 ± 0.15.431.5
3-GLP111.4 ± 0.25.5311.2 ± 0.16.431.2
4-GLP111.4 ± 0.57.4211.0 ± 0.417.122.3
5-GLP111.3 ± 0.14.8311.3 ± 0.15.031.0
6-GLP111.81.55111.53.2312.1
7-GLP111.5 ± 0.33.6211.5 ± 0.33.120.9
8-GLP111.17.2110.912.411.7
10-GLP111.4 ± 0.074.0311.4 ± 0.084.631.2
12-GLP111.2 ± 0.510.8210.9 ± 0.314.021.3
19-GLP17.53 ± 0.093.0 × 10426.91 ± 0.11.3 × 10524.3
Liraglutide11.2 ± 0.28.649.59 ± 0.1283432.9
2-Liraglutide11.0 ± 0.312.8311.1 ± 0.058.730.7
Exenatide11.5 ± 0.13.3311.3 ± 0.055.631.7
2-Exenatide11.4 ± 0.074.3311.4 ± 0.024.431.0
        
GIPR
GIP11.9 ± 0.081.439.77 ± 0.3226.53161.8
2-GIP12.2 ± 0.040.6312.1 ± 0.030.9031.5
        
GCGR
Glucagon11.3 ± 0.15.949.42 ± 0.48704147.5
2-Glucagon11.4 ± 0.24.9411.5 ± 0.23.740.8
        
GLP2R
GLP210.4 ± 0.0639.348.40 ± 0.024.0 × 1033101.8
2-GLP211.1 ± 0.29.6211.1 ± 0.059.020.9
a

Potency of synthesized peptides using HEK293 cells expressing GLP1R, GIPR, GCGR, or GLP2R and luciferase reporter system. Results are separated by target receptors. Peptides were incubated at 37 °C overnight with and without DPP4 before incubation of transfected cells.

b

See Figure 2 for structures of synthesized peptides.

c

EC50 is the concentration of peptide required for half maximal activity of the targeted receptor. pEC50 = −log(EC50) ± standard error of the mean (SEM) of independent experiments where applicable.

d

Number of independent experiments conducted in triplicate or quadruplicate.

e

Calculated by the ratio (EC50 with DPP4)/(EC50 without DPP4)

Because DPP4 is known to cleave multiple substrates, we interrogated the protease stability of secretin peptides: GIP, glucagon, and GLP2. Peptides were preincubated with DPP4, then titrated into a microtiter plate containing HEK293 cells overexpressing the receptor of interest (GIPR, GCGR, or GLP2R). Each peptide showed diminution in receptor stimulatory activity after DPP4 incubation. The tabulated loss for GIP was 162-fold, for glucagon 147-fold, and for GLP2 102-fold (Table 2, Figure 4c,d).
After determining the loss in potency upon DPP4 incubation, we assessed if this loss could be prevented by N-trifluoroethyl alkylation. All fluorinated peptides 2-GLP1, 2-liraglutide, 2-exenatide, 2-GIP, 2-glucagon, and 2-GLP2 showed retention of potency and efficacy upon preincubation with DPP4. An alternative to Aib protease protection in semaglutide is made available by these results, by retaining alanine at position 2 and alkylation of the N-terminus to give 2-semaglutide(Ala2) (Figure S3b,c). This pointed to a new, vigorous approach to prevent DPP4 proteolysis among various peptides of the secretin family without compromising receptor stimulatory function.

N-Terminal Modifications on GLP1 Extend beyond −CH2CF3

In order to establish the molecular parameters that best complement the receptor binding pocket in the membrane embedded region that is assumed to be elastic, we varied the alkyl tethers for various physicochemical attributes such as size (7-GLP1, 8-GLP1, 10-GLP1, and 19-GLP1), hydrophobicity (7-GLP1, 8-GLP1, and 10-GLP1), geometry (7-GLP1 and 8-GLP1), charge (12-GLP1, 13-GLP1, 14-GLP1, 15-GLP1, and 16-GLP1), polarizability (2-GLP1, 6-GLP1, 8-GLP1, 9-GLP1, 10-GLP1, and 11-GLP1), stereochemistry (12-GLP1, 13-GLP1, 14-GLP1, 15-GLP1, and 16-GLP1), and electronegativity (2-GLP1, 3-GLP1, and 6-GLP1). A polar appendage with multiple hydroxyl groups (mannitol, 19-GLP1) was also tested.
A structure activity relationship (SAR) approach was employed to explore an assortment of N-terminal decorations. The majority of N-alkyl groups were installed by reductive amination directly on resin by reaction of the α-amino group and the corresponding aldehyde. (50) After incubation of the peptide and aldehyde, the resulting imine was reduced with NaBH4 to afford the alkylated product creating a structurally diverse library of amine decorations (Figure 2).
The ethyl, propyl, and isobutyl modifications of GLP1 introduce small aliphatic groups on the N-terminus to afford 3-GLP1, 4-GLP1, and 5-GLP1. These modifications resulted in constructs that are close in potencies to native GLP1 and were able to stimulate the receptor with full efficacy. In addition, the largest group in 5-GLP1 resulted in the most potent compound of the group suggesting that larger nonpolar groups are able to penetrate the binding pocket and activate the receptor.
After observing the number of small aliphatic groups that GLP1R is able to accommodate, we investigated the size limit of these modifications. Introduction of a larger perfluoroalkyl group as in 6-GLP1 resulted in equipotent GLP1R activity (EC50 = 1.1 pM). We placed a large hydrophobic methyl adamantyl group on the N-terminus of GLP1 (7-GLP1), and surprisingly, this did not significantly diminish activation of GLP1R (∼3-fold loss in potency). Extension with a flat biphenyl aromatic ring system 8-GLP1 resulted in a 9-fold change in EC50 to 17.6 pM. Based on these observations, one can conclude that large nonpolar groups are compatible with GLP1R, but geometric attributes do play a role in receptor activation. We estimate that the receptor is able to accommodate aliphatic groups up to a volume of 130 Å3.
The observation that a greater reduction in potency with 8-GLP1 containing a biphenyl group led us to remove the second geometrically flat aromatic ring to yield a benzyl functionality (10-GLP1). The potency recovered from a 9-fold loss to only a 2-fold loss in potency compared to GLP1. The methyl imidazole containing 11-GLP1 was investigated to determine its effect and influence on receptor binding and activation. A priori, the expectation was an increase in potency because of the crucial role the methyl imidazole side chain of His7 had for receptor binding and activation; however, no increased potency was observed. The EC50 remained essentially unchanged compared to native GLP1 at 3 pM.
As the positively charged amino terminus of GLP1 was previously shown to be important for receptor binding and activation, we used alanine aldehyde to yield construct 12-GLP1. This resulted in essentially no change in potency at GLP1R (EC50 = 2.7 pM). More intriguingly, stereochemical differences play an important role in receptor activation. We used reductive amination with the R or S stereoisomers of Garner’s aldehyde to retain stereochemistry at the α-carbon and produce stereoisomeric constructs equivalent to those that would be obtained from the d-serine (13-GLP1) and l-serine (14-GLP1) aldehydes after cleavage from peptide resin. (51) Remarkably, there was a 6.3-fold difference in potencies, 4.0 pM and 25 pM, in favor of the S isomer. We also conducted a similar comparison with the aldehyde of l-phenylalanine (15-GLP1) and d-phenylalanine (16-GLP1). A difference of 3-fold was observed between the two stereoisomers, with the S chirality favored once again. This highlights the fact that stereochemical differences in the ligand can be exploited to fine-tune receptor activation and that the binding pocket has chirality that is responsive to changes in the three-dimensional disposition of functionality.
In order to gain a full understanding of the dynamics of this pocket, we synthesized two diazirine moieties to generate a probe capable of cross-linking with the receptor. We synthesized the commonly used trifluoromethylphenyl diazirine 17-GLP1 and assessed the potency at the receptor. We observed a modestly diminished potency (EC50 = 42 pM) which fell in line with the biphenyl compound 8-GLP1, suggesting that the large geometrically flat phenyl ring cannot bear too much steric bulk and still fit within the receptor pocket. This prompted the synthesis of a smaller alkyl diazirine, 18-GLP1, resulting in a construct with significantly improved potency of 8.4 pM.
Finally, we interrogated whether the receptor is tolerant of polar uncharged functionality. We tested the mannitol derivative, 19-GLP1, for potency and efficacy. With five hydroxyl groups positioned in the pocket, the potency of this construct was massively diminished with an EC50 of 33 nM (13,200-fold loss) while efficacy was fully retained. This suggests that 19-GLP1 is able to bind the receptor, but the appendage is unable to penetrate the receptor binding pocket in any meaningful way.
Overall, these peptide constructs provide a framework for understanding the types of modification that are topologically compatible with the GLP1R binding pocket, the volume that is available, the functional groups that are complementary, and the three-dimensional chiral space that the binding region presents to ligands. These observations should help guide design of a diverse library of high potency, full efficacy peptides that are also resistant to DPP4 catalyzed hydrolysis (Table 2). It is likely that subtle changes in the interaction of these ligands with the GLP1R binding pocket will result in peptides that trigger biased signaling.

Stability Conferred by Fluoroalkylation Extends to Protease Family Related to DPP4

DPP4 belongs to a family of homologous serine proteases that are active against the same N-terminal motif and cleave GLP1 resulting in the inactive fragment GLP1(9–37)NH2. (52) We interrogated if trifluoroethylation of GLP1 increases stability toward enzymes related to DPP4 (members of the S9B prolyl oligopeptidase subfamily that have similar arrangement of active sites and recognition of substrates by two glutamate residues), namely, dipeptidyl peptidase-9 (DPP9) and fibroblast activation protein α (FAP). Peptidases DPP4, DPP9, and FAP all cleave similar substrates: GLP1, GLP2, neuropeptide Y (NPY), and peptide YY (PYY). (53) Prior reports have documented the ability of DPP9 in cleaving GLP1 efficiently with a half-life of 6 min (54)in vitro as compared FAP, which gives GLP1 a t1/2 of 22 h. (53) After overnight incubation of GLP1 or 2-GLP1 peptides with vehicle, FAP, or DPP9, samples were titrated into a microtiter plate containing the luciferase reporter HEK293 cells overexpressing GLP1R. After the receptors were stimulated for 4–6 h, the production of luciferase was quantified as an index of GLP1R activation. GLP1 was cleaved by FAP and DPP9 with DPP9 cleaving more peptide due to the shorter half-life followed by FAP (Figure S7). This was not the case for 2-GLP1, where we did not observe any change in potency. This result indicates that 2-GLP1 is refractory to DPP9 and FAP action and expands the protective umbrella through this modification to other enzymes within the protease family.

In Vivo Glucose Tolerance Test

Blood glucose levels are regulated by the ability of GLP1 to promote insulin secretion, and we explored if our compounds achieve the same. We compared the ability of select peptides (2-GLP1, 7-GLP1, liraglutide, and 2-liraglutide) and GLP1 to stimulate insulin secretion and normalize glucose levels by performing in vivo glucose tolerance tests (Figure 5). Before peptide administration, the blood glucose of C57BL/6J mice was measured establishing an average fasting glucose level of 71 mg/dL. Mice were injected peritoneally with vehicle, GLP1, 2-GLP1, 7-GLP1, liraglutide, or 2-liraglutide at doses of 1 or 0.1 mg/kg. After 1 h, mice were administered an oral glucose bolus and mice administered vehicle showed a dramatic spike in blood glucose levels after 30 min (Figure 5a). All mice administered GLP1R agonists showed a less pronounced increase in blood glucose, which returned to basal glucose levels at about 120 min post glucose bolus. The area under the curve (AUC) between 0 and 120 min was significantly different for all GLP1R agonists compared to the vehicle (Figure 5b). To determine circulation longevity of each peptide, we administered a second bolus 5 h after the first glucose challenge. Lipidated analogues, liraglutide and 2-liraglutide, initially dosed at 1 mg/kg remained in circulation and prevented blood glucose level excursion post 30 min after the second glucose challenge (Figure 5c). No significant difference between GLP1, 2-GLP1, 7-GLP1, and vehicle was observed and 2-liraglutide performed just as well as liraglutide at both doses. This demonstrates the utility of N-alkylated peptides that are also lipidated to avoid renal clearance, in maintaining blood glucose levels to the same extent as compounds currently in clinical use. These findings also underscore the initial expectation that these minimal modifications do not affect in vivo biological function.

Figure 5

Figure 5. N-Trifluoroethyl alkylation and lipidation of 2-liraglutide performs as well as liraglutide at lowering blood sugar levels in vivo. (a) Measured glucose levels by tail vein prick for an oral glucose tolerance test (OGTT) of fasted mice treated intraperitoneally (i.p., dotted line) with vehicle, GLP1, 2-GLP1, 7-GLP1, liraglutide, or 2-liraglutide at (1 mg/kg or 0.1 mg/kg as noted). Glucose bolus was administered orally at time 0 and 240 min (gray, upward arrow). (b) Average area under the curve (AUC) calculated from 0 to 120 min in part a. (c) Glucose levels 30 min past second glucose challenge that occurred 5 h after first OGTT. Error represents the average ± SEM (n = 5). P-values compared to vehicle: **P < 0.01; ***P < 0.001; ****P < 0.0001.

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Serum Stability

Although Liraglutide has been shown to have increased serum stability, it is still metabolically processed by DPP4 (Figures 3c and 4b). Compound 2-liraglutide, which is only minimally modified from liraglutide with the sole addition of the N-terminal alkyl tether, but otherwise has the same sequence, linker, and lipid side chain, has a longer systemic half-life that is similar to semaglutide, but without the use of Aib at position 2. (25) Compounds were administered to rats via oral gavage (5 nmol/kg), and blood was collected sublingually to quantify the amount of peptide that remained in circulation via bioassay. The observed half-lives of liraglutide and 2-liraglutide were 3 and 5.5 h (Figure S8). These results indicate that the designed compounds survive in the bloodstream intact without damage by other metabolic processes and are active at the cognate receptor. The protracted lifetime is similar to semaglutide, the leading peptide-based compound in clinical use. (25)

Conclusion

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Decades of research on the design and modification of incretins has yielded functional templates that have resulted in several therapeutics for type 2 diabetes and related comorbidities. (21) The N-terminus of GLP1 has previously been assumed to be intolerant of modifications as it resides in a peptide domain crucial for intermolecular interactions with the receptor. This is mainly because the range of ligand modifications hitherto have relied primarily on acyl functionalities that the receptor has evolved to recognize as improperly processed fragments. We demonstrate in this study that not only is the N-terminal amenable to chemical modification but it is also tolerant to a wide range of functionality if the group is attached by alkylation, whereas acylation is deleterious to activity. The compounds described in this study are as efficacious and long-lived as the leading compounds in the clinic today. In addition, the strategy is generalizable to all templates for a range of receptors. Because the number of active compounds that can be generated for each template is in the hundreds, optimization of pharmacological properties (e.g., ligand bias and oral bioavailability) should be possible.
The use of native GLP1 as a therapeutic is hindered by its extreme lability, in that it is very short-lived with a half-life in vivo of less than 2 min, as it is degraded by the ubiquitous serine protease DPP4. We demonstrate in this study that the simultaneous goal of rendering substrates refractory to enzyme catalyzed hydrolysis and maintaining activity at the receptor is possible. We conducted activity assays (cAMP production) as well as LC ESI-MS to establish the resistance of the peptide constructs to DPP4. Through post-incubation assays, we show that the designed peptides have protracted lifetimes in vivo and are also able to restore normoglycemia over a prolonged period of time. We further show that the strategy is not only applicable to GLP1 but also to leading compounds currently in clinical use including liraglutide, exenatide, and semaglutide.
We extended the scope of such N-terminal modifications to other receptors within the class B family, namely, GCGR, GIPR, and GLP2R. These related receptors are also therapeutically relevant and provide avenues for design and development to a range of clinically useful peptide constructs. Collectively, this strategy is broadly applicable for modification of ligands which activate receptors that define the axis of glucose metabolism.
The results presented here open up an array of opportunities that have traditionally not been accessible. Class B G protein-coupled receptors (GPCRs) which encompass GLP1R, GIPR, GCGR, and GLP2R all interact with intracellular partner proteins upon activation by ligand. Several G proteins such as Gαs, Gαi, Gαq, Gαo, and arrestins (β-arrestin-1, β-arrestin-2) can serve in the effector role. (11,55,56) These partner/effector G proteins activate one or more signaling pathways such as mobilization of intracellular calcium and ERK1/2 phosphorylation (Gαs, Gαq, and Gαi/o or production of cAMP and insulin secretion (Gαs). (57,58) β-Arrestins control receptor internalization and/or mediate G protein independent signaling that lead to cell proliferation or apoptosis in part through activation of MAP kinases such as ERK1/2. (59) It is possible that binding of certain ligands to the receptor can selectively engage particular effectors over others resulting in “biased” signaling favoring one channel of activity over another. This has led to the idea that selective modulation of certain channels may result in ligands (therapeutics) that have fewer side effects sometimes accompanying receptor stimulation. (55) The molecular grafting method described in this study modifies the ligands at the crucial N-terminal residues and introduces new moieties in the receptor pocket interacting with a region of the receptor that is buried deep in the membrane. We believe this provides an avenue to achieve such signaling bias and studies along these lines are ongoing in our laboratories.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c01237.

  • Two-dimensional diagram of His7 interacting with GLP1R (Figure S1A), His7 mutations of GLP1 and calculated EC50 (Figure S1B), concentration response curve of 1-GLP1 with DPP4 incubation (Figure S2), concentration response curve of semaglutide and 2-Semaglutide(Ala2) with DPP4 incubation (Figure S3), Michaelis–Menten kinetics of DPP4 (Figure S4), concentration response curves of 2-triagonist(Ala2) (Figure S5), MetEnk and 2-MetEnk (Figure S6), GLP1 and 2-GLP1 incubated with FAP and DPP9 (Figure S7), and in vivo serum stability of 2-liraglutide (Figure S8); safety information; materials and methods; and analytical characterization of synthesized compounds and peptides (Table S1) (PDF)

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Author Information

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  • Corresponding Authors
    • Martin Beinborn - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States;  Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States;  Email: [email protected]
    • Krishna Kumar - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States;  Orcidhttp://orcid.org/0000-0002-0548-5014;  Email: [email protected]
  • Authors
    • Kathleen M. Sicinski - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    • Vittorio Montanari - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    • Venkata S. Raman - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    • Jamie R. Doyle - Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
    • Benjamin N. Harwood - Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
    • Yi Chi Song - Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
    • Micaella P. Fagan - Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
    • Maribel Rios - Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, United States
    • David R. Haines - Department of Chemistry, Wellesley College, Wellesley, Massachusetts 02481, United States
    • Alan S. Kopin - Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, United States
  • Notes
    The authors declare the following competing financial interest(s): K.K. is Scientific Founder of Velum, Inc. K.K., V.M., M.B., V.R. are named inventors in patent applications (WO2017075505 and WO2017075522 (additionally A.S.K.) to Tufts University) related to the work described in this paper. All other authors declare no competing interests.

Acknowledgments

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We thank Prof. Rebecca Scheck (Tufts University) for help with LC ESI-MS/MS. Thanks to Kathryn A. Lipford, Rebecca Roush and Dr. Michael J. Hanley for compound synthesis and assays described in Supplemental Figure S1B. The ESI-MS and NMR facilities at Tufts University were established by grants from the NSF (0320783 and 0821508). This work was supported in part by National Institutes of Health grants GM133272, GM130257 (to K.K.), and AG061909 (M.B.). B.N.H. was supported in part by NIH 5T32HL069770 (Karas). We thank Tufts University for ongoing and past support.

