Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli.Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity.Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints.We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm.Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor.As a rat's tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser.The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever.Each disruption of the laser was counted as a lick.Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy.Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness.All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue.The technology is capable of generating multiple concentration-response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.

Transient receptor potential M5 (TRPM5), a monovalent cation channel, is primarily activated by increases in intracellular calcium. However, we found unexpectedly that allyl isothiocyanate (AITC) and structural analogs triggered a membrane potential and calcium dye responses in TRPM5-HEK cells (AITC EC₅₀ = 9.0 ± 2.4 μM, n = 5). Although AITC and its analogs were more potent on transient receptor potential A1 (TRPA1)-HEK cells (AITC EC₅₀ = 0.23 ± 0.03 μM, n = 4), the rank order potency of these compounds were similar for TRPM5- and TRPA1-HEK cells. No response to these compounds was seen in parental HEK cells, TRPM5-CHO cells, and TRPM4b-, TRPM8-, or TRPV1-transfected HEK cells. An AITC-evoked current in TRPM5-HEK cells was confirmed in whole-cell voltage clamp recording. AITC elicited an intracellular calcium increase that was not dependent on phorpholipase C(β)₂ (PLC(β)₂) activation but was dependent on extracellular calcium concentration. TRPA1 mRNA was upregulated fourfold in TRPM5-HEK cells compared with parental cells. In contrast, TRPA1 was not upregulated in HEK cells transfected in a similar manner with TRPV1 or TRPM8 genes. The AITC response was blocked by a TRPA1 inhibitor and reduced by a TRPM5 inhibitor and by targeted TRPA1 siRNA. These results suggest that TRPM5 may play a role in upregulating endogenous expression of TRPA1, that TRPA1 activation may be an additional trigger for co-expressed calcium-dependent ion channels such as TRPM5, and that TRPM5 may amplify responses to TRPA1 ligands.

Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic β-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC₅₀ = 12 μM) and murine TRPM5 (IC₅₀ = 30 μM) heterologously expressed in HEK293 cells, but had no effect (up to 100 μM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)⁻ mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.

Chemosensory signaling by the tongue is a primary determinant of ingestive behavior and is mediated by specific interactions between tastant molecules and G protein-coupled and ion channel receptors. The functional relationship between tastant and receptor should be amenable to pharmacologic methods and manipulation. We have performed a pharmacologic characterization of the taste-directed licking of mice presented with solutions of capsaicin and other transient receptor potential vanilloid-1 (TRPV1) agonists using a brief access taste aversion assay. Dose-response functions for lick-rate suppression were established for capsaicin (EC50 = 0.5 μM), piperine (EC50 = 2 μM), and resiniferatoxin (EC50 = 0.02 μM). Little or no effect on lick rate was observed in response to the full TRPV1 agonist olvanil. Capsaicin lick rates of wild-type and transient receptor potential melastatin-5 (TRPM5) knockout mice were equivalent, indicating that TRPM5, a critical component of aversive signaling for many bitter tastants, did not contribute to the capsaicin taste response. The selective TRPV1 antagonists N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (10 μM) and (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (AMG9810) (10 μM) effectively blocked capsaicin- and piperine-mediated lick suppression. However, (E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)-N-phenylprop-2-enamide (SB 366791) and capsazepine, also TRPV1 antagonists, were without effect at test concentrations of up to 30 and 100 μM, respectively. Our results demonstrate that TRPV1-mediated oral aversiveness presents a pharmacologic profile differing from what has been reported previously for TRPV1 pain signaling and, furthermore, that aversive tastes can be evaluated and controlled pharmacologically.

Many orally administered pharmaceuticals are regarded by humans as aversive, most often described as 'bitter'. Taste aversiveness often leads to patient noncompliance and reduced treatment effectiveness. 'Bitter' taste is mediated by T2R G-protein coupled receptors through a peripheral signaling pathway critically dependent upon function of the TRPM5 ion channel. The brief-access taste aversion (BATA) assay operationally defines aversive taste as suppression of the rate at which a rodent licks from sipper tubes that deliver tastant solutions or suspensions. We have used a mouse BATA assay for rapid quantification of oral aversiveness from a set of 20 active pharmaceutical ingredients (APIs). Robust lick-rate dose–response functions were obtained from both C57BL/6J wild type (WT) and C57BL/6J/TRPM5−/− (TRPM5 knockout) mouse strains, generating reliable determinations of potency and relative maximal oral aversiveness for each API. A subset of APIs was also evaluated in a human bitterness assessment test; effective concentrations for half-maximum responses (EC50s) from both the human test and WT mouse BATA were equivalent. Relative to WT potencies, EC50s from TRPM5 knockout mice were right-shifted more than10-fold for most APIs. However, APIs were identified for which EC50s were essentially identical in both mouse strains, indicating a TRPM5-independent alternative aversive pathway. Our results suggest the BATA assay will facilitate formulation strategies and taste assessment of late development-phase APIs.

Thymol is a major component of the spice thyme. It has a distinctive pungent flavor mediated by receptor mechanisms in the nose and tongue in addition to having aversive properties. Using a voltage-sensitive fluorescent dye, we show that thymol specifically activates the human transient receptor potential channel A1 (TRPA1), EC50 12 μM, but not other taste sensory TRP channels (TRPV1 and TRPM5). Electrophysiology measurements further demonstrated that thymol activation was concentration-dependent, reversible, and rapidly desensitized TRPA1. A number of related phenols including 3-tert-butyl-4-hydroxyanisol (BHA), a food preservative, and propofol, an intravenous anesthetic, also selectively activated TRPA1 (EC50 3 and 13 μM, respectively). Phenols with less bulky carbon substitutions adjacent to the phenolic group, such as 2, 6-dimethlyphenol and 2-methlyphenol (o-cresol), were less potent. TRPA1-specific siRNA inhibited thymol activation, confirming the specificity of the response. In addition, thymol activation was blocked by camphor (IC50 200–500 μM) whereas other camphor analogs, e.g. camphorquinone, camphoric acid and limonene were TRPA1 agonists (EC50 0.3, 2, 3 mM, respectively). Our results suggest a role of TRPA1 activation in the reported pungent and aversive properties of some of the pharmaceutically important phenols.

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.