Small Molecule Glucagon-like Peptide-1 Receptor Agonists Research Articles

A novel approach to exploit Small-Molecule glucagon-like Peptide-1 receptor agonists with high potency

Small-molecule glucagon-like peptide-1 receptor (GLP-1R) agonists are recognized as promising therapeutics for type 2 diabetes mellitus (T2DM) and obesity. Danuglipron, an investigational small-molecule agonist, has demonstrated high efficacy in clinical trials. However, further development of danuglipron is challenged by a high rate of gastrointestinal adverse events. While these effects may be target-related, it is plausible that the carboxylic acid group present in danuglipron may also play a role in these outcomes by affecting the pharmacokinetic properties and dosing regimen of danuglipron, as well as by exerting direct gastrointestinal irritation. Therefore, this study aims to replace the problematic carboxylic acid group by exploring the internal binding cavity of danuglipron bound to GLP-1R using a water molecule displacement strategy. A series of novel triazole-containing compounds have been designed and synthesized during the structure–activity relationship (SAR) study. These efforts resulted in the discovery of compound 2j with high potency (EC50 = 0.065 nM). Moreover, docking simulations revealed that compound 2j directly interacts with the residue Glu387 within the internal cavity of GLP-1R, effectively displacing the structural water previously bound to Glu387. Subsequent in vitro and in vivo experiments demonstrated that compound 2j had comparable efficacy to danuglipron in enhancing insulin secretion and improving glycemic control. Collectively, this study offers a practicable approach for the discovery of novel small-molecule GLP-1R agonists based on danuglipron, and compound 2j may serve as a lead compound to further exploit the unoccupied internal cavity of danuglipron's binding pocket.

Newer pharmacological interventions directed at gut hormones for obesity.

The objective is to review the newer pharmacological interventions for obesity, specifically single, dual and triple incretin receptor agonists that are either available or in the pipeline for treatment of obesity. The three incretin receptor targets are glucagon like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and glucagon. There are several approved single or dual incretin agonists which can be administered subcutaneously daily (e.g., liraglutide) or weekly (e.g., semaglutide, dulaglutide, and exenatide QW), and other experimental dual or triple incretin agonists. Analogues of amylin, peptide YY and oxyntomodulin, as well as the combination of a GLP1R agonist and GIPR antagonist also are in development. Oral semaglutide (administered daily) is approved for type 2 diabetes mellitus and is on track for regulatory review for obesity. The review includes specifically perspectives on the effects of these mechanisms and pharmacological agents on gastric emptying, which contribute to satiation and weight loss, in addition to the established evidence on effects on central mechanisms controlling appetite. In the future, it is anticipated that small molecule GLP-1 receptor agonists (e.g., oral danuglipron) will be developed for treating obesity. These pharmacological agents are having significant impact on glycaemic control and obesity and on their co-morbidities.

Mollugin activates GLP-1R to improve cognitive dysfunction in type 2 diabetic mice

AimsThe incidence of diabetic cognitive dysfunction is increasing year by year, and it has gradually become a research hot spot. Studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists can improve cognitive dysfunction in diabetic patients. This study focuses on whether small molecule GLP-1R agonists from traditional Chinese medicine (TCM) can improve the diabetic cognitive dysfunction. Materials and methodsThe small molecules from TCM were screened by cell membrane chromatography (CMC) with GLP-1R-HEK293 cell membrane column. MTT assay, flow cytometry, immunofluorescence cytochemistry and other methods were used to determine the effects of mollugin on the apoptosis rate and reactive oxygen species (ROS) level of high glucose (HG)/hydrogen peroxide (H2O2) induced PC12 cells. Real-Time PCR was used to detect mRNA expression in mouse cerebral cortex. Water maze test was further used to confirm the effect of mollugin on cognitive dysfunction in T2DM mice. Key findingsMollugin bound to GLP-1R, promoted Ca2+ influx, increased insulin secretion and cAMP content in β-TC-6 cells. Mollugin enhanced the cell viability, ameliorated apoptosis, reduced intracellular ROS levels in HG/H2O2-injured PC12 cells. Mollugin reduced the T2DM mice's escape latency, improved neuronal cell damage, decreased the expression of Pik3ca, Akt1 and Mapk1 mRNA in the cerebral cortex tissue. SignificanceThe results suggest that mollugin could improve cognitive dysfunction in T2DM mice through activating GLP-1R/cAMP/PKA signal pathway.

