A structure-based approach towards the identification of novel antichagasic compounds: Trypanosoma cruzi carbonic anhydrase inhibitors.

Trypanosoma cruzi carbonic anhydrase (TcCA) has recently emerged as an interesting target for the design of new compounds to treat Chagas disease. In this study we report the results of a structure-based virtual screening campaign to identify novel and selective TcCA inhibitors. The combination of properly validated computational methodologies such as comparative modelling, molecular dynamics and docking simulations allowed us to find high potency hits, with KI values in the nanomolar range. The compounds also showed trypanocidal effects against T. cruzi epimastigotes and trypomastigotes. All the candidates are selective for inhibiting TcCA over the human isoform CA II, which is encouraging in terms of possible therapeutic safety and efficacy.

Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor.

Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

Anticonvulsant effect of sodium cyclamate and propylparaben on pentylenetetrazol-induced seizures in zebrafish.

Screening for novel anticonvulsant drugs requires appropriate animal seizure models. Zebrafish provide small, accessible, and cost-efficient preclinical models applicable to high-throughput small molecule screening. Based on previous results in rodents, we have here examined the effects of artificial sweetener sodium cyclamate and antimicrobial agent sodium propylparaben on a model of pentylenetetrazole (PTZ)-induced seizures in zebrafish. Sodium cyclamate reduced the bursts of hyperactivity, the spasms, increased the latency to spasms, and the latency to seizure, while propylparaben increased the latency to spasms. The results show the potential of zebrafish to detect novel anticonvulsant compounds while they also demonstrate the ability of two commonly ingested chemical compounds to modify the seizure threshold when were administrated at low concentration.

Anticonvulsant activity of artificial sweeteners: a structural link between sweet-taste receptor T1R3 and brain glutamate receptors.

A virtual screening campaign based on application of a topological discriminant function capable of identifying novel anticonvulsant agents indicated several widely-used artificial sweeteners as potential anticonvulsant candidates. Acesulfame potassium, cyclamate and saccharin were tested in the Maximal Electroshock Seizure model (mice, ip), showing moderate anticonvulsant activity. We hypothesized a probable structural link between the receptor responsible of sweet taste and anticonvulsant molecular targets. Bioinformatic tools confirmed a highly significant sequence-similarity between taste-related protein T1R3 and several metabotropic glutamate receptors from different species, including glutamate receptors upregulated in epileptogenesis and certain types of epilepsy.

Relationships among taste qualities assessed with response-context effects.

Psychophysical judgments often depend on stimulus context. For example, sugar solutions are judged sweeter when a tasteless fruity aroma has been added. Response context also matters; adding a fruity aroma to sugar increases the rated sweetness when only sweetness is considered but not when fruitiness is judged as well. The interaction between stimulus context and response context has been explored more extensively in taste-odor mixtures than in taste-taste mixtures. To address this issue, subjects in the current study rated the sourness of citric acid mixed with quinine (bitter), sodium chloride (salty), and cyclamate (sweet) (stimulus context). In one condition, subjects rated sourness alone. In another, subjects rated both sourness and the other salient quality (bitterness, saltiness, or sweetness) (response context). Sourness ratings were most sensitive to response context for sour-salty mixtures (i.e., ratings of sourness alone exceeded ratings of sourness made simultaneously with saltiness) and least sensitive to context for the sour-sweet mixtures (sourness ratings made under the 2 conditions were essentially identical). Response-context effects for the sour-bitter mixture were nominally intermediate. The magnitudes of these context effects were related to judgments of qualitative similarity between citric acid and the other stimuli, consistent with prior findings. These types of context effects are relevant to the study of taste-taste mixture interactions and should provide insight into the perceptual similarities among the taste qualities.

Anionic leak currents through the Na+/monocarboxylate cotransporter SMCT1.

SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular Cl(-) replacement with cyclamate(-) and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent Cl(-) dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion (MES(-)) was identified. The observed outward leak currents represented anionic influx and favored larger anions (NO(3)(-)>I(-)>Br(-)>Cl(-)); currents in excess of 1 muA (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external Na(+) was replaced by N-methyl-d-glucamine(+). More precisely, external Na(+) blocked the leak current in both directions, but its K(i) value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external Na(+) concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed.

