Novel Site-Specific Fatty Chain-Modified GLP-1 Receptor Agonist with Potent Antidiabetic Effects

Glucagon-like peptide-1 receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). Here, we designed a high-throughput GLP-1R extracellular domain (ECD)-based system that enabled the screening of high-potency receptor-biased GLP-1R agonists demonstrating new pharmacological virtues. Firstly, six 12-mer peptides (termed PEP01–06), screened from a large phage displayed peptide library were fused to the N-terminus of Exendin-4 (29–39) to generate PEP07–12. By the use of four lysine-altered PEP07 (PEP13–16) as the starting point, a series of fatty chain conjugates (PEP17–20) were synthesized and evaluated by in vitro GLP-1R-based cell assays. In addition, the acute and long-term in vivo effects on diet-induced obesity (DIO) mice were further evaluated. All four conjugates showed good receptor activation efficacy, and PEP20 was selected to undergo further assessment. Preclinical experiments in DIO mice demonstrated that PEP20 had significant insulinotropic activities and glucose-lowering abilities. Moreover, a prolonged antidiabetic effect of PEP20 was also observed by the hypoglycemic test in DIO mice. Furthermore, long-term treatment with PEP20 achieved beneficial effects on the food intake, weight gain, hemoglobin A1C (HbA1C) lowering activity, and glucose tolerance compared with the control and was similar to the Liraglutide. In conclusion, PEP20, a GLP-1R ECD-biased agonist, may provide a novel therapeutic approach to T2DM.


Introduction
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder characterized by hyperglycemia arising from insufficient insulin secretion and insulin resistance [1,2]. Glucagon-like peptide-1 (GLP-1) is a 30-amino-acid peptide hormone secreted from gut endocrine L-cells in response to nutrient ingestion, and plays an important role in glucose homeostasis and nutrient metabolism [3][4][5]. GLP-1 lowers postprandial glucose excursion by potentiating glucose stimulated insulin secretion from pancreatic B cells in a glucose-dependent mode. In addition, GLP-1 outperforms other oral hypoglycemic agents in promoting gastric emptying, weight loss and increasing insulin sensitivity in peripheral tissues. Hence, GLP-1-based therapies represent a strategy for the treatment of T2DM [6][7][8]. For example, Exendin-4, a 53% homology with native GLP-1, is a clinically-approved full agonist with a stronger affinity for GLP-1R and longer in vivo half-life [9][10][11][12].
GLP-1, or Exendin-4, exerts its action through the GLP-1R, which is expressed in a number of organs, including the pancreas, the central nervous system and the peripheral tissues. GLP-1R,

Discovery of Novel Glucagon-Like Peptide-1 Receptor Agonists
To discover the potential peptides with high affinity for GLP-1R a commercial peptide library, designed based on M13 phage and containing linear 12 random peptide sequences, was subjected for biopanning the GLP-1R ECD-binding domain ( Figure 1). Six peptides, with high affinity for GLP-1R ECD, were selected from peptide libraries using a phage display technique and defined as PEP01-PEP06.   The sequence GGPSSGAPPPS of Exendin-4, as the key C-terminal region which could enhance its affinity for the receptor, interacts with the N terminus ECD of GLP-1R, facilitating the receptor activation [20,25]. On basis of these Exendin-4 structure-activity insights, the selected short GLP-1R agonists (PEP01-06) peptide library consisting of random 12 amino acids fused to Exendin-4(29-39) was generated, which accounted for the increased receptor activation activity of Exendin-4 compared with GLP-1. As a result, six hybrid peptides termed PEP07-PEP12 were constructed after the fusion of two sequences. All of these hybrid peptides were identified to exhibit higher affinity for GLP-1R ECD after fusion (Table 1).
SPR measurements showed a significant higher binding affinity of PEP07 for GLP-1R ECD than either peptide, yielding a K d of 24 nM. Considering the predictable short half-life in vivo, these candidate molecules still should be further modified to enhance bioavailability. Therefore, PEP07 was selected as the lead peptides for further acylation.

