Oxidation of anti-thyroid drugs and their selenium analogs by ABTS radical cation.

Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.

Predominant Qualities Evoked by Quinine, Sucrose, and Capsaicin Associate With PROP Bitterness, but not TAS2R38 Genotype.

Genetic variability in the ability to taste thiourea compounds has been studied for 80+ years. Over the last 3 decades, many studies have reported perceived intensity of concentrated propylthiouracil (PROP) associates with greater intensity from a broad range of stimuli, including nonbitter tastants, irritants, and retronasally delivered odorants. Thus, PROP phenotype has become a common measure of individual differences in orosensation. Much, but not all, of the phenotypic variation in PROP bitterness is explained by TAS2R38 polymorphisms. While differences in PROP bitterness are clearly due to genetic variation, mechanistically it is challenging to envision how this receptor (narrowly tuned to the N-C=S moiety) relates to overall orosensory response. Here, we report data for 200+ individuals who had been genotyped for TAS2R38 and phenotyped for PROP in a laboratory setting. Participants also reported the intensity of quinine, capsaicin, and sucrose on a general Labeled Magnitude Scale. Our data recapitulate earlier reports associating PROP bitterness with the intensity of the predominant qualities of sucrose, quinine, and capsaicin; however, we also find correlations between the intensities of sucrose, quinine, and capsaicin were much stronger with each other than with PROP. As expected, TAS2R38 diplotype did not associate with the intensity of sucrose, quinine, or capsaicin. The strength of PROP-capsaicin and PROP-sucrose relationships increased after grouping participants by TAS2R38 diplotype, with the greatest increases in association observed within homozygotes. Collectively, this suggests the suprathreshold intensity of PROP is a confounded phenotype that captures both genetic variation specific to N-C=S compounds and overall orosensation.

A bioinspired in vitro bioelectronic tongue with human T2R38 receptor for high-specificity detection of N-C=S-containing compounds.

Detection and identification of bitter compounds draw great attention in pharmaceutical and food industry. Several well-known agonists of specific bitter taste receptors have been found to exhibit anti-cancer effects. For example, N-C=S-containing compounds, such as allyl-isothiocyanates, have shown cancer chemo-preventive effects. It is worth noting that human T2R38 receptor is specific for compounds containing N-C=S moiety. Here, a bioinspired cell-based bioelctronic tongue (BioET) is developed for the high-specificity isothiocyanate-induced bitter detection, utilizing human Caco-2 cells as a primary sensing element and interdigitated impedance sensor as a secondary transducer. As an intestinal carcinoma cell line, Caco-2 endogenously expresses human bitter receptor T2R38, and the activation of T2R38 induces the changes of cellular morphology which can be detected by electric cell-substrate impedance sensing (ECIS). After configuration and optimization of parameters including timing of compound administration and cell density, quantitative bitter evaluation models were built for two well-known bitter compounds, phenylthiocarbamide (PTC) and propylthiouracil (PROP). The bitter specific detection of this BioET is inhibited by probenecid and U-73122, and is not elicited by other taste modalities or bitter ligands that do not activate T2R38. Moreover, by combining different computational tools, we designed a ligand-based virtual screening (LBVS) protocol to select ligands that are likely to activate T2R38 receptor. Three computationally predicted agonists of T2R38 were selected using the LBVS protocol, and the BioET presented response to the predicted agonists, validating the capability of the LBVS protocol. This study suggests this unique cell-based BioET paves a general and promising way to specifically detect N-C=S-containing compounds that can be used for pharmaceutical study and drug development.

Antenatal/early postnatal hypothyroidism increases the contribution of Rho-kinase to contractile responses of mesenteric and skeletal muscle arteries in adult rats.

BACKGROUND: Maternal thyroid deficiency can increase Rho-kinase procontractile influence in arteries of 2-week-old progeny. Here we hypothesized that augmented role of Rho-kinase persists in arteries from adult progeny of hypothyroid rats. METHODS: Dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%) during pregnancy and 2 weeks postpartum; control (CON) females received PTU-free water. At the age of 10-12-weeks, serum T3/T4 levels did not differ between PTU and CON male offspring. Cutaneous (saphenous), mesenteric, and skeletal muscle (sural) arteries were studied by wire myography, qPCR, and Western blotting. RESULTS: Saphenous arteries of PTU and CON groups showed similar responses to α1-adrenoceptor agonist methoxamine and were equally suppressed by Rho-kinase inhibitor Y27632. Responses of mesenteric arteries also did not differ between PTU and CON, but the effects of Y27632 were more prominent in the PTU group. Sural arteries of PTU rats compared to CON demonstrated augmented responses to methoxamine, increased RhoA mRNA contents and higher levels of MYPT1 phosphorylation at Thr855. Intergroup differences in contractile responses and phospho-MYPT1-Thr855 were eliminated by Y27632. CONCLUSION: Rho-kinase contribution to contractile responses of mesenteric and especially sural arteries is augmented in adult PTU rats. Therefore, maternal thyroid deficiency may have long-term detrimental consequences for vasculature in adult offspring.