References

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    A review. Incretin peptides, principally GLP-1 and GIP, regulate islet hormone secretion, glucose concns., lipid metab., gut motility, appetite and body wt., and immune function, providing a scientific basis for utilizing incretin-based therapies in the treatment of type 2 diabetes. Activation of GLP-1 and GIP receptors also leads to nonglycemic effects in multiple tissues, through direct actions on tissues expressing incretin receptors and indirect mechanisms mediated through neuronal and endocrine pathways. Here we contrast the pharmacol. and physiol. of incretin hormones and review recent advances in mechanisms coupling incretin receptor signaling to pleiotropic metabolic actions in preclin. studies. We discuss whether mechanisms identified in preclin. studies have potential translational relevance for the treatment of human disease and highlight controversies and uncertainties in incretin biol. that require resoln. in future studies.
  2. 2
    Heinrich, G.; Gros, P.; Habener, J. F. Glucagon gene sequence. Four of six exons encode separate functional domains of rat pre-proglucagon. J. Biol. Chem. 1984, 259, 14082– 14087,  DOI: 10.1016/S0021-9258(18)89859-3
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    Glucagon gene sequence. Four of six exons encode separate functional domains of rat preproglucagon
    Heinrich, Gerhard; Gros, Philippe; Habener, Joel F.
    Journal of Biological Chemistry (1984), 259 (22), 14082-7CODEN: JBCHA3; ISSN:0021-9258.
    Glucagon [9007-92-5], a peptide of 29 amino acids that is produced and secreted by the pancreas, is a regulator of carbohydrate and protein metab. The nucleotide sequence of a mRNA of 1300 nucleotides that encodes rat preproglucagon [75432-63-2], a polyprotein precursor of glucagon, has been detd. The polyprotein contains the sequences of glucagon and 2 glucagon-like peptides arranged in tandem and sepd. by intervening peptides. The structure of the gene encoding rat preproglucagon was examd. The unique transcriptional unit of the gene spans 10 kilobase pairs and consists of 6 exons and 5 introns. Four of the 6 exons encode distinct functional domains of the preproglucagon. The signal sequence, glucagon, and 2 glucagon-like sequences arranged in tandem are each encoded by a sep. exon. A promoter sequence, TATAAA, is located 26 base pairs upstream from the mRNA cap site, and 2 polyadenylation signals (AATAAA) are present in the 3'-untranslated region of the encoded mRNA. The 3'-flanking region of the gene contains repetitive sequence DNA.
  3. 3
    Muller, T. D.; Finan, B.; Bloom, S. R.; D’Alessio, D.; Drucker, D. J.; Flatt, P. R.; Fritsche, A.; Gribble, F.; Grill, H. J.; Habener, J. F.; Holst, J. J.; Langhans, W.; Meier, J. J.; Nauck, M. A.; Perez-Tilve, D.; Pocai, A.; Reimann, F.; Sandoval, D. A.; Schwartz, T. W.; Seeley, R. J.; Stemmer, K.; Tang-Christensen, M.; Woods, S. C.; DiMarchi, R. D.; Tschop, M. H. Glucagon-like peptide 1 (GLP-1). Mol. Metab. 2019, 30, 72– 130,  DOI: 10.1016/j.molmet.2019.09.010
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    Glucagon-like peptide 1 (GLP-1)
    Muller T D; Finan B; Bloom S R; D'Alessio D; Drucker D J; Flatt P R; Fritsche A; Gribble F; Reimann F; Grill H J; Habener J F; Holst J J; Langhans W; Meier J J; Nauck M A; Perez-Tilve D; Pocai A; Sandoval D A; Seeley R J; Schwartz T W; Stemmer K; Tang-Christensen M; Woods S C; DiMarchi R D; Tschop M H
    Molecular metabolism (2019), 30 (), 72-130 ISSN:.
    BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent β-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.
  4. 4
    Vrang, N.; Larsen, P. J. Preproglucagon derived peptides GLP-1, GLP-2 and oxyntomodulin in the CNS: role of peripherally secreted and centrally produced peptides. Prog. Neurobiol. 2010, 92, 442– 462,  DOI: 10.1016/j.pneurobio.2010.07.003
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    Preproglucagon derived peptides GLP-1, GLP-2 and oxyntomodulin in the CNS: Role of peripherally secreted and centrally produced peptides
    Vrang, Niels; Larsen, Philip Just
    Progress in Neurobiology (Oxford, United Kingdom) (2010), 92 (3), 442-462CODEN: PGNBA5; ISSN:0301-0082. (Elsevier Ltd.)
    A review. The scientific understanding of preproglucagon derived peptides has provided people with type 2 diabetes with two novel classes of glucose lowering agents, the dipeptidyl peptidase IV (DPP-IV) inhibitors and GLP-1 receptor agonists. For the scientists, the novel GLP-1 agonists, and DPP-IV inhibitors have evolved as useful tools to understand the role of the preproglucagon derived peptides in normal physiol. and disease. However, the overwhelming interest attracted by GLP-1 analogs as potent incretins has somewhat clouded the efforts to understand the importance of preproglucagon derived peptides in other physiol. contexts. In particular, our neurobiol. understanding of the preproglucagon expressing neuronal pathways in the central nervous system as well as the degree to which central GLP-1 receptors are targeted by peripherally administered GLP-1 receptor agonists is still fairly limited. The role of GLP-1 as an anorectic neurotransmitter is well recognized, but clarification of the neuronal targets and physiol. basis of this response is further warranted, as is the mapping of GLP-1 sensitive neurons involved in a variety of neuroendocrine and behavioral responses. Further recent evidence points to GLP-1 as a central neuropeptide with neuroprotective capabilities potentially mitigating a wide array of neurodegenerative conditions. It is the aim of the present review to summarize our current understanding of preproglucagon derived peptides as neurotransmitters in the central nervous system.
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    Wu, T.; Rayner, C. K.; Horowitz, M. Incretins; Springer International Publishing: 2015; pp 137– 171.
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    Manandhar, B.; Ahn, J. M. Glucagon-like peptide-1 (GLP-1) analogs: recent advances, new possibilities, and therapeutic implications. J. Med. Chem. 2015, 58, 1020– 1037,  DOI: 10.1021/jm500810s
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    Glucagon-like Peptide-1 (GLP-1) Analogs: Recent Advances, New Possibilities, and Therapeutic Implications
    Manandhar, Bikash; Ahn, Jung-Mo
    Journal of Medicinal Chemistry (2015), 58 (3), 1020-1037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Glucagon-like peptide-1 (GLP-1) is an incretin that plays important physiol. roles in glucose homeostasis. Produced from intestine upon food intake, it stimulates insulin secretion and keeps pancreatic β-cells healthy and proliferating. Because of these beneficial effects, it has attracted a great deal of attention in the past decade, and an entirely new line of diabetic therapeutics has emerged based on the peptide. In addn. to the therapeutic applications, GLP-1 analogs have demonstrated a potential in mol. imaging of pancreatic β-cells; this may be useful in early detection of the disease and evaluation of therapeutic interventions, including islet transplantation. In this Perspective, we focus on GLP-1 analogs for their studies on improvement of biol. activities, enhancement of metabolic stability, investigation of receptor interaction, and visualization of the pancreatic islets.
  7. 7
    Kim, W.; Egan, J. M. The role of incretins in glucose homeostasis and diabetes treatment. Pharmacol. Rev. 2008, 60, 470– 512,  DOI: 10.1124/pr.108.000604
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    The role of incretins in glucose homeostasis and diabetes treatment
    Kim, Wook; Egan, Josephine M.
    Pharmacological Reviews (2008), 60 (4), 470-512CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
    A review. Incretins are gut hormones that are secreted from enteroendocrine cells into the blood within minutes after eating. One of their many physiol. roles is to regulate the amt. of insulin that is secreted after eating. In this manner, as well as others to be described in this review, their final common raison d'etre is to aid in disposal of the products of digestion. There are two incretins, known as glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1), that share many common actions in the pancreas but have distinct actions outside of the pancreas. Both incretins are rapidly deactivated by an enzyme called dipeptidyl peptidase 4 (DPP4). A lack of secretion of incretins or an increase in their clearance are not pathogenic factors in diabetes. However, in type 2 diabetes (T2DM), GIP no longer modulates glucose-dependent insulin secretion, even at supra-physiol. (pharmacol.) plasma levels, and therefore GIP incompetence is detrimental to β-cell function, esp. after eating. GLP-1, on the other hand, is still insulinotropic in T2DM, and this has led to the development of compds. that activate the GLP-1 receptor with a view to improving insulin secretion. Since 2005, two new classes of drugs based on incretin action have been approved for lowering blood glucose levels in T2DM: an incretin mimetic (exenatide, which is a potent long-acting agonist of the GLP-1 receptor) and an incretin enhancer (sitagliptin, which is a DPP4 inhibitor). Exenatide is injected s.c. twice daily and its use leads to lower blood glucose and higher insulin levels, esp. in the fed state. There is glucose-dependency to its insulin secretory capacity, making it unlikely to cause low blood sugars (hypoglycemia). DPP4 inhibitors are orally active and they increase endogenous blood levels of active incretins, thus leading to prolonged incretin action. The elevated levels of GLP-1 are thought to be the mechanism underlying their blood glucose-lowering effects.
  8. 8
    Knudsen, L. B. Glucagon-like peptide-1: the basis of a new class of treatment for type 2 diabetes. J. Med. Chem. 2004, 47, 4128– 4134,  DOI: 10.1021/jm030630m
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    Glucagon-like peptide-1:The basis of a new class of treatment for type 2 diabetes
    Knudsen, Lotte Bjerre
    Journal of Medicinal Chemistry (2004), 47 (17), 4128-4134CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Type 2 diabetes is increasingly becoming a epidemic worldwide. The author focuses on glucagon-like 1-peptide (GLP-1) peptide hormone as the basis for a potential new treatment paradigm for type 2 diabetes.
  9. 9
    Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.; Thogersen, H.; Wilken, M.; Johansen, N. L. Structure-activity and protraction relationship of long-acting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity, and bulkiness. J. Med. Chem. 2007, 50, 6126– 6132,  DOI: 10.1021/jm070861j
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    Structure-Activity and Protraction Relationship of Long-Acting Glucagon-like Peptide-1 Derivatives: Importance of Fatty Acid Length, Polarity, and Bulkiness
    Madsen, Kjeld; Knudsen, Lotte Bjerre; Agersoe, Henrik; Nielsen, Per Franklin; Thogersen, Henning; Wilken, Michael; Johansen, Nils Langeland
    Journal of Medicinal Chemistry (2007), 50 (24), 6126-6132CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We here report a series of derivs. describing the structure-activity relation around liraglutide, a once-daily human glucagon-like peptide-1 fatty acid deriv., with respect to potency as well as protraction in vivo. The spacer region between the fatty acid and the peptide is mostly important for potency, whereas the fatty acid or fatty acid mimetic is important for both potency and protraction. The length of the fatty acid is the most important parameter for protraction.
  10. 10
    Zhang, Y.; Sun, B.; Feng, D.; Hu, H.; Chu, M.; Qu, Q.; Tarrasch, J. T.; Li, S.; Sun Kobilka, T.; Kobilka, B. K.; Skiniotis, G. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 2017, 546, 248– 253,  DOI: 10.1038/nature22394
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    Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein
    Zhang, Yan; Sun, Bingfa; Feng, Dan; Hu, Hongli; Chu, Matthew; Qu, Qianhui; Tarrasch, Jeffrey T.; Li, Shane; Sun Kobilka, Tong; Kobilka, Brian K.; Skiniotis, Georgios
    Nature (London, United Kingdom) (2017), 546 (7657), 248-253CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metab. and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near at. resoln. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding.
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    Wootten, D.; Simms, J.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 5211– 5216,  DOI: 10.1073/pnas.1221585110
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    Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations
    Wootten, Denise; Simms, John; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (13), 5211-5216, S5211/1-S5211/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Recently, the concept of ligand-directed signaling - the ability of different ligands of an individual receptor to promote distinct patterns of cellular response - has gained much traction in the field of drug discovery, with the potential to sculpt biol. response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clin. used mimetic exendin-4.
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    Steensgaard, D. B.; Thomsen, J. K.; Olsen, H. B.; Knudsen, L. B. The molecular basis for the delayed absorption of the once-daily human GLP-1 analoge, liraglutide. Diabetes 2008, 57, A164– A164
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    Knudsen, L. B.; Nielsen, P. F.; Huusfeldt, P. O.; Johansen, N. L.; Madsen, K.; Pedersen, F. Z.; Thogersen, H.; Wilken, M.; Agerso, H. Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. J. Med. Chem. 2000, 43, 1664– 1669,  DOI: 10.1021/jm9909645
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    Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration
    Knudsen, Lotte B.; Nielsen, Per F.; Huusfeldt, Per O.; Johansen, Nils L.; Madsen, Kjeld; Pedersen, Freddy Z.; Thogersen, Henning; Wilken, Michael; Agerso, Henrik
    Journal of Medicinal Chemistry (2000), 43 (9), 1664-1669CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A series of very potent derivs. of the 30-amino acid peptide hormone glucagon-like peptide-1 (GLP-1) is described. The compds. were all derivatized with fatty acids in order to protract their action by facilitating binding to serum albumin. GLP-1 had a potency (EC50) of 55 pM for the cloned human GLP-1 receptor. Many of the compds. had similar or even higher potencies, despite quite large substituents. All compds. derivatized with fatty acids equal to or longer than 12 carbon atoms were very protracted compared to GLP-1 and thus seem suitable for once daily administration to type 2 diabetic patients. A structure-activity relationship was obtained. GLP-1 could be derivatized with linear fatty acids up to the length of 16 carbon atoms, sometimes longer, almost anywhere in the C-terminal part without considerable loss of potency. Derivatization with two fatty acid substituents led to a considerable loss of potency. A structure-activity relationship on derivatization of specific amino acids generally was obtained. It was found that the longer the fatty acid, the more potency was lost. Simultaneous modification of the N-terminus (in order to obtain better metabolic stability) interfered with fatty acid derivatization and led to loss of potency.
  14. 14
    Jimenez-Solem, E.; Rasmussen, M. H.; Christensen, M.; Knop, F. K. Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes. Curr. Opin. Mol. Ther. 2010, 12, 790– 797
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    Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes
    Jimenez-Solem, Espen; Rasmussen, Mette H.; Christensen, Mikkel; Knop, Filip K.
    Current Opinion in Molecular Therapeutics (2010), 12 (6), 790-797CODEN: CUOTFO; ISSN:2040-3445. (BioMed Central Ltd.)
    A review. Dulaglutide (LY-2189265) is a novel, long-acting glucagon-like peptide 1 (GLP-1) analog being developed by Eli Lilly for the treatment of type 2 diabetes mellitus (T2DM). Dulaglutide consists of GLP-l(7-37) covalently linked to an Fc fragment of human IgG4, thereby protecting the GLP-1 molety from inactivation by dipeptidyl peptidase 4. In vitro and in vivo studies on T2DM models demonstrated glucose-dependent insulin secretion stimulation. Pharmacokinetic studies demonstrated a t1/2 in humans of up to 90 h, making Dulaglutide an ideal candidate for once-weekly dosing. Clin. trials suggest that Dulaglutide reduces plasma glucose, and has an insulinotropic effect increasing insulin and C-peptide levels. Two phase II clin. trials demonstrated a dose-dependent redn. in glycated Hb (HbA1c) of up to 1.52% compared with placebo. Side effects assocd. with Dulaglutide administration were mainly gastrointestinal. To date, there have been no reports on the formation of antibodies against Dulaglutide, but, clearly, long-term data will be needed to asses this and other possible side effects. The results of several phase III clin. trials are awaited for clarification of the expected effects on HbA1c and body wt. If Dulaglutide possesses similar efficacy to other GLP-1 analogs, the once-weekly treatment will most likely be welcomed by patients with T2DM.
  15. 15
    Knudsen, L. B.; Lau, J. The Discovery and Development of Liraglutide and Semaglutide. Front. Endocrinol. (Lausanne, Switz.) 2019, 10, 155,  DOI: 10.3389/fendo.2019.00155
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    The Discovery and Development of Liraglutide and Semaglutide
    Knudsen Lotte Bjerre; Lau Jesper
    Frontiers in endocrinology (2019), 10 (), 155 ISSN:1664-2392.
    The discovery of glucagon-like peptide-1 (GLP-1), an incretin hormone with important effects on glycemic control and body weight regulation, led to efforts to extend its half-life and make it therapeutically effective in people with type 2 diabetes (T2D). The development of short- and then long-acting GLP-1 receptor agonists (GLP-1RAs) followed. Our article charts the discovery and development of the long-acting GLP-1 analogs liraglutide and, subsequently, semaglutide. We examine the chemistry employed in designing liraglutide and semaglutide, the human and non-human studies used to investigate their cellular targets and pharmacological effects, and ongoing investigations into new applications and formulations of these drugs. Reversible binding to albumin was used for the systemic protraction of liraglutide and semaglutide, with optimal fatty acid and linker combinations identified to maximize albumin binding while maintaining GLP-1 receptor (GLP-1R) potency. GLP-1RAs mediate their effects via this receptor, which is expressed in the pancreas, gastrointestinal tract, heart, lungs, kidneys, and brain. GLP-1Rs in the pancreas and brain have been shown to account for the respective improvements in glycemic control and body weight that are evident with liraglutide and semaglutide. Both liraglutide and semaglutide also positively affect cardiovascular (CV) outcomes in individuals with T2D, although the precise mechanism is still being explored. Significant weight loss, through an effect to reduce energy intake, led to the approval of liraglutide (3.0 mg) for the treatment of obesity, an indication currently under investigation with semaglutide. Other ongoing investigations with semaglutide include the treatment of non-alcoholic fatty liver disease (NASH) and its use in an oral formulation for the treatment of T2D. In summary, rational design has led to the development of two long-acting GLP-1 analogs, liraglutide and semaglutide, that have made a vast contribution to the management of T2D in terms of improvements in glycemic control, body weight, blood pressure, lipids, beta-cell function, and CV outcomes. Furthermore, the development of an oral formulation for semaglutide may provide individuals with additional benefits in relation to treatment adherence. In addition to T2D, liraglutide is used in the treatment of obesity, while semaglutide is currently under investigation for use in obesity and NASH.
  16. 16
    Davies, M. J.; D’Alessio, D. A.; Fradkin, J.; Kernan, W. N.; Mathieu, C.; Mingrone, G.; Rossing, P.; Tsapas, A.; Wexler, D. J.; Buse, J. B. Management of Hyperglycemia in Type 2 Diabetes, 2018. A Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care 2018, 41, 2669– 2701,  DOI: 10.2337/dci18-0033
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    Management of Hyperglycemia in Type 2 Diabetes, 2018. A Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD)
    Davies Melanie J; Davies Melanie J; D'Alessio David A; Fradkin Judith; Kernan Walter N; Mathieu Chantal; Mingrone Geltrude; Mingrone Geltrude; Rossing Peter; Rossing Peter; Tsapas Apostolos; Wexler Deborah J; Wexler Deborah J; Buse John B
    Diabetes care (2018), 41 (12), 2669-2701 ISSN:.
    The American Diabetes Association and the European Association for the Study of Diabetes convened a panel to update the prior position statements, published in 2012 and 2015, on the management of type 2 diabetes in adults. A systematic evaluation of the literature since 2014 informed new recommendations. These include additional focus on lifestyle management and diabetes self-management education and support. For those with obesity, efforts targeting weight loss, including lifestyle, medication, and surgical interventions, are recommended. With regards to medication management, for patients with clinical cardiovascular disease, a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a glucagon-like peptide 1 (GLP-1) receptor agonist with proven cardiovascular benefit is recommended. For patients with chronic kidney disease or clinical heart failure and atherosclerotic cardiovascular disease, an SGLT2 inhibitor with proven benefit is recommended. GLP-1 receptor agonists are generally recommended as the first injectable medication.
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    Trzaskalski, N. A.; Fadzeyeva, E.; Mulvihill, E. E. Dipeptidyl Peptidase-4 at the Interface Between Inflammation and Metabolism. Clin. Med. Insights: Endocrinol. Diabetes 2020, 13, 1– 10,  DOI: 10.1177/1179551420912972
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    Mulvihill, E. E.; Drucker, D. J. Pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors. Endocr. Rev. 2014, 35, 992– 1019,  DOI: 10.1210/er.2014-1035
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    Pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors
    Mulvihill, Erin E.; Drucker, Daniel J.
    Endocrine Reviews (2014), 35 (6), 992-1019CODEN: ERVIDP; ISSN:0163-769X. (Endocrine Society)
    A review. Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane mol. and a sol. circulating protein. Both membrane-assocd. and sol. DPP4 exert catalytic activity, cleaving proteins contg. a position 2 alanine or proline. DPP4-mediated enzymic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the mol. mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biol. of DPP4 with a focus on: (1) identification of pharmacol. vs physiol. DPP4 substrates; and (2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chem. redn. of DPP4 activity. We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclin. and clin. studies. Finally, we highlight exptl. pitfalls and tech. challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
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    Sebokova, E.; Christ, A. D.; Wang, H.; Sewing, S.; Dong, J. Z.; Taylor, J.; Cawthorne, M. A.; Culler, M. D. Taspoglutide, an analog of human glucagon-like Peptide-1 with enhanced stability and in vivo potency. Endocrinology 2010, 151, 2474– 2482,  DOI: 10.1210/en.2009-1459
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    Taspoglutide, an analog of human glucagon-like peptide-1 with enhanced stability and in vivo potency
    Sebokova, Elena; Christ, Andreas D.; Wang, Haiyan; Sewing, Sabine; Dong, Jesse Z.; Taylor, John; Cawthorne, Michael A.; Culler, Michael D.
    Endocrinology (2010), 151 (6), 2474-2482CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
    Taspoglutide is a novel analog of human glucagon-like peptide-1 [hGLP-1(7-36)NH2] in clin. development for the treatment of type 2 diabetes. Taspoglutide contains α-aminoisobutyric acid substitutions replacing Ala8 and Gly35 of hGLP-1(7-36)NH2. The binding affinity [radioligand binding assay using [125I]hGLP-1(7-36)NH2], potency (cAMP prodn. in CHO cells stably overexpressing hGLP-1 receptor), and in vitro plasma stability of taspoglutide compared with hGLP-1(7-36)NH2 have been evaluated. Effects on basal and glucose-stimulated insulin secretion were detd. in vitro in INS-1E cells and in vivo in normal rats. Taspoglutide has comparable affinity (affinity const. 1.1 ± 0.2 nM) to the natural ligand (affinity const. 1.5 ± 0.3 nM) for the hGLP-1 receptor and exhibits comparable potency in stimulating cAMP prodn. (EC50 Taspo 0.06 nM and EC50 hGLP-1(7-36)NH2 0.08 nM). Taspoglutide exerts insulinotropic action in vitro and in vivo and retains the glucoincretin property of hGLP-1(7-36)NH2. Stimulation of insulin secretion is concn. dependent and evident in the presence of high-glucose concns. (16.7 mM) with a taspoglutide concn. as low as 0.001 nM. Taspoglutide is fully resistant to dipeptidyl peptidase-4 cleavage (during 1 h incubation at room temp. with purified enzyme) and has an extended in vitro plasma half-life relative to hGLP-1(7-36)NH2 (9.8 h vs. 50 min). In vitro, taspoglutide does not inhibit dipeptidyl peptidase-4 activity. This study provides the biochem. and pharmacol. basis for the sustained plasma drug levels and prolonged therapeutic activity seen in early clin. trials of taspoglutide. Excellent stability and potency with substantial glucoincretin effects position taspoglutide as a promising new agent for treatment of type 2 diabetes.
  20. 20
    Rosenstock, J.; Balas, B.; Charbonnel, B.; Bolli, G. B.; Boldrin, M.; Ratner, R.; Balena, R. The fate of taspoglutide, a weekly GLP-1 receptor agonist, versus twice-daily exenatide for type 2 diabetes: the T-emerge 2 trial. Diabetes Care 2013, 36, 498– 504,  DOI: 10.2337/dc12-0709
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    The fate of taspoglutide, a weekly GLP-1 receptor agonist, versus twice-daily exenatide for type 2 diabetes: The T-emerge 2 trial
    Rosenstock, Julio; Balas, Bogdan; Charbonnel, Bernard; Bolli, Geremia B.; Boldrin, Mark; Ratner, Robert; Balena, Raffaella
    Diabetes Care (2013), 36 (3), 498-504CODEN: DICAD2; ISSN:0149-5992. (American Diabetes Association, Inc.)
