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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.


hFFA2-mediated calcium mobilization in human neutrophils. Human neutrophils from healthy volunteers were loaded with the calcium-sensitive dye Fluo4 and pretreated with either the hFF2 antagonist, N-CBT at 10 μmol/L (1e-5 mol/L), or vehicle. Transient intracellular calcium release evoked by the agonists C3 (A) or 4-CMTB (B) was measured by FLIPR. (C) Fluo4-loaded human neutrophils were challenged with N-thiazolylamide ligands 14, 9, 101, or 105: concentrations tested were 33, 11, 3.7, 1.2, 0.41, and 0.14 μmol/L. Data show mean ± SEM for n = 2 or 3 donors, (**P < 0.01; one-way ANOVA). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; ANOVA, analysis of variance; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
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fig05: hFFA2-mediated calcium mobilization in human neutrophils. Human neutrophils from healthy volunteers were loaded with the calcium-sensitive dye Fluo4 and pretreated with either the hFF2 antagonist, N-CBT at 10 μmol/L (1e-5 mol/L), or vehicle. Transient intracellular calcium release evoked by the agonists C3 (A) or 4-CMTB (B) was measured by FLIPR. (C) Fluo4-loaded human neutrophils were challenged with N-thiazolylamide ligands 14, 9, 101, or 105: concentrations tested were 33, 11, 3.7, 1.2, 0.41, and 0.14 μmol/L. Data show mean ± SEM for n = 2 or 3 donors, (**P < 0.01; one-way ANOVA). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; ANOVA, analysis of variance; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.

Mentions: Our data show that 14, 9, 101, and 105 are orthosteric ligands, binding to the same site in hFFA2 as C3, and having system-dependent efficacy. Compounds 14, 9, 101, and 105 do not compete for binding to the 4-CMTB site even though N-thiazolylamide is present in all these compounds. Since hFFA2 ligands are potential therapeutic agents in metabolic and immune diseases, we next studied ligand efficacy at FFA2 endogenously expressed in mouse and human cells. Neutrophils express high relative levels of FFA2 (Brown et al. 2003) and genetic deletion of mouse FFA2 abolishes both chemotaxis toward C3 and acetate-evoked intracellular Ca2+ release in bone marrow neutrophils (Maslowski et al. 2009; Vinolo et al. 2011). We measured intracellular Ca2+ mobilization in neutrophils purified from human donor blood, using FLIPR. C3 and 4-CMTB evoked robust concentration-dependent increases in intracellular Ca2+ (Fig.5A and B) of similar maximum magnitude to the control, leukotriene B4 (45 nmol/L; data not shown). Half-maximal effective concentrations were pEC50 = 4.0 ± 0.30 for C3 and pEC50 = 5.4 ± 0.28 for 4-CMTB (n = 8 donors across four separate occasions). Pretreatment with 10 μmol/L N-CBT blocked the Ca2+ response evoked by C3, confirming that hFFA2 mediates this effect (Fig.5A). N-CBT was also effective in reducing Ca2+ mobilization evoked by 4-CMTB, though to a lesser extent than with C3, possibly due to its allosteric antagonism of 4-CMTB (Fig.5B). Compound 14 appeared similarly effective to 4-CMTB in increasing neutrophil Ca2+, with the top four concentrations (1.2 to 33 μmol/L) causing significant Ca2+ mobilization (Fig.5C). However, 9, 101 and 105 were less effective, showing significant Ca2+ mobilization only at 33 and 10 μmol/L (101) or only at 33 μmol/L (9 and 105) (Fig.5C).


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)

hFFA2-mediated calcium mobilization in human neutrophils. Human neutrophils from healthy volunteers were loaded with the calcium-sensitive dye Fluo4 and pretreated with either the hFF2 antagonist, N-CBT at 10 μmol/L (1e-5 mol/L), or vehicle. Transient intracellular calcium release evoked by the agonists C3 (A) or 4-CMTB (B) was measured by FLIPR. (C) Fluo4-loaded human neutrophils were challenged with N-thiazolylamide ligands 14, 9, 101, or 105: concentrations tested were 33, 11, 3.7, 1.2, 0.41, and 0.14 μmol/L. Data show mean ± SEM for n = 2 or 3 donors, (**P < 0.01; one-way ANOVA). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; ANOVA, analysis of variance; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: hFFA2-mediated calcium mobilization in human neutrophils. Human neutrophils from healthy volunteers were loaded with the calcium-sensitive dye Fluo4 and pretreated with either the hFF2 antagonist, N-CBT at 10 μmol/L (1e-5 mol/L), or vehicle. Transient intracellular calcium release evoked by the agonists C3 (A) or 4-CMTB (B) was measured by FLIPR. (C) Fluo4-loaded human neutrophils were challenged with N-thiazolylamide ligands 14, 9, 101, or 105: concentrations tested were 33, 11, 3.7, 1.2, 0.41, and 0.14 μmol/L. Data show mean ± SEM for n = 2 or 3 donors, (**P < 0.01; one-way ANOVA). 4-CMTB, 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; ANOVA, analysis of variance; N-CBT, N-(4-Chlorobenzoyl)-l-tryptophan.
Mentions: Our data show that 14, 9, 101, and 105 are orthosteric ligands, binding to the same site in hFFA2 as C3, and having system-dependent efficacy. Compounds 14, 9, 101, and 105 do not compete for binding to the 4-CMTB site even though N-thiazolylamide is present in all these compounds. Since hFFA2 ligands are potential therapeutic agents in metabolic and immune diseases, we next studied ligand efficacy at FFA2 endogenously expressed in mouse and human cells. Neutrophils express high relative levels of FFA2 (Brown et al. 2003) and genetic deletion of mouse FFA2 abolishes both chemotaxis toward C3 and acetate-evoked intracellular Ca2+ release in bone marrow neutrophils (Maslowski et al. 2009; Vinolo et al. 2011). We measured intracellular Ca2+ mobilization in neutrophils purified from human donor blood, using FLIPR. C3 and 4-CMTB evoked robust concentration-dependent increases in intracellular Ca2+ (Fig.5A and B) of similar maximum magnitude to the control, leukotriene B4 (45 nmol/L; data not shown). Half-maximal effective concentrations were pEC50 = 4.0 ± 0.30 for C3 and pEC50 = 5.4 ± 0.28 for 4-CMTB (n = 8 donors across four separate occasions). Pretreatment with 10 μmol/L N-CBT blocked the Ca2+ response evoked by C3, confirming that hFFA2 mediates this effect (Fig.5A). N-CBT was also effective in reducing Ca2+ mobilization evoked by 4-CMTB, though to a lesser extent than with C3, possibly due to its allosteric antagonism of 4-CMTB (Fig.5B). Compound 14 appeared similarly effective to 4-CMTB in increasing neutrophil Ca2+, with the top four concentrations (1.2 to 33 μmol/L) causing significant Ca2+ mobilization (Fig.5C). However, 9, 101 and 105 were less effective, showing significant Ca2+ mobilization only at 33 and 10 μmol/L (101) or only at 33 μmol/L (9 and 105) (Fig.5C).

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.