Comprehensive structure-activity-relationship studies of sensory active compounds in licorice (Glycyrrhiza glabra).

Licorice saponins, the main constituents of Glycyrrhiza glabra L. roots, are highly appreciated by the consumer for their pleasant sweet and long lasting licorice taste. The objective of the present study was to understand the molecular features that contribute to bitter, sweet and licorice sensation of licorice roots, and whether individual compounds elicit more than one of these sensations. Therefore, a sensomics approach was conducted, followed by purification of the compounds with highest sensory impact, and by synthesis as well as full characterization via HRESIMS, ESIMS/MS and 1D/2D-NMR experiments. This led to the discovery and structure determination of 28 sweet, bitter and licorice tasting key phytochemicals, including two unknown compounds. A combination of sensorial, cell-based and computational analysis revealed distinct structural features, such as spatial arrangement of functional groups in the triterpenoid E-ring, driving to different taste sensations and sweet receptor hTAS1R2/R3 stimulation.

Sensory-Guided Multidimensional Exploration of Antisweet Principles from Gymnema sylvestre (Retz) Schult.

We report on activity-guided investigation of the key antisweet principles of Gymnema sylvestre. Orosensory-guided fractionation by means of solid phase extraction, preparative 2D-LC, and semipreparative HPLC followed by accurate MS and 1D/2D NMR experiments revealed six known and three previously unknown gymnemic acids as the key constituents of seven highly sensory-active fractions. Localized via a modified comparative taste dilution analysis (cTDA) and taste modulation probability (TMP) based screening techniques, a strong intrinsic bitterness was also observed for gymnemic acids. In addition, the suppressive effects of the most abundant acids on the response of the human sweet taste receptor to sucrose were verified by means of a functional hTAS1R2/hTAS1R3 sweet taste receptor assay. This in vitro screening revealed large differences in antisweet activity among the isolated compounds, where gymnemic acids XV and XIX showed the highest sweet suppressing activity. This broad-based molecular characterization of the sweet taste inhibiting activity of Gymnema sylvestre will enable further insight into the molecular basis of sweet taste modulation at the receptor level.

The Crystal Structure of Gurmarin, a Sweet Taste-Suppressing Protein: Identification of the Amino Acid Residues Essential for Inhibition.

Gurmarin is a highly specific sweet taste-suppressing protein in rodents that is isolated from the Indian plant Gymnema sylvestre. Gurmarin consists of 35 amino acid residues containing 3 intramolecular disulfide bridges that form a cystine knot. Here, we report the crystal structure of gurmarin at a 1.45 Å resolution and compare it with previously reported nuclear magnetic resonance solution structures. The atomic structure at this resolution allowed us to identify a very flexible region consisting of hydrophobic residues. Some of these amino acid residues had been identified as a putative binding site for the rat sweet taste receptor in a previous study. By combining alanine-scanning mutagenesis of the gurmarin molecule and a functional cell-based receptor assay, we confirmed that some single point mutations in these positions drastically affect sweet taste receptor inhibition by gurmarin.

The Odorant ( R)-Citronellal Attenuates Caffeine Bitterness by Inhibiting the Bitter Receptors TAS2R43 and TAS2R46.

Sensory studies showed the volatile fraction of lemon grass and its main constituent, the odor-active citronellal, to significantly decrease the perceived bitterness of a black tea infusion as well as caffeine solutions. Seven citronellal-related derivatives were synthesized and shown to inhibit the perceived bitterness of caffeine in a structure-dependent manner. The aldehyde function at carbon 1, the ( R)-configuration of the methyl-branched carbon 3, and a hydrophobic carbon chain were found to favor the bitter inhibitory activity of citronellal; for example, even low concentrations of 25 ppm were observed to reduce bitterness perception of caffeine solution (6 mmol/L) by 32%, whereas ( R)-citronellic acid (100 pm) showed a reduction of only 21% and ( R)-citronellol (100 pm) was completely inactive. Cell-based functional experiments, conducted with the human bitter taste receptors TAS2R7, TAS2R10, TAS2R14, TAS2R43, and TAS2R46 reported to be sensitive to caffeine, revealed ( R)-citronellal to completely block caffeine-induced calcium signals in TAS2R43-expressing cells, and, to a lesser extent, in TAS2R46-expressing cells. Stimulation of TAS2R43-expressing cells with structurally different bitter agonists identified ( R)-citronellal as a general allosteric inhibitor of TAS2R43. Further structure/activity studies indicated 3-methyl-branched aliphatic aldehydes with a carbon chain of ≥4 C atoms as best TAS2R43 antagonists. Whereas odor-taste interactions have been mainly interpreted in the literature to be caused by a central neuronal integration of odors and tastes, rather than by peripheral events at the level of reception, the findings of this study open up a new dimension regarding the interaction of the two chemical senses.