    OBJECTIVE: Taspoglutide is a long-acting glucagon-like peptide 1 receptor agonist developed for treatment of type 2 diabetes. The efficacy and safety of once-weekly taspoglutide was compared with twice-daily exenatide. RESEARCH DESIGN AND METHODS: Overweight adults with inadequately controlled type 2 diabetes on metformin ± a thiazolidinedione were randomized to s.c. taspoglutide 10 mg weekly (n = 399), taspoglutide 20 mg weekly (n = 398), or exenatide 10 μg twice daily (n = 392) in an open-label, multicenter trial. The primary end point was change in HbA1c after 24 wk. RESULTS: Mean baseline HbA1c was 8.1%. Both doses of taspoglutide reduced HbA1c significantly more than exenatide (taspoglutide 10 mg: -1.24% [SE 0.09], difference -0.26, 95% CI -0.37 to -0.15, P < 0.0001; taspoglutide 20 mg: -1.31% [0.08], difference -0.33, -0.44 to -0.22, P < 0.0001; exenatide: -0.98% [0.08]). Both taspoglutide doses reduced fasting plasma glucose significantly more than exenatide. Taspoglutide reduced body wt. (taspoglutide 10 mg, -1.6 kg; taspoglutide 20 mg, -2.3 kg) as did exenatide (-2.3 kg), which was greater than with taspoglutide 10 mg (P < 0.05). HbA1c and wt. effects were maintained after 52 wk. More adverse events with taspoglutide 10 and 20 mg than exenatide developed over time (nausea in 53, 59, and 35% and vomiting in 33, 37, and 16%, resp.). Allergic and injection-site reactions were more common with taspoglutide. Discontinuations were greater with taspoglutide. Antitaspoglutide antibodies were detected in 49% of patients. CONCLUSIONS: Once-weekly taspoglutide demonstrated greater glycemic control than twice-daily exenatide with comparable wt. loss, but with unacceptable levels of nausea/vomiting, injection-site reactions, and systemic allergic reactions.
  21. 21
    Jones, L. H.; Price, D. A. Medicinal Chemistry of Glucagon-Like Peptide Receptor Agonists. Prog. Med. Chem. 2013, 52, 45– 96,  DOI: 10.1016/B978-0-444-62652-3.00002-8
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    Medicinal chemistry of glucagon-like peptide receptor agonists
    Jones, Lyn H.; Price, David A.
    Progress in Medicinal Chemistry (2013), 52 (), 45-96CODEN: PMDCAY; ISSN:0079-6468. (Elsevier B.V.)
    A review. This article discusses the medicinal chem. of glucagon-like peptide receptor agonists. Applications of GLP-1 ligands as imaging agents in the diagnosis of diabetes and pancreatic cancer were discussed. Fundamental principles of GLP-1 injectable peptides medicinal chem. design and synthesis were discussed. The potential for alternative modes of drug delivery, such as oral and inhaled administration were discussed.
  22. 22
    Johnson, L. M.; Barrick, S.; Hager, M. V.; McFedries, A.; Homan, E. A.; Rabaglia, M. E.; Keller, M. P.; Attie, A. D.; Saghatelian, A.; Bisello, A.; Gellman, S. H. A potent alpha/beta-peptide analogue of GLP-1 with prolonged action in vivo. J. Am. Chem. Soc. 2014, 136, 12848– 12851,  DOI: 10.1021/ja507168t
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    A Potent α/β-Peptide Analogue of GLP-1 with Prolonged Action in Vivo
    Johnson, Lisa M.; Barrick, Stacey; Hager, Marlies V.; McFedries, Amanda; Homan, Edwin A.; Rabaglia, Mary E.; Keller, Mark P.; Attie, Alan D.; Saghatelian, Alan; Bisello, Alessandro; Gellman, Samuel H.
    Journal of the American Chemical Society (2014), 136 (37), 12848-12851CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic β cells. GLP-1R agonists are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. The authors describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained β-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogs of other peptide hormones.
  23. 23
    Meng, H.; Krishnaji, S. T.; Beinborn, M.; Kumar, K. Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1. J. Med. Chem. 2008, 51, 7303– 7307,  DOI: 10.1021/jm8008579
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    Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1
    Meng, He; Krishnaji, Subrahmanian Tarakkad; Beinborn, Martin; Kumar, Krishna
    Journal of Medicinal Chemistry (2008), 51 (22), 7303-7307CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogs of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogs displayed higher proteolytic stability with simultaneous retention of biol. activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions.
  24. 24
    Ueda, T.; Tomita, K.; Notsu, Y.; Ito, T.; Fumoto, M.; Takakura, T.; Nagatome, H.; Takimoto, A.; Mihara, S.; Togame, H.; Kawamoto, K.; Iwasaki, T.; Asakura, K.; Oshima, T.; Hanasaki, K.; Nishimura, S.; Kondo, H. Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity. J. Am. Chem. Soc. 2009, 131, 6237– 6245,  DOI: 10.1021/ja900261g
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    24
    Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity
    Ueda, Taichi; Tomita, Kazuyoshi; Notsu, Yoshihide; Ito, Takaomi; Fumoto, Masataka; Takakura, Tomoaki; Nagatome, Hirofumi; Takimoto, Akio; Mihara, Shin-Ichi; Togame, Hiroko; Kawamoto, Keiko; Iwasaki, Takanori; Asakura, Kenji; Oshima, Takeo; Hanasaki, Kohji; Nishimura, Shin-Ichiro; Kondo, Hirosato
    Journal of the American Chemical Society (2009), 131 (17), 6237-6245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogs with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and α2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepd. by chemoenzymic approaches. We assessed the receptor binding affinity and cAMP prodn. activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addn. of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic anal. of the degrdn. reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addn., the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogs with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogs of other bioactive peptides as desirable pharmaceuticals.
  25. 25
    Lau, J.; Bloch, P.; Schaffer, L.; Pettersson, I.; Spetzler, J.; Kofoed, J.; Madsen, K.; Knudsen, L. B.; McGuire, J.; Steensgaard, D. B.; Strauss, H. M.; Gram, D. X.; Knudsen, S. M.; Nielsen, F. S.; Thygesen, P.; Reedtz-Runge, S.; Kruse, T. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J. Med. Chem. 2015, 58, 7370– 7380,  DOI: 10.1021/acs.jmedchem.5b00726
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    25
    Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide
    Lau, Jesper; Bloch, Paw; Schaffer, Lauge; Pettersson, Ingrid; Spetzler, Jane; Kofoed, Jacob; Madsen, Kjeld; Knudsen, Lotte Bjerre; McGuire, James; Steensgaard, Dorte Bjerre; Strauss, Holger Martin; Gram, Dorte X.; Knudsen, Sanne Moeller; Nielsen, Flemming Seier; Thygesen, Peter; Reedtz-Runge, Steffen; Kruse, Thomas
    Journal of Medicinal Chemistry (2015), 58 (18), 7370-7380CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analog that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analog by increasing albumin affinity and secure full stability against metabolic degrdn. The fatty acid moiety and the linking chem. to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analog. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib8, Arg34) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clin. testing.
  26. 26
    Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C. J.; Harriott, P.; Greer, B.; O’Harte, F. P.; Flatt, P. R. N-terminal His(7)-modification of glucagon-like peptide-1(7–36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity. J. Endocrinol. 2004, 180, 379– 388,  DOI: 10.1677/joe.0.1800379
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    26
    N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity
    Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C. J.; Harriott, P.; Greer, B.; O'Harte, F. P. M.; Flatt, P. R.
    Journal of Endocrinology (2004), 180 (3), 379-388CODEN: JOENAK; ISSN:0022-0795. (Society for Endocrinology)
    Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analog of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesized and tested for DPP IV stability and biol. activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degrdn. of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degrdn. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32·9 and 6·7 nM, resp.) compared with native GLP-1 (IC50 0·37 nM). Similarly, both analogs stimulated cAMP prodn. with EC50 values of 16·3 and 27 nM resp. compared with GLP-1 (EC50 4·7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5·6 mM glucose (P<0·05 to P<0·001) similar to native GLP-1. Both analogs (25 nM/kg body wt.) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body wt.) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biol. potent forms of GLP-1. The particularly powerful antihyperglycemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.
  27. 27
    Wootten, D.; Reynolds, C. A.; Koole, C.; Smith, K. J.; Mobarec, J. C.; Simms, J.; Quon, T.; Coudrat, T.; Furness, S. G.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures. Mol. Pharmacol. 2016, 89, 335– 347,  DOI: 10.1124/mol.115.101246
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    27
    A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures
    Wootten, Denise; Reynolds, Christopher A.; Koole, Cassandra; Smith, Kevin J.; Mobarec, Juan C.; Simms, John; Quon, Tezz; Coudrat, Thomas; Furness, Sebastian G. B.; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.
    Molecular Pharmacology (2016), 89 (3), 335-347CODEN: MOPMA3; ISSN:1521-0111. (American Society for Pharmacology and Experimental Therapeutics)
    The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiol. significance of this is yet to be elucidated. Previous work has implicated R2.60190, N3.43240, Q7.49394, and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190, N3.43240, and Q7.49394 predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict addnl. key mol. interactions with these amino acids. We have introduced E6.53364A, N3.43240Q, Q7.49394N, and N3.43240Q/Q7.49394N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53364 and R2.60190 was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49394, but not R2.60190/E6.53364 was crit. for calcium mobilization for all three peptides. Mutation of N3.43240 and Q7.49394 had differential effects on individual peptides, providing evidence for mol. differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.
  28. 28
    Xiao, Q.; Giguere, J.; Parisien, M.; Jeng, W.; St-Pierre, S. A.; Brubaker, P. L.; Wheeler, M. B. Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo. Biochemistry 2001, 40, 2860– 2869,  DOI: 10.1021/bi0014498
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    Biological Activities of Glucagon-Like Peptide-1 Analogues in Vitro and in Vivo
    Xiao, Q.; Giguere, J.; Parisien, M.; Jeng, W.; St-Pierre, S. A.; Brubaker, P. L.; Wheeler, M. B.
    Biochemistry (2001), 40 (9), 2860-2869CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala2, its clin. application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogs. In this study, we investigated GLP-1 analogs including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala2]GLP-1 and [Gly2]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biol. effectiveness of [D-Ala2]GLP-1 was also obsd. in diabetic db/db mice. Similarly, improved biol. activity of acetyl- and hexenoic-His1-GLP-1, glucagon(1-5)-, glucagon(1-10)-, PACAP(1-5)-, VIP(1-5)-, and secretin(1-10)-GLP-1 was obsd., despite normal or lower receptor binding and activation in vitro. [Ala8/11/12/16] substitutions also increased biol. activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biol. activity. All other modified peptides examd. showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the α-amino group of His1 and replacement of Ala2 result in significantly increased biol. effects of GLP-1 in vivo, likely due to decreased degrdn. rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biol. activity.
  29. 29
    Buckley, D. I.; Habener, J. F.; Mallory, J. B.; Mojsov, S. GLP-1 Analogs Useful for Diabetes Treatment; European Patent EP 0512 042 B1; 1991.
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    Ohneda, A.; Ohneda, K.; Ohneda, M.; Koizumi, F.; Ohashi, S.; Kawai, K.; Suzuki, S. The structure-function relationship of GLP-1 related peptides in the endocrine function of the canine pancreas. Tohoku J. Exp. Med. 1991, 165, 209– 221,  DOI: 10.1620/tjem.165.209
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    The structure-function relationship of GLP-1 related peptides in the endocrine function of the canine pancreas
    Ohneda, Akira; Ohneda, Kinuko; Ohneda, Makoto; Koizumi, Fumiaki; Ohashi, Shinichi; Kawai, Koichi; Suzuki, Seiji
    Tohoku Journal of Experimental Medicine (1991), 165 (3), 209-21CODEN: TJEMAO; ISSN:0040-8727.
    In order to clarify the relationship between the structure and function of glucagon-like peptide (GLP) 1 in the endocrine function of the pancreas, the response of insulin and glucagon to various synthetic GLP-1-related peptides was investigated in anesthetized dogs. GLP-1-related peptides were administered in a dosage of 400 pmol within 10 min into the pancreatic artery during glucose or arginine infusion, and the changes in plasma insulin and glucagon in the pancreatic vein were studied. GLP-1 (7-36) and (7-37), as well as glucagon enhanced insulin release during glucose infusion, whereas neither GLP-1 (1-37), (7-20), (6-37), nor (8-37) stimulated insulin release. The administration of GLP-1 (1-37), (7-36), and (7-37) reduced glucagon release during glucose infusion. When arginine was infused, GLP-1 (7-20), (7-36), (7-37), and glucagon enhanced insulin release. In contrast, glucagon release was increased by the administration of GLP-1 (7-20), (8-37), and (7-37). The present study indicates that histidine at the 7th position of GLP-1 is important in eliciting biol. action and that only truncated GLP-1 (7-36), (7-37), and (7-20) showed an insulinotropic action as strong as glucagon in dogs. Furthermore, it is suggested that the response of insulin and glucagon to GLP-1 related peptides is dependent on a background condition.
  31. 31
    Gallwitz, B.; Ropeter, T.; Morys-Wortmann, C.; Mentlein, R.; Siegel, E. G.; Schmidt, W. E. GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro. Regul. Pept. 2000, 86, 103– 111,  DOI: 10.1016/S0167-0115(99)00095-6
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    GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro
    Gallwitz, B.; Ropeter, T.; Morys-Wortmann, C.; Mentlein, R.; Siegel, E. G.; Schmidt, W. E.
    Regulatory Peptides (2000), 86 (1-3), 103-111CODEN: REPPDY; ISSN:0167-0115. (Elsevier Science Ireland Ltd.)
    Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biol. half-time limits the therapeutic use of GLP-1. DPP IV requires an intact α-amino-group of the N-terminal histidine of GLP-1 in order to perform its enzymic activity. Therefore, the following GLP-1 analogs with alterations in the N-terminal position 1 were synthesized: N-methylated- (N-me-GLP-1), α-methylated (α-me-GLP-1), desamidated- (desamino-GLP-1) and imidazole-lactic-acid substituted GLP-1 (imi-GLP-1). All GLP-1 analogs except α-me-GLP-1 were hardly degraded by DPP IV in vitro. The GLP-1 analogs showed receptor affinity and in vitro biol. activity comparable to native GLP-1 in RINm5F cells. GLP-1 receptor affinity was highest for imi-GLP-1, followed by α-me-GLP-1 and N-me-GLP-1. Only desamino-GLP-1 showed a 15-fold loss of receptor affinity compared to native GLP-1. All analogs stimulated intracellular cAMP prodn. in RINm5F cells in concns. comparable to GLP-1. N-terminal modifications might therefore be useful in the development of long-acting GLP-1 analogs for type 2 diabetes therapy.
  32. 32
    Hareter, A.; Hoffmann, E.; Bode, H. P.; Goke, B.; Goke, R. The positive charge of the imidazole side chain of histidine(7) is crucial for GLP-1 action. Endocr. J. 1997, 44, 701– 705,  DOI: 10.1507/endocrj.44.701
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    The positive charge of the imidazole side chain of histidine7 is crucial for GLP-1 action
    Hareter, Alexandra; Hoffmann, Eike; Bode, Hans-Peter; Goke, Burkhard; Goke, Rudiger
    Endocrine Journal (Tokyo) (1997), 44 (5), 701-705CODEN: ENJOEO; ISSN:0918-8959. (Japan Endocrine Society)
    Glucagon-like peptide-1(7-36)amide/(7-37) (GLP-1) is an incretin hormone which plays an important role in postprandial glucose homeostasis. Since GLP-1 potentiates glucose-induced insulin secretion, stimulates insulin biosynthesis and inhibits glucagon release, it is a potential tool for the treatment of diabetes mellitus. For this, an exact understanding of the structural/functional moieties of the peptide is mandatory. The present study investigates the importance of structural features of histidine7 at the N-terminus for GLP-1 action. Based upon binding and activity data obtained from ten different GLP-1 analogs not the pos. charge of the free α-amino group but the pos. charge of the imidazole side chain of histidine is crucial for GLP-1 action. The presence of a ring structure and a basic function as well as the correct positioning of both seems to be decisive.
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    DesMarteau, D. D.; Montanari, V. Easy preparation of bioactive peptides from the novel N-alpha-trifluoroethyl amino acids. Chem. Lett. 2000, 29, 1052– 1053,  DOI: 10.1246/cl.2000.1052
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    Baggio, L. L.; Drucker, D. J. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007, 132, 2131– 2157,  DOI: 10.1053/j.gastro.2007.03.054
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    Biology of incretins: GLP-1 and GIP
    Baggio, Laurie L.; Drucker, Daniel J.
    Gastroenterology (2007), 132 (6), 2131-2157CODEN: GASTAB; ISSN:0016-5085. (Elsevier Inc.)
    A review. This review focuses on the mechanisms regulating the synthesis, secretion, biol. actions, and therapeutic relevance of the incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The published literature was reviewed, with emphasis on recent advances in our understanding of the biol. of GIP and GLP-1. GIP and GLP-1 are both secreted within minutes of nutrient ingestion and facilitate the rapid disposal of ingested nutrients. Both peptides share common actions on islet β-cells acting through structurally distinct yet related receptors. Incretin-receptor activation leads to glucose-dependent insulin secretion, induction of β-cell proliferation, and enhanced resistance to apoptosis. GIP also promotes energy storage via direct actions on adipose tissue, and enhances bone formation via stimulation of osteoblast proliferation and inhibition of apoptosis. In contrast, GLP-1 exerts glucoregulatory actions via slowing of gastric emptying and glucose-dependent inhibition of glucagon secretion. GLP-1 also promotes satiety and sustained GLP-1-receptor activation is assocd. with wt. loss in both preclin. and clin. studies. The rapid degrdn. of both GIP and GLP-1 by the enzyme dipeptidyl peptidase-4 has led to the development of degrdn.-resistant GLP-1-receptor agonists and dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes. These agents decrease Hb A1c (HbA1c) safely without wt. gain in subjects with type 2 diabetes. GLP-1 and GIP integrate nutrient-derived signals to control food intake, energy absorption, and assimilation. Recently approved therapeutic agents based on potentiation of incretin action provide new physiol. based approaches for the treatment of type 2 diabetes.
  35. 35
    Parthier, C.; Kleinschmidt, M.; Neumann, P.; Rudolph, R.; Manhart, S.; Schlenzig, D.; Fanghanel, J.; Rahfeld, J. U.; Demuth, H. U.; Stubbs, M. T. Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 13942– 13947,  DOI: 10.1073/pnas.0706404104
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    Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor
    Parthier, Christoph; Kleinschmidt, Martin; Neumann, Piotr; Rudolph, Rainer; Manhart, Susanne; Schlenzig, Dagmar; Fanghanel, Joerg; Rahfield, Hans-Ulrich; Stubbs, Milton T.
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (35), 13942-13947, S13942/1-S13942/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Incretins, endogenous polypeptide hormones released in response to food intake, potentiate insulin secretion from pancreatic β cells after oral glucose ingestion (the incretin effect). This response is signaled by the two peptide hormones glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) and glucagon-like peptide 1 through binding and activation of their cognate class 2 G protein-coupled receptors (GPCRs). Because the incretin effect is lost or significantly reduced in patients with type 2 diabetes mellitus, glucagon-like peptide 1 and GIP have attracted considerable attention for their potential in antidiabetic therapy. A paucity of structural information precludes a detailed understanding of the processes of hormone binding and receptor activation, hampering efforts to develop novel pharmaceuticals. Here we report the crystal structure of the complex of human GIP receptor extracellular domain (ECD) with its agonist, the incretin GIP1-42. The hormone binds in an a-helical conformation in a surface groove of the ECD largely through hydrophobic interactions. The N-terminal ligand residues would remain free to interact with other parts of the receptor. Thermodn. data suggest that binding is concomitant with structural organization of the hormone, resulting in a complex mode of receptor-ligand recognition. The presentation of a well structured, a-helical ligand by the ECD is expected to be conserved among other hormone receptors of this class.
  36. 36
    Underwood, C. R.; Parthier, C.; Reedtz-Runge, S. Structural Basis for Ligand Recognition of Incretin Receptors. Vitam. Horm. 2010, 84, 251– 278,  DOI: 10.1016/B978-0-12-381517-0.00009-6
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    Structural basis for ligand recognition of incretin receptors
    Underwood, Christina Rye; Parthier, Christoph; Reedtz-Runge, Steffen
    Vitamins and Hormones (San Diego, CA, United States) (2010), 84 (Incretins and Insulin Secretion), 251-278CODEN: VIHOAQ; ISSN:0083-6729. (Elsevier Inc.)
    A review. The glucose-dependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide-1 (GLP-1) receptor are homologous G-protein-coupled receptors (GPCRs). Incretin receptor agonists stimulate the synthesis and secretion of insulin from pancreatic β-cells and are therefore promising agents for the treatment of type 2 diabetes. It is well established that the N-terminal extracellular domain (ECD) of incretin receptors is important for ligand binding and ligand specificity, whereas the transmembrane domain is involved in receptor activation. Structures of the ligand-bound ECD of incretin receptors have been solved recently by X-ray crystallog. The crystal structures reveal a similar fold of the ECD and a similar mechanism of ligand binding, where the ligand adopts an α-helical conformation. Residues in the C-terminal part of the ligand interact directly with the ECD and hydrophobic interactions appear to be the main driving force for ligand binding to the ECD of incretin receptors. Obviously, the-still missing-structures of full-length incretin receptors are required to construct a complete picture of receptor function at the mol. level. However, the progress made recently in structural anal. of the ECDs of incretin receptors and related GPCRs has shed new light on the process of ligand recognition and binding and provided a basis to disclose some of the mechanisms underlying receptor activation at high resoln.
  37. 37
    Nauck, M. A.; Heimesaat, M. M.; Orskov, C.; Holst, J. J.; Ebert, R.; Creutzfeldt, W. Preserved incretin activity of glucagon-like peptide 1 [7–36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J. Clin. Invest. 1993, 91, 301– 307,  DOI: 10.1172/JCI116186
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    Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus
    Nauck M A; Heimesaat M M; Orskov C; Holst J J; Ebert R; Creutzfeldt W
    The Journal of clinical investigation (1993), 91 (1), 301-7 ISSN:0021-9738.
    In type-2 diabetes, the overall incretin effect is reduced. The present investigation was designed to compare insulinotropic actions of exogenous incretin hormones (gastric inhibitory peptide [GIP] and glucagon-like peptide 1 [GLP-1] [7-36 amide]) in nine type-2 diabetic patients (fasting plasma glucose 7.8 mmol/liter; hemoglobin A1c 6.3 +/- 0.6%) and in nine age- and weight-matched normal subjects. Synthetic human GIP (0.8 and 2.4 pmol/kg.min over 1 h each), GLP-1 [7-36 amide] (0.4 and 1.2 pmol/kg.min over 1 h each), and placebo were administered under hyperglycemic clamp conditions (8.75 mmol/liter) in separate experiments. Plasma GIP and GLP-1 [7-36 amide] concentrations (radioimmunoassay) were comparable to those after oral glucose with the low, and clearly supraphysiological with the high infusion rates. Both GIP and GLP-1 [7-36 amide] dose-dependently augmented insulin secretion (insulin, C-peptide) in both groups (P < 0.05). With GIP, the maximum effect in type-2 diabetic patients was significantly lower (by 54%; P < 0.05) than in normal subjects. With GLP-1 [7-36 amide] type-2 diabetic patients reached 71% of the increments in C-peptide of normal subjects (difference not significant). Glucagon was lowered during hyperglycemic clamps in normal subjects, but not in type-2 diabetic patients, and further by GLP-1 [7-36 amide] in both groups (P < 0.05), but not by GIP. In conclusion, in mild type-2 diabetes, GLP-1 [7-36 amide], in contrast to GIP, retains much of its insulinotropic activity. It also lowers glucagon concentrations.
  38. 38
    Holst, J. J.; Gromada, J.; Nauck, M. A. The pathogenesis of NIDDM involves a defective expression of the GIP receptor. Diabetologia 1997, 40, 984– 986,  DOI: 10.1007/s001250050779
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    The pathogenesis of NIDDM involves a defective expression of the GIP receptor
    Holst, J. J.; Gromada, J.; Nauck, M. A.
    Diabetologia (1997), 40 (8), 984-986CODEN: DBTGAJ; ISSN:0012-186X. (Springer)
    A review and discussion with 34 refs., describing decreased incretin effect in non-insulin-dependent diabetes mellitus (NIDDM) patients, full efficacy of glucagon-like peptide-1 (GLP-1) and lack of effect of Glc-dependent insulinotropic polypeptide (GIP) on insulin secretion, and absence of incretin effect (small loads of Glc) at normal GIP secretion in diabetic β-cells. The apparent polygenicity of NIDDM is hypothesized to be caused by genetically defective expression of the GIP receptor.
  39. 39