Stevia and saccharin preferences in rats and mice.

Use of natural noncaloric sweeteners in commercial foods and beverages has expanded recently to include compounds from the plant Stevia rebaudiana. Little is known about the responses of rodents, the animal models for many studies of taste systems and food intake, to stevia sweeteners. In the present experiments, preferences of female Sprague-Dawley rats and C57BL/6J mice for different stevia products were compared with those for the artificial sweetener saccharin. The stevia component rebaudioside A has the most sweetness and least off-tastes to human raters. In ascending concentration tests (48-h sweetener vs. water), rats and mice preferred a high-rebaudioside, low-stevioside extract as strongly as saccharin, but the extract stimulated less overdrinking and was much less preferred to saccharin in direct choice tests. Relative to the extract, mice drank more pure rebaudioside A and showed stronger preferences but still less than those for saccharin. Mice also preferred a commercial mixture of rebaudioside A and erythritol (Truvia). Similar tests of sweet receptor T1R3 knockout mice and brief-access licking tests with normal mice suggested that the preferences were based on sweet taste rather than post-oral effects. The preference response of rodents to stevia sweeteners is notable in view of their minimal response to some other noncaloric sweeteners (aspartame and cyclamate).

Evaluation of the effects of three sulfa sweeteners on the lifespan and intestinal fat deposition in C. elegans.

High sugar content in beverage or food can affect the aging process, and thus natural/artificial sweeteners are widely used as substitutes. However, whether sweeteners have such adverse effects as sugar remains to be clarified. Therefore, in the current study, three sulfa sweeteners, namely, saccharin sodium salt hydrate (SAC2), sodium cyclamate (CYC3) and acesulfame potassium (AceK4) were evaluated for their effects on the lifespan, deposition of lipofuscin, exercise activity, food intake, and intestinal fat deposition (IFD5) of Caenorhabditis elegans (C. elegans6). It was shown that SAC at 0.3 and 10 mg/mL shortened the lifespan of C. elegans and impaired the exercise capacity, while at other concentrations no significant effects were observed. In contrast, CYC at 0.1, 1 and 10 mg/mL prolonged the lifespan of C. elegans. On the other hand, AceK at 1 mg/mL increased the lifespan of C. elegans, and could decrease both lipofuscin deposition and IFD in a dose-dependent manner. Taken together, these results indicated that although SAC, CYC, and AceK all belong to the sulfa sweeteners, each has distinct effects on different physiological activities associated with aging, at least in C. elegans.

Noncaloric Sweeteners Induce Peripheral Serotonin Secretion via the T1R3-Dependent Pathway in Human Gastric Parietal Tumor Cells (HGT-1).

The role of sweet taste in energy intake and satiety regulation is still controversial. Noncaloric artificial sweeteners (NCSs) are thought to help reduce energy intake, although little is known about their impact on the satiating neurotransmitter serotonin (5-HT). In the gastrointestinal (GI) tract, 5-HT regulates gastric acid secretion and gastric motility, both part of the complex network of mechanisms regulating food intake and satiety. This study demonstrated a stimulating impact compared to controls (100%) on 5-HT release in human gastric tumor cells (HGT-1) by the NCSs cyclamate (50 mM, 157% ± 6.3%), acesulfame potassium (Ace K, 50 mM, 197% ± 8.6%), saccharin (50 mM, 147% ± 6.7%), sucralose (50 mM, 194% ± 11%), and neohesperidin dihydrochalcone (NHDC, 1 mM, 201% ± 13%). Although these effects were not associated with the sweet taste intensity of the NCSs tested, involvement of the sweet receptor subunit T1R3 in the NCS-evoked response was demonstrated by mRNA expression of TAS1R3, co-incubation experiments using the T1R3 receptor antagonist lactisole, and a TAS1R3 siRNA knockdown approach. Analysis of the downstream signaling revealed activation of the cAMP/ERK/Ca2+ cascade. Co-treatment experiments with 10 mM glucose enhanced the 5-HT release induced by cyclamate, Ace K, saccharin, and sucralose, thereby supporting the enhancing effect of glucose on a NCS-mediated response. Overall, the results obtained identify NCSs as potent inducers of 5-HT release via T1R3 in human gastric parietal cells in culture and warrant in vivo studies to demonstrate their efficacy.

