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WO2022192668A1 - Cellules dérivées du tissu du bourgeon du goût humain et leurs utilisations - Google Patents

Cellules dérivées du tissu du bourgeon du goût humain et leurs utilisations Download PDF

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WO2022192668A1
WO2022192668A1 PCT/US2022/019946 US2022019946W WO2022192668A1 WO 2022192668 A1 WO2022192668 A1 WO 2022192668A1 US 2022019946 W US2022019946 W US 2022019946W WO 2022192668 A1 WO2022192668 A1 WO 2022192668A1
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Prior art keywords
taste
htbec
cells
bitter
sweet
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Erik Schwiebert
John STREIFF
Grace SALZER
Nancy E. Rawson
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Monell Chemical Senses Center
Discoverybiomed Inc
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Monell Chemical Senses Center
Discoverybiomed Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0632—Cells of the oral mucosa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • BACKGROUND Mammalian taste cells include mammalian taste receptor-expressing cells that respond to taste stimuli or tastants. However, current taste receptor cells are limited in their uses, as they are typically pooled from several donors and are genetically heterogeneous.
  • pooled cells cannot be used to identify tastants or taste modulators for specific populations, for example, a population of a certain gender, race, ethnicity, and/or age to name a few. Therefore, individual donor-derived taste receptor cell lines, with a homogeneous genetic makeup, and methods of using same are necessary.
  • SUMMARY Provided herein is a method for producing a primary culture of human taste cells.
  • the method comprises comprising (a) isolating one or more human taste bud epithelial cells (hTBECs) from an individual human donor’s taste bud tissue; and (b) culturing the isolated one or more hTBECs in a culture dish or well under conditions for expanding the one or more cells to create a plurality of primary hTBECs, wherein the culture dish or well is coated with an extracellular matrix.
  • the extracellular matrix comprises human fibronectin and gelatin.
  • the ⁇ isolated one or more cells are placed on a ⁇ permeable filter support and expanded to create the primary cell culture.
  • the taste bud tissue comprises tongue epithelial tissue from the donor.
  • the taste cells comprise taste receptor cells.
  • the taste receptors are sweet taste receptors or bitter taste receptors.
  • the method further comprises passaging the cells.
  • the cells are passaged between about two and about fifteen times.
  • the method further comprises enriching the primary cell culture for an hTBEC subtype.
  • enriching comprises immunocapturing the hTBEC subtype and/or dilution cloning with targeted antibodies.
  • the hTBEC subtype is a sweet- responsive taste cell.
  • the immunocapturing comprises: (i) contacting the plurality of primary hTBECs with an antibody that specifically recognizes a cell-surface receptor on the sweet-responsive taste cells; and (ii) isolating the immunocaptured cells.
  • the receptor is TAS1R2 and TAS1R3.
  • the hTBEC subtype is a bitter-responsive taste cell, a sweet-responsive taste cell or a salt-responsive taste cell.
  • Some methods further comprise immortalizing the primary hTBECs.
  • the immortalization is performed using a viral oncogene, for example, SV40.
  • Some methods further comprise cryopreserving the cells.
  • the primary culture is a culture wherein the cells are produced by: (a) isolating one or more human taste bud epithelial cells (hTBECs) from an individual human donor’s taste bud tissue; and (b) culturing the isolated one or more hTBECs in a culture dish or well under conditions for expanding the one or more cells to create a plurality of primary (hTBECs), wherein the culture dish or well is coated with an extracellular matrix.
  • hTBECs human taste bud epithelial cells
  • hTBECs immortalized human taste bud epithelial cells
  • the hTBECs in the population are sweet-responsive taste cells (e.g. hTBECs that express TAS1R2 and/or TAS1R3), salt-responsive cells (e.g., hTBECs that express EnaC and/or CFTR), bitter-responsive taste cells (e.g., hTBECs that express TAS1R1, TAS2R39 and/or TAS2R14), umami-responsive taste cells (e.g., hTBECs that express TAS1R1), or sour-responsive taste cells (e.g., hTBECs that express H+activated channels).