    Vilsboll, T.; Krarup, T.; Madsbad, S.; Holst, J. J. Defective amplification of the late phase insulin response to glucose by GIP in obese Type II diabetic patients. Diabetologia 2002, 45, 1111– 1119,  DOI: 10.1007/s00125-002-0878-6
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    Defective amplification of the late phase insulin response to glucose by GIP in obese Type II diabetic patients
    Vilsboll, T.; Krarup, T.; Madsbad, S.; Holst, J. J.
    Diabetologia (2002), 45 (8), 1111-1119CODEN: DBTGAJ; ISSN:0012-186X. (Springer-Verlag)
    Aims/hypothesis. Glucagon-like-peptide-1 (GLP-1) is strongly insulinotropic in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas glucose-dependent insulinotropic polypeptide (GIP) is less effective. Our investigation evaluated "early" (protocol 1) - and "late phase" (protocol 2) insulin and C-peptide responses to GLP-1 and GIP stimulation in patients with Type II diabetes. Methods. Protocol 1: eight Type II diabetic patients and eight matched healthy subjects received i.v. bolus injections of GLP-1 (2.5 nmol) or GIP (7.5 nmol) concomitant with an increase of plasma glucose to 15 mmol/L. Protocol 2: eight Type II diabetic patients underwent a hyperglycemic clamp (15 mmol/L) with infusion (per kg/min) of either: 1 pmol GLP-1 (7-36) amide, 4 pmol GIP, 16 pmol, or no incretin hormone. For comparison, six matched healthy subjects were examd. Results. Protocol 1: Type II diabetic patients were characterized by a decreased "early phase" response to both stimuli, but their relative response to GIP vs. GLP-1 stimulation was exactly the same as in healthy subjects [insulin (C-peptide): patients 59% (74±6%) and healthy subjects 62% (71%)]. Protocol 2, "Early phase" (0-20 min) insulin response to glucose was delayed and reduced in the patients, but enhanced slightly and similarly by GIP and GLP-1. GLP-1 augmented the "late phase" (20-120 min) insulin secretion to levels similar to those obsd. in healthy subjects. In contrast, the "late phase" responses to both doses of GIP were not different from those obtained with glucose alone. Accordingly, glucose infusion rates required to maintain the hyperglycemic clamp in the "late phase" period (20-120 min) were similar with glucose alone and glucose plus GIP, whereas a doubling of the infusion rate was required during GLP-1 stimulation. Conclusion/interpretation. Lack of GIP amplification of the late phase insulin response to glucose, which contrasts markedly to the normalizing effect of GLP-1, could be a key defect in insulin secretion in Type II diabetic patients.
  40. 40
    Hojberg, P. V.; Vilsboll, T.; Rabol, R.; Knop, F. K.; Bache, M.; Krarup, T.; Holst, J. J.; Madsbad, S. Four weeks of near-normalisation of blood glucose improves the insulin response to glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes. Diabetologia 2009, 52, 199– 207,  DOI: 10.1007/s00125-008-1195-5
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    Four weeks of near-normalisation of blood glucose improves the insulin response to glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes
    Hojberg P V; Vilsboll T; Rabol R; Knop F K; Bache M; Krarup T; Holst J J; Madsbad S
    Diabetologia (2009), 52 (2), 199-207 ISSN:.
    OBJECTIVE: The incretin effect is attenuated in patients with type 2 diabetes mellitus, partly as a result of impaired beta cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The aim of the present study was to investigate whether 4 weeks of near-normalisation of the blood glucose level could improve insulin responses to GIP and GLP-1 in patients with type 2 diabetes. METHODS: Eight obese patients with type 2 diabetes with poor glycaemic control (HbA(1c) 8.6 +/- 1.3%), were investigated before and after 4 weeks of near-normalisation of blood glucose (mean blood glucose 7.4 +/- 1.2 mmol/l) using insulin treatment. Before and after insulin treatment the participants underwent three hyperglycaemic clamps (15 mmol/l) with infusion of GLP-1, GIP or saline. Insulin responses were evaluated as the incremental area under the plasma C-peptide curve. RESULTS: Before and after near-normalisation of blood glucose, the C-peptide responses did not differ during the early phase of insulin secretion (0-10 min). The late phase C-peptide response (10-120 min) increased during GIP infusion from 33.0 +/- 8.5 to 103.9 +/- 24.2 (nmol/l) x (110 min)(-1) (p < 0.05) and during GLP-1 infusion from 48.7 +/- 11.8 to 126.6 +/- 32.5 (nmol/l) x (110 min)(-1) (p < 0.05), whereas during saline infusion the late-phase response did not differ before vs after near-normalisation of blood glucose (40.2 +/- 11.2 vs 46.5 +/- 12.7 [nmol/l] x [110 min](-1)). CONCLUSIONS: Near-normalisation of blood glucose for 4 weeks improves beta cell responsiveness to both GLP-1 and GIP by a factor of three to four. No effect was found on beta cell responsiveness to glucose alone. CLINICALTRIALS.GOV ID NO.: NCT 00612950. FUNDING: This study was supported by The Novo Nordisk Foundation, The Medical Science Research Foundation for Copenhagen.
  41. 41
    Habegger, K. M.; Heppner, K. M.; Geary, N.; Bartness, T. J.; DiMarchi, R.; Tschop, M. H. The metabolic actions of glucagon revisited. Nat. Rev. Endocrinol. 2010, 6, 689– 697,  DOI: 10.1038/nrendo.2010.187
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    The metabolic actions of glucagon revisited
    Habegger, Kirk M.; Heppner, Kristy M.; Geary, Nori; Bartness, Timothy J.; DiMarchi, Richard; Tschoep, Matthias H.
    Nature Reviews Endocrinology (2010), 6 (12), 689-697CODEN: NREABD; ISSN:1759-5029. (Nature Publishing Group)
    A review. The diabetogenic effect of glucagon has long overshadowed the potential of this pancreatic hormone as an endogenous satiety and anti-obesity factor. This Review discusses the role of glucagon as a beneficial endocrine factor in lipid and energy metab. and its potential as a therapeutic agent on the basis of studies that combine the agonism of glucagon receptor and glucagon-like peptide 1 receptor. The initial identification of glucagon as a counter-regulatory hormone to insulin revealed this hormone to be of largely singular physiol. and pharmacol. purpose. Glucagon agonism, however, has also been shown to exert effects on lipid metab., energy balance, body adipose tissue mass and food intake. The ability of glucagon to stimulate energy expenditure, along with its hypolipidemic and satiating effects, in particular, make this hormone an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Studies that describe novel preclin. applications of glucagon, alone and in concert with glucagon-like peptide 1 agonism, have revealed potential benefits of glucagon agonism in the treatment of the metabolic syndrome. Collectively, these observations challenge us to thoroughly investigate the physiol. and therapeutic potential of insulin's long-known opponent.
  42. 42
    Finan, B.; Yang, B.; Ottaway, N.; Smiley, D. L.; Ma, T.; Clemmensen, C.; Chabenne, J.; Zhang, L.; Habegger, K. M.; Fischer, K.; Campbell, J. E.; Sandoval, D.; Seeley, R. J.; Bleicher, K.; Uhles, S.; Riboulet, W.; Funk, J.; Hertel, C.; Belli, S.; Sebokova, E.; Conde-Knape, K.; Konkar, A.; Drucker, D. J.; Gelfanov, V.; Pfluger, P. T.; Muller, T. D.; Perez-Tilve, D.; DiMarchi, R. D.; Tschop, M. H. A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat. Med. 2015, 21, 27– 36,  DOI: 10.1038/nm.3761
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    A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents
    Finan, Brian; Yang, Bin; Ottaway, Nickki; Smiley, David L.; Ma, Tao; Clemmensen, Christoffer; Chabenne, Joe; Zhang, Lianshan; Habegger, Kirk M.; Fischer, Katrin; Campbell, Jonathan E.; Sandoval, Darleen; Seeley, Randy J.; Bleicher, Konrad; Uhles, Sabine; Riboulet, William; Funk, Juergen; Hertel, Cornelia; Belli, Sara; Sebokova, Elena; Conde-Knape, Karin; Konkar, Anish; Drucker, Daniel J.; Gelfanov, Vasily; Pfluger, Paul T.; Mueller, Timo D.; Perez-Tilve, Diego; Di Marchi, Richard D.; Tschoep, Matthias H.
    Nature Medicine (New York, NY, United States) (2015), 21 (1), 27-36CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)
    We report the discovery of a new monomeric peptide that reduces body wt. and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiol. potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimol. triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body wt., enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacol. blockade and selective chem. knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclin. studies suggest that, so far, this unimol., polypharmaceutical strategy has potential to be the most effective pharmacol. approach to reversing obesity and related metabolic disorders.
  43. 43
    Pospisilik, J. A.; Hinke, S. A.; Pederson, R. A.; Hoffmann, T.; Rosche, F.; Schlenzig, D.; Glund, K.; Heiser, U.; McIntosh, C. H.; Demuth, H. Metabolism of glucagon by dipeptidyl peptidase IV (CD26). Regul. Pept. 2001, 96, 133– 141,  DOI: 10.1016/S0167-0115(00)00170-1
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    Metabolism of glucagon by dipeptidyl peptidase IV (CD26)
    Pospisilik, J. A.; Hinke, S. A.; Pederson, R. A.; Hoffmann, T.; Rosche, F.; Schlenzig, D.; Glund, K.; Heiser, U.; McIntosh, C. H. S.; Demuth, H.-U.
    Regulatory Peptides (2001), 96 (3), 133-141CODEN: REPPDY; ISSN:0167-0115. (Elsevier Science Ireland Ltd.)
    Glucagon is a 29-amino acid polypeptide released from pancreatic islet α-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymic metab. of glucagon was assessed using sensitive mass spectrometric techniques to identify the mol. products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential prodn. of glucagon3-29 and glucagon5-29. In human serum, degrdn. to glucagon3-29 was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degrdn., monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degrdn. and inactivation of glucagon. These findings have important implications for the detn. of glucagon levels in human plasma.
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    Jaspan, J. B.; Polonsky, K. S.; Lewis, M.; Pensler, J.; Pugh, W.; Moossa, A. R.; Rubenstein, A. H. Hepatic-Metabolism of Glucagon in the Dog - Contribution of the Liver to Overall Metabolic Disposal of Glucagon. Am. J. Physiol. 1981, 240, E233– E244,  DOI: 10.1152/ajpendo.1981.240.3.E233
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    Hepatic metabolism of glucagon in the dog: contribution of the liver to overall metabolic disposal of glucagon
    Jaspan, J. B.; Polonsky, K. S.; Lewis, M.; Pensler, J.; Pugh, W.; Moossa, A. R.; Rubenstein, A. H.
    American Journal of Physiology (1981), 240 (3), E233-E244CODEN: AJPHAP; ISSN:0002-9513.
    The hepatic extn. (HE) of glucagon (I) [9007-92-5] and insulin (II) [9004-10-8] was measured in dogs, by peripheral infusion of the hormones following pancreatectomy or somatostatin [51110-01-1] infusion. HE of I was 22.5% and that of II was 45.1%. Somatostatin did not affect HE of either hormone. HE of endogenous II was similar to that of exogenously infused II. HE I was nonsaturable in the physiologic and pathophysiologic range of plasma I levels, but there was evidence of saturability in the pharmacologic range. Comparison of simultaneously measured parameters of II and I metab. indicated independence of the metabolic processes of these 2 islet hormones, despite distinct similarities in their overall patterns of metabolic disposal. Apparently, the liver is an important site for I removal.
  45. 45
    Unson, C. G.; Macdonald, D.; Merrifield, R. B. The role of histidine-1 in glucagon action. Arch. Biochem. Biophys. 1993, 300, 747– 750,  DOI: 10.1006/abbi.1993.1103
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    The role of histidine-1 in glucagon action
    Unson, Cecilia G.; Macdonald, Douglas; Merrifield, R. B.
    Archives of Biochemistry and Biophysics (1993), 300 (2), 747-50CODEN: ABBIA4; ISSN:0003-9861.
    The identification of position 9 aspartic acid in glucagon as a crit. residue for transduction reinforced the notion that specific residues in the peptide sequence dictate either receptor recognition or biol. activity. It was evident from previous studies that Asp9 operates in conjunction with His1 as part of the activation mechanism that follows binding to the glucagon receptor. This investigation was conducted to delineate structural features of histidine that contribute to its role in glucagon action. The authors report, based on binding and activity data from 10 replacement analogs, that the imidazole ring of His1 furnishes an arom. determinant for receptor binding affinity and that its protonatable imidazole nitrogen is important for transduction.
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    Drucker, D. J. The Discovery of GLP-2 and Development of Teduglutide for Short Bowel Syndrome. ACS Pharmacol. Transl. Sci. 2019, 2, 134– 142,  DOI: 10.1021/acsptsci.9b00016
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    The Discovery of GLP-2 and Development of Teduglutide for Short Bowel Syndrome
    Drucker, Daniel J.
    ACS Pharmacology & Translational Science (2019), 2 (2), 134-142CODEN: APTSFN; ISSN:2575-9108. (American Chemical Society)
    A review. The proglucagon gene encodes multiple structurally-related peptides with overlapping actions promoting the absorption and assimilation of ingested energy. Notably, glucagon has been developed pharmaceutically to treat hypoglycemia and glucagon-like peptide-1 (GLP-1) receptor agonists are used for the therapy of type 2 diabetes and obesity. Here I describe the discovery of glucagon-like peptide-2 (GLP-2), a 33 amino acid peptide co-secreted together with GLP-1 from gut endocrine cells. GLP-2 was found to exhibit robust intestinal growth-promoting activity, following serendipitous observations that proglucagon-producing tumors induced intestinal growth in mice. Key developments in the pharmaceutical development of GLP-2 included the cloning of the GLP-2 receptor, and the recognition of the importance of dipeptidyl peptidase-4 as a crit. determinant of GLP-2 bioactivity. A therapeutic focus on short bowel syndrome, a serious medical disorder with compelling unmet medical need, enabled the pharmaceutical development of a simple GLP-2 analog, teduglutide, suitable for once daily administration.
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    Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discovery Today 2015, 20, 122– 128,  DOI: 10.1016/j.drudis.2014.10.003
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    Peptide therapeutics: current status and future directions
    Fosgerau, Keld; Hoffmann, Torsten
    Drug Discovery Today (2015), 20 (1), 122-128CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)
    A review. Peptides are recognized for being highly selective and efficacious and, at the same time, relatively safe and well tolerated. Consequently, there is an increased interest in peptides in pharmaceutical research and development (R&D), and approx. 140 peptide therapeutics are currently being evaluated in clin. trials. Given that the low-hanging fruits in the form of obvious peptide targets have already been picked, it has now become necessary to explore new routes beyond traditional peptide design. Examples of such approaches are multifunctional and cell penetrating peptides, as well as peptide drug conjugates. Here, we discuss the current status, strengths, and weaknesses of peptides as medicines and the emerging new opportunities in peptide drug design and development.
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    Marini, M.; Urbani, A.; Trani, E.; Bongiorno, L.; Roda, L. G. Interindividual variability of enkephalin-degrading enzymes in human plasma. Peptides 1997, 18, 741– 748,  DOI: 10.1016/S0196-9781(97)00129-0
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    Interindividual variability of enkephalin-degrading enzymes in human plasma
    Marini, Mario; Urbani, Alessandra; Trani, Eugenia; Bongiorno, Lucilla; Roda, L. Giorgio
    Peptides (Tarrytown, New York) (1997), 18 (5), 741-748CODEN: PPTDD5; ISSN:0196-9781. (Elsevier)
    The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis byproducts has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class - as well as the expression of the single enzyme species within each class - appears to be individually detd. Interindividual variability obsd. seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low mol. wt. plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the compn. of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and const. in blood obtained from successive drawings, at least within a relatively short period of time.
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    Yakovleva, A. A.; Zolotov, N. N.; Sokolov, O. Y.; Kost, N. V.; Kolyasnikova, K. N.; Micheeva, I. G. Dipeptidylpeptidase 4 (DPP4, CD26) activity in the blood serum of term and preterm neonates with cerebral ischemia. Neuropeptides 2015, 52, 113– 117,  DOI: 10.1016/j.npep.2015.05.001
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    Dipeptidylpeptidase 4 (DPP4, CD26) activity in the blood serum of term and preterm neonates with cerebral ischemia
    Yakovleva, A. A.; Zolotov, N. N.; Sokolov, O. Yu.; Kost, N. V.; Kolyasnikova, K. N.; Micheeva, I. G.
    Neuropeptides (Oxford, United Kingdom) (2015), 52 (), 113-117CODEN: NRPPDD; ISSN:0143-4179. (Elsevier Ltd.)
    To investigate the mechanisms of inflammation in neonates after cerebral ischemia (CI), we evaluated the DPP4 activity in their blood sera and compared these values with clin. indicators. The activity of DPP4 was detd. in blood serum by a fluorescent method. We studied the correlation between the blood serum DPP4 activity and clin., neurol. and biochem. parameters in neonates with CI. No correlation between the DPP4 activity in umbilical blood and the venous blood of mothers was discovered. Increased blood serum DPP4 activity in full-term and pre-term newborns with CI is demonstrated. The interrelation between serum DPP4 activity and the functional disturbances of CNS (such as depression or excitement) was found in mature but not in premature newborns. Enzyme activity was still elevated at 2-3 wk after birth. It is possible that in neonates this enzymic system operates independently from mothers. It is assumed that increased DPP4 activity in newborns with CI is apparently connected with immune system activation in response to hypoxic stress. The obtained data support the participation of DPP4 in adaptive reactions of newborns and its regulating influence during hypoxemic damage of the CNS due to inflammation and neurodegeneration.
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    Pels, K.; Kodadek, T. Solid-phase synthesis of diverse peptide tertiary amides by reductive amination. ACS Comb. Sci. 2015, 17, 152– 155,  DOI: 10.1021/acscombsci.5b00007
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    Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination
    Pels, Kevin; Kodadek, Thomas
    ACS Combinatorial Science (2015), 17 (3), 152-155CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)
    The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chem. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3-strain interactions. Here, the authors report an improved synthesis of these species on solid supports through the use of reductive amination chem. using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chem. is demonstrated by the synthesis of a library of 10 000 mixed peptoid-PTA oligomers.
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    Xing, X.; Fichera, A.; Kumar, K. A novel synthesis of enantiomerically pure 5,5,5,5′,5′,5′-hexafluoroleucine. Org. Lett. 2001, 3, 1285– 1286,  DOI: 10.1021/ol015567e
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    A Novel Synthesis of Enantiomerically Pure 5,5,5,5',5',5'-Hexafluoroleucine
    Xing, Xuechao; Fichera, Alfio; Kumar, Krishna
    Organic Letters (2001), 3 (9), 1285-1286CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A novel, short, and efficient synthesis of (S)-5,5,5,5',5',5'-hexafluoroleucine (6) in greater than 99% ee was carried out starting from Garner's aldehyde, a protected oxazolidine aldehyde. The enantiomeric excess of the product was calcd. from an NMR anal. of a dipeptide formed by reaction with a protected L-serine deriv. Furthermore, a racemic sample of N-acylated hexafluoroleucine was enzymically resolved by treatment with porcine kidney acylase I and was found to have the same optical rotation as a synthetic sample of 6.
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    Bušek, P.; Malík, R.; Šedo, A. Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer. Int. J. Biochem. Cell Biol. 2004, 36, 408– 421,  DOI: 10.1016/S1357-2725(03)00262-0
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    Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer
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    A review. Post-translational modification of proteins is an important regulatory event. Numerous biol. active peptides that play an essential role in cancerogenesis contain an evolutionary conserved proline residue as a proteolytic-processing regulatory element. Proline-specific proteases could therefore be viewed as important "check-points". Limited proteolysis of such peptides may lead to quant. but, importantly, due to the change of receptor preference, also qual. changes of their signaling potential. Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with CD26) was for many years believed to be a unique cell membrane protease cleaving X-Pro dipeptides from the N-terminal end of peptides and proteins. Subsequently, a no. of other mols. were discovered, exhibiting various degree of structural homol. and DPP-IV-like enzyme activity, capable of cleaving similar set of substrates. These comprise for example, seprase, fibroblast activation protein α, DPP6, DPP8, DPP9, attractin, N-acetylated-α-linked-acidic dipeptidases I, II and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-β. It is tempting to speculate their potential participation on DPP-IV biol. function(s). Disrupted expression and enzymic activity of "DPP-IV activity and/or structure homologs" (DASH) might corrupt the message carried by their substrates, promoting abnormal cell behavior. Consequently, modulation of particular enzyme activity using e.g. DASH inhibitors, specific antibodies or DASH expression modification may be an attractive therapeutic concept in cancer treatment. This review summarizes recent information on the interactions between DASH members and their substrates with respect to their possible role in cancer biol.
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    Biochemical Journal (2006), 396 (2), 391-399CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 resp. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar Ki values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.
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    Hager, M. V.; Johnson, L. M.; Wootten, D.; Sexton, P. M.; Gellman, S. H. beta-Arrestin-Biased Agonists of the GLP-1 Receptor from beta-Amino Acid Residue Incorporation into GLP-1 Analogues. J. Am. Chem. Soc. 2016, 138, 14970– 14979,  DOI: 10.1021/jacs.6b08323
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    Journal of the American Chemical Society (2016), 138 (45), 14970-14979CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Activation of a G protein-coupled receptor (GPCR) causes recruitment of multiple intracellular proteins, each of which can activate distinct signaling pathways. This complexity has engendered interest in agonists that preferentially stimulate subsets among the natural signaling pathways ("biased agonists"). We have examd. analogs of glucagon-like peptide-1 (GLP-1) contg. β-amino acid residues in place of native α residues at selected sites and found that some analogs differ from GLP-1 in terms of their relative abilities to promote G protein activation (as monitored via cAMP prodn.) vs. β-arrestin recruitment (as monitored via BRET assays). The α → β replacements generally cause modest declines in stimulation of cAMP prodn. and β-arrestin recruitment, but for some replacement sets cAMP prodn. is more strongly affected than is β-arrestin recruitment. The central portion of GLP-1 appears to be crit. for achieving bias toward β-arrestin recruitment. These results suggest that backbone modification via α → β residue replacement may be a versatile source of agonists with biased GLP-1R activation profiles.
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    Made, V.; Babilon, S.; Jolly, N.; Wanka, L.; Bellmann-Sickert, K.; Diaz Gimenez, L. E.; Morl, K.; Cox, H. M.; Gurevich, V. V.; Beck-Sickinger, A. G. Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias. Angew. Chem., Int. Ed. 2014, 53, 10067– 10071,  DOI: 10.1002/anie.201403750
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    Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.
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    Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells
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    Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gsα, Gq/11α, and Gi1,2α, but not Gi3α. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), resp. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, resp. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.
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    Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (2001), 1546 (1), 79-86CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
    It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Gαs proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP prodn. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Gαs but also to the Gαi/Gαo type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.
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    Quoyer, J.; Longuet, C.; Broca, C.; Linck, N.; Costes, S.; Varin, E.; Bockaert, J.; Bertrand, G.; Dalle, S. GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells. J. Biol. Chem. 2010, 285, 1989– 2002,  DOI: 10.1074/jbc.M109.067207
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    GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a β-Arrestin 1-mediated ERK1/2 Activation in Pancreatic β-Cells
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    Journal of Biological Chemistry (2010), 285 (3), 1989-2002CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gαs/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacol. inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the assocn. of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