Odor-Cued Bitter Taste Avoidance.

In the course of our ongoing studies of odor-cued taste avoidance (OCTA) to measure olfactory capabilities in animals, we observed that mice could rapidly learn to use the vapor of the classical bitterant quinine hydrochloride to avoid contact with the tastant. Here we expand on this observation to determine which among several compounds generally classed as bitter could be detected at a distance. Since mice were initially naïve we were able to assess whether the vapors of the bitter compounds tested were innately aversive as are their tastes. CD-1 mice could readily use vapor cues from quinine hydrochloride, denatonium benzoate (DB), and 6-propyl-2-thiouracil to avoid their taste. Although mice did not hesitate to make contact with these solutions on their first exposure, they did learn to do so typically after only 1 or 2 exposures. Bilaterally bulbectomized mice did not learn or retain the ability to avoid quinine and DB solutions by vapor alone, implicating olfaction as the mode of detection. Saturated aqueous solutions of sucrose octaacetate and caffeine which are bitter to humans and some strains of mice were not aversive in our studies. The very low vapor concentrations of the 3 bitterant solutions that mice detected at a distance, suggest that impurities in the reagent grade solutions, rather than the bitter molecules themselves were the basis of detection. Implications of these findings for taste testing and the role of odor in food acceptance/rejections decisions are discussed.

Electrochemical versus Enzymatic in Vitro Oxidations of 6-propyl-2-thiouracil: Identification, Detection, and Characterization of Metabolites.

6-Propylthiouracil, PTU, is a well-known antithyroid drug that has been the mainstay of treatment of Graves' disease. It is, however, also associated with liver toxicity and idiosyncratic toxicity. These toxicities are generally associated with metabolites derived from its bioactivation. In this manuscript, bioactivation of PTU was studied via two separate techniques: electrochemical oxidation and through the use of human liver microsomes. The aim of this work was to compare the bioactivation products of these two techniques. The electrochemical technique was studied online with a mass spectrometer, EC/ESI/MS. The microsomal oxidations were studied in tandem with liquid chromatography. The EC/ESI/MS technique was devoid of the normal reducing biological matrix prevalent in microsomal incubations. The predominant product at 400 mV was the dimeric PTU species with negligible formation of other metabolites. At higher potentials, complete desulfurization of PTU was observed with formation of sulfate. No sulfonic acid was observed, suggesting that the cleavage of the C-S bond was effected at the sulfinic acid stage, releasing a highly reducing sulfur species which is known to give rise to genotoxicity. The microsomal oxidations, surprisingly, showed formation of the unstable sulfenic acid, the S-oxide. Further incubation showed both the sulfinic and sulfonic acids. None of the systems showed any adducts with nucleophiles such as glutathione, showing that none of the reactive metabolites were stable enough to be adducted to nucleophiles in both the biological matrix and the electrochemical oxidizing environment.

Factors Influencing the Phenotypic Characterization of the Oral Marker, PROP.

In the last several decades, the genetic ability to taste the bitter compound, 6-n-propyltiouracil (PROP) has attracted considerable attention as a model for understanding individual differences in taste perception, and as an oral marker for food preferences and eating behavior that ultimately impacts nutritional status and health. However, some studies do not support this role. This review describes common factors that can influence the characterization of this phenotype including: (1) changes in taste sensitivity with increasing age; (2) gender differences in taste perception; and (3) effects of smoking and obesity. We suggest that attention to these factors during PROP screening could strengthen the associations between this phenotype and a variety of health outcomes ranging from variation in body composition to oral health and cancer risk.

Stability of Acetazolamide, Baclofen, Dipyridamole, Mebeverine Hydrochloride, Propylthiouracil, Quinidine Sulfate, and Topiramate Oral Suspensions in SyrSpend SF PH4.