  • sweet-responsive taste cells e.g. hTBECs that express TAS1R2 and/or TAS1R3
  • salt-responsive cells e.g., hTBECs that express EnaC and/or CFTR
  • bitter-responsive taste cells e.g., hTBECs that express TAS1R1, TAS2R39 and/or TAS2R
  • hTBECs immortalized human taste bud epithelial cells derived from an individual donor’s taste bud tissue
  • the population of hTBECS is produced by any of the methods provided herein that comprise an immortalization step.
  • a method for identifying a bitter taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a bitter taste receptor; and b) measuring bitter taste receptor activity, wherein a change in bitter taste receptor activity, as compared to a control, indicates that the test compound modulates bitter taste receptor activity.
  • a method for identifying a bitter taste receptor modulator comprising: a) contacting the hTBEC with a test compound and a bitter tastant; and b) measuring bitter taste receptor activity, wherein a change in bitter taste receptor activity, as compared to bitter taste receptor activity in the presence of the bitter tastant alone, indicates the test compound modulates the activity of the bitter tastant.
  • the test compound inhibits the activity of the bitter tastant on the bitter taste receptor.
  • the bitter taste receptor activity is measured by detecting the level of ATP secreted by the hTBEC into the media supernatant.
  • the level of secreted ATP is detected using a luciferase-luciferin system.
  • the bitter taster receptor activity is measured by detecting the level of an intracellular second messenger in the hTBEC.
  • the intracellular second messenger is intracellular calcium. Also provided is a method for identifying a sweet taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a sweet taste receptor; b) measuring sweet taste receptor activity, wherein an increase in sweet taste receptor activity, as compared to a control, indicates that the test compound is a sweet tastant.
  • the sweet taste receptor activity is measured by measured by detecting the level of ATP secreted by the hTBEC into the media supernatant. In some methods, the level of ATP secreted by the hTBEC into the media supernatant is detected using a luciferase-luciferin system. In some methods, the sweet taste receptor activity is measured by detecting the level of an intracellular second messenger in the hTBEC. In some methods, the intracellular second messenger is intracellular calcium.
  • a method for identifying a salt taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a salt taste receptor; b) measuring salt taste receptor activity, wherein an increase in salt taste receptor activity, as compared to a control, indicates that the test compound is a salt tastant.
  • the salt taste receptor activity is measured by detecting the level of an intracellular second messenger in the hTBEC.
  • the intracellular second messenger is intracellular calcium.
  • the salt taste receptor activity is measured by measured by detecting the level of ATP secreted by the hTBEC into the media supernatant.
  • the level of ATP secreted by the hTBEC into the media supernatant is detected using a luciferase-luciferin system.
  • a population of hTBEC cells is contacted with the test compound, wherein the population of hTBEC cells is a population of any of the cells described herein.
  • FIGS.1A-B show that primary human taste cell-based screening led to identification of 6- methylflavone as a suppressor of TAF-induced taste cell activity.
  • TAF activates a limited number of bitter taste receptors.
  • HEK293 cells transfected with human TAS2Rs, along with G ⁇ 16-gust44 were assayed for their responses to 0.1 mM (A, left) and 1 mM TAF (A, right). Quantitative analysis of responses were presented. Data are percentage change (mean ⁇ SD) in fluorescence (peak RFU ⁇ baseline RFU, denoted ⁇ F) from baseline fluorescence (denoted F) averaged from triplicates. The experiment was replicated one more time.
  • FIG. 3 shows that 6-methylflavone and its analogs block the responses of TAS2R39 to TAF and denatonium.
  • HEK293 cells were transfected with TAS2R39 and G ⁇ 16-gust44 and assayed for responses to 6-methylflavone, its analogs, and other putative blockers identified in taste cell-based screening (FIG. 1).
  • Data are averaged from triplicates. The experiment was replicated one more time.
  • FIG.4 shows perceived bitterness of TAF with and without 1 mM 6-methylflavone as pre- rinse and admixture in two subjects.
  • the top 2 graphs are ratings by one subject.
  • the bottom 2 graphs are ratings by another individual subject.
