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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.


Positive modulation between hFFA2 agonists. Orthosteric agonist (Propionate; C3), allosteric agonist (4-CMTB) and the acid N-thiazolylamide compound 14 were combined in the hFFA2 yeast assay: (A) C3 plus 4-CMTB, (B) C3 plus compound 14, and (C) 4-CMTB plus compound 14 (representative experiments are shown). (D) Comparison of ΔpEC50 for agonist combinations C3 and 14 and 4-CMTB and 14 (n = 4 or 5 experiments). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
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fig03: Positive modulation between hFFA2 agonists. Orthosteric agonist (Propionate; C3), allosteric agonist (4-CMTB) and the acid N-thiazolylamide compound 14 were combined in the hFFA2 yeast assay: (A) C3 plus 4-CMTB, (B) C3 plus compound 14, and (C) 4-CMTB plus compound 14 (representative experiments are shown). (D) Comparison of ΔpEC50 for agonist combinations C3 and 14 and 4-CMTB and 14 (n = 4 or 5 experiments). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.

Mentions: Next, we investigated allosteric cooperativity of hFFA2 agonists, using the yeast assay. As expected, 4-CMTB increased the potency of C3 (Fig.3A). The extent of shift of the C3 concentration–response curve (ΔpEC50) was 0.17 ± 0.06, 0.47 ± 0.08, 0.82 ± 0.02, and 0.94 ± 0.07 log units in the presence of 30, 100, 300 nmol/L, and 1 μmol/L (3e-8, 1e-7, 3e-7 and 1e-6 mol/L) 4-CMTB, respectively, comparing with the pEC50 for C3 alone (n = 2). This cooperativity of C3 and 4-CMTB at hFFA2 has not previously been shown in yeast, but has been published from studies of recombinant hFFA2 measuring Ca2+ mobilization, cAMP generation and [35S]GTPγS-incorporation (Lee et al. 2008; Hudson et al. 2013a), and lipolysis in FFA2-expressing mouse 3T3 cells (Lee et al. 2008). No cooperativity between C3 and compound 14 was observed, since increasing concentrations of 14 did not increase C3 potency (Fig.3B and D), consistent with 14 binding to the orthosteric (C3) site in hFFA2. This behavior is similar to a structurally related hFFA2 agonist, also containing both acid and N-thiazolylamide groups, which was shown to interact with the carboxylate-binding histidines within FFA2 (Hudson et al. 2013a). For the final pair of agonists, 14 and 4-CMTB, potency of 4-CMTB increased with increasing concentration of 14, indicating cooperativity (Fig.3C). ΔpEC50 values illustrate that whereas compound 14 caused no significant positive displacement of C3 pEC50, concentration-dependent positive displacement of 4-CMTB pEC50 was observed (Fig.3D). Allosteric modulation of 14 and 4-CMTB is consistent with binding of 4-CMTB and 14 to distinct hFFA2 sites. Hudson et al. (2013a) were unable to show allosteric cooperativity between 4-CMTB and a different acid N-thiazolylamide hFFA2 agonist (described as compound 2), which they attributed to probe dependence.


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)

Positive modulation between hFFA2 agonists. Orthosteric agonist (Propionate; C3), allosteric agonist (4-CMTB) and the acid N-thiazolylamide compound 14 were combined in the hFFA2 yeast assay: (A) C3 plus 4-CMTB, (B) C3 plus compound 14, and (C) 4-CMTB plus compound 14 (representative experiments are shown). (D) Comparison of ΔpEC50 for agonist combinations C3 and 14 and 4-CMTB and 14 (n = 4 or 5 experiments). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
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fig03: Positive modulation between hFFA2 agonists. Orthosteric agonist (Propionate; C3), allosteric agonist (4-CMTB) and the acid N-thiazolylamide compound 14 were combined in the hFFA2 yeast assay: (A) C3 plus 4-CMTB, (B) C3 plus compound 14, and (C) 4-CMTB plus compound 14 (representative experiments are shown). (D) Comparison of ΔpEC50 for agonist combinations C3 and 14 and 4-CMTB and 14 (n = 4 or 5 experiments). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide.
Mentions: Next, we investigated allosteric cooperativity of hFFA2 agonists, using the yeast assay. As expected, 4-CMTB increased the potency of C3 (Fig.3A). The extent of shift of the C3 concentration–response curve (ΔpEC50) was 0.17 ± 0.06, 0.47 ± 0.08, 0.82 ± 0.02, and 0.94 ± 0.07 log units in the presence of 30, 100, 300 nmol/L, and 1 μmol/L (3e-8, 1e-7, 3e-7 and 1e-6 mol/L) 4-CMTB, respectively, comparing with the pEC50 for C3 alone (n = 2). This cooperativity of C3 and 4-CMTB at hFFA2 has not previously been shown in yeast, but has been published from studies of recombinant hFFA2 measuring Ca2+ mobilization, cAMP generation and [35S]GTPγS-incorporation (Lee et al. 2008; Hudson et al. 2013a), and lipolysis in FFA2-expressing mouse 3T3 cells (Lee et al. 2008). No cooperativity between C3 and compound 14 was observed, since increasing concentrations of 14 did not increase C3 potency (Fig.3B and D), consistent with 14 binding to the orthosteric (C3) site in hFFA2. This behavior is similar to a structurally related hFFA2 agonist, also containing both acid and N-thiazolylamide groups, which was shown to interact with the carboxylate-binding histidines within FFA2 (Hudson et al. 2013a). For the final pair of agonists, 14 and 4-CMTB, potency of 4-CMTB increased with increasing concentration of 14, indicating cooperativity (Fig.3C). ΔpEC50 values illustrate that whereas compound 14 caused no significant positive displacement of C3 pEC50, concentration-dependent positive displacement of 4-CMTB pEC50 was observed (Fig.3D). Allosteric modulation of 14 and 4-CMTB is consistent with binding of 4-CMTB and 14 to distinct hFFA2 sites. Hudson et al. (2013a) were unable to show allosteric cooperativity between 4-CMTB and a different acid N-thiazolylamide hFFA2 agonist (described as compound 2), which they attributed to probe dependence.

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.