Synthesis and Characterization of the Fatty Chain-Modified Hybrid Glucagon-Like Peptide-1 Receptor Agonists
Lysine scanning mutagenesis was performed to determine a suitable acylation site. The four consecutive amino acids (YLET) of PEP07 were individually replaced by lysine to generate four peptides ( Figure 2, named PEP13-16). The associating affinity of Lys-PEP07 peptides for GLP-1R ECD are shown in Table 2. Although lysine mutagenesis on a few sites led to decreased affinity (e.g., PEP15), lysine replacement at some sites did not affect the receptor binding potency, such as PEP14 and PEP16. Particularly, PEP16, with highest associating affinity for GLP-1R ECD (25 nM), was selected for further site specific acylation.    As shown in Table 3, the selected lysine-altered peptides (PEP13-16) were reacted with fatty chains to give four fatty chain-modified conjugates (PEP17-20) which were purified by preparative reverse phase high-performance liquid chromatography (RP-HPLC) and further identified by liquid chromatrography tandem mass spectrometry (LC-MS).

In Vitro Activity of Hybrid Glucagon-Like Peptide-1 Receptor Agonist
As shown in Table 4, slight reduction in the activation potency was observed due to the fatty chain modifications. Notably, the modified conjugates (PEP17 and PEP18), close to a consensus motif of "YSSPXA", which could enhance its affinity for the receptor, were less potent to the modified conjugates, such as PEP20, which were relatively far away from the probable active domain. Since the EC 50 of PEP20 in both human or mouse GLP-1R activation was similar to the reference peptide Exendin-4, and better than the native GLP-1 or Liraglutide, we selected this molecule for the further efficacy evaluation in vivo.

Glucoregulatory and Insulin Secretion Assay
Oral glucose tolerance test was performed at 1 h after diet-induced obesity (DIO) mice received 25 nmol/kg PEP20, Exendine-4 or Liraglutide to evaluate the glucose-lowering and insulinotropic effects ( Figure 3A,B). Half an hour after a glucose challenge, the blood glucose level (BGL) in the vehicle treated group rapidly increased to 23.6 ± 3.1 mmol/L while in the PEP20-, Exendin-4-, and Liraglutide-treated mice, dramatically reduced to 10.1, 9.8, and 12.2 mmol/L, respectively. Moreover, a glucose-lowering percentage of 60.2% was showed in PEP20 treated group, while the Exendine-4 or Liraglutide group showed a reduction of 66.1% or 46.1%, respectively, compared with the vehicle control group.
Similar time courses for plasma insulin concentrations in PEP20, Liraglutide or Exendin-4 treated group were observed and all plasma insulin concentrations from 15 to 60 min were significantly greater than those of the control group. In particular, PEP20 showed similar and comparable insulin secretion promoting ability than Exendin-4 and Liraglutide, respectively ( Figure 3C,D).
or Liraglutide group showed a reduction of 66.1% or 46.1%, respectively, compared with the vehicle control group.
Similar time courses for plasma insulin concentrations in PEP20, Liraglutide or Exendin-4 treated group were observed and all plasma insulin concentrations from 15 to 60 min were significantly greater than those of the control group. In particular, PEP20 showed similar and comparable insulin secretion promoting ability than Exendin-4 and Liraglutide, respectively ( Figure  3C,D). Results are presented as means ± SD (n = 6 each group). *** p < 0.001.

Hypoglycemic Duration Test
The antihyperglycemic efficacy of PEP20 was investigated in male DIO mice. The BGLs of PEP20 group decreased rapidly to the normoglycemic state (<15 mmol/L) in 1 h after the administration of 25 nmol/kg. The lowest BGLs in the PEP20 group were similar to the Liraglutide group ( Figure 4A). Moreover, the glucose-lowering effect of PEP20 was maintained up to at least 96 h, but only about 24 h in Liraglutide treated group. Furthermore, PEP20 significantly lowered the glucose level of 63.8% for 0-96 h compared with the vehicle group ( Figure 4B), while it was only 23.2% after the Liraglutide Results are presented as means ± SD (n = 6 each group). *** p < 0.001.

Hypoglycemic Duration Test
The antihyperglycemic efficacy of PEP20 was investigated in male DIO mice. The BGLs of PEP20 group decreased rapidly to the normoglycemic state (<15 mmol/L) in 1 h after the administration of 25 nmol/kg. The lowest BGLs in the PEP20 group were similar to the Liraglutide group ( Figure 4A). Moreover, the glucose-lowering effect of PEP20 was maintained up to at least 96 h, but only about 24 h in Liraglutide treated group. Furthermore, PEP20 significantly lowered the glucose level of 63.8% for 0-96 h compared with the vehicle group ( Figure 4B), while it was only 23.2% after the Liraglutide treatment. These results revealed PEP20 has better antihyperglycemic efficacy and hypoglycemic duration performance than the commercial Liraglutide at the same dose. treatment. These results revealed PEP20 has better antihyperglycemic efficacy and hypoglycemic duration performance than the commercial Liraglutide at the same dose.

