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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.


Related in: MedlinePlus

Inhibition of lipolysis by FFA2 agonists in primary human adipocytes. (A) Primary human adipocytes were treated with isoproterenol (Iso) or insulin (both at 200 nmol/L) or FFA2 agonists (10–100 μmol/L; only 50 μmol/L treatment is shown) and lipolysis determined as above. Bars represent mean ± SD (n = 4 determinations across two experiment occasions). (B) Primary human adipocytes were pretreated with PTX or vehicle before drug treatment. Bars represent mean ± SD from three experiments with each condition determined in duplicate in each experiment. (C) Primary human adipocytes were pretreated with the hFFA2 antagonist N-CBT (10–50 μmol/L) prior to 4-CMTB treatment. Data show mean ± SD from two experiments, each condition was determined n = 2–4 per experiment. *P < 0.05; **P < 0.01. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
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fig07: Inhibition of lipolysis by FFA2 agonists in primary human adipocytes. (A) Primary human adipocytes were treated with isoproterenol (Iso) or insulin (both at 200 nmol/L) or FFA2 agonists (10–100 μmol/L; only 50 μmol/L treatment is shown) and lipolysis determined as above. Bars represent mean ± SD (n = 4 determinations across two experiment occasions). (B) Primary human adipocytes were pretreated with PTX or vehicle before drug treatment. Bars represent mean ± SD from three experiments with each condition determined in duplicate in each experiment. (C) Primary human adipocytes were pretreated with the hFFA2 antagonist N-CBT (10–50 μmol/L) prior to 4-CMTB treatment. Data show mean ± SD from two experiments, each condition was determined n = 2–4 per experiment. *P < 0.05; **P < 0.01. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.

Mentions: On primary human adipocytes, 4-CMTB (50 μmol/L), and insulin significantly reduced lipolysis as expected (Fig.7A). The magnitude of this reduction was similar to the effect of 4-CMTB on mouse adipose explants (Fig.6A). Compounds 14, 9, 101, and 105 were each tested at 10, 25, 50, and 100 μmol/L, however, none significantly altered lipolysis at any concentration tested (only data for 50 μmol/L is shown; Fig.7A). To confirm the involvement of Gi-proteins, we showed that pretreatment with PTX prevented the inhibition of lipolysis by 4-CMTB (Fig.7B). Finally, to confirm involvement of hFFA2, we pretreated primary human adipocytes with N-CBT. N-CBT alone showed a trend toward increased basal lipolysis, which did not reach statistical significance (Fig.7C). The effect of 4-CMTB on lipolysis was significantly attenuated by 50 μmol/L N-CBT (Fig.7C). Considering 4-CMTB is a selective FFA2 agonist, and its effect on human adipocytes is PTX sensitive and is blocked by an FFA2-selective antagonist, this confirms the presence of functional hFFA2 in the primary human adipocytes. Hence, compound 14 is unable to inhibit lipolysis via hFFA2 on human adipocytes in this assay, even though it behaves as an agonist in recombinant assays of hFFA2, and is able to inhibit lipolysis via FFA2 in mouse adipose explants.


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)

Inhibition of lipolysis by FFA2 agonists in primary human adipocytes. (A) Primary human adipocytes were treated with isoproterenol (Iso) or insulin (both at 200 nmol/L) or FFA2 agonists (10–100 μmol/L; only 50 μmol/L treatment is shown) and lipolysis determined as above. Bars represent mean ± SD (n = 4 determinations across two experiment occasions). (B) Primary human adipocytes were pretreated with PTX or vehicle before drug treatment. Bars represent mean ± SD from three experiments with each condition determined in duplicate in each experiment. (C) Primary human adipocytes were pretreated with the hFFA2 antagonist N-CBT (10–50 μmol/L) prior to 4-CMTB treatment. Data show mean ± SD from two experiments, each condition was determined n = 2–4 per experiment. *P < 0.05; **P < 0.01. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
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fig07: Inhibition of lipolysis by FFA2 agonists in primary human adipocytes. (A) Primary human adipocytes were treated with isoproterenol (Iso) or insulin (both at 200 nmol/L) or FFA2 agonists (10–100 μmol/L; only 50 μmol/L treatment is shown) and lipolysis determined as above. Bars represent mean ± SD (n = 4 determinations across two experiment occasions). (B) Primary human adipocytes were pretreated with PTX or vehicle before drug treatment. Bars represent mean ± SD from three experiments with each condition determined in duplicate in each experiment. (C) Primary human adipocytes were pretreated with the hFFA2 antagonist N-CBT (10–50 μmol/L) prior to 4-CMTB treatment. Data show mean ± SD from two experiments, each condition was determined n = 2–4 per experiment. *P < 0.05; **P < 0.01. 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
Mentions: On primary human adipocytes, 4-CMTB (50 μmol/L), and insulin significantly reduced lipolysis as expected (Fig.7A). The magnitude of this reduction was similar to the effect of 4-CMTB on mouse adipose explants (Fig.6A). Compounds 14, 9, 101, and 105 were each tested at 10, 25, 50, and 100 μmol/L, however, none significantly altered lipolysis at any concentration tested (only data for 50 μmol/L is shown; Fig.7A). To confirm the involvement of Gi-proteins, we showed that pretreatment with PTX prevented the inhibition of lipolysis by 4-CMTB (Fig.7B). Finally, to confirm involvement of hFFA2, we pretreated primary human adipocytes with N-CBT. N-CBT alone showed a trend toward increased basal lipolysis, which did not reach statistical significance (Fig.7C). The effect of 4-CMTB on lipolysis was significantly attenuated by 50 μmol/L N-CBT (Fig.7C). Considering 4-CMTB is a selective FFA2 agonist, and its effect on human adipocytes is PTX sensitive and is blocked by an FFA2-selective antagonist, this confirms the presence of functional hFFA2 in the primary human adipocytes. Hence, compound 14 is unable to inhibit lipolysis via hFFA2 on human adipocytes in this assay, even though it behaves as an agonist in recombinant assays of hFFA2, and is able to inhibit lipolysis via FFA2 in mouse adipose explants.

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