A nonpeptidic agonist of glucagon-like peptide 1 receptors with efficacy in diabetic db/db mice

Abstract

Peptidic mimics of the gut hormone glucagon-like peptide (GLP) 1, exemplified by the recently approved drug exenatide, show promise as therapies for type 2 diabetes. Such "incretin mimetics" regulate glucose appearance in the plasma and can restore glucose-stimulated insulin secretion without excess risk of hypoglycemia. The need for injection, which may limit the use of peptidic GLP-1 receptor (GLP-1R) agonists, has driven largely unsuccessful efforts to find smaller molecules. The failure to identify orally effective agonists has instead promoted the indirect approach of inhibiting the GLP-1-degrading enzyme dipeptidyl peptidase IV. Here we report a nonpeptidic GLP-1R agonist with sufficient activity to evoke effects in whole animals, including antidiabetic efficacy in db/db mice. Two substituted cyclobutanes (S4P and Boc5) were developed after screening a compound library against a cell line stably cotransfected with GLP-1R and a cAMP-responsive reporter. Each bound to GLP-1R and increased intracellular cAMP. Agonist effects were blocked by the GLP-1R antagonist exendin(9-39). Boc5 amplified glucose-stimulated insulin secretion in isolated rat islets. Both i.p. and oral administration of Boc5 dose-dependently inhibited food intake in mice, an effect that could be blocked by pretreatment with exendin(9-39). Daily injections of Boc5 into db/db mice reduced HbA1c to nondiabetic values, an effect not observed in ad libitum-fed or pair-fed diabetic controls. Thus, Boc5 behaved as a full GLP-1 mimetic in vitro and in vivo. The chemical genus represented by Boc5 may prompt the exploration of orally available GLP-1R agonists with potential utility in diabetes and obesity.

Fig. 1.

Structures of SH14800, NC133908, NC133909, S4P, and Boc5.

Fig. 2.

The effects of the substituted cyclobutanes are mediated through GLP-1R. (a) cAMP response element-driven luciferase activities were induced by different concentrations of GLP-1, S4P, and Boc5 in the HEK293-rGLP-1R cells. Estimated EC₅₀ values were 68 pM, 1.08 μM, and 2.73 μM, respectively. (b) GLP-1R-mediated luciferase responses to Boc5 (10 μM), S4P (10 μM), and GLP-1 (0.05 nM) were each dose-dependently blocked by exendin(9–39). (c) Competitive inhibition of [¹²⁵I]GLP-1(7–36) amide binding to GLP-1R by GLP-1, S4P, or Boc5. Estimated K_i values were 0.66 nM, 287 nM, and 1.47 μM, respectively. (d) The effect of Boc5 on the glucose-stimulated insulin secretion in rat isolated pancreatic islets. Symbols are means ± SEM in all panels. n = 4 measurements per symbol in a and d; n = 3 measurements per symbol in b and c.

Fig. 3.

The acute effect of Boc5 on food intake in female C57BL/6 mice. (a) Cumulative food intake up to 12 h after various i.p. doses of Boc5. Exenatide (1 μg) was used as control. (b) Dose–response characteristics. ED₅₀ was estimated based on 9–10 measurements per group as a shared parameter for all time points. Hill slope was also shared. (c) Blockade of the 30-min anorectic effect of i.p. Boc5 at 1 mg by prior i.p. injection of exendin(9–39) at 10 μg. (d and e) Time course (d) and dose–response (e) of gavaged Boc5 on cumulative food intake. (f) Blockade of the 90-min anorectic effect of gavaged Boc5 (10 mg) by prior i.p. injection of exendin(9–39) at 15 μg. Symbols are means ± SEM, and asterisks indicate P < 0.05 in all panels. Ex(9–39), exendin(9–39).

Fig. 4.

The effects of chronically administered Boc5 in diabetic db/db mice. (a) HbA1c measured weekly. (b) Body weight. (c) Excursion of plasma glucose after i.p. administration of 2 g of d-glucose after 6 weeks of treatment. (d) Fasting insulin concentration after 6 weeks of treatment. Symbols are means ± SEM in all panels. IPGTT, i.p. glucose tolerance testing.