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Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin.

Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PA, Rucker JB - PLoS ONE (2011)

Bottom Line: Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin.Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs.Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function.

View Article: PubMed Central - PubMed

Affiliation: Integral Molecular, Inc, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

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Identification of hTAS2R16 residues required for probenecid inhibition.(A) HEK-293T cells were transiently transfected with wild type hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium flux was measured for cells that were challenged with 3 mM salicin in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Salicin response to mock transfected (vector alone) HEK-293T cells is shown for comparison. (B, C) HEK-293T cells were similarly transfected with hTAS2R16 variants containing the mutations N96T or P44T/H113R, and challenged with 3 mM salicin in the presence or absence of probenecid (1 mM; 1 hour pre-incubation). N96T and P44T/H113R mutants showed decreased sensitivity to probenecid. A separate clone containing the single point mutant H113R was also tested to rule out this residue (Figure S1). Error bars represent standard deviations (n = 4).
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pone-0020123-g007: Identification of hTAS2R16 residues required for probenecid inhibition.(A) HEK-293T cells were transiently transfected with wild type hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium flux was measured for cells that were challenged with 3 mM salicin in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Salicin response to mock transfected (vector alone) HEK-293T cells is shown for comparison. (B, C) HEK-293T cells were similarly transfected with hTAS2R16 variants containing the mutations N96T or P44T/H113R, and challenged with 3 mM salicin in the presence or absence of probenecid (1 mM; 1 hour pre-incubation). N96T and P44T/H113R mutants showed decreased sensitivity to probenecid. A separate clone containing the single point mutant H113R was also tested to rule out this residue (Figure S1). Error bars represent standard deviations (n = 4).

Mentions: To determine whether probenecid directly interacts with hTAS2R16, we screened a random mutation library of hTAS2R16 for mutations that caused a loss of inhibition by probenecid. We identified two clones containing a total of three mutations, N96T, P44T, and H113R, which were significantly insensitive to probenecid inhibition while maintaining wild-type levels of responsiveness to salicin (Fig. 7, p<0.001). Since one of the probenecid insensitive clones contained two point mutations (P44T and H113R), we also analyzed an additional clone containing only the single point mutation H113R (Figure S1). The H113R mutant demonstrates wild type calcium flux in the presence of salicin and complete inhibition in the presence of probenecid, strongly suggesting that P44T is the mutation that confers probenecid insensitivity. These data thus define two amino acid residues required for probenecid interaction and suggest a direct interaction between probenecid and hTAS2R16 that is consistent with probenecid's rapid activity. Interestingly, both P44 and N96 are predicted to be located in or near the intracellular regions of hTAS2R16 [36], [37], consistent with our dose-response profile of probenecid's mechanism of action, which suggests allosteric inhibition. Taken together, the presence of mutations in hTAS2R16 that desensitize the receptor to probenecid but not the ligand, the rapid mechanism of action of probenecid, and the dose-response profiles of hTAS2R16 to salicin in the presence of increasing amounts of probenecid suggest that probenecid interacts directly with hTAS2R16 and behaves as a negative allosteric modulator of hTAS2R16 function.


Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin.

Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PA, Rucker JB - PLoS ONE (2011)

Identification of hTAS2R16 residues required for probenecid inhibition.(A) HEK-293T cells were transiently transfected with wild type hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium flux was measured for cells that were challenged with 3 mM salicin in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Salicin response to mock transfected (vector alone) HEK-293T cells is shown for comparison. (B, C) HEK-293T cells were similarly transfected with hTAS2R16 variants containing the mutations N96T or P44T/H113R, and challenged with 3 mM salicin in the presence or absence of probenecid (1 mM; 1 hour pre-incubation). N96T and P44T/H113R mutants showed decreased sensitivity to probenecid. A separate clone containing the single point mutant H113R was also tested to rule out this residue (Figure S1). Error bars represent standard deviations (n = 4).
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pone-0020123-g007: Identification of hTAS2R16 residues required for probenecid inhibition.(A) HEK-293T cells were transiently transfected with wild type hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium flux was measured for cells that were challenged with 3 mM salicin in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Salicin response to mock transfected (vector alone) HEK-293T cells is shown for comparison. (B, C) HEK-293T cells were similarly transfected with hTAS2R16 variants containing the mutations N96T or P44T/H113R, and challenged with 3 mM salicin in the presence or absence of probenecid (1 mM; 1 hour pre-incubation). N96T and P44T/H113R mutants showed decreased sensitivity to probenecid. A separate clone containing the single point mutant H113R was also tested to rule out this residue (Figure S1). Error bars represent standard deviations (n = 4).
Mentions: To determine whether probenecid directly interacts with hTAS2R16, we screened a random mutation library of hTAS2R16 for mutations that caused a loss of inhibition by probenecid. We identified two clones containing a total of three mutations, N96T, P44T, and H113R, which were significantly insensitive to probenecid inhibition while maintaining wild-type levels of responsiveness to salicin (Fig. 7, p<0.001). Since one of the probenecid insensitive clones contained two point mutations (P44T and H113R), we also analyzed an additional clone containing only the single point mutation H113R (Figure S1). The H113R mutant demonstrates wild type calcium flux in the presence of salicin and complete inhibition in the presence of probenecid, strongly suggesting that P44T is the mutation that confers probenecid insensitivity. These data thus define two amino acid residues required for probenecid interaction and suggest a direct interaction between probenecid and hTAS2R16 that is consistent with probenecid's rapid activity. Interestingly, both P44 and N96 are predicted to be located in or near the intracellular regions of hTAS2R16 [36], [37], consistent with our dose-response profile of probenecid's mechanism of action, which suggests allosteric inhibition. Taken together, the presence of mutations in hTAS2R16 that desensitize the receptor to probenecid but not the ligand, the rapid mechanism of action of probenecid, and the dose-response profiles of hTAS2R16 to salicin in the presence of increasing amounts of probenecid suggest that probenecid interacts directly with hTAS2R16 and behaves as a negative allosteric modulator of hTAS2R16 function.

Bottom Line: Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin.Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs.Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function.

View Article: PubMed Central - PubMed

Affiliation: Integral Molecular, Inc, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

Show MeSH
Related in: MedlinePlus