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.


Related in: MedlinePlus

Agonist and inverse agonist effects of a panel of hFFA2 ligands. Propionate (C3), acetate (C2), 4-CMTB, and acid N-thiazolylamides 14, 9, 101, and 105 (free base) were tested for their ability to activate hFFA2. (A) and (B) Stimulation of hFFA2-mediated [35S]-GTPγS incorporation. (C) and (D) Inhibition of forskolin-stimulated cAMP production. (E) to (H) modulation of yeast growth via effects on the FUS1-HIS3 gene-reporter. Conditions for the yeast assay were 5 mmol/L 3AT (E), (F) and (G), which is the default used for hFFA2 yeast assays, or 1 mmol/L 3AT (H). Panels (F) and (G) present identical data but with an expanded y-axis in (G) to illustrate inverse agonism of compounds 9, 101, and 105. Panels show data from representative experiments; for clarity, error bars were omitted from panel (B). Mean pEC50 values and experimental replication are presented in Table1. Curves shown were separately fitted to four-parameter nonlinear regression isotherms but data in panels (E–H) were also subjected to simultaneous curve-fitting by application of an operational model (Figs. S1, S2). 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

fig02: Agonist and inverse agonist effects of a panel of hFFA2 ligands. Propionate (C3), acetate (C2), 4-CMTB, and acid N-thiazolylamides 14, 9, 101, and 105 (free base) were tested for their ability to activate hFFA2. (A) and (B) Stimulation of hFFA2-mediated [35S]-GTPγS incorporation. (C) and (D) Inhibition of forskolin-stimulated cAMP production. (E) to (H) modulation of yeast growth via effects on the FUS1-HIS3 gene-reporter. Conditions for the yeast assay were 5 mmol/L 3AT (E), (F) and (G), which is the default used for hFFA2 yeast assays, or 1 mmol/L 3AT (H). Panels (F) and (G) present identical data but with an expanded y-axis in (G) to illustrate inverse agonism of compounds 9, 101, and 105. Panels show data from representative experiments; for clarity, error bars were omitted from panel (B). Mean pEC50 values and experimental replication are presented in Table1. Curves shown were separately fitted to four-parameter nonlinear regression isotherms but data in panels (E–H) were also subjected to simultaneous curve-fitting by application of an operational model (Figs. S1, S2). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.

Mentions: We synthesized four reported FFA2 agonists, designated 9, 14, 101, and 105 using the same numbering system as (Hoyveda et al. 2010). These compounds contain both carboxylic acid and N-thiazolylamide chemical moieties, as shown in Figure1. To confirm agonist activity, we measured [35S]-GTPγS incorporation in membranes prepared from HEKF-Go host cells transfected with hFFA2. Compounds 9, 14, 101, and 105 each activated hFFA2 resulting in similar maximum levels of [35S]-GTPγS incorporation to the standard FFA2 agonists C3 and 4-CMTB (Fig.2A and B). This suggests they all behave as full agonists under the conditions of this test. Half-maximal effective concentrations for compounds 9 and 101 were similar to those reported previously, whereas 14 and 105 appeared ∼1 log unit more potent in our experiments, compared with the previous report (Hoyveda et al. 2010). C3 and 4-CMTB were also more potent than in previous reports (Smith et al. 2011), by ∼0.4 log units (pEC50 values are presented in Table1). Next we tested hFFA2 activation by measuring cAMP production using LANCE™ competition immunoassays. hFFA2 was introduced into human U2 osteosarcoma (U2OS) cells by baculovirus-mediated transduction, and cells were treated with forskolin to elevate intracellular cAMP before exposure to test compound. As expected, C3 and 4-CMTB caused concentration-dependent decrease in cAMP (Fig.2C; Table1). Acetate (C2) also inhibited cAMP production (pEC50 = 3.9 ± 0.07; n = 2). 4-CMTB caused the greatest maximum decrease in cAMP of 118 ± 4% relative to C3, which was included in each experiment as normalizing standard (100%). (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (Compound 14) was approximately equipotent to 4-CMTB (Fig.2D; Table1), which contrasts with the [35S]-GTPγS incorporation assay where compound 14 appeared more potent in activating hFFA2 than 4-CMTB. The trend of lower potency in cAMP compared with [35S]-GTPγS assays was also apparent for the other acid N-thiazolylamides tested. Compounds 105 and 101 were ∼2 log units less potent in the cAMP assay compared with [35S]-GTPγS incorporation. hFFA2 activation by 9 was only detected at the top concentrations tested (30 and 100 μmol/L) in the cAMP assay, and a pEC50 value for 9 was estimated at ∼4. The maximum cAMP reduction achieved by compounds 14 and 105 was close to that of 4-CMTB indicating that they behaved as full agonists. The maximum cAMP reduction achieved by compound 101 was significantly less (P < 0.05 compared with 4-CMTB, according to one-way ANOVA), showing behavior as a partial agonist.