Evidence for a Transient Additional Ligand Binding Site in the TAS2R46 Bitter Taste Receptor.

Most human G protein coupled receptors (GPCRs) are activated by small molecules binding to their 7-transmembrane (7-TM) helix bundle. They belong to basally diverging branches: the 25 bitter taste 2 receptors and most members of the very large rhodopsin-like/class A GPCRs subfamily. Some members of the class A branch have been suggested to feature not only an orthosteric agonist-binding site but also a more extracellular or "vestibular" site, involved in the binding process. Here we use a hybrid molecular mechanics/coarse-grained (MM/CG) molecular dynamics approach on a widely studied bitter taste receptor (TAS2R46) receptor in complex with its agonist strychnine. Three ∼1 µs molecular simulation trajectories find two sites hosting the agonist, which together elucidate experimental data measured previously and in this work. This mechanism shares similarities with the one suggested for the evolutionarily distant class A GPCRs. It might be instrumental for the remarkably broad but specific spectrum of agonists of these chemosensory receptors.

Human taste and umami receptor responses to chemosensorica generated by Maillard-type N²-alkyl- and N²-arylthiomethylation of guanosine 5'-monophosphates.

Structural modification of the exocyclic amino function of guanosine 5'-monophosphate (5'-GMP) by Maillard-type reactions with reducing carbohydrates was recently found to increase the umami-enhancing activity of the nucleotide upon S-N(2)-1-carboxyalkylation and S-N(2)-(1-alkylamino)carbonylalkylation, respectively. Since the presence of sulfur atoms in synthetic N(2)-alkylated nucleotides was reported to be beneficial for sensory activity, a versatile Maillard-type modification of 5'-GMP upon reaction with glycine's Strecker aldehyde formaldehyde and organic thiols was performed in the present study. A series of N(2)-(alkylthiomethyl)guanosine and N(2)-(arylthiomethyl)guanosine 5'-monophosphates was generated and the compounds were evaluated to what extent they enhance the umami response to monosodium L-glutamate in vivo by a paired-choice comparison test using trained human volunteers and in vitro by means of cell-based umami taste receptor assay. Associated with a high umami-enhancing activity (ß-value 5.1), N(2)-(propylthiomethyl)guanosine 5'-monophosphate could be generated when 5'-GMP reacted with glucose, glycine, and the onion-derived odorant 1-propanethiol, thus opening a valuable avenue to produce high-potency umami-enhancing chemosensorica from food-derived natural products by kitchen-type chemistry.

Sensomics analysis of taste compounds in balsamic vinegar and discovery of 5-acetoxymethyl-2-furaldehyde as a novel sweet taste modulator.

Sensory-directed fractionation of traditional balsamic vinegar of Modena (TBV) led to the identification of the sweet-bitter tasting hexose acetates 6-O-acetyl-α/ß-d-glucopyranose and 1-O-acetyl-ß-d-fructopyranose as well as the previously unknown sweetness modulator 5-acetoxymethyl-2-furaldehyde. Taste re-engineering experiments and sensory time-intensity studies confirmed 5-acetoxymethyl-2-furaldehyde to contribute to the typical long-lasting sweet taste quality of TBV. Moreover, the response of the sweet taste receptor to this furaldehyde was verified by means of a functional hTAS1R2/hTAS1R3 receptor assay. Quantitative analysis of a total of 59 nonvolatile sensometabolites and taste modulators revealed higher concentrations of the sweet-modulating 5-acetoxymethyl-2-furaldehyde, nonvolatile organic acids and polyphenols such as wood-derived ellagitannins, and lower concentrations of acetic acid in the premium quality TBV when compared to balsamic vinegar of Modena (BV). Quantitative monitoring of sensometabolites throughout TBV manufacturing, followed by agglomerative hierarchical clustering and sensomics heatmapping, gave molecular insights into the taste alterations occurring during TBV maturation.

Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.

Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

Sweet-taste-suppressing compounds: current knowledge and perspectives of application.

Sweet-tasting compounds are recognized by a heterodimeric receptor composed of the taste receptor, type 1, members 2 (T1R2) and 3 (T1R3) located in the mouth. This receptor is also expressed in the gut where it is involved in intestinal absorption, metabolic regulation, and glucose homeostasis. These metabolic functions make the sweet taste receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions such as diabetes. Existing sweet taste inhibitors or blockers that are still in development would constitute promising therapeutic agents. In this review, we will summarize the current knowledge of sweet taste inhibitors, including a sweet-taste-suppressing protein named gurmarin, which is only active on rodent sweet taste receptors but not on that of humans. In addition, their potential applications as therapeutic tools are discussed.

Human psychometric and taste receptor responses to steviol glycosides.

Steviol glycosides, the sweet principle of Stevia Rebaudiana (Bertoni) Bertoni, have recently been approved as a food additive in the EU. The herbal non-nutritive high-potency sweeteners perfectly meet the rising consumer demand for natural food ingredients in Europe. We have characterized the organoleptic properties of the most common steviol glycosides by an experimental approach combining human sensory studies and cell-based functional taste receptor expression assays. On the basis of their potency to elicit sweet and bitter taste sensations, we identified glycone chain length, pyranose substitution, and the C16 double bond as the structural features giving distinction to the gustatory profile of steviol glycosides. A comprehensive screening of 25 human bitter taste receptors revealed that two receptors, hTAS2R4 and hTAS2R14, mediate the bitter off-taste of steviol glycosides. For some test substances, e.g., stevioside, we observed a decline in sweet intensity at supra-maximum concentrations. This effect did not arise from allosteric modulation of the hTAS1R2/R3 sweet taste receptor but might be explained by intramolecular cross-modal suppression between the sweet and bitter taste component of steviol glycosides. These results might contribute to the production of preferentially sweet and least bitter tasting Stevia extracts by an optimization of breeding and postharvest downstream processing.

Optimization of the production of gurmarin, a sweet-taste-suppressing protein, secreted by the methylotrophic yeast Pichia pastoris.

Gurmarin, a 35-residue polypeptide, is known to selectively inhibit responses to sweet substances in rodents without affecting responses to other basic taste stimuli, such as NaCl, HCl, and quinine. Here, we report the heterologous expression of gurmarin using the methylotrophic yeast Pichia pastoris. Gurmarin was secreted into the buffered minimal medium using the α-factor preprosequence without the EAEA spacer peptide of Saccharomyces cerevisiae and was under the control of the methanol-inducible alcohol oxidase promoter. We found that gurmarin accumulated in the yeast culture medium reaching 5 mg per liter of culture over an expression period of 4 days. To compare the production level and the signal peptide processing, the N-terminal amino acid of gurmarin was substituted by a glutamic acid residue. This construct resulted in a 6-fold increase in the level of gurmarin secretion leading to 30 mg of purified protein per liter of culture. Purified recombinant gurmarin resulting from both constructs was characterized using mass spectrometry. Circular dichroism and NMR spectroscopy revealed that recombinant gurmarin was properly folded and had secondary and tertiary structures. We also confirmed its capability to inhibit the rat heterodimeric sweet taste T1R2/T1R3 receptor by functional expression in human embryonic kidney HEK293T cells. The high level of fully active gurmarin obtained in P. pastoris makes this expression system attractive for fermentor growth and pharmacological investigations of taste receptor and gurmarin functions.

Functional characterization of an allatotropin receptor expressed in the corpora allata of mosquitoes.