The objective of this study was to evaluate the stability of 7 commonly used active pharmaceutical ingredients compounded in oral suspensions using an internationally used suspending vehicle (SyrSpend SF PH4): acetazolamide 25.0 mg/mL, baclofen 10.0 mg/mL, dipyridamole 10.0 mg/mL, mebeverine hydrochloride 10.0 mg/mL, propylthiouracil 5.0 mg/mL, quinidine sulfate 10.0 mg/mL, and topiramate 5.0 mg/mL. All suspensions were stored both at controlled refrigerated (2°C to 8°C) and room temperature (20°C to 25°C). Stability was assessed by measuring the percentage recovery at varying time points throughout a 90-day period. Active pharmaceutical ingredient quantification was performed by ultraviolet (UV) high-performance liquid chromatography, via a stability-indicating method. Given the percentage of recovery of the active pharmaceutical ingredients within the suspensions, the beyond-use date of the final products (active pharmaceutical ingredient + vehicle) was at least 90 days for all suspensions with regards to both temperatures. This suggests that SyrSpend SF PH4 is suitable for compounding active pharmaceutical ingredients from different pharmacological classes.

6-Propyl-2-thiouracil versus 6-methoxymethyl-2-thiouracil: enhancing the hydrogen-bonded synthon motif by replacement of a methylene group with an O atom.

The understanding of intermolecular interactions is a key objective of crystal engineering in order to exploit the derived knowledge for the rational design of new molecular solids with tailored physical and chemical properties. The tools and theories of crystal engineering are indispensable for the rational design of (pharmaceutical) cocrystals. The results of cocrystallization experiments of the antithyroid drug 6-propyl-2-thiouracil (PTU) with 2,4-diaminopyrimidine (DAPY), and of 6-methoxymethyl-2-thiouracil (MOMTU) with DAPY and 2,4,6-triaminopyrimidine (TAPY), respectively, are reported. PTU and MOMTU show a high structural similarity and differ only in the replacement of a methylene group (-CH2-) with an O atom in the side chain, thus introducing an additional hydrogen-bond acceptor in MOMTU. Both molecules contain an ADA hydrogen-bonding site (A = acceptor and D = donor), while the coformers DAPY and TAPY both show complementary DAD sites and therefore should be capable of forming a mixed ADA/DAD synthon with each other, i.e. N-H...O, N-H...N and N-H...S hydrogen bonds. The experiments yielded one solvated cocrystal salt of PTU with DAPY, four different solvates of MOMTU, one ionic cocrystal of MOMTU with DAPY and one cocrystal salt of MOMTU with TAPY, namely 2,4-diaminopyrimidinium 6-propyl-2-thiouracilate-2,4-diaminopyrimidine-N,N-dimethylacetamide-water (1/1/1/1) (the systematic name for 6-propyl-2-thiouracilate is 6-oxo-4-propyl-2-sulfanylidene-1,2,3,6-tetrahydropyrimidin-1-ide), C4H7N4(+)·C7H9N2OS(-)·C4H6N4·C4H9NO·H2O, (I), 6-methoxymethyl-2-thiouracil-N,N-dimethylformamide (1/1), C6H8N2O2S·C3H7NO, (II), 6-methoxymethyl-2-thiouracil-N,N-dimethylacetamide (1/1), C6H8N2O2S·C4H9NO, (III), 6-methoxymethyl-2-thiouracil-dimethyl sulfoxide (1/1), C6H8N2O2S·C2H6OS, (IV), 6-methoxymethyl-2-thiouracil-1-methylpyrrolidin-2-one (1/1), C6H8N2O2S·C5H9NO, (V), 2,4-diaminopyrimidinium 6-methoxymethyl-2-thiouracilate (the systematic name for 6-methoxymethyl-2-thiouracilate is 4-methoxymethyl-6-oxo-2-sulfanylidene-1,2,3,6-tetrahydropyrimidin-1-ide), C4H7N4(+)·C6H7N2O2S(-), (VI), and 2,4,6-triaminopyrimidinium 6-methoxymethyl-2-thiouracilate-6-methoxymethyl-2-thiouracil (1/1), C4H8N5(+)·C6H7N2O2S(-)·C6H8N2O2S, (VII). Whereas in (I) only an AA/DD hydrogen-bonding interaction was formed, the structures of (VI) and (VII) both display the desired ADA/DAD synthon. Conformational studies on the side chains of PTU and MOMTU also revealed a significant deviation for cocrystals (VI) and (VII), leading to the desired enhancement of the hydrogen-bond pattern within the crystal.

Global diversity in the TAS2R38 bitter taste receptor: revisiting a classic evolutionary PROPosal.

