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Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

Sloop KW, Willard FS, Brenner MB, Ficorilli J, Valasek K, Showalter AD, Farb TB, Cao JX, Cox AL, Michael MD, Gutierrez Sanfeliciano SM, Tebbe MJ, Coghlan MJ - Diabetes (2010)

Bottom Line: 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.In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals.These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Discovery, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. sloop_kyle_w@lilly.com

ABSTRACT

Objective: The 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 methods: Screening 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.

Results: Novel 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.

Conclusions: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

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Related in: MedlinePlus

Compound B increases glucose-dependent insulin secretion from SD rat islets. A: Insulin concentrations from static cultures of SD rat islets incubated in media containing low glucose (2.8 mmol/l) and either GLP-1 (100 nmol/l), compound B (3 μmol/l), or the sulfonylurea glibenclamide (5 μmol/l). B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (0.3–10 μmol/l). All islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.
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Figure 3: Compound B increases glucose-dependent insulin secretion from SD rat islets. A: Insulin concentrations from static cultures of SD rat islets incubated in media containing low glucose (2.8 mmol/l) and either GLP-1 (100 nmol/l), compound B (3 μmol/l), or the sulfonylurea glibenclamide (5 μmol/l). B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (0.3–10 μmol/l). All islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.

Mentions: Compound B also showed activity in rat and human islets (Figs. 3B and 7A–C, respectively), enabling in vivo profiling of the compound. Additional details of the structure-activity relationship will be published in due course. To further evaluate compound B, assays were performed using the GLP-1 receptor peptide antagonist, exendin 9-39 (exendin-49-39) (24,25). This competitive antagonist binds the GLP-1 receptor ECD, but lacks the amino acids needed for interaction with extracellular loop regions to induce intracellular signaling (25,26). Treatment of GLP-1 receptor expressing HEK293 cells with exendin 9-39 blunted GLP-1 activity; however, the antagonist had no inhibitory effect on compound B-induced GLP-1 receptor signaling (Fig. 2B). These results suggest compound B activates the GLP-1 receptor via a different mechanism than native GLP-1. Consistent with these findings, compound B was unable to displace [125I] GLP-1 binding to cell membranes expressing the human GLP-1 receptor (data not shown). Additional in vitro studies were performed to further evaluate the mechanistic differences between GLP-1 and compound B using a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (deletion of amino acids 1–138; referred to as Δ–ECD-GLP-1 receptor). In line with the established requirement of the ECD for GLP-1 binding and receptor activation, GLP-1 (tested at concentrations as high as 300 nmol/l) was not active in cells expressing the Δ–ECD-GLP-1 receptor (Fig. 2C); however, compound B did show activation of the truncated receptor (Fig. 2C), indicating the ECD is not required to induce GLP-1 receptor signaling by this small molecule. Based on these results, compound B likely interacts with an allosteric site proximal to or within the GLP-1 receptor transmembrane domains. A similar hypothesis was proposed previously for the quinoxaline series of GLP-1 receptor agonists (13,16).


Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

Sloop KW, Willard FS, Brenner MB, Ficorilli J, Valasek K, Showalter AD, Farb TB, Cao JX, Cox AL, Michael MD, Gutierrez Sanfeliciano SM, Tebbe MJ, Coghlan MJ - Diabetes (2010)

Compound B increases glucose-dependent insulin secretion from SD rat islets. A: Insulin concentrations from static cultures of SD rat islets incubated in media containing low glucose (2.8 mmol/l) and either GLP-1 (100 nmol/l), compound B (3 μmol/l), or the sulfonylurea glibenclamide (5 μmol/l). B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (0.3–10 μmol/l). All islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.
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Related In: Results  -  Collection

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Figure 3: Compound B increases glucose-dependent insulin secretion from SD rat islets. A: Insulin concentrations from static cultures of SD rat islets incubated in media containing low glucose (2.8 mmol/l) and either GLP-1 (100 nmol/l), compound B (3 μmol/l), or the sulfonylurea glibenclamide (5 μmol/l). B: Insulin concentrations from static cultures of SD rat islets incubated in media containing high glucose (11.2 mmol/l) and either GLP-1 (100 nmol/l) or various concentrations of compound B (0.3–10 μmol/l). All islet treatments were performed for 90 min. Results are expressed as mean ± SEM, *P < 0.05.
Mentions: Compound B also showed activity in rat and human islets (Figs. 3B and 7A–C, respectively), enabling in vivo profiling of the compound. Additional details of the structure-activity relationship will be published in due course. To further evaluate compound B, assays were performed using the GLP-1 receptor peptide antagonist, exendin 9-39 (exendin-49-39) (24,25). This competitive antagonist binds the GLP-1 receptor ECD, but lacks the amino acids needed for interaction with extracellular loop regions to induce intracellular signaling (25,26). Treatment of GLP-1 receptor expressing HEK293 cells with exendin 9-39 blunted GLP-1 activity; however, the antagonist had no inhibitory effect on compound B-induced GLP-1 receptor signaling (Fig. 2B). These results suggest compound B activates the GLP-1 receptor via a different mechanism than native GLP-1. Consistent with these findings, compound B was unable to displace [125I] GLP-1 binding to cell membranes expressing the human GLP-1 receptor (data not shown). Additional in vitro studies were performed to further evaluate the mechanistic differences between GLP-1 and compound B using a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (deletion of amino acids 1–138; referred to as Δ–ECD-GLP-1 receptor). In line with the established requirement of the ECD for GLP-1 binding and receptor activation, GLP-1 (tested at concentrations as high as 300 nmol/l) was not active in cells expressing the Δ–ECD-GLP-1 receptor (Fig. 2C); however, compound B did show activation of the truncated receptor (Fig. 2C), indicating the ECD is not required to induce GLP-1 receptor signaling by this small molecule. Based on these results, compound B likely interacts with an allosteric site proximal to or within the GLP-1 receptor transmembrane domains. A similar hypothesis was proposed previously for the quinoxaline series of GLP-1 receptor agonists (13,16).

Bottom Line: 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.In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals.These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Discovery, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. sloop_kyle_w@lilly.com

ABSTRACT

Objective: The 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 methods: Screening 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.

Results: Novel 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.

Conclusions: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Show MeSH
Related in: MedlinePlus