Chronic In Vivo Studies
To further examine the therapeutic potential of PEP20, chronic in vivo studies were performed by a daily subcutaneous (s.c.) injection of PEP20 (25nmol/kg) in DIO mice for 4 weeks. As is indicated in Figure 5A,B, the body weight gains and food intake amounts were effectively suppressed in PEP20 treated mice, to a comparable extent to Liraglutide treated ones. Moreover, as is showed in Figure   Figure 4. Glucose-lowering and stabilizing effects of Liraglutide and PEP20 were determined by hypoglycemic duration test in DIO mice. (A) Antihyperglycemic efficacies of Liraglutide and PEP20 in DIO mice pretreated with each sample (25 nmol/kg) for 96 h. (B) Hypoglycemic effects of control, Liraglutide and PEP20 based on AUC 0-96 h . Results are presented as means ± SD (n = 6 each group). *** p < 0.001.

Chronic In Vivo Studies
To further examine the therapeutic potential of PEP20, chronic in vivo studies were performed by a daily subcutaneous (s.c.) injection of PEP20 (25 nmol/kg) in DIO mice for 4 weeks. As is indicated in Figure 5A,B, the body weight gains and food intake amounts were effectively suppressed in PEP20 treated mice, to a comparable extent to Liraglutide treated ones. Moreover, as is showed in Figure 5C, both PEP20 and Liraglutide treatment resulted in a reduction in HbA1c values (p < 0.001, n = 6) than that in control group.
OGTT tests were performed to evaluate whether a long-term treatment of PEP20 helped to improve the glucose metabolism in DIO mice. The results, graphed in Figure 5D, revealed that there is no significant difference between the area under the curve (AUC) of saline treated group before or after 4 weeks administration. However, we found that the AUC of PEP20 and Liraglutide treated group at 4 weeks decreased 22.1% or 38.2% compared with week 0. These results showed that PEP20 exerted a significant improvement in the glucose metabolism of DIO mice which was similar to Liraglutide, a commercialized GLP-1R agonist. Results are presented as means ± SD (n = 6 each group). * p < 0.05, ** p < 0.02, *** p < 0.001.

Discussion
G protein-coupled receptor (GPCR) peptide ligands have always been noted for their selectivity, potency, and rapid optimization [26]. Although typically peptides can be developed by chemical synthesis, they have obvious disadvantages, such as a low-throughput process that intrinsically lacks diversity. To address this, we have developed affinity-based screening technologies from a random peptide library with a storage capacity of more than one billion peptides. In the proof-of concept experiments presented here, we show how this methodology allows one to detect and identify novel GLP-1R ligands with unique pharmacology.
GLP-1, an endogenous polypeptide hormone released in response to food intake, potentiate insulin secretion from pancreatic cells through binding and activation of its receptor, and GLP-1R agonists have emerged as treatment options for type 2 diabetes mellitus. [6,18,27]. Although the treatments with GLP-1R agonists demonstrate the significant acute or long-term antidiabetic activities [28,29], there were a great many problems such as unavoidable side effects and the homogenization because almost all the related drugs were developed according to the only two native GLP-1R agonists (GLP-1 and Exendin-4) [30][31][32]. Recently, a novel concept of 'biasing seventransmembrane receptors', for the developing drugs have been getting increased attention for its