A Bitter Tale of Sweet Synergy.

Some sweeteners show a synergistic enhancement of perceived sweetness when they are tasted as binary mixtures. In this issue of Cell Chemical Biology, Behrens et al. (2017) find that a surprising explanation for this classic observation may lie in their reciprocal inhibition of bitter taste receptors.

Differential modulation of the lactisole 'Sweet Water Taste' by sweeteners.

Pre-exposure to taste stimuli and certain chemicals can cause water to have a taste. Here we studied further the 'sweet water taste' (SWT) perceived after exposure to the sweet taste inhibitor lactisole. Experiment 1 investigated an incidental observation that presenting lactisole in mixture with sucrose reduced the intensity of the SWT. The results confirmed this observation and also showed that rinsing with sucrose after lactisole could completely eliminate the SWT. The generalizability of these findings was investigated in experiment 2 by presenting 5 additional sweeteners before, during, or after exposure to lactisole. The results found with sucrose were replicated with fructose and cyclamate, but the 3 other sweeteners were less effective suppressors of the SWT, and the 2 sweeteners having the highest potency initially enhanced it. A third experiment investigated these interactions on the tongue tip and found that the lactisole SWT was perceived only when water was actively flowed across the tongue. The same experiment yielded evidence against the possibility that suppression of the SWT following exposure to sweeteners is an aftereffect of receptor activation while providing additional support for a role of sweetener potency. Collectively these results provide new evidence that complex inhibitory and excitatory interactions occur between lactisole and agonists of the sweet taste receptor TAS1R2-TAS1R3. Receptor mechanisms that may be responsible for these interactions are discussed in the context of the current model of the SWT and the possible contribution of allosteric modulation.

Blends of Non-caloric Sweeteners Saccharin and Cyclamate Show Reduced Off-Taste due to TAS2R Bitter Receptor Inhibition.

Non-caloric sweeteners are widely used for the formulation of calorie-reduced beverages for health-conscious consumers. However, disadvantages such as undesired off-tastes limit the use of non-nutritive sweeteners. Therefore, the food industry is constantly searching for novel sweeteners and frequently resorts to using blends combining non-caloric sweeteners in a single formulation. The earliest blend allowing higher sweetness levels with reduced bitter off-taste combined saccharin with cyclamate. However, the mechanism by which sweetener blends become superior to single compounds remained obscure. By functional expression of human bitter taste receptors, we found the explanation for the phenomenon observed ∼60 years ago. We demonstrate that cyclamate potently blocks the receptors responsible for saccharin's bitter off-taste. This effect occurs at concentrations where cyclamate itself does not elicit a side taste. Intriguingly, also saccharin inhibits cyclamate-activated bitter receptors. Our experiments demonstrate that heterologous assays are useful for understanding perceptual phenomena and the development of novel tastant formulations.

Inhibition of iodinated nerve growth factor binding by the suspected tumor promoters saccharin and cyclamate.

The strong correlation between tumor-promoting activity and the capacity to inhibit nerve growth factor-induced neurite outgrowth for a number of compounds prompted studies on the suspected bladder tumor promoters saccharin and cyclamate. These artificial sweeteners reversibly inhibited neurite outgrowth and binding of [125I]nerve growth factor in embryonic chick sensory ganglia cells. Curves showing the dose-dependent inhibition of response and the dose-dependent inhibition of binding to high affinity sites were closely parallel. The inhibition appeared to be due to a reduction in the affinity rather than to a change in the number of binding sites, as indicated by the extent of reduction in the association rate. These results suggest that alteration of cellular differentiation by tumor promoters may result from interactions with receptor systems that regulate specialized cell functions.

Effect of the suspected tumor promoters saccharin, cyclamate, and phenol on nerve growth factor binding and response in cultured embryonic chick ganglia.

The suspected bladder tumor promoters saccharin and cyclamate reversibly inhibited nerve growth factor-induced neurite outgrowth in embryonic chick sensory ganglia, and active concentrations of these artificial sweeteners inhibited binding of 125I-labeled mouse submaxillary gland nerve growth factor as well. The skin tumor promoter phenol also reversibly inhibited neurite outgrowth, while comparable concentrations of the nonpromoting but structurally related compounds benzene and fluorobenzene did not. However, in contrast to findings with saccharin and cyclamate, phenol had little, if any, effect on binding of radioactive nerve growth factor.

