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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.


Orthosteric and allosteric antagonism of hFFA2. Responses to orthosteric agonist (Propionate; C3) and allosteric agonist (4-CMTB) were antagonized with N-CBT or acid N-thiazolylamide compounds 9, 101, and 105 in the hFFA2 yeast assay. Data show sets of concentration–response curves (left) and corresponding Schild plots with linear unit-slope fits (right) from the combinations (A): C3 and N-CBT; (B): 4-CMTB and N-CBT; (C): C3 and 9 and (D): 4-CMTB and 9. Schild analysis for antagonism of C3 by 101 and 105 is also shown in (C) but full sets of concentration–response curves for C3 and 101 and C3 and 105 are not presented. Unconstrained Schild slopes in (A) and (C) were not significantly different to unity. In (B) and (D), Schild plots were fitted with linear unit-slope lines to lower antagonist concentrations only, to illustrate the deviation from linearity at higher concentration characteristic of allosteric antagonists. Representative data are shown; for experimental replication (n) and estimated mean values of pA2, pKB, and α, see Results. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
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fig04: Orthosteric and allosteric antagonism of hFFA2. Responses to orthosteric agonist (Propionate; C3) and allosteric agonist (4-CMTB) were antagonized with N-CBT or acid N-thiazolylamide compounds 9, 101, and 105 in the hFFA2 yeast assay. Data show sets of concentration–response curves (left) and corresponding Schild plots with linear unit-slope fits (right) from the combinations (A): C3 and N-CBT; (B): 4-CMTB and N-CBT; (C): C3 and 9 and (D): 4-CMTB and 9. Schild analysis for antagonism of C3 by 101 and 105 is also shown in (C) but full sets of concentration–response curves for C3 and 101 and C3 and 105 are not presented. Unconstrained Schild slopes in (A) and (C) were not significantly different to unity. In (B) and (D), Schild plots were fitted with linear unit-slope lines to lower antagonist concentrations only, to illustrate the deviation from linearity at higher concentration characteristic of allosteric antagonists. Representative data are shown; for experimental replication (n) and estimated mean values of pA2, pKB, and α, see Results. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.

Mentions: Next, we investigated whether hFFA2 inverse agonists 9, 101, and 105 could also antagonize C3 and 4-CMTB, in yeast. N-CBT (Fig.1; Table S1) is a tool hFFA2 antagonist that lacks the N-thiazolylamide group. N-CBT was originally described as a CCK antagonist (Bill 1990) and its activity at hFFA2 has not previously been published. However N-CBT is from the same chemotype as another hFFA2 antagonist, (S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid (CATPB) (Hudson et al. 2012). Increasing concentrations of N-CBT caused sequential shift of the C3 concentration–response curve (Fig.4A). Schild analysis showed a linear relationship across the full range of N-CBT concentrations tested with calculated pA2 = 6.3 (for slope of unity), agreeing with estimated pKB = 6.3 ± 0.16 (n = 8) and pKB =  6.3 ± 0.27 (n = 12) from yeast and [35S]-GTPγS incorporation assays, respectively (see Table S1). Antagonism of 4-CMTB by N-CBT differed in that lower concentrations of N-CBT caused sequential shift of the 4-CMTB concentration–response curve but at 16 and 50 μmol/L (1.6e-5 and 5e-5 mol/L) the extent of shift reduced and approached a maximum (Fig.4B). This observation is unlikely to be due to limiting solubility since N-CBT shows apparent competitive behavior with C3 at the same concentrations. Figure4B shows a linear unit-slope Schild plot fitted to lower N-CBT concentrations only, showing the deviation from linearity at higher concentrations which is characteristic of allosteric antagonism (Kenakin 2009). Using the Schild equation for allosteric antagonism gave estimated pKB = 6.5 ± 0.14, and α = 0.017 ± 0.003 (n = 2). hFFA2 antagonism by N-CBT in yeast is similar to the behavior of CATPB in ERK phosphorylation assays, in that CATPB shows orthosteric competitive antagonism of C3, but allosteric nonsurmountable antagonism of 4-CMTB (Hudson et al. 2013a). Together, this is consistent with N-CBT and CATPB (which both contain carboxylate groups) binding in the same site as C3 in hFFA2 and distinct from the 4-CMTB-binding site.


