Probenecid

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.

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

BackgroundThere are no therapies shown to improve outcome after severe traumatic brain injury (TBI) in humans, a leading cause of morbidity and mortality. We sought to verify brain exposure of the systemically administered antioxidant N-acetylcysteine (NAC) and the synergistic adjuvant probenecid, and identify adverse effects of this drug combination after severe TBI in children.MethodsIRB-approved, randomized, double-blind, placebo controlled Phase I study in children 2 to 18 years-of-age admitted to a Pediatric Intensive Care Unit after severe TBI (Glasgow Coma Scale [GCS] score ≤8) requiring an externalized ventricular drain for measurement of intracranial pressure (ICP). Patients were recruited from November 2011-August 2013. Fourteen patients (n = 7/group) were randomly assigned after obtaining informed consent to receive probenecid (25 mg/kg load, then 10 mg/kg/dose q6h×11 doses) and NAC (140 mg/kg load, then 70 mg/kg/dose q4h×17 doses), or placebos via naso/orogastric tube. Serum and CSF samples were drawn pre-bolus and 1-96 h after randomization and drug concentrations were measured via UPLC-MS/MS. Glasgow Outcome Scale (GOS) score was assessed at 3 months.ResultsThere were no adverse events attributable to drug treatment. One patient in the placebo group was withdrawn due to adverse effects. In the treatment group, NAC concentrations ranged from 16,977.3±2,212.3 to 16,786.1±3,285.3 in serum and from 269.3±113.0 to 467.9±262.7 ng/mL in CSF, at 24 to 72 h post-bolus, respectively; and probenecid concentrations ranged from 75.4.3±10.0 to 52.9±25.8 in serum and 5.4±1.0 to 4.6±2.1 μg/mL in CSF, at 24 to 72 h post-bolus, respectively (mean±SEM). Temperature, mean arterial pressure, ICP, use of ICP-directed therapies, surveillance serum brain injury biomarkers, and GOS at 3 months were not different between groups.ConclusionsTreatment resulted in detectable concentrations of NAC and probenecid in CSF and was not associated with undesirable effects after TBI in children.Trial registrationClinicalTrials.gov NCT01322009.

IntroductionUremic toxins, including indoxyl sulfate (IS) and p-cresyl sulfate (PCS), contribue to cardiac fibrosis and maladaptive remodeling in chronic kidney disease (CKD). Although their roles in ventricular and vascular pathology are well established, their effects on sinus node cells and the associated molecular alterations remain poorly defined. Understanding how the uremic milieu affects sinus node cell homeostasis may provide insight into CKD-associated conduction abnormalities.MethodsTo investigate uremic toxin-induced stress responses, H9C2 cardiomyocytes were used as a comparative cardiac stress-model, whereas primary sinus node cells served as the principal pacemaker-relevant model. Both cell types were treated with IS and PCS. Profibrotic, pro-apoptotic, and stress-related signaling pathways were evaluated by protein and mRNA analyses, together p38 and ERK activation. In parallel, a CKD mouse model was generated by 2-week adenine feeding, and sinus node cells were isolated for molecular analysis. The effects of probenecid were examined in cultured cells and ex vivo using primary sinus node cells isolated from adenine-fed mice. Results IS and PCS induced profibrotic, pro-apoptotic, and remodeling-associated signaling in both H9C2 cardiomyocytes and primary sinus node cells, indicating activation of shared cardiac stress pathways. Conclusions regarding pacemaker-relevant molecular alterations were based primarily on primary sinus node cell findings, including cells isolated from 2-week adenine-fed mice., In primary sinus node cells, uremic toxin exposure increased fibronectin accumulation, altered the Bax/Bcl-2 balance, and activated p38 and ERK signaling, whereas TBX3 expression remained unchanged. changes occurred without overt loss of this pacemaker identity marker. These findings indicate apoptosis-associated, profibrotic, and stress-responsive molecular alterations rather than direct evidence of functional pacemaker impairment. Sinus node cells isolated from adenine-fed mice showed similar remodeling-associated molecular marker changes. Probenecid attenuated fibronectin accumulation, Bax/Bcl-2 imbalance, and stress kinase activation in primary sinus node cells, with similar effects observed ex vivo in cells isolated from adenine-fed mice.ConclusionThese findings suggest that uremic toxin-driven stress signaling is associated with molecular remodeling signatures in sinus node cells under CKD-related conditions. Although the functional consequences of these molecular alterations were not assessed, probenecid attenuated uremic toxin-associated profibrotic, pro-apoptotic, and stress kinase signaling, suggesting that targeting molecular stress pathways may help strategy sinus node cell homeostasis in CKD.