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  • Abstract

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    Figure 1

    Figure 1. (a) Cryo-EM structure of the GLP1:GLP1R complex (PDB: 5VAI). (10) GLP1 (ribbon, gold) bound to cognate G protein-coupled receptor, GLP1R (ribbon and surface, gray), with solid gray lines representing approximate locations of the cellular membrane that separate the extracellular domain (ECD) from the transmembrane domain (TMD) of GLP1R. (b) Illustration of receptor amino acids within 4 Å of the N-terminal histidine of GLP1. Residue numbers denote the Wootten nomenclature (11) for class B GPCRs. To note, human GLP1R contains R3105.40, but this PDB structure contains an alanine mutation at this position. The dashed pink circle indicates approximate space where N-terminal decorations may reside. (c) Flattened 2D rendering of the interactions of GLP1R with His7 of GLP1. R299ECL2 forms two hydrogen bonds with the N-terminal histidine (dashed line, gray) and a putative cation−π interaction of the guanidine group of R299ECL2 with the imidazole of His7 of GLP1 (dotted line, maroon). Select neighboring side chains of the receptor are shown at approximate positions relative to GLP1. (d) 2D depiction of DPP4 active site, with a known inhibitor bound (valine–pyrrolidide, blue, PDB: 1N1M). Important electrostatic interactions (dashed lines, gray) occur between the primary amine of the substrate and Glu205 and Glu206 of DPP4 (highlighted yellow). The carbonyl of the first amide bond is anchored by a hydrogen bond to Asn710 (dashed line, gray). The catalytic triad (Ser630, His710, and Asp708) forms a hydrogen bonding network (dashed lines, gray) and is positioned proximal to the pyrrole ring. If the structure bound were a peptide substrate, the labile amide bond would be located close to the canonical nucleophile Ser630.

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    Figure 2

    Figure 2. Library of N-terminally modified peptides. (a) Alignment of GLP1 and related peptides with positions and numbering above each residue (gray). GLP1 starts with amino acid 7 based on established convention. (3) Blue residues are homologous to GLP1, and residues highlighted yellow are conserved between all peptides. Liraglutide and triagonist contain a lysine (K, maroon) modified with a γ-glutamic acid spacer and by palmitoylation (right). Semaglutide contains a modified lysine (K, orange) with two oliogoethylene glycol (OEG) spacers, γ-glutamic acid, and octadecanedioic acid (right). “X” denotes the noncanonical amino acid, aminoisobutyric acid (Aib, bottom right). (b) Native amino acid sequences are modified with N-terminal chemical modifications 1–19 resulting in a library of peptides, nominally “R-Peptide” where “R” is the number referencing the N-terminus modification and “Peptide” indicates the template sequence as in (a). Semaglutide and triagonist peptides were also assembled with Aib2Ala mutation denoted as R-semaglutide(Ala2) and R-triagonist(Ala2), respectively.

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    Figure 3

    Figure 3. (a) LC-MS/MS total ion chromatogram depicting the stability of GLP1 (maroon) and 2-GLP1 (pink) with (shaded) and without (nonshaded) DPP4. GLP1 incubated with DPP4 shows the same retention time as control GLP1(9–36) (gray) indicating excision of dipeptide His7Ala8 to give cleaved, c, peptide. 2-GLP1 incubated with DPP4 exhibits no change in retention time or mass. (b) LC-MS/MS total ion chromatogram depicting the stability of exenatide (purple) and 2-exenatide (light purple) with (shaded) and without DPP4 (nonshaded). Exenatide incubated with DPP4 results in a mixture of cleaved, c, and native (unreacted), n, exenatide. The retention time of cleaved exenatide, c, is the same as control exenatide(3–39) (gray). 2-Exenatide incubated with DPP4 is unreactive with no change in retention time or mass. (c) LC-MS/MS total ion chromatogram depicting the stability of liraglutide (navy) and 2-liraglutide (light blue) with (shaded) and without DPP4 (nonshaded). Liraglutide incubated with DPP4 results in a mixture of cleaved, c, and native (unreacted), n, liraglutide. 2-Liraglutide incubated with DPP4 does not undergo reaction with unchanged retention time and mass.

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    Figure 4

    Figure 4. Representative concentration–response curves of unmodified peptides (GLP1, liraglutide, GIP, and glucagon; a–d) or N-trifluoroethyl analogues (2-GLP1, 2-liraglutide, 2-GIP, and 2-glucagon; e–h) incubated overnight with DPP4 or vehicle prior to diluting into microtiter plates containing HEK293 cells overly expressing receptors (GLP1R, GIPR, or GCGR) and reporter CRE6x-luciferase. Luciferase production corresponds directly to activation of cognate GPCR via a cAMP dependent pathway, normalized to 100% maximal activity, and resultant fold-loss in EC50 upon DPP4 incubation is listed when applicable. Error bars represent SEM for three independent experiments (n = 3).

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    Figure 5

    Figure 5. N-Trifluoroethyl alkylation and lipidation of 2-liraglutide performs as well as liraglutide at lowering blood sugar levels in vivo. (a) Measured glucose levels by tail vein prick for an oral glucose tolerance test (OGTT) of fasted mice treated intraperitoneally (i.p., dotted line) with vehicle, GLP1, 2-GLP1, 7-GLP1, liraglutide, or 2-liraglutide at (1 mg/kg or 0.1 mg/kg as noted). Glucose bolus was administered orally at time 0 and 240 min (gray, upward arrow). (b) Average area under the curve (AUC) calculated from 0 to 120 min in part a. (c) Glucose levels 30 min past second glucose challenge that occurred 5 h after first OGTT. Error represents the average ± SEM (n = 5). P-values compared to vehicle: **P < 0.01; ***P < 0.001; ****P < 0.0001.