6-Azaspiro[2.5]octanes as small molecule agonists of the human glucagon-like peptide-1 receptor

Activation of the glucagon-like peptide-1 (GLP-1) receptor stimulates insulin release, lowers plasma glucose levels, delays gastric emptying, increases satiety, suppresses food intake, and affords weight loss in humans. These beneficial attributes have made peptide-based agonists valuable tools for the treatment of type 2 diabetes mellitus and obesity. However, efficient, and consistent delivery of peptide agents generally requires subcutaneous injection, which can reduce patient utilization. Traditional orally absorbed small molecules for this target may offer improved patient compliance as well as the opportunity for co-formulation with other oral therapeutics. Herein, we describe an SAR investigation leading to small-molecule GLP-1 receptor agonists that represent a series that parallels the recently reported clinical candidate danuglipron. In the event, identification of a benzyloxypyrimidine lead, using a sensitized high-throughput GLP-1 agonist assay, was followed by optimization of the SAR using substituent modifications analogous to those discovered in the danuglipron series. A new series of 6-azaspiro[2.5]octane molecules was optimized into potent GLP-1 agonists. Information gleaned from cryogenic electron microscope structures was used to rationalize the SAR of the optimized compounds.

760-P: Discovery of GSBR-1290, a Highly Potent, Orally Available, Novel Small Molecule GLP-1 Receptor Agonist

Peptidic glucagon-like peptide 1 receptor agonists (GLP-1RAs) are established treatments for patients with Type 2 Diabetes Mellitus (T2DM) and obesity. However, the need for injection and cold chain storage may limit the utility of most peptidic GLP-1RAs. An oral small molecule GLP-1RA that offers enhanced bioavailability and stability could be more convenient and accessible to patients. Here we report the discovery of GSBR-1290, a highly potent, orally available, novel small molecule GLP-1RA and the characterization of its in vitro and in vivo pharmacology profiles. GSBR-1290 is a small molecule GLP-1RA with high binding affinity to human GLP-1R. GSBR-1290 strongly activated GLP-1R Gαs cAMP pathway without inducing measurable β-arrestin recruitment signaling, which indicates it is a fully biased agonist. The insulin secretion stimulation effect of GSBR-1290 was evaluated in a functional human pancreatic beta cell line, in which GSBR-1290 showed dose dependent induction of insulin secretion. The in vivo efficacy of GSBR-1290 on insulin secretion, glucose control and food intake were evaluated in nonhuman primates (NHPs). In an acute intravenous glucose tolerance test (ivGTT), a single dose of GSBR-1290 strongly induced insulin secretion and glucose clearance. In a repeated dosing study, GSBR-1290 administered orally once daily for 7-day demonstrated robust increase in insulin secretion and glucose clearance in ivGTT and a dose dependent reduction of food intake and body weight. Overall, GSBR-1290 demonstrated potent in vivo efficacy in stimulating insulin secretion, improving glucose tolerance, and reducing food intake and body weight. In conclusion, GSBR-1290 is a highly potent, orally available, fully biased GLP-1RA. Human clinical trials are underway to further evaluate GSBR-1290 as a potential therapy for T2DM and obesity. Disclosure T.Mao: Employee; Structuretx. X.Lin: Employee; Structure Therapeutics. Q.Meng: Employee; Beijing Stonewise Technology, Structure Therapeutics Inc. H.Zhang: Employee; Structuretx. J.J.Zhang: Employee; Structure Therapeutics Inc. S.Shi: Employee; Structure Therapeutics Inc. Z.Guan: Employee; Structure Therapeutics. X.Jiang: Employee; Structure Therapeutics. F.Zhang: Employee; Structure Therapeutics, Eternity BioSciences, Stock/Shareholder; Novo Nordisk. H.Lei: Employee; Structure Therapeutics.

Cinchonine, a Potential Oral Small-Molecule Glucagon-Like Peptide-1 Receptor Agonist, Lowers Blood Glucose and Ameliorates Non-Alcoholic Steatohepatitis.

