Limits...
Pharmacological properties of acid N-thiazolylamide FFA2 agonists.

Brown AJ, Tsoulou C, Ward E, Gower E, Bhudia N, Chowdhury F, Dean TW, Faucher N, Gangar A, Dowell SJ - Pharmacol Res Perspect (2015)

Bottom Line: These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide.Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule.Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, GlaxoSmithKline Stevenage, United Kingdom.

ABSTRACT
FFA2 is a receptor for short-chain fatty acids. Propionate (C3) and 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), the prototypical synthetic FFA2 agonist, evoke calcium mobilization in neutrophils and inhibit lipolysis in adipocytes via this G-protein-coupled receptor. 4-CMTB contains an N-thiazolylamide motif but no acid group, and 4-CMTB and C3 bind to different sites on FFA2 and show allosteric cooperativity. Recently, FFA2 agonists have been described that contain both N-thiazolylamide and carboxylate groups, reminiscent of bitopic ligands. These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide. Here, we describe the characterization of four FFA2 ligands containing both N-thiazolylamide and carboxylate. (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (compound 14) exhibits allosteric agonism with 4-CMTB but not C3. Three other compounds agonize FFA2 in [(35)S]GTPγS-incorporation or cAMP assays but behave as inverse agonists in yeast-based gene-reporter assays, showing orthosteric antagonism of C3 responses but allosteric antagonism of 4-CMTB responses. Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule. Compound 14 activates FFA2 on human neutrophils and mouse adipocytes, but appears not to inhibit lipolysis upon treatment of human primary adipocytes in spite of the presence of a functional FFA2 receptor in these cells. Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.

No MeSH data available.


Effects of FFA2 agonists on lipolysis and GLP-1 release in mouse cells. (A) Adipose tissue explants from FFA2−/− mice and matched wild-type littermates were treated in vitro with vehicle, C3 (20 mmol/L), 4-CMTB, or 14 (75 μmol/L) and lipolysis determined by measurement of glycerol release. Data represent mean ± SEM (n = 6–11 across two experiment occasions; **P < 0.01). (B) Cultured mouse enteroendocrine STC-1 cells were treated with 4-CMTB or 14 (0–50 μmol/L) and GLP-1 secretion was measured by an ELISA-based method (MSD). Bars show mean ± SD; experiment repeated 2–5 times for each compound and representative experiment shown. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492757&req=5

fig06: Effects of FFA2 agonists on lipolysis and GLP-1 release in mouse cells. (A) Adipose tissue explants from FFA2−/− mice and matched wild-type littermates were treated in vitro with vehicle, C3 (20 mmol/L), 4-CMTB, or 14 (75 μmol/L) and lipolysis determined by measurement of glycerol release. Data represent mean ± SEM (n = 6–11 across two experiment occasions; **P < 0.01). (B) Cultured mouse enteroendocrine STC-1 cells were treated with 4-CMTB or 14 (0–50 μmol/L) and GLP-1 secretion was measured by an ELISA-based method (MSD). Bars show mean ± SD; experiment repeated 2–5 times for each compound and representative experiment shown. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.

Mentions: Finally, we studied agonist efficacy at FFA2 in adipose tissue. Explants from epididymal adipose fat pads of wild-type and FFA2−/− mice were tested. Lipolysis resulted in glycerol release into the media, and was attenuated by insulin and by an agonist of hydroxycarboxylic acid receptor-2, and stimulated by isoproterenol, as expected. The effects of these agents were comparable between wild-type and FFA2-deficient explants (data not shown). Potency of 4-CMTB has been shown to be similar at mouse FFA2 (mFFA2) and hFFA2 (Lee et al. 2008). Moreover, 4-CMTB causes PTX-sensitive inhibition of lipolysis in differentiated mouse 3T3L1 adipocytes (Lee et al. 2008). Treatment of wild-type explants with either 4-CMTB, 14 (75 μmol/L) or C3 (20 mmol/L) resulted in significant inhibition of lipolysis, by approximately half in each case (Fig.6A). Treatment of explants from FFA2−/− animals caused no significant effects, confirming the involvement of FFA2 (Fig.6A). We also examined the capacity of 4-CMTB and 14 to stimulate GLP-1 secretion using mouse enteroendocrine STC-1 cells (Hudson et al. 2013a). Both 4-CMTB and 14 stimulated GLP-1 secretion but different potencies were observed. 4-CMTB (10 μmol/L) increased GLP-1 secretion ∼10-fold over basal, which was not further increased by higher concentrations of 4-CMTB, (25 and 50 μmol/L; Fig.6B). In contrast, 10 μmol/L 14 had no significant effect on GLP-1 secretion and higher concentrations were required to increase GLP-1. Even at the highest tested concentration (50 μmol/L), 14 increased GLP-1 secretion only to approximately sixfold over basal levels. Thus, 14 is less potent than 4-CMTB in evoking GLP-1 release from mouse STC-1 cells, possibly by fivefold or more.