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      Muller, T. D.; Finan, B.; Bloom, S. R.; D’Alessio, D.; Drucker, D. J.; Flatt, P. R.; Fritsche, A.; Gribble, F.; Grill, H. J.; Habener, J. F.; Holst, J. J.; Langhans, W.; Meier, J. J.; Nauck, M. A.; Perez-Tilve, D.; Pocai, A.; Reimann, F.; Sandoval, D. A.; Schwartz, T. W.; Seeley, R. J.; Stemmer, K.; Tang-Christensen, M.; Woods, S. C.; DiMarchi, R. D.; Tschop, M. H. Glucagon-like peptide 1 (GLP-1). Mol. Metab. 2019, 30, 72– 130,  DOI: 10.1016/j.molmet.2019.09.010
      3
      Glucagon-like peptide 1 (GLP-1)
      Muller T D; Finan B; Bloom S R; D'Alessio D; Drucker D J; Flatt P R; Fritsche A; Gribble F; Reimann F; Grill H J; Habener J F; Holst J J; Langhans W; Meier J J; Nauck M A; Perez-Tilve D; Pocai A; Sandoval D A; Seeley R J; Schwartz T W; Stemmer K; Tang-Christensen M; Woods S C; DiMarchi R D; Tschop M H
      Molecular metabolism (2019), 30 (), 72-130 ISSN:.
      BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent β-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.
    4. 4
      Vrang, N.; Larsen, P. J. Preproglucagon derived peptides GLP-1, GLP-2 and oxyntomodulin in the CNS: role of peripherally secreted and centrally produced peptides. Prog. Neurobiol. 2010, 92, 442– 462,  DOI: 10.1016/j.pneurobio.2010.07.003
      4
      Preproglucagon derived peptides GLP-1, GLP-2 and oxyntomodulin in the CNS: Role of peripherally secreted and centrally produced peptides
      Vrang, Niels; Larsen, Philip Just
      Progress in Neurobiology (Oxford, United Kingdom) (2010), 92 (3), 442-462CODEN: PGNBA5; ISSN:0301-0082. (Elsevier Ltd.)
      A review. The scientific understanding of preproglucagon derived peptides has provided people with type 2 diabetes with two novel classes of glucose lowering agents, the dipeptidyl peptidase IV (DPP-IV) inhibitors and GLP-1 receptor agonists. For the scientists, the novel GLP-1 agonists, and DPP-IV inhibitors have evolved as useful tools to understand the role of the preproglucagon derived peptides in normal physiol. and disease. However, the overwhelming interest attracted by GLP-1 analogs as potent incretins has somewhat clouded the efforts to understand the importance of preproglucagon derived peptides in other physiol. contexts. In particular, our neurobiol. understanding of the preproglucagon expressing neuronal pathways in the central nervous system as well as the degree to which central GLP-1 receptors are targeted by peripherally administered GLP-1 receptor agonists is still fairly limited. The role of GLP-1 as an anorectic neurotransmitter is well recognized, but clarification of the neuronal targets and physiol. basis of this response is further warranted, as is the mapping of GLP-1 sensitive neurons involved in a variety of neuroendocrine and behavioral responses. Further recent evidence points to GLP-1 as a central neuropeptide with neuroprotective capabilities potentially mitigating a wide array of neurodegenerative conditions. It is the aim of the present review to summarize our current understanding of preproglucagon derived peptides as neurotransmitters in the central nervous system.
    5. 5
      Wu, T.; Rayner, C. K.; Horowitz, M. Incretins; Springer International Publishing: 2015; pp 137– 171.
      There is no corresponding record for this reference.
    6. 6
      Manandhar, B.; Ahn, J. M. Glucagon-like peptide-1 (GLP-1) analogs: recent advances, new possibilities, and therapeutic implications. J. Med. Chem. 2015, 58, 1020– 1037,  DOI: 10.1021/jm500810s
      6
      Glucagon-like Peptide-1 (GLP-1) Analogs: Recent Advances, New Possibilities, and Therapeutic Implications
      Manandhar, Bikash; Ahn, Jung-Mo
      Journal of Medicinal Chemistry (2015), 58 (3), 1020-1037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Glucagon-like peptide-1 (GLP-1) is an incretin that plays important physiol. roles in glucose homeostasis. Produced from intestine upon food intake, it stimulates insulin secretion and keeps pancreatic β-cells healthy and proliferating. Because of these beneficial effects, it has attracted a great deal of attention in the past decade, and an entirely new line of diabetic therapeutics has emerged based on the peptide. In addn. to the therapeutic applications, GLP-1 analogs have demonstrated a potential in mol. imaging of pancreatic β-cells; this may be useful in early detection of the disease and evaluation of therapeutic interventions, including islet transplantation. In this Perspective, we focus on GLP-1 analogs for their studies on improvement of biol. activities, enhancement of metabolic stability, investigation of receptor interaction, and visualization of the pancreatic islets.
    7. 7
      Kim, W.; Egan, J. M. The role of incretins in glucose homeostasis and diabetes treatment. Pharmacol. Rev. 2008, 60, 470– 512,  DOI: 10.1124/pr.108.000604
      7
      The role of incretins in glucose homeostasis and diabetes treatment
      Kim, Wook; Egan, Josephine M.
      Pharmacological Reviews (2008), 60 (4), 470-512CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
      A review. Incretins are gut hormones that are secreted from enteroendocrine cells into the blood within minutes after eating. One of their many physiol. roles is to regulate the amt. of insulin that is secreted after eating. In this manner, as well as others to be described in this review, their final common raison d'etre is to aid in disposal of the products of digestion. There are two incretins, known as glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1), that share many common actions in the pancreas but have distinct actions outside of the pancreas. Both incretins are rapidly deactivated by an enzyme called dipeptidyl peptidase 4 (DPP4). A lack of secretion of incretins or an increase in their clearance are not pathogenic factors in diabetes. However, in type 2 diabetes (T2DM), GIP no longer modulates glucose-dependent insulin secretion, even at supra-physiol. (pharmacol.) plasma levels, and therefore GIP incompetence is detrimental to β-cell function, esp. after eating. GLP-1, on the other hand, is still insulinotropic in T2DM, and this has led to the development of compds. that activate the GLP-1 receptor with a view to improving insulin secretion. Since 2005, two new classes of drugs based on incretin action have been approved for lowering blood glucose levels in T2DM: an incretin mimetic (exenatide, which is a potent long-acting agonist of the GLP-1 receptor) and an incretin enhancer (sitagliptin, which is a DPP4 inhibitor). Exenatide is injected s.c. twice daily and its use leads to lower blood glucose and higher insulin levels, esp. in the fed state. There is glucose-dependency to its insulin secretory capacity, making it unlikely to cause low blood sugars (hypoglycemia). DPP4 inhibitors are orally active and they increase endogenous blood levels of active incretins, thus leading to prolonged incretin action. The elevated levels of GLP-1 are thought to be the mechanism underlying their blood glucose-lowering effects.
    8. 8
      Knudsen, L. B. Glucagon-like peptide-1: the basis of a new class of treatment for type 2 diabetes. J. Med. Chem. 2004, 47, 4128– 4134,  DOI: 10.1021/jm030630m
      8
      Glucagon-like peptide-1:The basis of a new class of treatment for type 2 diabetes
      Knudsen, Lotte Bjerre
      Journal of Medicinal Chemistry (2004), 47 (17), 4128-4134CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Type 2 diabetes is increasingly becoming a epidemic worldwide. The author focuses on glucagon-like 1-peptide (GLP-1) peptide hormone as the basis for a potential new treatment paradigm for type 2 diabetes.
    9. 9
      Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.; Thogersen, H.; Wilken, M.; Johansen, N. L. Structure-activity and protraction relationship of long-acting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity, and bulkiness. J. Med. Chem. 2007, 50, 6126– 6132,  DOI: 10.1021/jm070861j
      9
      Structure-Activity and Protraction Relationship of Long-Acting Glucagon-like Peptide-1 Derivatives: Importance of Fatty Acid Length, Polarity, and Bulkiness
      Madsen, Kjeld; Knudsen, Lotte Bjerre; Agersoe, Henrik; Nielsen, Per Franklin; Thogersen, Henning; Wilken, Michael; Johansen, Nils Langeland
      Journal of Medicinal Chemistry (2007), 50 (24), 6126-6132CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We here report a series of derivs. describing the structure-activity relation around liraglutide, a once-daily human glucagon-like peptide-1 fatty acid deriv., with respect to potency as well as protraction in vivo. The spacer region between the fatty acid and the peptide is mostly important for potency, whereas the fatty acid or fatty acid mimetic is important for both potency and protraction. The length of the fatty acid is the most important parameter for protraction.
    10. 10
      Zhang, Y.; Sun, B.; Feng, D.; Hu, H.; Chu, M.; Qu, Q.; Tarrasch, J. T.; Li, S.; Sun Kobilka, T.; Kobilka, B. K.; Skiniotis, G. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 2017, 546, 248– 253,  DOI: 10.1038/nature22394
      10
      Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein
      Zhang, Yan; Sun, Bingfa; Feng, Dan; Hu, Hongli; Chu, Matthew; Qu, Qianhui; Tarrasch, Jeffrey T.; Li, Shane; Sun Kobilka, Tong; Kobilka, Brian K.; Skiniotis, Georgios
      Nature (London, United Kingdom) (2017), 546 (7657), 248-253CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metab. and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near at. resoln. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding.
    11. 11
      Wootten, D.; Simms, J.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 5211– 5216,  DOI: 10.1073/pnas.1221585110
      11
      Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations
      Wootten, Denise; Simms, John; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (13), 5211-5216, S5211/1-S5211/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Recently, the concept of ligand-directed signaling - the ability of different ligands of an individual receptor to promote distinct patterns of cellular response - has gained much traction in the field of drug discovery, with the potential to sculpt biol. response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clin. used mimetic exendin-4.
    12. 12
      Steensgaard, D. B.; Thomsen, J. K.; Olsen, H. B.; Knudsen, L. B. The molecular basis for the delayed absorption of the once-daily human GLP-1 analoge, liraglutide. Diabetes 2008, 57, A164– A164
      There is no corresponding record for this reference.
    13. 13
      Knudsen, L. B.; Nielsen, P. F.; Huusfeldt, P. O.; Johansen, N. L.; Madsen, K.; Pedersen, F. Z.; Thogersen, H.; Wilken, M.; Agerso, H. Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. J. Med. Chem. 2000, 43, 1664– 1669,  DOI: 10.1021/jm9909645
      13
      Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration
      Knudsen, Lotte B.; Nielsen, Per F.; Huusfeldt, Per O.; Johansen, Nils L.; Madsen, Kjeld; Pedersen, Freddy Z.; Thogersen, Henning; Wilken, Michael; Agerso, Henrik
      Journal of Medicinal Chemistry (2000), 43 (9), 1664-1669CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A series of very potent derivs. of the 30-amino acid peptide hormone glucagon-like peptide-1 (GLP-1) is described. The compds. were all derivatized with fatty acids in order to protract their action by facilitating binding to serum albumin. GLP-1 had a potency (EC50) of 55 pM for the cloned human GLP-1 receptor. Many of the compds. had similar or even higher potencies, despite quite large substituents. All compds. derivatized with fatty acids equal to or longer than 12 carbon atoms were very protracted compared to GLP-1 and thus seem suitable for once daily administration to type 2 diabetic patients. A structure-activity relationship was obtained. GLP-1 could be derivatized with linear fatty acids up to the length of 16 carbon atoms, sometimes longer, almost anywhere in the C-terminal part without considerable loss of potency. Derivatization with two fatty acid substituents led to a considerable loss of potency. A structure-activity relationship on derivatization of specific amino acids generally was obtained. It was found that the longer the fatty acid, the more potency was lost. Simultaneous modification of the N-terminus (in order to obtain better metabolic stability) interfered with fatty acid derivatization and led to loss of potency.
    14. 14
      Jimenez-Solem, E.; Rasmussen, M. H.; Christensen, M.; Knop, F. K. Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes. Curr. Opin. Mol. Ther. 2010, 12, 790– 797
      14
      Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes
      Jimenez-Solem, Espen; Rasmussen, Mette H.; Christensen, Mikkel; Knop, Filip K.
      Current Opinion in Molecular Therapeutics (2010), 12 (6), 790-797CODEN: CUOTFO; ISSN:2040-3445. (BioMed Central Ltd.)
      A review. Dulaglutide (LY-2189265) is a novel, long-acting glucagon-like peptide 1 (GLP-1) analog being developed by Eli Lilly for the treatment of type 2 diabetes mellitus (T2DM). Dulaglutide consists of GLP-l(7-37) covalently linked to an Fc fragment of human IgG4, thereby protecting the GLP-1 molety from inactivation by dipeptidyl peptidase 4. In vitro and in vivo studies on T2DM models demonstrated glucose-dependent insulin secretion stimulation. Pharmacokinetic studies demonstrated a t1/2 in humans of up to 90 h, making Dulaglutide an ideal candidate for once-weekly dosing. Clin. trials suggest that Dulaglutide reduces plasma glucose, and has an insulinotropic effect increasing insulin and C-peptide levels. Two phase II clin. trials demonstrated a dose-dependent redn. in glycated Hb (HbA1c) of up to 1.52% compared with placebo. Side effects assocd. with Dulaglutide administration were mainly gastrointestinal. To date, there have been no reports on the formation of antibodies against Dulaglutide, but, clearly, long-term data will be needed to asses this and other possible side effects. The results of several phase III clin. trials are awaited for clarification of the expected effects on HbA1c and body wt. If Dulaglutide possesses similar efficacy to other GLP-1 analogs, the once-weekly treatment will most likely be welcomed by patients with T2DM.
    15. 15
      Knudsen, L. B.; Lau, J. The Discovery and Development of Liraglutide and Semaglutide. Front. Endocrinol. (Lausanne, Switz.) 2019, 10, 155,  DOI: 10.3389/fendo.2019.00155
      15
      The Discovery and Development of Liraglutide and Semaglutide
      Knudsen Lotte Bjerre; Lau Jesper
      Frontiers in endocrinology (2019), 10 (), 155 ISSN:1664-2392.
      The discovery of glucagon-like peptide-1 (GLP-1), an incretin hormone with important effects on glycemic control and body weight regulation, led to efforts to extend its half-life and make it therapeutically effective in people with type 2 diabetes (T2D). The development of short- and then long-acting GLP-1 receptor agonists (GLP-1RAs) followed. Our article charts the discovery and development of the long-acting GLP-1 analogs liraglutide and, subsequently, semaglutide. We examine the chemistry employed in designing liraglutide and semaglutide, the human and non-human studies used to investigate their cellular targets and pharmacological effects, and ongoing investigations into new applications and formulations of these drugs. Reversible binding to albumin was used for the systemic protraction of liraglutide and semaglutide, with optimal fatty acid and linker combinations identified to maximize albumin binding while maintaining GLP-1 receptor (GLP-1R) potency. GLP-1RAs mediate their effects via this receptor, which is expressed in the pancreas, gastrointestinal tract, heart, lungs, kidneys, and brain. GLP-1Rs in the pancreas and brain have been shown to account for the respective improvements in glycemic control and body weight that are evident with liraglutide and semaglutide. Both liraglutide and semaglutide also positively affect cardiovascular (CV) outcomes in individuals with T2D, although the precise mechanism is still being explored. Significant weight loss, through an effect to reduce energy intake, led to the approval of liraglutide (3.0 mg) for the treatment of obesity, an indication currently under investigation with semaglutide. Other ongoing investigations with semaglutide include the treatment of non-alcoholic fatty liver disease (NASH) and its use in an oral formulation for the treatment of T2D. In summary, rational design has led to the development of two long-acting GLP-1 analogs, liraglutide and semaglutide, that have made a vast contribution to the management of T2D in terms of improvements in glycemic control, body weight, blood pressure, lipids, beta-cell function, and CV outcomes. Furthermore, the development of an oral formulation for semaglutide may provide individuals with additional benefits in relation to treatment adherence. In addition to T2D, liraglutide is used in the treatment of obesity, while semaglutide is currently under investigation for use in obesity and NASH.
    16. 16
      Davies, M. J.; D’Alessio, D. A.; Fradkin, J.; Kernan, W. N.; Mathieu, C.; Mingrone, G.; Rossing, P.; Tsapas, A.; Wexler, D. J.; Buse, J. B. Management of Hyperglycemia in Type 2 Diabetes, 2018. A Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care 2018, 41, 2669– 2701,  DOI: 10.2337/dci18-0033
      16
      Management of Hyperglycemia in Type 2 Diabetes, 2018. A Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD)
      Davies Melanie J; Davies Melanie J; D'Alessio David A; Fradkin Judith; Kernan Walter N; Mathieu Chantal; Mingrone Geltrude; Mingrone Geltrude; Rossing Peter; Rossing Peter; Tsapas Apostolos; Wexler Deborah J; Wexler Deborah J; Buse John B
      Diabetes care (2018), 41 (12), 2669-2701 ISSN:.
      The American Diabetes Association and the European Association for the Study of Diabetes convened a panel to update the prior position statements, published in 2012 and 2015, on the management of type 2 diabetes in adults. A systematic evaluation of the literature since 2014 informed new recommendations. These include additional focus on lifestyle management and diabetes self-management education and support. For those with obesity, efforts targeting weight loss, including lifestyle, medication, and surgical interventions, are recommended. With regards to medication management, for patients with clinical cardiovascular disease, a sodium-glucose cotransporter 2 (SGLT2) inhibitor or a glucagon-like peptide 1 (GLP-1) receptor agonist with proven cardiovascular benefit is recommended. For patients with chronic kidney disease or clinical heart failure and atherosclerotic cardiovascular disease, an SGLT2 inhibitor with proven benefit is recommended. GLP-1 receptor agonists are generally recommended as the first injectable medication.
    17. 17
      Trzaskalski, N. A.; Fadzeyeva, E.; Mulvihill, E. E. Dipeptidyl Peptidase-4 at the Interface Between Inflammation and Metabolism. Clin. Med. Insights: Endocrinol. Diabetes 2020, 13, 1– 10,  DOI: 10.1177/1179551420912972
      There is no corresponding record for this reference.
    18. 18
      Mulvihill, E. E.; Drucker, D. J. Pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors. Endocr. Rev. 2014, 35, 992– 1019,  DOI: 10.1210/er.2014-1035
      18
      Pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors
      Mulvihill, Erin E.; Drucker, Daniel J.
      Endocrine Reviews (2014), 35 (6), 992-1019CODEN: ERVIDP; ISSN:0163-769X. (Endocrine Society)
      A review. Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane mol. and a sol. circulating protein. Both membrane-assocd. and sol. DPP4 exert catalytic activity, cleaving proteins contg. a position 2 alanine or proline. DPP4-mediated enzymic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the mol. mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biol. of DPP4 with a focus on: (1) identification of pharmacol. vs physiol. DPP4 substrates; and (2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chem. redn. of DPP4 activity. We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclin. and clin. studies. Finally, we highlight exptl. pitfalls and tech. challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
    19. 19
      Sebokova, E.; Christ, A. D.; Wang, H.; Sewing, S.; Dong, J. Z.; Taylor, J.; Cawthorne, M. A.; Culler, M. D. Taspoglutide, an analog of human glucagon-like Peptide-1 with enhanced stability and in vivo potency. Endocrinology 2010, 151, 2474– 2482,  DOI: 10.1210/en.2009-1459
      19
      Taspoglutide, an analog of human glucagon-like peptide-1 with enhanced stability and in vivo potency
      Sebokova, Elena; Christ, Andreas D.; Wang, Haiyan; Sewing, Sabine; Dong, Jesse Z.; Taylor, John; Cawthorne, Michael A.; Culler, Michael D.
      Endocrinology (2010), 151 (6), 2474-2482CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
      Taspoglutide is a novel analog of human glucagon-like peptide-1 [hGLP-1(7-36)NH2] in clin. development for the treatment of type 2 diabetes. Taspoglutide contains α-aminoisobutyric acid substitutions replacing Ala8 and Gly35 of hGLP-1(7-36)NH2. The binding affinity [radioligand binding assay using [125I]hGLP-1(7-36)NH2], potency (cAMP prodn. in CHO cells stably overexpressing hGLP-1 receptor), and in vitro plasma stability of taspoglutide compared with hGLP-1(7-36)NH2 have been evaluated. Effects on basal and glucose-stimulated insulin secretion were detd. in vitro in INS-1E cells and in vivo in normal rats. Taspoglutide has comparable affinity (affinity const. 1.1 ± 0.2 nM) to the natural ligand (affinity const. 1.5 ± 0.3 nM) for the hGLP-1 receptor and exhibits comparable potency in stimulating cAMP prodn. (EC50 Taspo 0.06 nM and EC50 hGLP-1(7-36)NH2 0.08 nM). Taspoglutide exerts insulinotropic action in vitro and in vivo and retains the glucoincretin property of hGLP-1(7-36)NH2. Stimulation of insulin secretion is concn. dependent and evident in the presence of high-glucose concns. (16.7 mM) with a taspoglutide concn. as low as 0.001 nM. Taspoglutide is fully resistant to dipeptidyl peptidase-4 cleavage (during 1 h incubation at room temp. with purified enzyme) and has an extended in vitro plasma half-life relative to hGLP-1(7-36)NH2 (9.8 h vs. 50 min). In vitro, taspoglutide does not inhibit dipeptidyl peptidase-4 activity. This study provides the biochem. and pharmacol. basis for the sustained plasma drug levels and prolonged therapeutic activity seen in early clin. trials of taspoglutide. Excellent stability and potency with substantial glucoincretin effects position taspoglutide as a promising new agent for treatment of type 2 diabetes.
    20. 20
      Rosenstock, J.; Balas, B.; Charbonnel, B.; Bolli, G. B.; Boldrin, M.; Ratner, R.; Balena, R. The fate of taspoglutide, a weekly GLP-1 receptor agonist, versus twice-daily exenatide for type 2 diabetes: the T-emerge 2 trial. Diabetes Care 2013, 36, 498– 504,  DOI: 10.2337/dc12-0709
      20
      The fate of taspoglutide, a weekly GLP-1 receptor agonist, versus twice-daily exenatide for type 2 diabetes: The T-emerge 2 trial
      Rosenstock, Julio; Balas, Bogdan; Charbonnel, Bernard; Bolli, Geremia B.; Boldrin, Mark; Ratner, Robert; Balena, Raffaella
      Diabetes Care (2013), 36 (3), 498-504CODEN: DICAD2; ISSN:0149-5992. (American Diabetes Association, Inc.)
      OBJECTIVE: Taspoglutide is a long-acting glucagon-like peptide 1 receptor agonist developed for treatment of type 2 diabetes. The efficacy and safety of once-weekly taspoglutide was compared with twice-daily exenatide. RESEARCH DESIGN AND METHODS: Overweight adults with inadequately controlled type 2 diabetes on metformin ± a thiazolidinedione were randomized to s.c. taspoglutide 10 mg weekly (n = 399), taspoglutide 20 mg weekly (n = 398), or exenatide 10 μg twice daily (n = 392) in an open-label, multicenter trial. The primary end point was change in HbA1c after 24 wk. RESULTS: Mean baseline HbA1c was 8.1%. Both doses of taspoglutide reduced HbA1c significantly more than exenatide (taspoglutide 10 mg: -1.24% [SE 0.09], difference -0.26, 95% CI -0.37 to -0.15, P < 0.0001; taspoglutide 20 mg: -1.31% [0.08], difference -0.33, -0.44 to -0.22, P < 0.0001; exenatide: -0.98% [0.08]). Both taspoglutide doses reduced fasting plasma glucose significantly more than exenatide. Taspoglutide reduced body wt. (taspoglutide 10 mg, -1.6 kg; taspoglutide 20 mg, -2.3 kg) as did exenatide (-2.3 kg), which was greater than with taspoglutide 10 mg (P < 0.05). HbA1c and wt. effects were maintained after 52 wk. More adverse events with taspoglutide 10 and 20 mg than exenatide developed over time (nausea in 53, 59, and 35% and vomiting in 33, 37, and 16%, resp.). Allergic and injection-site reactions were more common with taspoglutide. Discontinuations were greater with taspoglutide. Antitaspoglutide antibodies were detected in 49% of patients. CONCLUSIONS: Once-weekly taspoglutide demonstrated greater glycemic control than twice-daily exenatide with comparable wt. loss, but with unacceptable levels of nausea/vomiting, injection-site reactions, and systemic allergic reactions.
    21. 21
      Jones, L. H.; Price, D. A. Medicinal Chemistry of Glucagon-Like Peptide Receptor Agonists. Prog. Med. Chem. 2013, 52, 45– 96,  DOI: 10.1016/B978-0-444-62652-3.00002-8
      21
      Medicinal chemistry of glucagon-like peptide receptor agonists
      Jones, Lyn H.; Price, David A.
      Progress in Medicinal Chemistry (2013), 52 (), 45-96CODEN: PMDCAY; ISSN:0079-6468. (Elsevier B.V.)
      A review. This article discusses the medicinal chem. of glucagon-like peptide receptor agonists. Applications of GLP-1 ligands as imaging agents in the diagnosis of diabetes and pancreatic cancer were discussed. Fundamental principles of GLP-1 injectable peptides medicinal chem. design and synthesis were discussed. The potential for alternative modes of drug delivery, such as oral and inhaled administration were discussed.
    22. 22
      Johnson, L. M.; Barrick, S.; Hager, M. V.; McFedries, A.; Homan, E. A.; Rabaglia, M. E.; Keller, M. P.; Attie, A. D.; Saghatelian, A.; Bisello, A.; Gellman, S. H. A potent alpha/beta-peptide analogue of GLP-1 with prolonged action in vivo. J. Am. Chem. Soc. 2014, 136, 12848– 12851,  DOI: 10.1021/ja507168t
      22
      A Potent α/β-Peptide Analogue of GLP-1 with Prolonged Action in Vivo
      Johnson, Lisa M.; Barrick, Stacey; Hager, Marlies V.; McFedries, Amanda; Homan, Edwin A.; Rabaglia, Mary E.; Keller, Mark P.; Attie, Alan D.; Saghatelian, Alan; Bisello, Alessandro; Gellman, Samuel H.