The glucagon-like peptide-1 receptor (GLP-1R) is an effective therapeutic target for type 2 diabetes mellitus (T2DM) and non-alcoholic steatohepatitis (NASH). Research has focused on small-molecule GLP-1R agonists because of their ease of use in oral formulations and improved patient compliance. However, no small-molecule GLP-1R agonists are currently available in the market. We aimed to screen for a potential oral small-molecule GLP-1R agonist and evaluated its effect on blood glucose and NASH. The Connectivity map database was used to screen for candidate small-molecule compounds. Molecular docking was performed using SYBYL software. Rat pancreatic islets were incubated in different concentrations glucose solutions, with cinchonine or Exendin (9-39) added to determine insulin secretion levels. C57BL/6 mice, GLP-1R-/- mice and hGLP-1R mice were used to conduct oral glucose tolerance test. In addition, we fed ob/ob mice with the GAN diet to induce the NASH model. Cinchonine (50 mg/kg or 100 mg/kg) was administered orally twice daily to the mice. Serum liver enzymes were measured using biochemical analysis. Liver tissues were examined using Hematoxylin-eosin staining, Oil Red O staining and Sirius Red staining. Based on the small intestinal transcriptome of geniposide, a recognized small-molecule GLP-1R agonist, we identified that cinchonine exerted GLP-1R agonist-like effects. Cinchonine had a good binding affinity for GLP-1R. Cinchonine promoted glucose-dependent insulin secretion, which could be attenuated significantly by Exendin (9-39), a specific GLP-1R antagonist. Moreover, cinchonine could reduce blood glucose in C57BL/6 and hGLP-1R mice, an effect that could be inhibited with GLP-1R knockout. In addition, cinchonine reduced body weight gain and food intake in ob/ob-GAN NASH mice dose-dependently. 100 mg/kg cinchonine significantly improved liver function by reducing the ALT, ALP and LDH levels. Importantly, 100 mg/kg cinchonine ameliorated hepatic steatosis and fibrosis in NASH mice. Cinchonine, a potential oral small-molecule GLP-1R agonist, could reduce blood glucose and ameliorate NASH, providing a strategy for developing small-molecule GLP-1R agonists.

Structural basis of peptidomimetic agonism revealed by small-molecule GLP-1R agonists Boc5 and WB4-24

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.

Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor

An absolute or relative deficiency of pancreatic β-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect β-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces β-cell replication and attenuate β-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic β-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional β-cell mass.

Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist S6 Stimulates Insulin Secretion From Rat Islets.

Glucagon-like peptide-1 receptor (GLP-1R) agonist-based therapeutics for type 2 diabetes mellitus have attracted worldwide attention. However, there are challenges in the development of small molecule GLP-1R agonists owing to the complexity of ligand recognition and signal induction mechanisms. Here, we attained S6 using virtual screening and fluorescent imaging plate reader (FLIPR)-based calcium assays. The purpose of this study was to identify and characterize S6, a novel small molecule GLP-1R agonist. Data from cellular thermal shift assay (CETSA) and Bio-Layer Interferometry (BLI) indicated that S6 could bind potently with GLP-1R. Radioimmunoassay data showed that S6 potentiated insulin secretion in a glucose-dependent manner and the insulinotropic effect was mediated by GLP-1R. Calcium imaging techniques suggested that S6 elevated the intracellular calcium concentration [(Ca2+)i] by activating GLP-1R. In patch-clamp experiments, we demonstrated that S6 inhibited voltage-dependent K+ (Kv) channels in a GLP-1R-dependent fashion. Besides, S6 significantly prolonged action potential duration but had no effect on voltage-dependent Ca2+ channels. In summary, these findings indicate that S6 stimulates glucose-dependent insulin secretion mainly by acting on GLP-1R, inhibiting Kv channels, increasing (Ca2+)i. This study will provide direction for the screening and development of novel small-molecule agents targeting GLP-1R in the future.

In silico prediction of GLP-1R agonists using machine learning approach

Glucagon-like peptide 1 receptor (GLP-1R) is a well-known drug target for the treatment of type 2 diabetes mellitus (T2DM). However, the currently marketed peptidyl GLP-1R agonist drugs are restricted by the requirement of injection. Hence, there is a continued need to develop orally bioavailable small molecule GLP-1R agonist drugs that could be beneficial for the treatment of T2DM. In this study, we report a new strategy to predict small molecule GLP-1R agonists with machine learning approach. Several regression and classification models were built based on support vector machine algorithm and diverse compounds with molecular properties and structural fingerprints as descriptors. For regression models, the ten-fold cross-validation squared correlation coefficient (q2, for training sets) and determination coefficient (r2, for test sets) of the optimized models were greater than 0.6, respectively. For classification models, the overall predictive accuracies were around or over 90% (for test sets). The results demonstrated that these reliable models could be used to identify highly active agonists for the purpose of virtual screening. The important properties and structural fragments for GLP-1R agonists derived from these models can be used for the novel GLP-1R agonist scaffold design.

