Limits...
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.

Show MeSH

Related in: MedlinePlus

Low molecular weight GLP-1 receptor agonists activate GLP-1 receptor signaling. A: Compound A and compound B induced GLP-1 receptor-mediated signaling in HEK293 cells coexpressing the human GLP-1 receptor and a 3x-cAMP response element-luciferase reporter, but were not active in cells lacking the GLP-1 receptor. In the human GLP-1 receptor HEK293 cells, the EC50 values for compounds A and B were 1.6 μmol/l and 0.66 μmol/l, respectively; data are presented as percentages of stimulation of a maximum concentration of human GLP-1. B: The competitive GLP-1 receptor peptide antagonist exendin (9-39) blunted GLP-1 peptide activity but did not reduce compound B-induced signaling. C: Compound B was active in HEK293 cells expressing a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (Δ–ECD-GLP-1 receptor); the native GLP-1 peptide had no effect in cells expressing the Δ–ECD-GLP-1 receptor. D: Compound B was not active in HEK293 cells expressing the glucagon-like peptide-2 receptor (GLP-2R), glucose-dependent insulinotropic polypeptide receptor (GIP-R), glucagon receptor (GCG-R), or parathyroid hormone receptor (PTH-R).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992771&req=5

Figure 2: Low molecular weight GLP-1 receptor agonists activate GLP-1 receptor signaling. A: Compound A and compound B induced GLP-1 receptor-mediated signaling in HEK293 cells coexpressing the human GLP-1 receptor and a 3x-cAMP response element-luciferase reporter, but were not active in cells lacking the GLP-1 receptor. In the human GLP-1 receptor HEK293 cells, the EC50 values for compounds A and B were 1.6 μmol/l and 0.66 μmol/l, respectively; data are presented as percentages of stimulation of a maximum concentration of human GLP-1. B: The competitive GLP-1 receptor peptide antagonist exendin (9-39) blunted GLP-1 peptide activity but did not reduce compound B-induced signaling. C: Compound B was active in HEK293 cells expressing a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (Δ–ECD-GLP-1 receptor); the native GLP-1 peptide had no effect in cells expressing the Δ–ECD-GLP-1 receptor. D: Compound B was not active in HEK293 cells expressing the glucagon-like peptide-2 receptor (GLP-2R), glucose-dependent insulinotropic polypeptide receptor (GIP-R), glucagon receptor (GCG-R), or parathyroid hormone receptor (PTH-R).

Mentions: Since identification of small molecule activators of class B GPCRs remains largely elusive, we surmised that assessment of additional chemical space associated with several literature reports of GLP-1 receptor agonists (13,17,21–23) would provide a more rational group of molecules for evaluation versus high throughput screening. Accordingly, a series of three-dimensional pharmacophore models was created, leading to the identification of a small (∼2000 compound) library gleaned from a variety of internal and commercial sources. This library was designed to assess key hypotheses associated with novel chemical space aiming to identify new GLP-1 receptor ligands. Compound A (Fig. 1A) was discovered via screening of this library using HEK293 cells stably cotransfected with the human GLP-1 receptor (NM_002062) and a 3X-cAMP response element (CRE) luciferase reporter. Compound A, which is commercially available (CAS registry number: 870083-94-6), increased luciferase in GLP-1 receptor HEK293 cells, but was inactive in parent HEK293 cells. Compound A displayed an EC50 value of 1.6 μmol/l with 46% efficacy relative to maximum stimulation by GLP-1 (Fig. 2A). Although this compound represented a viable starting point with moderate in vitro activity and some insulinotropic capability in ex vivo rat islet assays, structural modifications were introduced to improve potency to warrant in vivo studies. Through examination of the various regions of the molecule, Compound B (Fig. 1B) with a benzyloxy substituent introduced onto the phenyl ring of compound A in the meta position, along with deletion of the chlorine atom and a change of the oxidation state of the sulfur from sulfone to sulfoxide, resulted in an improved EC50 value of 0.66 μmol/l and 99% efficacy (similar data were obtained using the rat GLP-1 receptor; EC50 = 0.755 μmol/l, 85% efficacy of maximum GLP-1). Neither compound A nor compound B were active in cells expressing the GLP-2, GIP, PTH, or glucagon receptors (Fig. 2D and data not shown).