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)

Agonist and inverse agonist effects of a panel of hFFA2 ligands. Propionate (C3), acetate (C2), 4-CMTB, and acid N-thiazolylamides 14, 9, 101, and 105 (free base) were tested for their ability to activate hFFA2. (A) and (B) Stimulation of hFFA2-mediated [35S]-GTPγS incorporation. (C) and (D) Inhibition of forskolin-stimulated cAMP production. (E) to (H) modulation of yeast growth via effects on the FUS1-HIS3 gene-reporter. Conditions for the yeast assay were 5 mmol/L 3AT (E), (F) and (G), which is the default used for hFFA2 yeast assays, or 1 mmol/L 3AT (H). Panels (F) and (G) present identical data but with an expanded y-axis in (G) to illustrate inverse agonism of compounds 9, 101, and 105. Panels show data from representative experiments; for clarity, error bars were omitted from panel (B). Mean pEC50 values and experimental replication are presented in Table1. Curves shown were separately fitted to four-parameter nonlinear regression isotherms but data in panels (E–H) were also subjected to simultaneous curve-fitting by application of an operational model (Figs. S1, S2). 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

fig02: Agonist and inverse agonist effects of a panel of hFFA2 ligands. Propionate (C3), acetate (C2), 4-CMTB, and acid N-thiazolylamides 14, 9, 101, and 105 (free base) were tested for their ability to activate hFFA2. (A) and (B) Stimulation of hFFA2-mediated [35S]-GTPγS incorporation. (C) and (D) Inhibition of forskolin-stimulated cAMP production. (E) to (H) modulation of yeast growth via effects on the FUS1-HIS3 gene-reporter. Conditions for the yeast assay were 5 mmol/L 3AT (E), (F) and (G), which is the default used for hFFA2 yeast assays, or 1 mmol/L 3AT (H). Panels (F) and (G) present identical data but with an expanded y-axis in (G) to illustrate inverse agonism of compounds 9, 101, and 105. Panels show data from representative experiments; for clarity, error bars were omitted from panel (B). Mean pEC50 values and experimental replication are presented in Table1. Curves shown were separately fitted to four-parameter nonlinear regression isotherms but data in panels (E–H) were also subjected to simultaneous curve-fitting by application of an operational model (Figs. S1, S2). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
Mentions: We synthesized four reported FFA2 agonists, designated 9, 14, 101, and 105 using the same numbering system as (Hoyveda et al. 2010). These compounds contain both carboxylic acid and N-thiazolylamide chemical moieties, as shown in Figure1. To confirm agonist activity, we measured [35S]-GTPγS incorporation in membranes prepared from HEKF-Go host cells transfected with hFFA2. Compounds 9, 14, 101, and 105 each activated hFFA2 resulting in similar maximum levels of [35S]-GTPγS incorporation to the standard FFA2 agonists C3 and 4-CMTB (Fig.2A and B). This suggests they all behave as full agonists under the conditions of this test. Half-maximal effective concentrations for compounds 9 and 101 were similar to those reported previously, whereas 14 and 105 appeared ∼1 log unit more potent in our experiments, compared with the previous report (Hoyveda et al. 2010). C3 and 4-CMTB were also more potent than in previous reports (Smith et al. 2011), by ∼0.4 log units (pEC50 values are presented in Table1). Next we tested hFFA2 activation by measuring cAMP production using LANCE™ competition immunoassays. hFFA2 was introduced into human U2 osteosarcoma (U2OS) cells by baculovirus-mediated transduction, and cells were treated with forskolin to elevate intracellular cAMP before exposure to test compound. As expected, C3 and 4-CMTB caused concentration-dependent decrease in cAMP (Fig.2C; Table1). Acetate (C2) also inhibited cAMP production (pEC50 = 3.9 ± 0.07; n = 2). 4-CMTB caused the greatest maximum decrease in cAMP of 118 ± 4% relative to C3, which was included in each experiment as normalizing standard (100%). (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (Compound 14) was approximately equipotent to 4-CMTB (Fig.2D; Table1), which contrasts with the [35S]-GTPγS incorporation assay where compound 14 appeared more potent in activating hFFA2 than 4-CMTB. The trend of lower potency in cAMP compared with [35S]-GTPγS assays was also apparent for the other acid N-thiazolylamides tested. Compounds 105 and 101 were ∼2 log units less potent in the cAMP assay compared with [35S]-GTPγS incorporation. hFFA2 activation by 9 was only detected at the top concentrations tested (30 and 100 μmol/L) in the cAMP assay, and a pEC50 value for 9 was estimated at ∼4. The maximum cAMP reduction achieved by compounds 14 and 105 was close to that of 4-CMTB indicating that they behaved as full agonists. The maximum cAMP reduction achieved by compound 101 was significantly less (P < 0.05 compared with 4-CMTB, according to one-way ANOVA), showing behavior as a partial agonist.

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.


Related in: MedlinePlus