Allatotropin is an insect neuropeptide with pleiotropic actions on a variety of different tissues. In the present work we describe the identification, cloning and functional and molecular characterization of an Aedes aegypti allatotropin receptor (AeATr) and provide a detailed quantitative study of the expression of the AeATr gene in the adult mosquito. Analysis of the tissue distribution of AeATr mRNA in adult female revealed high transcript levels in the nervous system (brain, abdominal, thoracic and ventral ganglia), corpora allata-corpora cardiaca complex and ovary. The receptor is also expressed in heart, hindgut and male testis and accessory glands. Separation of the corpora allata (CA) and corpora cardiaca followed by analysis of gene expression in the isolated glands revealed expression of the AeATr primarily in the CA. In the female CA, the AeATr mRNA levels were low in the early pupae, started increasing 6h before adult eclosion and reached a maximum 24h after female emergence. Blood feeding resulted in a decrease in transcript levels. The pattern of changes of AeATr mRNA resembles the changes in JH biosynthesis. Fluorometric Imaging Plate Reader recordings of calcium transients in HEK293 cells expressing the AeATr showed a selective response to A. aegypti allatotropin stimulation in the low nanomolar concentration range. Our studies suggest that the AeATr play a role in the regulation of JH synthesis in mosquitoes.

Receptor agonism and antagonism of dietary bitter compounds.

Food contains complex blends of structurally diverse bitter compounds that trigger bitterness through activation of one or more of the ∼25 human TAS2 bitter taste receptors. It remains unsolved, however, whether the perceived bitterness of binary bitter-compound mixtures can be considered an additive function of all bitter-inducing chemicals in the mouth, suggesting that little mutual interaction takes place among bitter substances or if mixture suppression and synergism occurs. Here we report on two natural sesquiterpene lactones from edible plants, which stimulate distinct sets of hTAS2Rs in transfected cells. Both chemicals also robustly inhibit different but overlapping subsets of agonist-activated hTAS2Rs. These findings demonstrate that mixtures of bitter compounds, because they normally occur in human foodstuff, likely elicit bitter perception in a complex and not in a merely additive manner. An unexpected implication of this discovery is that, during evolution, the naturally occurring bitter taste receptor antagonists have shaped some of the pharmacological properties of the receptors, such as overlapping recognition profiles and breadth of tuning.

Stereoselective synthesis of amides sharing the guanosine 5'-monophosphate scaffold and Umami enhancement studies using human sensory and hT1R1/rT1R3 receptor assays.

Recent studies led to the identification of umami-enhancing (S)-N(2)-(1-carboxyethyl)- and (S)-N(2)-(1-alkylamino)carbonylalkyl)guanosine 5'-monophosphates that, together with their sensorially inactive (R)-stereoisomers, were found to be formed upon Maillard-type glycation of guanosine 5'-monophosphate (5'-GMP) with 1,3-dihydroxyacetone or glyceraldehyde, respectively. As the efficiency of this Maillard-type procedure to generate the amidated derivatives is limited by the low solubility and reactivity of long-chain alkyl amines as well as by the tedious separation of the diastereomers formed, a versatile synthesis for the (R)- and (S)-configured amides of N(2)-carboxyalkylated guanosine 5'-monophosphate was developed. Sensory evaluation of a series of N(2)-(1-alkylamino)carbonylalkyl)guanosine 5'-monophosphates revealed ß-values for umami enhancement between 0.1 and 7.7 and identified a strong influence of the stereochemistry as well as the chain length of the N(2)-substituent on the umami-enhancing activity. The observed sensory impact of the (S)-configured isomers was confirmed by recording the enhancing effect of these nucleotides on the l-glutamate-induced response of the functionally expressed T1R1/T1R3 umami receptor in a cell-based assay, thus underscoring the stereospecifity of the umami taste receptor binding site.

Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor.

Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.

Modulation of bitter taste perception by a small molecule hTAS2R antagonist.

Human bitter taste is mediated by the hTAS2R family of G protein-coupled receptors. The discovery of the hTAS2Rs enables the potential to develop specific bitter receptor antagonists that could be beneficial as chemical probes to examine the role of bitter receptor function in gustatory and nongustatory tissues. In addition, they could have widespread utility in food and beverages fortified with vitamins, antioxidants, and other nutraceuticals, because many of these have unwanted bitter aftertastes. We employed a high-throughput screening approach to discover a novel bitter receptor antagonist (GIV3727) that inhibits activation of hTAS2R31 (formerly hTAS2R44) by saccharin and acesulfame K, two common artificial sweeteners. Pharmacological analyses revealed that GIV3727 likely acts as an orthosteric, insurmountable antagonist of hTAS2R31. Surprisingly, we also found that this compound could inhibit five additional hTAS2Rs, including the closely related receptor hTAS2R43. Molecular modeling and site-directed mutagenesis studies suggest that two residues in helix 7 are important for antagonist activity in hTAS2R31 and hTAS2R43. In human sensory trials, GIV3727 significantly reduced the bitterness associated with the two sulfonamide sweeteners, indicating that hTAS2R antagonists are active in vivo. Our results demonstrate that small molecule bitter receptor antagonists can effectively reduce the bitter taste qualities of foods, beverages, and pharmaceuticals.

