Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin

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.

Principal Component Analysis of Binding Energies for Single-Point Mutants of hT2R16 Bound to an Agonist Correlate with Experimental Mutant Cell Response

“…T2Rs are GPCRs with short N-terminal domains, and there are an estimated of 25 functional T2Rs in humans (Zuker et al, 2000;Lindemann, 2001). Human bitter taste receptor 16 (hT2R16) is a broadly tuned member of the human T2R family capable of detecting various molecules structurally similar to b-glucopyranosides (Bufe et al, 2002;Behrens et al, 2007;Greene et al, 2011). Greene and coauthors recently obtained experimentally a functional map of single-point amino acid replacements for hT2R16 using shotgun mutagenesis (Greene et al, 2011).…”

“…Human bitter taste receptor 16 (hT2R16) is a broadly tuned member of the human T2R family capable of detecting various molecules structurally similar to b-glucopyranosides (Bufe et al, 2002;Behrens et al, 2007;Greene et al, 2011). Greene and coauthors recently obtained experimentally a functional map of single-point amino acid replacements for hT2R16 using shotgun mutagenesis (Greene et al, 2011). Their initial analysis focused on mutations that affected probenecid response without affecting response to salicin.…”

“…Our approach is validated using published experimental data that correlate surface expression and agonist response for 424 single-point mutants (Greene et al, 2011), and that cross-compares mutant response to four ligands relative to wild type (Anu Thomas et al, in preparation). The latter identifies mutations that primarily influence ligand binding, and not expression or trafficking.…”

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation

“…4A, top). While probenecid inhibits the organic anion transporters of the kidney and the human bitter taste receptor (TASR16) (14,30), these receptors are not found in heart and therefore the effects of probenecid on cardiac myocytes are most likely its closure of Panx1 channels. Here, low level of dye uptake is most likely due to pinocytosis or endocytosis.…”

Insights into the binding of agonist and antagonist to TAS2R16 receptor: a molecular simulation study

“…Salicin was selected as an agonist since TAS2R16 could specifically respond to it. Probenecid, which has been proved to inhibit TAS2R16 and suppresses bitter perception of salicin [12], was selected as an antagonist for calculation.…”