Pharmacological properties of acid N-thiazolylamide FFA2 agonists.

Brown AJ, Tsoulou C, Ward E, Gower E, Bhudia N, Chowdhury F, Dean TW, Faucher N, Gangar A, Dowell SJ - Pharmacol Res Perspect (2015)

Effects of FFA2 agonists on lipolysis and GLP-1 release in mouse cells. (A) Adipose tissue explants from FFA2−/− mice and matched wild-type littermates were treated in vitro with vehicle, C3 (20 mmol/L), 4-CMTB, or 14 (75 μmol/L) and lipolysis determined by measurement of glycerol release. Data represent mean ± SEM (n = 6–11 across two experiment occasions; **P < 0.01). (B) Cultured mouse enteroendocrine STC-1 cells were treated with 4-CMTB or 14 (0–50 μmol/L) and GLP-1 secretion was measured by an ELISA-based method (MSD). Bars show mean ± SD; experiment repeated 2–5 times for each compound and representative experiment shown. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492757&req=5

fig06: Effects of FFA2 agonists on lipolysis and GLP-1 release in mouse cells. (A) Adipose tissue explants from FFA2−/− mice and matched wild-type littermates were treated in vitro with vehicle, C3 (20 mmol/L), 4-CMTB, or 14 (75 μmol/L) and lipolysis determined by measurement of glycerol release. Data represent mean ± SEM (n = 6–11 across two experiment occasions; **P < 0.01). (B) Cultured mouse enteroendocrine STC-1 cells were treated with 4-CMTB or 14 (0–50 μmol/L) and GLP-1 secretion was measured by an ELISA-based method (MSD). Bars show mean ± SD; experiment repeated 2–5 times for each compound and representative experiment shown. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
Mentions: Finally, we studied agonist efficacy at FFA2 in adipose tissue. Explants from epididymal adipose fat pads of wild-type and FFA2−/− mice were tested. Lipolysis resulted in glycerol release into the media, and was attenuated by insulin and by an agonist of hydroxycarboxylic acid receptor-2, and stimulated by isoproterenol, as expected. The effects of these agents were comparable between wild-type and FFA2-deficient explants (data not shown). Potency of 4-CMTB has been shown to be similar at mouse FFA2 (mFFA2) and hFFA2 (Lee et al. 2008). Moreover, 4-CMTB causes PTX-sensitive inhibition of lipolysis in differentiated mouse 3T3L1 adipocytes (Lee et al. 2008). Treatment of wild-type explants with either 4-CMTB, 14 (75 μmol/L) or C3 (20 mmol/L) resulted in significant inhibition of lipolysis, by approximately half in each case (Fig.6A). Treatment of explants from FFA2−/− animals caused no significant effects, confirming the involvement of FFA2 (Fig.6A). We also examined the capacity of 4-CMTB and 14 to stimulate GLP-1 secretion using mouse enteroendocrine STC-1 cells (Hudson et al. 2013a). Both 4-CMTB and 14 stimulated GLP-1 secretion but different potencies were observed. 4-CMTB (10 μmol/L) increased GLP-1 secretion ∼10-fold over basal, which was not further increased by higher concentrations of 4-CMTB, (25 and 50 μmol/L; Fig.6B). In contrast, 10 μmol/L 14 had no significant effect on GLP-1 secretion and higher concentrations were required to increase GLP-1. Even at the highest tested concentration (50 μmol/L), 14 increased GLP-1 secretion only to approximately sixfold over basal levels. Thus, 14 is less potent than 4-CMTB in evoking GLP-1 release from mouse STC-1 cells, possibly by fivefold or more.

Bottom Line: These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide.Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule.Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, GlaxoSmithKline Stevenage, United Kingdom.

ABSTRACT
FFA2 is a receptor for short-chain fatty acids. Propionate (C3) and 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), the prototypical synthetic FFA2 agonist, evoke calcium mobilization in neutrophils and inhibit lipolysis in adipocytes via this G-protein-coupled receptor. 4-CMTB contains an N-thiazolylamide motif but no acid group, and 4-CMTB and C3 bind to different sites on FFA2 and show allosteric cooperativity. Recently, FFA2 agonists have been described that contain both N-thiazolylamide and carboxylate groups, reminiscent of bitopic ligands. These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide. Here, we describe the characterization of four FFA2 ligands containing both N-thiazolylamide and carboxylate. (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (compound 14) exhibits allosteric agonism with 4-CMTB but not C3. Three other compounds agonize FFA2 in [(35)S]GTPγS-incorporation or cAMP assays but behave as inverse agonists in yeast-based gene-reporter assays, showing orthosteric antagonism of C3 responses but allosteric antagonism of 4-CMTB responses. Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule. Compound 14 activates FFA2 on human neutrophils and mouse adipocytes, but appears not to inhibit lipolysis upon treatment of human primary adipocytes in spite of the presence of a functional FFA2 receptor in these cells. Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.

No MeSH data available.