The putative binding site and SAR rationalization of small molecules against glucagon-like peptide-1 receptor using homology model and crystal structures: a comparative study

Glucagon-like peptide-1 (GLP-1) is involved in glucose-stimulated insulin secretion and weight regulating actions through the activation of the GLP-1 receptor (GLP-1R). Clinical effectiveness of GLP-1 mimetics is effective in improving glucose control in patients. Thus, identifying and developing orally active small-molecule agonists are highly desirable. This study summarizes the structure-function relationship of hGLP-1R through computational approaches and search of small molecule GLP-1R agonists. We carried out mutation guided data-driven study, for developing the GLP-1R model to explore and validate the putative site for quinoxaline analogues. The developed GLP-1R homology model was subjected to 500 ns MD simulation for validation. Various snapshots were considered to identify the best structure of GLP-1R based on correlation between experimental pEC50 and various theoretical parameters (docking score, MM-GBSA ΔG bind, WM/MM ΔG bind). The putative binding site (Sitemap and WaterMap has been predicted and it matched well with the available data. Excellent correlation (R2 =0.94), between pEC50 and WM/MM ΔG bind for the snapshot at 350 ns was observed after including induced-fit docking results of the most potent molecule. Enrichment calculation indicates better AUC (=0.75) for predicted complex structure. A comparison of the developed GLP-1R model with the available crystal structure shows excellent similarities and it was used for virtual screening to find small molecule agonists. The good correlation of our model with crystal structures of GLP-1R may help to understand the structure-function relationship of other secretin families.

Synthesis and in vitro biological evaluation of new pyrimidines as glucagon-like peptide-1 receptor agonists

The therapeutic success of peptide glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small-molecule GLP-1 receptor agonists. In this study, two series of new pyrimidine derivatives were designed and synthesized using an efficient route, and were evaluated in terms of GLP-1 receptor agonist activity. In the first series, novel pyrimidines substituted at positions 2 and 4 with groups varying in size and electronic properties were synthesized in a good yield (78–90%). In the second series, the designed pyrimidine templates included both urea and Schiff base linkers, and these compounds were successfully produced with yields of 77–84%. In vitro experiments with cultured cells showed that compounds 3a and 10a (10−15–10−9M) significantly increased insulin secretion compared to that of the control cells in both the absence and presence of 2.8mM glucose; compound 8b only demonstrated significance in the absence of glucose. These findings represent a valuable starting point for the design and discovery of small-molecule GLP-1 receptor agonists that can be administered orally.

Small Molecule Drug Discovery at the Glucagon-Like Peptide-1 Receptor

The therapeutic success of peptide glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small molecule GLP-1 receptor agonists. Although the GLP-1 receptor is a member of the structurally complex class B1 family of GPCRs, in recent years, a diverse array of orthosteric and allosteric nonpeptide ligands has been reported. These compounds include antagonists, agonists, and positive allosteric modulators with intrinsic efficacy. In this paper, a comprehensive review of currently disclosed small molecule GLP-1 receptor ligands is presented. In addition, examples of “ligand bias” and “probe dependency” for the GLP-1 receptor are discussed; these emerging concepts may influence further optimization of known molecules or persuade designs of expanded screening strategies to identify novel chemical starting points for GLP-1 receptor drug discovery.

Two small molecule agonists of glucagon-like peptide-1 receptor modulate the receptor activation response differently

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9–36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.

Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

OBJECTIVEThe clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.RESEARCH DESIGN AND METHODSScreening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.RESULTSNovel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.CONCLUSIONSThese studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Comparative Effects of the Endogenous Agonist Glucagon-Like Peptide-1 (GLP-1)-(7-36) Amide and the Small-Molecule Ago-Allosteric Agent “Compound 2” at the GLP-1 Receptor

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

Measurement of shear stress on endothelium in a flow chamber and its gene expression response

The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9–36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists.

Wolfenden Report

<h3>Abstract</h3> Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited by the requirement for injection. Here we describe the first effective, orally bioavailable small molecule GLP-1R agonists. A sensitized high-throughput screen identified a series of small molecule GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nM potency. These small molecule agonists increased insulin levels in primates but not rodents, which is explained by a cryo-EM structure that revealed a binding pocket requiring primate-specific tryptophan 33. Importantly, oral administration of agonist PF-06882961 to healthy humans produced dose-dependent declines in serum glucose (NCT03309241). This opens the door to a new era of oral small molecule therapies that target the well-validated GLP-1R pathway for metabolic health. <h3>One Sentence Summary</h3> PF-06882961 is an orally administered small molecule that activates the GLP-1 receptor to lower blood glucose in humans.