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)

Low molecular weight GLP-1 receptor agonists activate GLP-1 receptor signaling. A: Compound A and compound B induced GLP-1 receptor-mediated signaling in HEK293 cells coexpressing the human GLP-1 receptor and a 3x-cAMP response element-luciferase reporter, but were not active in cells lacking the GLP-1 receptor. In the human GLP-1 receptor HEK293 cells, the EC50 values for compounds A and B were 1.6 μmol/l and 0.66 μmol/l, respectively; data are presented as percentages of stimulation of a maximum concentration of human GLP-1. B: The competitive GLP-1 receptor peptide antagonist exendin (9-39) blunted GLP-1 peptide activity but did not reduce compound B-induced signaling. C: Compound B was active in HEK293 cells expressing a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (Δ–ECD-GLP-1 receptor); the native GLP-1 peptide had no effect in cells expressing the Δ–ECD-GLP-1 receptor. D: Compound B was not active in HEK293 cells expressing the glucagon-like peptide-2 receptor (GLP-2R), glucose-dependent insulinotropic polypeptide receptor (GIP-R), glucagon receptor (GCG-R), or parathyroid hormone receptor (PTH-R).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992771&req=5

Figure 2: Low molecular weight GLP-1 receptor agonists activate GLP-1 receptor signaling. A: Compound A and compound B induced GLP-1 receptor-mediated signaling in HEK293 cells coexpressing the human GLP-1 receptor and a 3x-cAMP response element-luciferase reporter, but were not active in cells lacking the GLP-1 receptor. In the human GLP-1 receptor HEK293 cells, the EC50 values for compounds A and B were 1.6 μmol/l and 0.66 μmol/l, respectively; data are presented as percentages of stimulation of a maximum concentration of human GLP-1. B: The competitive GLP-1 receptor peptide antagonist exendin (9-39) blunted GLP-1 peptide activity but did not reduce compound B-induced signaling. C: Compound B was active in HEK293 cells expressing a modified form of the GLP-1 receptor lacking the NH2-terminal ECD (Δ–ECD-GLP-1 receptor); the native GLP-1 peptide had no effect in cells expressing the Δ–ECD-GLP-1 receptor. D: Compound B was not active in HEK293 cells expressing the glucagon-like peptide-2 receptor (GLP-2R), glucose-dependent insulinotropic polypeptide receptor (GIP-R), glucagon receptor (GCG-R), or parathyroid hormone receptor (PTH-R).
Mentions: Since identification of small molecule activators of class B GPCRs remains largely elusive, we surmised that assessment of additional chemical space associated with several literature reports of GLP-1 receptor agonists (13,17,21–23) would provide a more rational group of molecules for evaluation versus high throughput screening. Accordingly, a series of three-dimensional pharmacophore models was created, leading to the identification of a small (∼2000 compound) library gleaned from a variety of internal and commercial sources. This library was designed to assess key hypotheses associated with novel chemical space aiming to identify new GLP-1 receptor ligands. Compound A (Fig. 1A) was discovered via screening of this library using HEK293 cells stably cotransfected with the human GLP-1 receptor (NM_002062) and a 3X-cAMP response element (CRE) luciferase reporter. Compound A, which is commercially available (CAS registry number: 870083-94-6), increased luciferase in GLP-1 receptor HEK293 cells, but was inactive in parent HEK293 cells. Compound A displayed an EC50 value of 1.6 μmol/l with 46% efficacy relative to maximum stimulation by GLP-1 (Fig. 2A). Although this compound represented a viable starting point with moderate in vitro activity and some insulinotropic capability in ex vivo rat islet assays, structural modifications were introduced to improve potency to warrant in vivo studies. Through examination of the various regions of the molecule, Compound B (Fig. 1B) with a benzyloxy substituent introduced onto the phenyl ring of compound A in the meta position, along with deletion of the chlorine atom and a change of the oxidation state of the sulfur from sulfone to sulfoxide, resulted in an improved EC50 value of 0.66 μmol/l and 99% efficacy (similar data were obtained using the rat GLP-1 receptor; EC50 = 0.755 μmol/l, 85% efficacy of maximum GLP-1). Neither compound A nor compound B were active in cells expressing the GLP-2, GIP, PTH, or glucagon receptors (Fig. 2D and data not shown).

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