Response properties of macaque monkey chorda tympani fibers.

Many of the chorda tympani fibers of crab-eating monkeys respond to more than one of the four basic stimuli (NaCl, sucrose, HCl, and quinine hydrochloride) as well as cooling or warming of the tongue. Fibers could be classified into four categories depending on their best sensitivity to any one of the four basic stimuli. Sucrose-best and quinine-best fibers are rather specifically sensitive to sucrose and quinine, respectively, while salt-best and acid-best fibers respond relatively well to HCl and NaCl, respectively. Saccharin, dulcin, and Pb acetate produce a good response in sucrose-best fibers, but quinine-best and salt-best fibers also respond to saccharin. Highly significant positive correlations exist between amounts of responses to sucrose and those to saccharin, dulcin, and Pb acetate, indicating that these substances produce in the monkey a taste quality similar to that produced by sucrose. Compared with chroda tympani fibers of rats, hamsters, and squirrel monkeys, macaque monkey taste fibers are more narrowly tuned to one of the four basic taste stimuli and more highly developed in sensitivity to various sweet-tasting substances. Also LiCl and NaCl are more effective stimuli for gustatory receptors in macaque monkeys than NH4Cl and KCl. This contrasts with a higher sensitivity to KCl and NH4Cl than to NaCl in chorda tympani fibers of squirrel monkeys.

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium.

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.

Effects of artificial sweeteners on insulin release and cationic fluxes in rat pancreatic islets.

Beta-L-glucose pentaacetate, but not alpha-D-galactose pentaacetate, was recently reported to taste bitter and to stimulate insulin release. This finding led, in the present study, to the investigation of the effects of both bitter and non-bitter artificial sweeteners on insulin release and cationic fluxes in isolated rat pancreatic islets. Sodium saccharin (1.0-10.0 mM), sodium cyclamate (5.0-10.0 mM), stevioside (1.0 mM) and acesulfame-K (1.0-15.0 mM), all of which display a bitter taste, augmented insulin release from islets incubated in the presence of 7.0 mM D-glucose. In contrast, aspartame (1.0-10.0 mM), which is devoid of bitter taste, failed to affect insulin secretion. A positive secretory response to acesulfame-K was still observed when the extracellular K+ concentration was adjusted to the same value as that in control media. No major changes in 86Rb and 45Ca outflow from pre-labelled perifused islets could be attributed to the saccharin, cyclamic or acesulfame anions. It is proposed that the insulinotropic action of some artificial sweeteners and, possibly, that of selected hexose pentaacetate esters may require G-protein-coupled receptors similar to those operative in the recognition of bitter compounds by taste buds.

Modulation of sweet taste by umami compounds via sweet taste receptor subunit hT1R2.

Although the five basic taste qualities-sweet, sour, bitter, salty and umami-can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5'-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.

Aspartame: effects on learning, behavior, and mood.

The effect of aspartame on the learning, behavior, and mood of children was evaluated in two experiments. After an overnight fast and a standard breakfast, 20 healthy 9- to 10-year-old children were given the treatments in a double-blind crossover design at 10:30 AM. Lunch was served at 12:00 noon. In experiment 1, the treatment consisted of an ice slurry of strawberry Kool-Aid containing 1.75 g/kg of carbohydrate (polycose) plus either aspartame (34 mg/kg) or the equivalent sweetness as sodium cyclamate and amino acids as alanine. In experiment 2, the treatment consisted of a drink of cold unsweetened strawberry Kool-Aid, containing either 1.75 g/kg of sucrose or 9.7 mg/kg of aspartame. Measures of associative learning, arithmetic calculation, activity level, social interaction, and mood were unaffected by treatment in experiment 1. In experiment 2, the only significant treatment effect was that on the frequency of minor and gross motor behaviors, which were less frequent after the consumption of sucrose than after aspartame. Thus, the effect of aspartame on the short-term behavior of healthy 9- to 10-year-old children appears to be related to its absence of metabolic consequences rather than to its amino acid composition and putative neurochemical impact.

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium.

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.