Structural requirements of bitter taste receptor activation.

An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception.

Allatostatin-C receptors in mosquitoes.

In the present work we describe the functional and molecular characterization of two Aedes aegypti allatostatin-C receptor paralogs (AeAS-CrA and AeAS-CrB) and provide a detailed quantitative study of the expression of the AS-C receptor genes in an adult insect. The tissue distribution of the two AS-C receptors differed significantly; the mRNA levels of AeAS-CrB in the Malpighian tubules were the highest detected, while transcripts for AeAS-CrA were relatively low in this tissue. In addition, the transcript levels of both receptors were different in the thoracic and abdominal ganglia, corpora allata (CA) and the testis of the male. In the CA, the AeAS-CrB mRNA levels were constant from 0 to 72 h after female emergence, while the AeAS-CrA levels increased at 72 h. To complement the receptor expression studies, we analyzed the tissue specificity for allatostatin-C mRNA in female mosquitoes. Expression was high in abdominal ganglia and brain. Transcript levels of allatostatin-C in the head of females were elevated at eclosion and there were no major changes during the first week of adult life or after blood feeding. Fluorometric Imaging Plate Reader (FLIPR) recordings of calcium transients in HEK293T cells transiently expressing both putative receptors showed that they both responded selectively to allatostatin-C stimulation in the nanomolar concentration range. However, the peptide showed slightly greater affinity for AeAS-CrB than AeAS-CrA. Our studies suggest that some of the pleiotropic effects of allatostatin-C in mosquitoes could be mediated by the different receptor paralogs. Transcriptional regulation of the AS-C receptors may not have a critical role in the changes of CA responsiveness to the peptide that we previously described.

The molecular receptive ranges of human TAS2R bitter taste receptors.

Humans perceive thousands of compounds as bitter. In sharp contrast, only approximately 25 taste 2 receptors (TAS2R) bitter taste receptors have been identified, raising the question as to how the vast array of bitter compounds can be detected by such a limited number of sensors. To address this issue, we have challenged 25 human taste 2 receptors (hTAS2Rs) with 104 natural or synthetic bitter chemicals in a heterologous expression system. Thirteen cognate bitter compounds for 5 orphan receptors and 64 new compounds for previously identified receptors were discovered. Whereas some receptors recognized only few agonists, others displayed moderate or extreme tuning broadness. Thus, 3 hTAS2Rs together were able to detect approximately 50% of the substances used. Conversely, though 63 bitter substances activated only 1-3 receptors, 19 compounds stimulated up to 15 hTAS2Rs. Our data suggest that the detection of the numerous bitter chemicals is related to the molecular receptive ranges of hTAS2Rs.

The human bitter taste receptor hTAS2R50 is activated by the two natural bitter terpenoids andrographolide and amarogentin.

Bitterness perception in mammals is mediated through activation of dedicated bitter taste receptors located in the oral cavity. Genomic analyses revealed the existence of orthologous mammalian bitter taste receptor genes, which presumably recognize the same compounds in different species, as well as species-specific receptor gene expansions believed to fulfill a critical role during evolution. In man, 8 of the 25 bitter taste receptors (hTAS2Rs) are closely related members of such an expanded subfamily of receptor genes. This study identified two natural bitter terpenoids, andrographolide and amarogentin, that are agonists for the orphan receptor hTAS2R50, the most distant member of the subfamily. This paper presents the pharmacological characterization of this receptor and analyzes its functional relationship with the previously deorphaned hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. Insights into the general breadth of tuning, functional redundancies, and relationships between pharmacological activation patterns and amino acid homologies for this receptor subfamily are presented.