Sweet proteins trigger sweet taste perception through interactions with the human T1R2/R3 sweet taste receptor. To date, relatively few proteins have been identified as causing sweet taste perception, and the four most studied proteins: monellin, brazzein, thaumatin, and honey truffle active component (HT-AC), have minimal sequence homology or structural similarities aside from positively charged surface sites. Sweet taste perception has also been found to be readily perturbed by minor changes in the protein structure, such as natural isoforms inherent to heterologous expression of the protein, and synthetic amino acid substitutions. This study uses ab initio rigid-body docking to predict the interactions of known sweet proteins and variants with a recently resolved cryo-EM structure of the T1R2/R3 sweet taste receptor, incorporating comparative analyses between apo-, holo-, and a potentially transient conformation of the receptor. HT-AC mediated activation of the sweet taste receptor is confirmed by in vitro cell-based assays, and results from in silico docking of various sweet proteins are used to derive additional insights regarding sweet taste perception. Perturbations of HT-AC due to naturally occurring post-translational modifications and synthetic modifications are evaluated using in vitro and in silico methods to determine robustness of the interaction between T1R2/R3 and sweet proteins with primary focuses on HT-AC.

Several non-caloric sweeteners exhibit a delay in sweetness onset and a sweetness linger after sampling. These temporal properties are thought to be the result of non-specific interactions with cell membranes and proteins in the oral cavity. Data and analysis presented in this report also support the potential involvement of receptor affinity and binding kinetics to this phenomenon. In general, affected sweeteners exhibit distinctly higher binding affinity compared to carbohydrate sweeteners, which do not have temporal issues. In addition, binding kinetic simulations illustrate much slower receptor binding association and dissociation kinetics for a set of non-caloric sweeteners presenting temporal issues, in comparison to carbohydrate sweeteners. So, the higher affinity of some non-caloric sweeteners, dictating lower use levels, and affecting binding kinetics, could contribute to their delay and linger in sweetness perception. Simple pharmacology principles could explain, at least in part, some of the temporal issues of sweeteners.

Umami, the fifth taste, has been recognized as a legitimate taste modality only recently relative to the other tastes. Dozens of compounds from vastly different chemical classes elicit a savory (also called umami) taste. The prototypical umami substance glutamic acid or its salt monosodium glutamate (MSG) is present in numerous savory food sources or ingredients such as kombu (edible kelp), beans, soy sauce, tomatoes, cheeses, mushrooms, and certain meats and fish. Derivatives of glutamate (Glu), other amino acids, nucleotides, and small peptides can also elicit or modulate umami taste. In addition, many potent umami tasting compounds structurally unrelated to amino acids, nucleotides, and MSG have been either synthesized or discovered as naturally occurring in plants and other substances. Over the last 20 years several receptors have been suggested to mediate umami taste, including members of the metabotropic and ionotropic Glu receptor families, and more recently, the heterodimeric G protein-coupled receptor, T1R1/T1R3. Careful assessment of representative umami tasting molecules from several different chemical classes shows activation of T1R1/T1R3 with the expected rank order of potency in cell-based assays. Moreover, 5'-ribonucleotides, molecules known to enhance the savory note of Glu, considerably enhance the effect of MSG on T1R1/T1R3 in vitro. Binding sites are found on at least 4 distinct locations on T1R1/T1R3, explaining the propensity of the receptor to being activated or modulated by many structurally distinct compounds and these binding sites allosterically interact to modulate receptor activity. Activation of T1R1/T1R3 by all known umami substances evaluated and the receptor's pharmacological properties are sufficient to explain the basic human sensory experience of savory taste and it is therefore unlikely that other receptors are involved.

The sweet taste receptor is rather unique, recognizing a diverse repertoire of natural or synthetic ligands, with a surprisingly large structural diversity, and with potencies stretching over more than six orders of magnitude. Yet, it is not clear if different cell-based assays can faithfully report the relative potencies and efficacies of these molecules. Indeed, up to now, sweet taste receptor agonists have been almost exclusively characterized using cell-based assays developed with overexpressed and promiscuous G proteins. This non-physiological coupling has allowed the quantification of receptor activity via phospholipase C activation and calcium mobilization measurements in heterologous cells on a FLIPR system, for example. Here, we developed a novel assay for the human sweet taste receptor where endogenous G proteins and signaling pathways are recruited by the activated receptor. The effects of several sweet taste receptor agonists and other types of modulators were recorded by measuring changes in dynamic mass redistribution (DMR) using an Epic® reader. Potency and efficacy values obtained in the DMR assay were compared to those results obtained with the classical FLIPR assay. Results demonstrate that for some ligands, the two assay systems provide similar information. However, a clear bias for the FLIPR assay was observed for one third of the agonists evaluated, suggesting that the use of non-physiological coupling may influence the potency and efficacy of sweet taste receptor ligands. Replacing the promiscuous G protein with a chimeric G protein containing the C-terminal tail 25 residues of the physiologically relevant G protein subunit Gαgustducin reduced or abrogated bias.

