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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.


Structures of N-thiazolylamide and synthetic FFA2 ligands used in this study.
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fig01: Structures of N-thiazolylamide and synthetic FFA2 ligands used in this study.

Mentions: Unlike FFA1, the first synthetic agonists of FFA2 did not resemble physiological agonists as they lacked carboxylate groups. The prototype synthetic FFA2 agonist is 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), which contains a characteristic N-thiazolylamide group (Fig.1) and was identified by Amgen (Wang et al. 2010). 4-CMTB is highly selective for FFA2 over FFA3 and FFA1 (Lee et al. 2008) and acts as a chemoattractant for mouse bone marrow neutrophils (Maslowski et al. 2009). 4-CMTB activates FFA2 and also has a cooperative effect in combination with SCFAs such as propionate (C3) (Lee et al. 2008). The allosteric nature of its interaction was confirmed by showing that FFA2 residues are required for agonism by SCFAs, Arg(5.39) and Arg(7.35) (Stoddart et al. 2008) are not required for agonism by 4-CMTB (Smith et al. 2011). The 4-CMTB binding site has been modeled but not yet validated, although extracellular loop 2 is shown to support communication between SCFA and 4-CMTB sites (Smith et al. 2011). Recently patented FFA2 agonists are reminiscent of “bitopic” ligands: compounds containing structural elements of both orthosteric and allosteric ligands oriented to engage both sites simultaneously (Lane et al. 2013). These new FFA2 agonists contain both carboxylate (orthosteric) and N-thiazolylamide (allosteric), though there appears to be no “linker”, setting them apart from most rationally designed bitopic ligands. A key question, therefore, is whether acid N-thiazolylamides have a bitopic mode of action, binding simultaneously to FFA2 residues that bind SCFAs and those that bind allosteric agonists such as 4-CMTB. Previously they were shown to engage the same basic residues as C3, that is, they are orthosteric ligands (Hudson et al. 2013a). Preliminary evidence was presented that binding was not mutually competitive with 4-CMTB, which argued against a bitopic mode of action, however, in this study 4-CMTB did not show allosteric cooperativity with the particular acid N-thiazolylamides tested. Here, we have synthesized four further acid N-thiazolylamide FFA2 agonists. We show that each has an allosteric interaction with 4-CMTB, which we interpret in the context of potential bitopic and nonbitopic modes of action. Finally, we use these allosteric and orthosteric tools to characterize FFA2 signaling in immune and adipose primary human cells.


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)

Structures of N-thiazolylamide and synthetic FFA2 ligands used in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Structures of N-thiazolylamide and synthetic FFA2 ligands used in this study.
Mentions: Unlike FFA1, the first synthetic agonists of FFA2 did not resemble physiological agonists as they lacked carboxylate groups. The prototype synthetic FFA2 agonist is 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), which contains a characteristic N-thiazolylamide group (Fig.1) and was identified by Amgen (Wang et al. 2010). 4-CMTB is highly selective for FFA2 over FFA3 and FFA1 (Lee et al. 2008) and acts as a chemoattractant for mouse bone marrow neutrophils (Maslowski et al. 2009). 4-CMTB activates FFA2 and also has a cooperative effect in combination with SCFAs such as propionate (C3) (Lee et al. 2008). The allosteric nature of its interaction was confirmed by showing that FFA2 residues are required for agonism by SCFAs, Arg(5.39) and Arg(7.35) (Stoddart et al. 2008) are not required for agonism by 4-CMTB (Smith et al. 2011). The 4-CMTB binding site has been modeled but not yet validated, although extracellular loop 2 is shown to support communication between SCFA and 4-CMTB sites (Smith et al. 2011). Recently patented FFA2 agonists are reminiscent of “bitopic” ligands: compounds containing structural elements of both orthosteric and allosteric ligands oriented to engage both sites simultaneously (Lane et al. 2013). These new FFA2 agonists contain both carboxylate (orthosteric) and N-thiazolylamide (allosteric), though there appears to be no “linker”, setting them apart from most rationally designed bitopic ligands. A key question, therefore, is whether acid N-thiazolylamides have a bitopic mode of action, binding simultaneously to FFA2 residues that bind SCFAs and those that bind allosteric agonists such as 4-CMTB. Previously they were shown to engage the same basic residues as C3, that is, they are orthosteric ligands (Hudson et al. 2013a). Preliminary evidence was presented that binding was not mutually competitive with 4-CMTB, which argued against a bitopic mode of action, however, in this study 4-CMTB did not show allosteric cooperativity with the particular acid N-thiazolylamides tested. Here, we have synthesized four further acid N-thiazolylamide FFA2 agonists. We show that each has an allosteric interaction with 4-CMTB, which we interpret in the context of potential bitopic and nonbitopic modes of action. Finally, we use these allosteric and orthosteric tools to characterize FFA2 signaling in immune and adipose primary human cells.

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.