Discussion
G protein-coupled receptor (GPCR) peptide ligands have always been noted for their selectivity, potency, and rapid optimization [26]. Although typically peptides can be developed by chemical synthesis, they have obvious disadvantages, such as a low-throughput process that intrinsically lacks diversity. To address this, we have developed affinity-based screening technologies from a random peptide library with a storage capacity of more than one billion peptides. In the proof-of concept experiments presented here, we show how this methodology allows one to detect and identify novel GLP-1R ligands with unique pharmacology.
GLP-1, an endogenous polypeptide hormone released in response to food intake, potentiate insulin secretion from pancreatic cells through binding and activation of its receptor, and GLP-1R agonists have emerged as treatment options for type 2 diabetes mellitus. [6,18,27]. Although the treatments with GLP-1R agonists demonstrate the significant acute or long-term antidiabetic activities [28,29], there were a great many problems such as unavoidable side effects and the homogenization because almost all the related drugs were developed according to the only two native GLP-1R agonists (GLP-1 and Exendin-4) [30][31][32]. Recently, a novel concept of 'biasing seven-transmembrane receptors', for the developing drugs have been getting increased attention for its potential greater therapeutic effects while possibly avoiding unwanted side effects [33]. The GLP-1R consists of a predicted signal sequence, the ECD of approximately 120 residues, seven membrane-spanning α-helices connected by three extracellular and three intracellular loops (the TM domain), and a C-terminal intracellular domain [20]. It is well established that the N-terminal ECD of incretin receptors is important for ligand binding and ligand specificity, whereas the transmembrane domain is involved in receptor activation [15]. Moreover, the GLP-1R ECD, with ligand binding function, can be expressed in both eukaryotic and prokaryotic systems, such as Escherichia coli and mammalian cells [34,35].
Here, a GLP-1R ECD-based system to screen the large phage displayed 12-mer peptide library with the aim of identifying receptor-biased GLP-1R agonists was successfully established (Figure 1). As a result, six 12-mer peptides (PEP01-06) were successfully screened from a large phage displayed peptide library (Table 1). Interestingly, three of the peptides contained a motif of YSSPXA (where X is S, A, or T), which could enhance its affinity for the receptor. Then the fusion of six selected peptides to the Exendin-4(29-39) was confirmed to significantly enhance their affinity for GLP-1R (Table 1). Considering the predictable short half-life in vivo, we selected the PEP07, with the highest affinity for GLP-1R ECD, as the leader peptide for the further lysine mutagenesis and side chain modification of fatty acid to determine whether different conjugation sites affected the in vitro activity ( Figure 2 and Table 2). A series of fatty chain conjugates (PEP17-20) were synthesized and evaluated by in vitro GLP-1R-based cell assays and results PEP20 showed similar effects on the activation of both human and mouse GLP-1R compared to Exendin-4 (Table 4).
It was not only that PEP20 showed similar and comparable promoting insulin secretion ability than Exendin-4 and Liraglutide, respectively, correspond with the in vitro results (Figure 3). The long-acting glucose-lowering effects of PEP20 were further confirmed by the hypoglycemic duration in DIO mice. As we expect, the glucose-lowering effect of PEP20 was maintained up to at least 96 h, which was significant longer than Liraglutide, a commercial GLP-1R agonist (Figure 4). Chronic in vivo studies on DIO mice showed that the body weight gains and food intake amounts were effectively suppressed in PEP20 treated group, to a comparable extent to Liraglutide treated ones ( Figure 5A,B). Moreover, as is showed in Figure 5C, both PEP20 and Liraglutide treatment resulted in a reduction in HbA1c values (p < 0.001, n = 6) than that in control group. At week 0, glucose tolerance in both control, Liraglutide and PEP20 treatment group is similar, but at 4 weeks, effect of PEP20 on improvement of impaired glucose tolerance in DIO mice was predominantly compared with vehicle control group ( Figure 5D).
Moreover, what deserves more investigation is that PEP20 showed significant better hypoglycemic duration than Liraglutide while both the improvement on the HbA1c lowering and glucose tolerance of DIO mice were a little weaker than Liraglutide. We hypothesized that might be possibly because that newly designed PEP20 owns a lower sequence homology than Liraglutide compared with native GLP-1 or the coupled fatty chain which lead to a weaker long-term effects on DIO mice. Considering that PEP20, in spite of its prolonged glucose lowering ability, may not the best choice to be a candidate to bring to clinics, we did not further investigate the pharmacokinetic properties of it. Somewhere along the line, we will further to evaluate PEP20 in some other animal model, such as db/db mice or optimize the sequence combination (e.g., PEP07 and GLP-1 (9-37)) and fatty chain of PEP20.
In summary, GLP-1R-based binding screening of phage display peptide libraries enabled the discovery of high-potency GLP-1R agonists. A series of preclinical studies demonstrates that the fusion of selected GLP-1R agonist and Exendin-4 (29-39) showed a significant enhancement in the affinity for GLP-1R ECD. Then the further side fatty chain modified hybrid peptide PEP20 showed well