The ability to taste phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP) is a polymorphic trait mediated by the TAS2R38 bitter taste receptor gene. It has long been hypothesized that global genetic diversity at this locus evolved under pervasive pressures from balancing natural selection. However, recent high-resolution population genetic studies of TAS2Rs suggest that demographic events have played a critical role in the evolution of these genes. We here utilized the largest TAS2R38 database yet analyzed, consisting of 5,589 individuals from 105 populations, to examine natural selection, haplotype frequencies and linkage disequilibrium to estimate the effects of both selection and demography on contemporary patterns of variation at this locus. We found signs of an ancient balancing selection acting on this gene but no post Out-Of-Africa departures from neutrality, implying that the current observed patterns of variation can be predominantly explained by demographic, rather than selective events. In addition, we found signatures of ancient selective forces acting on different African TAS2R38 haplotypes. Collectively our results provide evidence for a relaxation of recent selective forces acting on this gene and a revised hypothesis for the origins of the present-day worldwide distribution of TAS2R38 haplotypes.

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine.

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva.

Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability.

NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 µM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 µM) and ZINC39395747 (IC50 = 9.14 µM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development.

Mode of binding of the antithyroid drug propylthiouracil to mammalian haem peroxidases.

The mammalian haem peroxidase superfamily consists of myeloperoxidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). These enzymes catalyze a number of oxidative reactions of inorganic substrates such as Cl(-), Br(-), I(-) and SCN(-) as well as of various organic aromatic compounds. To date, only structures of MPO and LPO are known. The substrate-binding sites in these enzymes are located on the distal haem side. Propylthiouracil (PTU) is a potent antithyroid drug that acts by inhibiting the function of TPO. It has also been shown to inhibit the action of LPO. However, its mode of binding to mammalian haem peroxidases is not yet known. In order to determine the mode of its binding to peroxidases, the structure of the complex of LPO with PTU has been determined. It showed that PTU binds to LPO in the substrate-binding site on the distal haem side. The IC50 values for the inhibition of LPO and TPO by PTU are 47 and 30 µM, respectively. A comparision of the residues surrounding the substrate-binding site on the distal haem side in LPO with those in TPO showed that all of the residues were identical except for Ala114 (LPO numbering scheme), which is replaced by Thr205 (TPO numbering scheme) in TPO. A threonine residue in place of alanine in the substrate-binding site may affect the affinity of PTU for peroxidases.

Propylthiouracil quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry: application in a bioequivalence study.

UNLABELLED: A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS) in negative mode (ES-). Chromatography was performed using a Phenomenex Gemini C18 5µm analytical column (4.6mm×150mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v)+0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 (V), -26 (eV) and -5 (V), respectively. The method had a chromatographic run time of 2.5min and a linear calibration curve over the range 20-5000ng/mL. The limit of quantification was 20ng/mL. The stability tests indicated no significant degradation. This HPLC-MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63-115.25%) and 115.60% (109.03-122.58%) for Cmax, 103.31% (100.74-105.96%) and 103.40% (101.03-105.84) for AUClast. CONCLUSION: This method offers advantages over those previously reported, in terms of both a simple liquid-liquid extraction without clean-up procedures, as well as a faster run time (2.5min). The LOQ of 20ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the Tmax and decreased the bioavailability (Cmax and AUC).

Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression.

Fetal/neonatal iron (Fe) and iodine/TH deficiencies lead to similar brain developmental abnormalities and often coexist in developing countries. We recently demonstrated that fetal/neonatal Fe deficiency results in a mild neonatal thyroidal impairment, suggesting that TH insufficiency contributes to the neurodevelopmental abnormalities associated with Fe deficiency. We hypothesized that combining Fe deficiency with an additional mild thyroidal perturbation (6-propyl-2-thiouracil [PTU]) during development would more severely impair neonatal thyroidal status and brain TH-responsive gene expression than either deficiency alone. Early gestation pregnant rats were assigned to 7 different treatment groups: control, Fe deficient (FeD), mild TH deficient (1 ppm PTU), moderate TH deficient (3 ppm PTU), severe TH deficient (10 ppm PTU), FeD/1 ppm PTU, or FeD/3 ppm PTU. FeD or 1 ppm PTU treatment alone reduced postnatal day 15 serum total T4 concentrations by 64% and 74%, respectively, without significantly altering serum total T3 concentrations. Neither treatment alone significantly altered postnatal day 16 cortical or hippocampal T3 concentrations. FeD combined with 1 ppm PTU treatment produced a more severe effect, reducing serum total T4 by 95%, and lowering hippocampal and cortical T3 concentrations by 24% and 31%, respectively. Combined FeD/PTU had a more severe effect on brain TH-responsive gene expression than either treatment alone, significantly altering Pvalb, Dio2, Mbp, and Hairless hippocampal and/or cortical mRNA levels. FeD/PTU treatment more severely impacted cortical and hippocampal parvalbumin protein expression compared with either individual treatment. These data suggest that combining 2 mild thyroidal insults during development significantly disrupts thyroid function and impairs TH-regulated brain gene expression.

Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally-validated detailed structural prediction of agonist binding.

Bitter molecules in humans are detected by ∼25 G protein-coupled receptors (GPCRs). The lack of atomic resolution structure for any of them is complicating an in depth understanding of the molecular mechanisms underlying bitter taste perception. Here, we investigate the molecular determinants of the interaction of the TAS2R38 bitter taste receptor with its agonists phenylthiocarbamide (PTC) and propylthiouracil (PROP). We use the recently developed hybrid Molecular Mechanics/Coarse Grained (MM/CG) method tailored specifically for GPCRs. The method, through an extensive exploration of the conformational space in the binding pocket, allows the identification of several residues important for agonist binding that would have been very difficult to capture from the standard bioinformatics/docking approach. Our calculations suggest that both agonists bind to Asn103, Phe197, Phe264 and Trp201, whilst they do not interact with the so-called extra cellular loop 2, involved in cis-retinal binding in the GPCR rhodopsin. These predictions are consistent with data sets based on more than 20 site-directed mutagenesis and functional calcium imaging experiments of TAS2R38. The method could be readily used for other GPCRs for which experimental information is currently lacking.

Marked increase in PROP taste responsiveness following oral supplementation with selected salivary proteins or their related free amino acids.

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by (1)H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.(1)H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.

3D structure prediction of TAS2R38 bitter receptors bound to agonists phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP).

The G protein-coupled receptor (GPCR) TAS2R38 is a bitter taste receptor that can respond to bitter compounds such as phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP). This receptor was chosen because its four haplotypes (based on three residue site polymorphism) hTAS2R38PAV, hTAS2R38AVI, hTAS2R38AAI, and hTAS2R38PVV are known to have dramatically different responses to PTC and PROP. We aimed to identify the protein-ligand interaction features that determine whether the bitter taste signal from this receptor is sent to the cortex. To do this we predicted the 3D structures of the TAS2R38 bitter taste receptor using our new BiHelix and SuperBiHelix Monte Carlo methods (No experimental determinations of the 3D structure have been reported for any taste receptors.). We find that residue 262 (2nd position in the polymorphism) is involved in the interhelical hydrogen bond network stabilizing the GPCR structure in tasters (hTAS2R38PAV, hTAS2R38AAI, and hTAS2R38PVV), while it is not in the nontaster (hTAS2R38AVI). This suggests that the hydrogen bond interactions between TM3 and TM6 or between TM5 and TM6 may play a role in activating this GPCR. To further validate these structures, we used the DarwinDock method to predict the binding sites and 3D structures for PTC and PROP bound to hTAS2R38PAV, hTAS2R38AVI, hTAS2R38AAI, and hTAS2R38PVV, respectively. Our results show that PTC and PROP can form H-bonds with the backbone of residue 262 in the tasters (hTAS2R38PAV, hTAS2R38AAI, and hTAS2R38PVV) but not in the nontaster (hTAS2R38AVI). Thus it appears that the hydrogen bond interaction between TM3 and TM6 may activate the receptor to pass the ligand binding signal to intracellular processes and that the H-bond between agonists and residue 262 in tasters is involved in the bitter tasting. This is in agreement with experimental observations, providing validation of the predicted ligand-protein complexes and also a potential activation mechanism for the TAS2R38 receptor.

Cocrystals of 6-propyl-2-thiouracil: N-H···O versus N-H···S hydrogen bonds.

In order to investigate the relative stability of N-H···O and N-H···S hydrogen bonds, we cocrystallized the antithyroid drug 6-propyl-2-thiouracil with two complementary heterocycles. In the cocrystal pyrimidin-2-amine-6-propyl-2-thiouracil (1/2), C(4)H(5)N(3)·2C(7)H(10)N(2)OS, (I), the `base pair' is connected by one N-H···S and one N-H···N hydrogen bond. Homodimers of 6-propyl-2-thiouracil linked by two N-H···S hydrogen bonds are observed in the cocrystal N-(6-acetamidopyridin-2-yl)acetamide-6-propyl-2-thiouracil (1/2), C(9)H(11)N(3)O(2)·2C(7)H(10)N(2)OS, (II). The crystal structure of 6-propyl-2-thiouracil itself, C(7)H(10)N(2)OS, (III), is stabilized by pairwise N-H···O and N-H···S hydrogen bonds. In all three structures, N-H···S hydrogen bonds occur only within R(2)(2)(8) patterns, whereas N-H···O hydrogen bonds tend to connect the homo- and heterodimers into extended networks. In agreement with related structures, the hydrogen-bonding capability of C=O and C=S groups seems to be comparable.