      Journal of the American Chemical Society (2014), 136 (37), 12848-12851CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic β cells. GLP-1R agonists are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. The authors describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained β-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogs of other peptide hormones.
    23. 23
      Meng, H.; Krishnaji, S. T.; Beinborn, M.; Kumar, K. Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1. J. Med. Chem. 2008, 51, 7303– 7307,  DOI: 10.1021/jm8008579
      23
      Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1
      Meng, He; Krishnaji, Subrahmanian Tarakkad; Beinborn, Martin; Kumar, Krishna
      Journal of Medicinal Chemistry (2008), 51 (22), 7303-7307CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogs of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogs displayed higher proteolytic stability with simultaneous retention of biol. activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions.
    24. 24
      Ueda, T.; Tomita, K.; Notsu, Y.; Ito, T.; Fumoto, M.; Takakura, T.; Nagatome, H.; Takimoto, A.; Mihara, S.; Togame, H.; Kawamoto, K.; Iwasaki, T.; Asakura, K.; Oshima, T.; Hanasaki, K.; Nishimura, S.; Kondo, H. Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity. J. Am. Chem. Soc. 2009, 131, 6237– 6245,  DOI: 10.1021/ja900261g
      24
      Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity
      Ueda, Taichi; Tomita, Kazuyoshi; Notsu, Yoshihide; Ito, Takaomi; Fumoto, Masataka; Takakura, Tomoaki; Nagatome, Hirofumi; Takimoto, Akio; Mihara, Shin-Ichi; Togame, Hiroko; Kawamoto, Keiko; Iwasaki, Takanori; Asakura, Kenji; Oshima, Takeo; Hanasaki, Kohji; Nishimura, Shin-Ichiro; Kondo, Hirosato
      Journal of the American Chemical Society (2009), 131 (17), 6237-6245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogs with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and α2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepd. by chemoenzymic approaches. We assessed the receptor binding affinity and cAMP prodn. activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addn. of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic anal. of the degrdn. reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addn., the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogs with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogs of other bioactive peptides as desirable pharmaceuticals.
    25. 25
      Lau, J.; Bloch, P.; Schaffer, L.; Pettersson, I.; Spetzler, J.; Kofoed, J.; Madsen, K.; Knudsen, L. B.; McGuire, J.; Steensgaard, D. B.; Strauss, H. M.; Gram, D. X.; Knudsen, S. M.; Nielsen, F. S.; Thygesen, P.; Reedtz-Runge, S.; Kruse, T. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J. Med. Chem. 2015, 58, 7370– 7380,  DOI: 10.1021/acs.jmedchem.5b00726
      25
      Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide
      Lau, Jesper; Bloch, Paw; Schaffer, Lauge; Pettersson, Ingrid; Spetzler, Jane; Kofoed, Jacob; Madsen, Kjeld; Knudsen, Lotte Bjerre; McGuire, James; Steensgaard, Dorte Bjerre; Strauss, Holger Martin; Gram, Dorte X.; Knudsen, Sanne Moeller; Nielsen, Flemming Seier; Thygesen, Peter; Reedtz-Runge, Steffen; Kruse, Thomas
      Journal of Medicinal Chemistry (2015), 58 (18), 7370-7380CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analog that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analog by increasing albumin affinity and secure full stability against metabolic degrdn. The fatty acid moiety and the linking chem. to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analog. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib8, Arg34) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clin. testing.
    26. 26
      Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C. J.; Harriott, P.; Greer, B.; O’Harte, F. P.; Flatt, P. R. N-terminal His(7)-modification of glucagon-like peptide-1(7–36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity. J. Endocrinol. 2004, 180, 379– 388,  DOI: 10.1677/joe.0.1800379
      26
      N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity
      Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C. J.; Harriott, P.; Greer, B.; O'Harte, F. P. M.; Flatt, P. R.
      Journal of Endocrinology (2004), 180 (3), 379-388CODEN: JOENAK; ISSN:0022-0795. (Society for Endocrinology)
      Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analog of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesized and tested for DPP IV stability and biol. activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degrdn. of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degrdn. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32·9 and 6·7 nM, resp.) compared with native GLP-1 (IC50 0·37 nM). Similarly, both analogs stimulated cAMP prodn. with EC50 values of 16·3 and 27 nM resp. compared with GLP-1 (EC50 4·7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5·6 mM glucose (P<0·05 to P<0·001) similar to native GLP-1. Both analogs (25 nM/kg body wt.) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body wt.) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biol. potent forms of GLP-1. The particularly powerful antihyperglycemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.
    27. 27
      Wootten, D.; Reynolds, C. A.; Koole, C.; Smith, K. J.; Mobarec, J. C.; Simms, J.; Quon, T.; Coudrat, T.; Furness, S. G.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures. Mol. Pharmacol. 2016, 89, 335– 347,  DOI: 10.1124/mol.115.101246
      27
      A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures
      Wootten, Denise; Reynolds, Christopher A.; Koole, Cassandra; Smith, Kevin J.; Mobarec, Juan C.; Simms, John; Quon, Tezz; Coudrat, Thomas; Furness, Sebastian G. B.; Miller, Laurence J.; Christopoulos, Arthur; Sexton, Patrick M.
      Molecular Pharmacology (2016), 89 (3), 335-347CODEN: MOPMA3; ISSN:1521-0111. (American Society for Pharmacology and Experimental Therapeutics)
      The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiol. significance of this is yet to be elucidated. Previous work has implicated R2.60190, N3.43240, Q7.49394, and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190, N3.43240, and Q7.49394 predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict addnl. key mol. interactions with these amino acids. We have introduced E6.53364A, N3.43240Q, Q7.49394N, and N3.43240Q/Q7.49394N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53364 and R2.60190 was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49394, but not R2.60190/E6.53364 was crit. for calcium mobilization for all three peptides. Mutation of N3.43240 and Q7.49394 had differential effects on individual peptides, providing evidence for mol. differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.
    28. 28
      Xiao, Q.; Giguere, J.; Parisien, M.; Jeng, W.; St-Pierre, S. A.; Brubaker, P. L.; Wheeler, M. B. Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo. Biochemistry 2001, 40, 2860– 2869,  DOI: 10.1021/bi0014498
      28
      Biological Activities of Glucagon-Like Peptide-1 Analogues in Vitro and in Vivo
      Xiao, Q.; Giguere, J.; Parisien, M.; Jeng, W.; St-Pierre, S. A.; Brubaker, P. L.; Wheeler, M. B.
      Biochemistry (2001), 40 (9), 2860-2869CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala2, its clin. application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogs. In this study, we investigated GLP-1 analogs including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala2]GLP-1 and [Gly2]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biol. effectiveness of [D-Ala2]GLP-1 was also obsd. in diabetic db/db mice. Similarly, improved biol. activity of acetyl- and hexenoic-His1-GLP-1, glucagon(1-5)-, glucagon(1-10)-, PACAP(1-5)-, VIP(1-5)-, and secretin(1-10)-GLP-1 was obsd., despite normal or lower receptor binding and activation in vitro. [Ala8/11/12/16] substitutions also increased biol. activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biol. activity. All other modified peptides examd. showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the α-amino group of His1 and replacement of Ala2 result in significantly increased biol. effects of GLP-1 in vivo, likely due to decreased degrdn. rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biol. activity.
    29. 29
      Buckley, D. I.; Habener, J. F.; Mallory, J. B.; Mojsov, S. GLP-1 Analogs Useful for Diabetes Treatment; European Patent EP 0512 042 B1; 1991.
      There is no corresponding record for this reference.
    30. 30
      Ohneda, A.; Ohneda, K.; Ohneda, M.; Koizumi, F.; Ohashi, S.; Kawai, K.; Suzuki, S. The structure-function relationship of GLP-1 related peptides in the endocrine function of the canine pancreas. Tohoku J. Exp. Med. 1991, 165, 209– 221,  DOI: 10.1620/tjem.165.209
      30
      The structure-function relationship of GLP-1 related peptides in the endocrine function of the canine pancreas
      Ohneda, Akira; Ohneda, Kinuko; Ohneda, Makoto; Koizumi, Fumiaki; Ohashi, Shinichi; Kawai, Koichi; Suzuki, Seiji
      Tohoku Journal of Experimental Medicine (1991), 165 (3), 209-21CODEN: TJEMAO; ISSN:0040-8727.
      In order to clarify the relationship between the structure and function of glucagon-like peptide (GLP) 1 in the endocrine function of the pancreas, the response of insulin and glucagon to various synthetic GLP-1-related peptides was investigated in anesthetized dogs. GLP-1-related peptides were administered in a dosage of 400 pmol within 10 min into the pancreatic artery during glucose or arginine infusion, and the changes in plasma insulin and glucagon in the pancreatic vein were studied. GLP-1 (7-36) and (7-37), as well as glucagon enhanced insulin release during glucose infusion, whereas neither GLP-1 (1-37), (7-20), (6-37), nor (8-37) stimulated insulin release. The administration of GLP-1 (1-37), (7-36), and (7-37) reduced glucagon release during glucose infusion. When arginine was infused, GLP-1 (7-20), (7-36), (7-37), and glucagon enhanced insulin release. In contrast, glucagon release was increased by the administration of GLP-1 (7-20), (8-37), and (7-37). The present study indicates that histidine at the 7th position of GLP-1 is important in eliciting biol. action and that only truncated GLP-1 (7-36), (7-37), and (7-20) showed an insulinotropic action as strong as glucagon in dogs. Furthermore, it is suggested that the response of insulin and glucagon to GLP-1 related peptides is dependent on a background condition.
    31. 31
      Gallwitz, B.; Ropeter, T.; Morys-Wortmann, C.; Mentlein, R.; Siegel, E. G.; Schmidt, W. E. GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro. Regul. Pept. 2000, 86, 103– 111,  DOI: 10.1016/S0167-0115(99)00095-6
      31
      GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro
      Gallwitz, B.; Ropeter, T.; Morys-Wortmann, C.; Mentlein, R.; Siegel, E. G.; Schmidt, W. E.
      Regulatory Peptides (2000), 86 (1-3), 103-111CODEN: REPPDY; ISSN:0167-0115. (Elsevier Science Ireland Ltd.)
      Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biol. half-time limits the therapeutic use of GLP-1. DPP IV requires an intact α-amino-group of the N-terminal histidine of GLP-1 in order to perform its enzymic activity. Therefore, the following GLP-1 analogs with alterations in the N-terminal position 1 were synthesized: N-methylated- (N-me-GLP-1), α-methylated (α-me-GLP-1), desamidated- (desamino-GLP-1) and imidazole-lactic-acid substituted GLP-1 (imi-GLP-1). All GLP-1 analogs except α-me-GLP-1 were hardly degraded by DPP IV in vitro. The GLP-1 analogs showed receptor affinity and in vitro biol. activity comparable to native GLP-1 in RINm5F cells. GLP-1 receptor affinity was highest for imi-GLP-1, followed by α-me-GLP-1 and N-me-GLP-1. Only desamino-GLP-1 showed a 15-fold loss of receptor affinity compared to native GLP-1. All analogs stimulated intracellular cAMP prodn. in RINm5F cells in concns. comparable to GLP-1. N-terminal modifications might therefore be useful in the development of long-acting GLP-1 analogs for type 2 diabetes therapy.
    32. 32
      Hareter, A.; Hoffmann, E.; Bode, H. P.; Goke, B.; Goke, R. The positive charge of the imidazole side chain of histidine(7) is crucial for GLP-1 action. Endocr. J. 1997, 44, 701– 705,  DOI: 10.1507/endocrj.44.701
      32
      The positive charge of the imidazole side chain of histidine7 is crucial for GLP-1 action
      Hareter, Alexandra; Hoffmann, Eike; Bode, Hans-Peter; Goke, Burkhard; Goke, Rudiger
      Endocrine Journal (Tokyo) (1997), 44 (5), 701-705CODEN: ENJOEO; ISSN:0918-8959. (Japan Endocrine Society)
      Glucagon-like peptide-1(7-36)amide/(7-37) (GLP-1) is an incretin hormone which plays an important role in postprandial glucose homeostasis. Since GLP-1 potentiates glucose-induced insulin secretion, stimulates insulin biosynthesis and inhibits glucagon release, it is a potential tool for the treatment of diabetes mellitus. For this, an exact understanding of the structural/functional moieties of the peptide is mandatory. The present study investigates the importance of structural features of histidine7 at the N-terminus for GLP-1 action. Based upon binding and activity data obtained from ten different GLP-1 analogs not the pos. charge of the free α-amino group but the pos. charge of the imidazole side chain of histidine is crucial for GLP-1 action. The presence of a ring structure and a basic function as well as the correct positioning of both seems to be decisive.
    33. 33
      DesMarteau, D. D.; Montanari, V. Easy preparation of bioactive peptides from the novel N-alpha-trifluoroethyl amino acids. Chem. Lett. 2000, 29, 1052– 1053,  DOI: 10.1246/cl.2000.1052
      There is no corresponding record for this reference.
    34. 34
      Baggio, L. L.; Drucker, D. J. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007, 132, 2131– 2157,  DOI: 10.1053/j.gastro.2007.03.054
      34
      Biology of incretins: GLP-1 and GIP
      Baggio, Laurie L.; Drucker, Daniel J.
      Gastroenterology (2007), 132 (6), 2131-2157CODEN: GASTAB; ISSN:0016-5085. (Elsevier Inc.)
      A review. This review focuses on the mechanisms regulating the synthesis, secretion, biol. actions, and therapeutic relevance of the incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The published literature was reviewed, with emphasis on recent advances in our understanding of the biol. of GIP and GLP-1. GIP and GLP-1 are both secreted within minutes of nutrient ingestion and facilitate the rapid disposal of ingested nutrients. Both peptides share common actions on islet β-cells acting through structurally distinct yet related receptors. Incretin-receptor activation leads to glucose-dependent insulin secretion, induction of β-cell proliferation, and enhanced resistance to apoptosis. GIP also promotes energy storage via direct actions on adipose tissue, and enhances bone formation via stimulation of osteoblast proliferation and inhibition of apoptosis. In contrast, GLP-1 exerts glucoregulatory actions via slowing of gastric emptying and glucose-dependent inhibition of glucagon secretion. GLP-1 also promotes satiety and sustained GLP-1-receptor activation is assocd. with wt. loss in both preclin. and clin. studies. The rapid degrdn. of both GIP and GLP-1 by the enzyme dipeptidyl peptidase-4 has led to the development of degrdn.-resistant GLP-1-receptor agonists and dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes. These agents decrease Hb A1c (HbA1c) safely without wt. gain in subjects with type 2 diabetes. GLP-1 and GIP integrate nutrient-derived signals to control food intake, energy absorption, and assimilation. Recently approved therapeutic agents based on potentiation of incretin action provide new physiol. based approaches for the treatment of type 2 diabetes.
    35. 35
      Parthier, C.; Kleinschmidt, M.; Neumann, P.; Rudolph, R.; Manhart, S.; Schlenzig, D.; Fanghanel, J.; Rahfeld, J. U.; Demuth, H. U.; Stubbs, M. T. Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 13942– 13947,  DOI: 10.1073/pnas.0706404104
      35
      Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor
      Parthier, Christoph; Kleinschmidt, Martin; Neumann, Piotr; Rudolph, Rainer; Manhart, Susanne; Schlenzig, Dagmar; Fanghanel, Joerg; Rahfield, Hans-Ulrich; Stubbs, Milton T.
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (35), 13942-13947, S13942/1-S13942/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Incretins, endogenous polypeptide hormones released in response to food intake, potentiate insulin secretion from pancreatic β cells after oral glucose ingestion (the incretin effect). This response is signaled by the two peptide hormones glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) and glucagon-like peptide 1 through binding and activation of their cognate class 2 G protein-coupled receptors (GPCRs). Because the incretin effect is lost or significantly reduced in patients with type 2 diabetes mellitus, glucagon-like peptide 1 and GIP have attracted considerable attention for their potential in antidiabetic therapy. A paucity of structural information precludes a detailed understanding of the processes of hormone binding and receptor activation, hampering efforts to develop novel pharmaceuticals. Here we report the crystal structure of the complex of human GIP receptor extracellular domain (ECD) with its agonist, the incretin GIP1-42. The hormone binds in an a-helical conformation in a surface groove of the ECD largely through hydrophobic interactions. The N-terminal ligand residues would remain free to interact with other parts of the receptor. Thermodn. data suggest that binding is concomitant with structural organization of the hormone, resulting in a complex mode of receptor-ligand recognition. The presentation of a well structured, a-helical ligand by the ECD is expected to be conserved among other hormone receptors of this class.
    36. 36
      Underwood, C. R.; Parthier, C.; Reedtz-Runge, S. Structural Basis for Ligand Recognition of Incretin Receptors. Vitam. Horm. 2010, 84, 251– 278,  DOI: 10.1016/B978-0-12-381517-0.00009-6
      36
      Structural basis for ligand recognition of incretin receptors
      Underwood, Christina Rye; Parthier, Christoph; Reedtz-Runge, Steffen
      Vitamins and Hormones (San Diego, CA, United States) (2010), 84 (Incretins and Insulin Secretion), 251-278CODEN: VIHOAQ; ISSN:0083-6729. (Elsevier Inc.)
      A review. The glucose-dependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide-1 (GLP-1) receptor are homologous G-protein-coupled receptors (GPCRs). Incretin receptor agonists stimulate the synthesis and secretion of insulin from pancreatic β-cells and are therefore promising agents for the treatment of type 2 diabetes. It is well established that the N-terminal extracellular domain (ECD) of incretin receptors is important for ligand binding and ligand specificity, whereas the transmembrane domain is involved in receptor activation. Structures of the ligand-bound ECD of incretin receptors have been solved recently by X-ray crystallog. The crystal structures reveal a similar fold of the ECD and a similar mechanism of ligand binding, where the ligand adopts an α-helical conformation. Residues in the C-terminal part of the ligand interact directly with the ECD and hydrophobic interactions appear to be the main driving force for ligand binding to the ECD of incretin receptors. Obviously, the-still missing-structures of full-length incretin receptors are required to construct a complete picture of receptor function at the mol. level. However, the progress made recently in structural anal. of the ECDs of incretin receptors and related GPCRs has shed new light on the process of ligand recognition and binding and provided a basis to disclose some of the mechanisms underlying receptor activation at high resoln.
    37. 37
      Nauck, M. A.; Heimesaat, M. M.; Orskov, C.; Holst, J. J.; Ebert, R.; Creutzfeldt, W. Preserved incretin activity of glucagon-like peptide 1 [7–36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J. Clin. Invest. 1993, 91, 301– 307,  DOI: 10.1172/JCI116186
      37
      Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus
      Nauck M A; Heimesaat M M; Orskov C; Holst J J; Ebert R; Creutzfeldt W
      The Journal of clinical investigation (1993), 91 (1), 301-7 ISSN:0021-9738.
      In type-2 diabetes, the overall incretin effect is reduced. The present investigation was designed to compare insulinotropic actions of exogenous incretin hormones (gastric inhibitory peptide [GIP] and glucagon-like peptide 1 [GLP-1] [7-36 amide]) in nine type-2 diabetic patients (fasting plasma glucose 7.8 mmol/liter; hemoglobin A1c 6.3 +/- 0.6%) and in nine age- and weight-matched normal subjects. Synthetic human GIP (0.8 and 2.4 pmol/kg.min over 1 h each), GLP-1 [7-36 amide] (0.4 and 1.2 pmol/kg.min over 1 h each), and placebo were administered under hyperglycemic clamp conditions (8.75 mmol/liter) in separate experiments. Plasma GIP and GLP-1 [7-36 amide] concentrations (radioimmunoassay) were comparable to those after oral glucose with the low, and clearly supraphysiological with the high infusion rates. Both GIP and GLP-1 [7-36 amide] dose-dependently augmented insulin secretion (insulin, C-peptide) in both groups (P < 0.05). With GIP, the maximum effect in type-2 diabetic patients was significantly lower (by 54%; P < 0.05) than in normal subjects. With GLP-1 [7-36 amide] type-2 diabetic patients reached 71% of the increments in C-peptide of normal subjects (difference not significant). Glucagon was lowered during hyperglycemic clamps in normal subjects, but not in type-2 diabetic patients, and further by GLP-1 [7-36 amide] in both groups (P < 0.05), but not by GIP. In conclusion, in mild type-2 diabetes, GLP-1 [7-36 amide], in contrast to GIP, retains much of its insulinotropic activity. It also lowers glucagon concentrations.
    38. 38
      Holst, J. J.; Gromada, J.; Nauck, M. A. The pathogenesis of NIDDM involves a defective expression of the GIP receptor. Diabetologia 1997, 40, 984– 986,  DOI: 10.1007/s001250050779
      38
      The pathogenesis of NIDDM involves a defective expression of the GIP receptor
      Holst, J. J.; Gromada, J.; Nauck, M. A.
      Diabetologia (1997), 40 (8), 984-986CODEN: DBTGAJ; ISSN:0012-186X. (Springer)
      A review and discussion with 34 refs., describing decreased incretin effect in non-insulin-dependent diabetes mellitus (NIDDM) patients, full efficacy of glucagon-like peptide-1 (GLP-1) and lack of effect of Glc-dependent insulinotropic polypeptide (GIP) on insulin secretion, and absence of incretin effect (small loads of Glc) at normal GIP secretion in diabetic β-cells. The apparent polygenicity of NIDDM is hypothesized to be caused by genetically defective expression of the GIP receptor.
    39. 39
      Vilsboll, T.; Krarup, T.; Madsbad, S.; Holst, J. J. Defective amplification of the late phase insulin response to glucose by GIP in obese Type II diabetic patients. Diabetologia 2002, 45, 1111– 1119,  DOI: 10.1007/s00125-002-0878-6
      39
      Defective amplification of the late phase insulin response to glucose by GIP in obese Type II diabetic patients
      Vilsboll, T.; Krarup, T.; Madsbad, S.; Holst, J. J.
      Diabetologia (2002), 45 (8), 1111-1119CODEN: DBTGAJ; ISSN:0012-186X. (Springer-Verlag)
      Aims/hypothesis. Glucagon-like-peptide-1 (GLP-1) is strongly insulinotropic in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas glucose-dependent insulinotropic polypeptide (GIP) is less effective. Our investigation evaluated "early" (protocol 1) - and "late phase" (protocol 2) insulin and C-peptide responses to GLP-1 and GIP stimulation in patients with Type II diabetes. Methods. Protocol 1: eight Type II diabetic patients and eight matched healthy subjects received i.v. bolus injections of GLP-1 (2.5 nmol) or GIP (7.5 nmol) concomitant with an increase of plasma glucose to 15 mmol/L. Protocol 2: eight Type II diabetic patients underwent a hyperglycemic clamp (15 mmol/L) with infusion (per kg/min) of either: 1 pmol GLP-1 (7-36) amide, 4 pmol GIP, 16 pmol, or no incretin hormone. For comparison, six matched healthy subjects were examd. Results. Protocol 1: Type II diabetic patients were characterized by a decreased "early phase" response to both stimuli, but their relative response to GIP vs. GLP-1 stimulation was exactly the same as in healthy subjects [insulin (C-peptide): patients 59% (74±6%) and healthy subjects 62% (71%)]. Protocol 2, "Early phase" (0-20 min) insulin response to glucose was delayed and reduced in the patients, but enhanced slightly and similarly by GIP and GLP-1. GLP-1 augmented the "late phase" (20-120 min) insulin secretion to levels similar to those obsd. in healthy subjects. In contrast, the "late phase" responses to both doses of GIP were not different from those obtained with glucose alone. Accordingly, glucose infusion rates required to maintain the hyperglycemic clamp in the "late phase" period (20-120 min) were similar with glucose alone and glucose plus GIP, whereas a doubling of the infusion rate was required during GLP-1 stimulation. Conclusion/interpretation. Lack of GIP amplification of the late phase insulin response to glucose, which contrasts markedly to the normalizing effect of GLP-1, could be a key defect in insulin secretion in Type II diabetic patients.
    40. 40
      Hojberg, P. V.; Vilsboll, T.; Rabol, R.; Knop, F. K.; Bache, M.; Krarup, T.; Holst, J. J.; Madsbad, S. Four weeks of near-normalisation of blood glucose improves the insulin response to glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes. Diabetologia 2009, 52, 199– 207,  DOI: 10.1007/s00125-008-1195-5
      40
      Four weeks of near-normalisation of blood glucose improves the insulin response to glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes
      Hojberg P V; Vilsboll T; Rabol R; Knop F K; Bache M; Krarup T; Holst J J; Madsbad S
      Diabetologia (2009), 52 (2), 199-207 ISSN:.