Humans perceive sweet taste via activation of a specific taste receptor expressed at the surface of taste receptor cells located on the tongue and soft palate papillae. The sweet taste receptor functions as an obligate heterodimer, comprising two different class C GPCR subunits. This receptor is unique in that it is activated or modulated by a plethora of ligands from highly diverse chemical classes, from small molecules to peptides and proteins and interacting with topologically distinct sites on each of its subunits. Modulators acting at separate functional domains of the sweet taste receptor can behave as full agonists. However, contrary to observations made with other class C GPCRs such as the metabotropic glutamate receptors and the γ-aminobutyric acid type B receptor (GABAB) receptor, modulators interacting within the allosteric sites in the transmembrane domains of the sweet taste receptor only exert a relatively small effect on the affinity and efficacy of the agonist interacting at the orthosteric binding site located within the Venus fly trap domain (VFD). Newly identified potent and efficacious positive allosteric modulators (PAM)s of the sweet taste receptor rather interact at a site in close proximity to the agonist, within the VFD, display significant probe dependence, and markedly increase the affinity of the orthosteric ligand. Several sweet taste receptor inhibitors have also been characterized. Functional studies reveal a complex relationship between different ligands. Whether the antagonist will be surmountable or insurmountable and will act competitively or non-competitively largely depends on the agonist being studied and the location of its interaction site on the receptor.

In humans, bitter taste is mediated by 25 TAS2Rs. Many compounds, including certain active pharmaceutical ingredients, excipients, and nutraceuticals, impart their bitter taste (or in part) through TAS2R8 activation. However, effective TAS2R8 blockers that can either suppress or reduce the bitterness of these compounds have not been described. We are hereby reporting a series of novel 3-(pyrazol-4-yl) imidazolidine-2,4-diones as potent and selective TAS2R8 antagonists. In human sensory tests, S6821 and S7958, two of the most potent analogues from the series, demonstrated efficacy in blocking TAS2R8-mediated bitterness and were selected for development. Following data evaluation by expert panels of a number of national and multinational regulatory bodies, including the US, the EU, and Japan, S6821 and S7958 were approved as safe under conditions of intended use as bitter taste blockers.

The paper presents the activity trends for a novel series of phenoxyacetyl amides as human TRPM8 receptor agonists. This series encompasses in vitro activity values ranging from the micromolar to the picomolar levels. Sensory evaluation of these molecules highlights their relevance as cooling agents for oral applications. The positive outcome of the complete evaluation of N-(1H-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)-2-(p-tolyloxy)acetamide resulted in its approval for Generally Recognized As Safe (GRAS) status by the Flavor & Extract Manufacturer Association (FEMA) as FEMA 4809.

In gradients of external chemo-attractant, mammalian neutrophilic leukocytes (neutrophils)(1) and Dictyostelium discoideum amoebae 2 adopt a polarized morphology and selectively accumulate lipid products of phosphatidylinositol-3-OH kinases (PI(3) Ks), including PtdIns(3,4,5) P-3, at their up-gradient edges; the internal PtdIns(3,4,5) P-3 gradient substantially exceeds that of the external attractant. An accompanying report 3 presents evidence for a positive feedback loop that amplifies the gradient of internal signal: PtdIns(3,4,5) P-3 at the leading edge stimulates its own accumulation by inducing activation of one or more Rho GTPases (Rac, Cdc42, and/or Rho), which in turn increase PtdIns(3,4,5) P-3 accumulation. Here we show that interruption of this feedback by treatment with PI(3) K inhibitors reduces the size and stability of pseudopods and causes cells to migrate in jerky trajectories that deviate more from the up-gradient direction than do those of controls. Moreover, amplification of the internal PtdIns(3,4,5) P-3 gradient is markedly impaired by latrunculin or jasplakinolide, toxins that inhibit polymerization (4,5) or depolymerization(6) of actin, respectively. Thus reciprocal interplay between PtdIns(3,4,5) P-3 and polymerized actin initiates and maintains the asymmetry of intracellular signals responsible for cell polarity and directed motility.

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT(2)), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of I-125-[Bpa(1)]AngII . AT(2) receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of I-125-[Bpa(1)]AngII within amino acids 330 of AT(2) receptor. Treatment of I-125-[Bpa(8)]AngII . AT(2) receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT(2) receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of I-125-[Bpa(8)]AngII within residues 129-138 of AT(2) receptor. Our results indicate that the aminoterminal tail of the AT(2) receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT(2) receptor interacts with the carboxyl-terminal end of AngII.