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)

Orthosteric and allosteric antagonism of hFFA2. Responses to orthosteric agonist (Propionate; C3) and allosteric agonist (4-CMTB) were antagonized with N-CBT or acid N-thiazolylamide compounds 9, 101, and 105 in the hFFA2 yeast assay. Data show sets of concentration–response curves (left) and corresponding Schild plots with linear unit-slope fits (right) from the combinations (A): C3 and N-CBT; (B): 4-CMTB and N-CBT; (C): C3 and 9 and (D): 4-CMTB and 9. Schild analysis for antagonism of C3 by 101 and 105 is also shown in (C) but full sets of concentration–response curves for C3 and 101 and C3 and 105 are not presented. Unconstrained Schild slopes in (A) and (C) were not significantly different to unity. In (B) and (D), Schild plots were fitted with linear unit-slope lines to lower antagonist concentrations only, to illustrate the deviation from linearity at higher concentration characteristic of allosteric antagonists. Representative data are shown; for experimental replication (n) and estimated mean values of pA2, pKB, and α, see Results. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
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fig04: Orthosteric and allosteric antagonism of hFFA2. Responses to orthosteric agonist (Propionate; C3) and allosteric agonist (4-CMTB) were antagonized with N-CBT or acid N-thiazolylamide compounds 9, 101, and 105 in the hFFA2 yeast assay. Data show sets of concentration–response curves (left) and corresponding Schild plots with linear unit-slope fits (right) from the combinations (A): C3 and N-CBT; (B): 4-CMTB and N-CBT; (C): C3 and 9 and (D): 4-CMTB and 9. Schild analysis for antagonism of C3 by 101 and 105 is also shown in (C) but full sets of concentration–response curves for C3 and 101 and C3 and 105 are not presented. Unconstrained Schild slopes in (A) and (C) were not significantly different to unity. In (B) and (D), Schild plots were fitted with linear unit-slope lines to lower antagonist concentrations only, to illustrate the deviation from linearity at higher concentration characteristic of allosteric antagonists. Representative data are shown; for experimental replication (n) and estimated mean values of pA2, pKB, and α, see Results. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
Mentions: Next, we investigated whether hFFA2 inverse agonists 9, 101, and 105 could also antagonize C3 and 4-CMTB, in yeast. N-CBT (Fig.1; Table S1) is a tool hFFA2 antagonist that lacks the N-thiazolylamide group. N-CBT was originally described as a CCK antagonist (Bill 1990) and its activity at hFFA2 has not previously been published. However N-CBT is from the same chemotype as another hFFA2 antagonist, (S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid (CATPB) (Hudson et al. 2012). Increasing concentrations of N-CBT caused sequential shift of the C3 concentration–response curve (Fig.4A). Schild analysis showed a linear relationship across the full range of N-CBT concentrations tested with calculated pA2 = 6.3 (for slope of unity), agreeing with estimated pKB = 6.3 ± 0.16 (n = 8) and pKB =  6.3 ± 0.27 (n = 12) from yeast and [35S]-GTPγS incorporation assays, respectively (see Table S1). Antagonism of 4-CMTB by N-CBT differed in that lower concentrations of N-CBT caused sequential shift of the 4-CMTB concentration–response curve but at 16 and 50 μmol/L (1.6e-5 and 5e-5 mol/L) the extent of shift reduced and approached a maximum (Fig.4B). This observation is unlikely to be due to limiting solubility since N-CBT shows apparent competitive behavior with C3 at the same concentrations. Figure4B shows a linear unit-slope Schild plot fitted to lower N-CBT concentrations only, showing the deviation from linearity at higher concentrations which is characteristic of allosteric antagonism (Kenakin 2009). Using the Schild equation for allosteric antagonism gave estimated pKB = 6.5 ± 0.14, and α = 0.017 ± 0.003 (n = 2). hFFA2 antagonism by N-CBT in yeast is similar to the behavior of CATPB in ERK phosphorylation assays, in that CATPB shows orthosteric competitive antagonism of C3, but allosteric nonsurmountable antagonism of 4-CMTB (Hudson et al. 2013a). Together, this is consistent with N-CBT and CATPB (which both contain carboxylate groups) binding in the same site as C3 in hFFA2 and distinct from the 4-CMTB-binding site.

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.