      OBJECTIVE: The incretin effect is attenuated in patients with type 2 diabetes mellitus, partly as a result of impaired beta cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The aim of the present study was to investigate whether 4 weeks of near-normalisation of the blood glucose level could improve insulin responses to GIP and GLP-1 in patients with type 2 diabetes. METHODS: Eight obese patients with type 2 diabetes with poor glycaemic control (HbA(1c) 8.6 +/- 1.3%), were investigated before and after 4 weeks of near-normalisation of blood glucose (mean blood glucose 7.4 +/- 1.2 mmol/l) using insulin treatment. Before and after insulin treatment the participants underwent three hyperglycaemic clamps (15 mmol/l) with infusion of GLP-1, GIP or saline. Insulin responses were evaluated as the incremental area under the plasma C-peptide curve. RESULTS: Before and after near-normalisation of blood glucose, the C-peptide responses did not differ during the early phase of insulin secretion (0-10 min). The late phase C-peptide response (10-120 min) increased during GIP infusion from 33.0 +/- 8.5 to 103.9 +/- 24.2 (nmol/l) x (110 min)(-1) (p < 0.05) and during GLP-1 infusion from 48.7 +/- 11.8 to 126.6 +/- 32.5 (nmol/l) x (110 min)(-1) (p < 0.05), whereas during saline infusion the late-phase response did not differ before vs after near-normalisation of blood glucose (40.2 +/- 11.2 vs 46.5 +/- 12.7 [nmol/l] x [110 min](-1)). CONCLUSIONS: Near-normalisation of blood glucose for 4 weeks improves beta cell responsiveness to both GLP-1 and GIP by a factor of three to four. No effect was found on beta cell responsiveness to glucose alone. CLINICALTRIALS.GOV ID NO.: NCT 00612950. FUNDING: This study was supported by The Novo Nordisk Foundation, The Medical Science Research Foundation for Copenhagen.
    41. 41
      Habegger, K. M.; Heppner, K. M.; Geary, N.; Bartness, T. J.; DiMarchi, R.; Tschop, M. H. The metabolic actions of glucagon revisited. Nat. Rev. Endocrinol. 2010, 6, 689– 697,  DOI: 10.1038/nrendo.2010.187
      41
      The metabolic actions of glucagon revisited
      Habegger, Kirk M.; Heppner, Kristy M.; Geary, Nori; Bartness, Timothy J.; DiMarchi, Richard; Tschoep, Matthias H.
      Nature Reviews Endocrinology (2010), 6 (12), 689-697CODEN: NREABD; ISSN:1759-5029. (Nature Publishing Group)
      A review. The diabetogenic effect of glucagon has long overshadowed the potential of this pancreatic hormone as an endogenous satiety and anti-obesity factor. This Review discusses the role of glucagon as a beneficial endocrine factor in lipid and energy metab. and its potential as a therapeutic agent on the basis of studies that combine the agonism of glucagon receptor and glucagon-like peptide 1 receptor. The initial identification of glucagon as a counter-regulatory hormone to insulin revealed this hormone to be of largely singular physiol. and pharmacol. purpose. Glucagon agonism, however, has also been shown to exert effects on lipid metab., energy balance, body adipose tissue mass and food intake. The ability of glucagon to stimulate energy expenditure, along with its hypolipidemic and satiating effects, in particular, make this hormone an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Studies that describe novel preclin. applications of glucagon, alone and in concert with glucagon-like peptide 1 agonism, have revealed potential benefits of glucagon agonism in the treatment of the metabolic syndrome. Collectively, these observations challenge us to thoroughly investigate the physiol. and therapeutic potential of insulin's long-known opponent.
    42. 42
      Finan, B.; Yang, B.; Ottaway, N.; Smiley, D. L.; Ma, T.; Clemmensen, C.; Chabenne, J.; Zhang, L.; Habegger, K. M.; Fischer, K.; Campbell, J. E.; Sandoval, D.; Seeley, R. J.; Bleicher, K.; Uhles, S.; Riboulet, W.; Funk, J.; Hertel, C.; Belli, S.; Sebokova, E.; Conde-Knape, K.; Konkar, A.; Drucker, D. J.; Gelfanov, V.; Pfluger, P. T.; Muller, T. D.; Perez-Tilve, D.; DiMarchi, R. D.; Tschop, M. H. A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat. Med. 2015, 21, 27– 36,  DOI: 10.1038/nm.3761
      42
      A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents
      Finan, Brian; Yang, Bin; Ottaway, Nickki; Smiley, David L.; Ma, Tao; Clemmensen, Christoffer; Chabenne, Joe; Zhang, Lianshan; Habegger, Kirk M.; Fischer, Katrin; Campbell, Jonathan E.; Sandoval, Darleen; Seeley, Randy J.; Bleicher, Konrad; Uhles, Sabine; Riboulet, William; Funk, Juergen; Hertel, Cornelia; Belli, Sara; Sebokova, Elena; Conde-Knape, Karin; Konkar, Anish; Drucker, Daniel J.; Gelfanov, Vasily; Pfluger, Paul T.; Mueller, Timo D.; Perez-Tilve, Diego; Di Marchi, Richard D.; Tschoep, Matthias H.
      Nature Medicine (New York, NY, United States) (2015), 21 (1), 27-36CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)
      We report the discovery of a new monomeric peptide that reduces body wt. and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiol. potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimol. triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body wt., enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacol. blockade and selective chem. knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclin. studies suggest that, so far, this unimol., polypharmaceutical strategy has potential to be the most effective pharmacol. approach to reversing obesity and related metabolic disorders.
    43. 43
      Pospisilik, J. A.; Hinke, S. A.; Pederson, R. A.; Hoffmann, T.; Rosche, F.; Schlenzig, D.; Glund, K.; Heiser, U.; McIntosh, C. H.; Demuth, H. Metabolism of glucagon by dipeptidyl peptidase IV (CD26). Regul. Pept. 2001, 96, 133– 141,  DOI: 10.1016/S0167-0115(00)00170-1
      43
      Metabolism of glucagon by dipeptidyl peptidase IV (CD26)
      Pospisilik, J. A.; Hinke, S. A.; Pederson, R. A.; Hoffmann, T.; Rosche, F.; Schlenzig, D.; Glund, K.; Heiser, U.; McIntosh, C. H. S.; Demuth, H.-U.
      Regulatory Peptides (2001), 96 (3), 133-141CODEN: REPPDY; ISSN:0167-0115. (Elsevier Science Ireland Ltd.)
      Glucagon is a 29-amino acid polypeptide released from pancreatic islet α-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymic metab. of glucagon was assessed using sensitive mass spectrometric techniques to identify the mol. products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential prodn. of glucagon3-29 and glucagon5-29. In human serum, degrdn. to glucagon3-29 was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degrdn., monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degrdn. and inactivation of glucagon. These findings have important implications for the detn. of glucagon levels in human plasma.
    44. 44
      Jaspan, J. B.; Polonsky, K. S.; Lewis, M.; Pensler, J.; Pugh, W.; Moossa, A. R.; Rubenstein, A. H. Hepatic-Metabolism of Glucagon in the Dog - Contribution of the Liver to Overall Metabolic Disposal of Glucagon. Am. J. Physiol. 1981, 240, E233– E244,  DOI: 10.1152/ajpendo.1981.240.3.E233
      44
      Hepatic metabolism of glucagon in the dog: contribution of the liver to overall metabolic disposal of glucagon
      Jaspan, J. B.; Polonsky, K. S.; Lewis, M.; Pensler, J.; Pugh, W.; Moossa, A. R.; Rubenstein, A. H.
      American Journal of Physiology (1981), 240 (3), E233-E244CODEN: AJPHAP; ISSN:0002-9513.
      The hepatic extn. (HE) of glucagon (I) [9007-92-5] and insulin (II) [9004-10-8] was measured in dogs, by peripheral infusion of the hormones following pancreatectomy or somatostatin [51110-01-1] infusion. HE of I was 22.5% and that of II was 45.1%. Somatostatin did not affect HE of either hormone. HE of endogenous II was similar to that of exogenously infused II. HE I was nonsaturable in the physiologic and pathophysiologic range of plasma I levels, but there was evidence of saturability in the pharmacologic range. Comparison of simultaneously measured parameters of II and I metab. indicated independence of the metabolic processes of these 2 islet hormones, despite distinct similarities in their overall patterns of metabolic disposal. Apparently, the liver is an important site for I removal.
    45. 45
      Unson, C. G.; Macdonald, D.; Merrifield, R. B. The role of histidine-1 in glucagon action. Arch. Biochem. Biophys. 1993, 300, 747– 750,  DOI: 10.1006/abbi.1993.1103
      45
      The role of histidine-1 in glucagon action
      Unson, Cecilia G.; Macdonald, Douglas; Merrifield, R. B.
      Archives of Biochemistry and Biophysics (1993), 300 (2), 747-50CODEN: ABBIA4; ISSN:0003-9861.
      The identification of position 9 aspartic acid in glucagon as a crit. residue for transduction reinforced the notion that specific residues in the peptide sequence dictate either receptor recognition or biol. activity. It was evident from previous studies that Asp9 operates in conjunction with His1 as part of the activation mechanism that follows binding to the glucagon receptor. This investigation was conducted to delineate structural features of histidine that contribute to its role in glucagon action. The authors report, based on binding and activity data from 10 replacement analogs, that the imidazole ring of His1 furnishes an arom. determinant for receptor binding affinity and that its protonatable imidazole nitrogen is important for transduction.
    46. 46
      Drucker, D. J. The Discovery of GLP-2 and Development of Teduglutide for Short Bowel Syndrome. ACS Pharmacol. Transl. Sci. 2019, 2, 134– 142,  DOI: 10.1021/acsptsci.9b00016
      46
      The Discovery of GLP-2 and Development of Teduglutide for Short Bowel Syndrome
      Drucker, Daniel J.
      ACS Pharmacology & Translational Science (2019), 2 (2), 134-142CODEN: APTSFN; ISSN:2575-9108. (American Chemical Society)
      A review. The proglucagon gene encodes multiple structurally-related peptides with overlapping actions promoting the absorption and assimilation of ingested energy. Notably, glucagon has been developed pharmaceutically to treat hypoglycemia and glucagon-like peptide-1 (GLP-1) receptor agonists are used for the therapy of type 2 diabetes and obesity. Here I describe the discovery of glucagon-like peptide-2 (GLP-2), a 33 amino acid peptide co-secreted together with GLP-1 from gut endocrine cells. GLP-2 was found to exhibit robust intestinal growth-promoting activity, following serendipitous observations that proglucagon-producing tumors induced intestinal growth in mice. Key developments in the pharmaceutical development of GLP-2 included the cloning of the GLP-2 receptor, and the recognition of the importance of dipeptidyl peptidase-4 as a crit. determinant of GLP-2 bioactivity. A therapeutic focus on short bowel syndrome, a serious medical disorder with compelling unmet medical need, enabled the pharmaceutical development of a simple GLP-2 analog, teduglutide, suitable for once daily administration.
    47. 47
      Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discovery Today 2015, 20, 122– 128,  DOI: 10.1016/j.drudis.2014.10.003
      47
      Peptide therapeutics: current status and future directions
      Fosgerau, Keld; Hoffmann, Torsten
      Drug Discovery Today (2015), 20 (1), 122-128CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)
      A review. Peptides are recognized for being highly selective and efficacious and, at the same time, relatively safe and well tolerated. Consequently, there is an increased interest in peptides in pharmaceutical research and development (R&D), and approx. 140 peptide therapeutics are currently being evaluated in clin. trials. Given that the low-hanging fruits in the form of obvious peptide targets have already been picked, it has now become necessary to explore new routes beyond traditional peptide design. Examples of such approaches are multifunctional and cell penetrating peptides, as well as peptide drug conjugates. Here, we discuss the current status, strengths, and weaknesses of peptides as medicines and the emerging new opportunities in peptide drug design and development.
    48. 48
      Marini, M.; Urbani, A.; Trani, E.; Bongiorno, L.; Roda, L. G. Interindividual variability of enkephalin-degrading enzymes in human plasma. Peptides 1997, 18, 741– 748,  DOI: 10.1016/S0196-9781(97)00129-0
      48
      Interindividual variability of enkephalin-degrading enzymes in human plasma
      Marini, Mario; Urbani, Alessandra; Trani, Eugenia; Bongiorno, Lucilla; Roda, L. Giorgio
      Peptides (Tarrytown, New York) (1997), 18 (5), 741-748CODEN: PPTDD5; ISSN:0196-9781. (Elsevier)
      The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis byproducts has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class - as well as the expression of the single enzyme species within each class - appears to be individually detd. Interindividual variability obsd. seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low mol. wt. plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the compn. of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and const. in blood obtained from successive drawings, at least within a relatively short period of time.
    49. 49
      Yakovleva, A. A.; Zolotov, N. N.; Sokolov, O. Y.; Kost, N. V.; Kolyasnikova, K. N.; Micheeva, I. G. Dipeptidylpeptidase 4 (DPP4, CD26) activity in the blood serum of term and preterm neonates with cerebral ischemia. Neuropeptides 2015, 52, 113– 117,  DOI: 10.1016/j.npep.2015.05.001
      49
      Dipeptidylpeptidase 4 (DPP4, CD26) activity in the blood serum of term and preterm neonates with cerebral ischemia
      Yakovleva, A. A.; Zolotov, N. N.; Sokolov, O. Yu.; Kost, N. V.; Kolyasnikova, K. N.; Micheeva, I. G.
      Neuropeptides (Oxford, United Kingdom) (2015), 52 (), 113-117CODEN: NRPPDD; ISSN:0143-4179. (Elsevier Ltd.)
      To investigate the mechanisms of inflammation in neonates after cerebral ischemia (CI), we evaluated the DPP4 activity in their blood sera and compared these values with clin. indicators. The activity of DPP4 was detd. in blood serum by a fluorescent method. We studied the correlation between the blood serum DPP4 activity and clin., neurol. and biochem. parameters in neonates with CI. No correlation between the DPP4 activity in umbilical blood and the venous blood of mothers was discovered. Increased blood serum DPP4 activity in full-term and pre-term newborns with CI is demonstrated. The interrelation between serum DPP4 activity and the functional disturbances of CNS (such as depression or excitement) was found in mature but not in premature newborns. Enzyme activity was still elevated at 2-3 wk after birth. It is possible that in neonates this enzymic system operates independently from mothers. It is assumed that increased DPP4 activity in newborns with CI is apparently connected with immune system activation in response to hypoxic stress. The obtained data support the participation of DPP4 in adaptive reactions of newborns and its regulating influence during hypoxemic damage of the CNS due to inflammation and neurodegeneration.
    50. 50
      Pels, K.; Kodadek, T. Solid-phase synthesis of diverse peptide tertiary amides by reductive amination. ACS Comb. Sci. 2015, 17, 152– 155,  DOI: 10.1021/acscombsci.5b00007
      50
      Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination
      Pels, Kevin; Kodadek, Thomas
      ACS Combinatorial Science (2015), 17 (3), 152-155CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)
      The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chem. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3-strain interactions. Here, the authors report an improved synthesis of these species on solid supports through the use of reductive amination chem. using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chem. is demonstrated by the synthesis of a library of 10 000 mixed peptoid-PTA oligomers.
    51. 51
      Xing, X.; Fichera, A.; Kumar, K. A novel synthesis of enantiomerically pure 5,5,5,5′,5′,5′-hexafluoroleucine. Org. Lett. 2001, 3, 1285– 1286,  DOI: 10.1021/ol015567e
      51
      A Novel Synthesis of Enantiomerically Pure 5,5,5,5',5',5'-Hexafluoroleucine
      Xing, Xuechao; Fichera, Alfio; Kumar, Krishna
      Organic Letters (2001), 3 (9), 1285-1286CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A novel, short, and efficient synthesis of (S)-5,5,5,5',5',5'-hexafluoroleucine (6) in greater than 99% ee was carried out starting from Garner's aldehyde, a protected oxazolidine aldehyde. The enantiomeric excess of the product was calcd. from an NMR anal. of a dipeptide formed by reaction with a protected L-serine deriv. Furthermore, a racemic sample of N-acylated hexafluoroleucine was enzymically resolved by treatment with porcine kidney acylase I and was found to have the same optical rotation as a synthetic sample of 6.
    52. 52
      Bušek, P.; Malík, R.; Šedo, A. Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer. Int. J. Biochem. Cell Biol. 2004, 36, 408– 421,  DOI: 10.1016/S1357-2725(03)00262-0
      52
      Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer
      Busek, Petr; Malik, Radek; Sedo, Aleksi
      International Journal of Biochemistry & Cell Biology (2004), 36 (3), 408-421CODEN: IJBBFU; ISSN:1357-2725. (Elsevier Science B.V.)
      A review. Post-translational modification of proteins is an important regulatory event. Numerous biol. active peptides that play an essential role in cancerogenesis contain an evolutionary conserved proline residue as a proteolytic-processing regulatory element. Proline-specific proteases could therefore be viewed as important "check-points". Limited proteolysis of such peptides may lead to quant. but, importantly, due to the change of receptor preference, also qual. changes of their signaling potential. Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with CD26) was for many years believed to be a unique cell membrane protease cleaving X-Pro dipeptides from the N-terminal end of peptides and proteins. Subsequently, a no. of other mols. were discovered, exhibiting various degree of structural homol. and DPP-IV-like enzyme activity, capable of cleaving similar set of substrates. These comprise for example, seprase, fibroblast activation protein α, DPP6, DPP8, DPP9, attractin, N-acetylated-α-linked-acidic dipeptidases I, II and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-β. It is tempting to speculate their potential participation on DPP-IV biol. function(s). Disrupted expression and enzymic activity of "DPP-IV activity and/or structure homologs" (DASH) might corrupt the message carried by their substrates, promoting abnormal cell behavior. Consequently, modulation of particular enzyme activity using e.g. DASH inhibitors, specific antibodies or DASH expression modification may be an attractive therapeutic concept in cancer treatment. This review summarizes recent information on the interactions between DASH members and their substrates with respect to their possible role in cancer biol.
    53. 53
      Keane, F. M.; Nadvi, N. A.; Yao, T. W.; Gorrell, M. D. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-alpha. FEBS J. 2011, 278, 1316– 1332,  DOI: 10.1111/j.1742-4658.2011.08051.x
      53
      Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α
      Keane, Fiona M.; Nadvi, Naveed A.; Yao, Tsun-Wen; Gorrell, Mark D.
      FEBS Journal (2011), 278 (8), 1316-1332CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Fibroblast activation protein-α (FAP) is a cell surface-expressed and sol. enzyme of the prolyl oligopeptidase family, which includes dipeptidyl peptidase 4 (DPP4). FAP is not generally expressed in normal adult tissues, but is found at high levels in activated myofibroblasts and hepatic stellate cells in fibrosis and in stromal fibroblasts of epithelial tumors. FAP possesses a rare catalytic activity, hydrolysis of the post-proline bond two or more residues from the N-terminus of target substrates. α2-Antiplasmin is an important physiol. substrate of FAP endopeptidase activity. This study reports the first natural substrates of FAP dipeptidyl peptidase activity. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY were the most efficiently hydrolyzed substrates and the first hormone substrates of FAP to be identified. In addn., FAP slowly hydrolyzed other hormone peptides, such as the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are efficient DPP4 substrates. FAP showed negligible or no hydrolysis of eight chemokines that are readily hydrolyzed by DPP4. This novel identification of FAP substrates furthers our understanding of this unique protease by indicating potential roles in cardiac function and neurobiol.
    54. 54
      Bjelke, J. R.; Christensen, J.; Nielsen, P. F.; Branner, S.; Kanstrup, A. B.; Wagtmann, N.; Rasmussen, H. B. Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV. Biochem. J. 2006, 396, 391– 399,  DOI: 10.1042/BJ20060079
      54
      Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV
      Bjelke, Jais R.; Christensen, Jesper; Nielsen, Per F.; Branner, Sven; Kanstrup, Anders B.; Wagtmann, Nicolai; Rasmussen, Hanne B.
      Biochemical Journal (2006), 396 (2), 391-399CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 resp. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar Ki values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.
    55. 55
      Hager, M. V.; Johnson, L. M.; Wootten, D.; Sexton, P. M.; Gellman, S. H. beta-Arrestin-Biased Agonists of the GLP-1 Receptor from beta-Amino Acid Residue Incorporation into GLP-1 Analogues. J. Am. Chem. Soc. 2016, 138, 14970– 14979,  DOI: 10.1021/jacs.6b08323
      55
      β-Arrestin-Biased Agonists of the GLP-1 Receptor from β-Amino Acid Residue Incorporation into GLP-1 Analogues
      Hager, Marlies V.; Johnson, Lisa M.; Wootten, Denise; Sexton, Patrick M.; Gellman, Samuel H.
      Journal of the American Chemical Society (2016), 138 (45), 14970-14979CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Activation of a G protein-coupled receptor (GPCR) causes recruitment of multiple intracellular proteins, each of which can activate distinct signaling pathways. This complexity has engendered interest in agonists that preferentially stimulate subsets among the natural signaling pathways ("biased agonists"). We have examd. analogs of glucagon-like peptide-1 (GLP-1) contg. β-amino acid residues in place of native α residues at selected sites and found that some analogs differ from GLP-1 in terms of their relative abilities to promote G protein activation (as monitored via cAMP prodn.) vs. β-arrestin recruitment (as monitored via BRET assays). The α → β replacements generally cause modest declines in stimulation of cAMP prodn. and β-arrestin recruitment, but for some replacement sets cAMP prodn. is more strongly affected than is β-arrestin recruitment. The central portion of GLP-1 appears to be crit. for achieving bias toward β-arrestin recruitment. These results suggest that backbone modification via α → β residue replacement may be a versatile source of agonists with biased GLP-1R activation profiles.
    56. 56
      Made, V.; Babilon, S.; Jolly, N.; Wanka, L.; Bellmann-Sickert, K.; Diaz Gimenez, L. E.; Morl, K.; Cox, H. M.; Gurevich, V. V.; Beck-Sickinger, A. G. Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias. Angew. Chem., Int. Ed. 2014, 53, 10067– 10071,  DOI: 10.1002/anie.201403750
      56
      Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias
      Made Veronika; Babilon Stefanie; Jolly Navjeet; Wanka Lizzy; Bellmann-Sickert Kathrin; Diaz Gimenez Luis E; Morl Karin; Cox Helen M; Gurevich Vsevolod V; Beck-Sickinger Annette G
      Angewandte Chemie (International ed. in English) (2014), 53 (38), 10067-71 ISSN:.
      Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.
    57. 57
      Montrose-Rafizadeh, C.; Avdonin, P.; Garant, M. J.; Rodgers, B. D.; Kole, S.; Yang, H.; Levine, M. A.; Schwindinger, W.; Bernier, M. Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells. Endocrinology 1999, 140, 1132– 1140,  DOI: 10.1210/endo.140.3.6550
      57
      Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells
      Montrose-Rafizadeh, Chahrzad; Avdonin, Pavel; Garant, Michael J.; Rodgers, Buel D.; Kole, Sutapa; Yang, Huan; Levine, Michael A.; Schwindinger, William; Bernier, Michel
      Endocrinology (1999), 140 (3), 1132-1140CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
      Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gsα, Gq/11α, and Gi1,2α, but not Gi3α. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), resp. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, resp. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.
    58. 58
      Hallbrink, M.; Holmqvist, T.; Olsson, M.; Ostenson, C. G.; Efendic, S.; Langel, U. Different domains in the third intracellular loop of the GLP-1 receptor are responsible for G alpha(s) and G alpha(i)/G alpha(o) activation. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 2001, 1546, 79– 86,  DOI: 10.1016/S0167-4838(00)00270-3
      58
      Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Gαs and Gαi/Gαo activation
      Hallbrink, M.; Holmqvist, T.; Olsson, M.; Ostenson, C.-G.; Efendic, S.; Langel, U.
      Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (2001), 1546 (1), 79-86CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
      It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Gαs proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP prodn. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Gαs but also to the Gαi/Gαo type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.
    59. 59
      Quoyer, J.; Longuet, C.; Broca, C.; Linck, N.; Costes, S.; Varin, E.; Bockaert, J.; Bertrand, G.; Dalle, S. GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells. J. Biol. Chem. 2010, 285, 1989– 2002,  DOI: 10.1074/jbc.M109.067207
      59
      GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a β-Arrestin 1-mediated ERK1/2 Activation in Pancreatic β-Cells
      Quoyer, Julie; Longuet, Christine; Broca, Christophe; Linck, Nathalie; Costes, Safia; Varin, Elodie; Bockaert, Joel; Bertrand, Gyslaine; Dalle, Stephane
      Journal of Biological Chemistry (2010), 285 (3), 1989-2002CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gαs/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacol. inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the assocn. of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c01237.

    • Two-dimensional diagram of His7 interacting with GLP1R (Figure S1A), His7 mutations of GLP1 and calculated EC50 (Figure S1B), concentration response curve of 1-GLP1 with DPP4 incubation (Figure S2), concentration response curve of semaglutide and 2-Semaglutide(Ala2) with DPP4 incubation (Figure S3), Michaelis–Menten kinetics of DPP4 (Figure S4), concentration response curves of 2-triagonist(Ala2) (Figure S5), MetEnk and 2-MetEnk (Figure S6), GLP1 and 2-GLP1 incubated with FAP and DPP9 (Figure S7), and in vivo serum stability of 2-liraglutide (Figure S8); safety information; materials and methods; and analytical characterization of synthesized compounds and peptides (Table S1) (PDF)


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