  • the graphs on the left are Session 1 and the graphs on the right are Session 2.
  • TAF was dosed at 0.5 mg/mL.6-methylflavone was dosed at 1 mM.
  • FIG. 5 shows perceived bitterness of TAF with and without 1 mM 6-Methylflavone as pre-rinse and admixture in all subjects.
  • the graph on the top shows the average perceived bitter intensity of 6-methylflavone (green bar), TAF (blue bar) and TAF with 6-methylflavone as pre- rinse and admixture (orange bar) for all 16 subjects with two replications.
  • TAF was dosed at 0.5 mg/mL and 6-methylflavone was dosed at 1 mM.
  • the solution volume placed in the mouth was 10 ml.
  • the error bars are standard errors of the mean.
  • FIGS. 6A-D shows that 6-methylflavone does not block the responses of TAS2R1, TAS2R8, TAS2R14 to TAF and its analogs do not block the responses of TAS2R1 to TAF.
  • HEK293 cells were transfected with TAS2R1, and G ⁇ 16-gust44 and assayed for responses to 6- methylflavone, its analogs, and other putative blockers identified in taste cell-based screening . Bar graph showing the responses of TAS2R1 to TAF (1 mM) in the presence of 6-methylflavone, its analogs, and other putative blockers (0.1 mM). None of them blocked the response of TAS2R1 to TAF.
  • B-D HEK293 cells were transfected with TAS2R1,TAS2R8, TAS2R14 and G ⁇ 16-gust44 and assayed for responses to 6-methylflavone.
  • FIG. 7 shows human sensory testing ratings for donors, for different tastants. Arrows reflect the specific individual donor 00H (also referred to as Donor H), which responded the most vigorously to the bitter ligands denatonium benzoate (DB), phenylthiocarbamate (PTC) and quinine. Interestingly, this donor also responded well to sucrose and salt and also rated water as modestly bitter.
  • Donor H also referred to as Donor H
  • Donor H was a younger donor, and 11 different primary cultures, out of twelve possible wells, were isolated from donor H taste bud tissue.
  • FIG.8 is a schematic illustrating how individual human taste buds are processed and seeded into primary culture to obtain invididual, donor-derived hTBEC cultures.
  • FIG. 9 is a schematic illustrating a magnetic immunocapture method for enrichment of specific hTBEC cultures.
  • a magnetic immunocapture method used antibodies to predicted external epitopes of the sweet taste receptor proteins TAS1R2 and TAS1R3 to enrich the cultures in sweet-responsive cells.
  • FIG. 10 shows the results of quantitative RT-PCR (qRT-PCR) for TAS1R3 sweet taste receptor.
  • FIG. 11 shows immunocytochemistry confirmation of a sweet-responsive clonal cell line.
  • the 56.SV.18 sweet clonal cell line was tested via immunocytochemical analysis for key sweet taste transduction targets.
  • FIG. 12 shows immunocytochemistry confirmation of a sweet-responsive clonal cell line.
  • T1R2 staining was much stronger and uniform across most, if not all, of the cells in each field.
  • Staining for Kir6.1 and Kir6.2, inwardly rectifying potassium channels also implicated in glucose sensing was also robust and some co-localization was also observed.
  • Staining for the sulfonylurea receptor (SUR), for GLUT glucose transporters and for the insulin receptor was also observed in this sweet clonal cell line.
  • SUR sulfonylurea receptor
  • FIG.13 is a schematic of of taste signaling in the context of an hTBEC cell, as well as the methods used to perform dual assays (for example, detection of intracellular calcium and detection of secreted ATP) measuring cell signaling at different points in the taste signal transduction cascade.
  • FIG. 14A is an exemplary 96-well load plate used in the methods described herein.
  • FIG. 14B is is an exemplary 384-well load plate used in the methods described herein.
  • FIG.15 is an exemplary graph showing inhibition of luciferase by sodium salt. Equation 1 is used to correct experimental data.
  • FIG.16A is an exemplary graph showing an uncorrected sodium gluconate ATP response.
  • FIG. 14A is an exemplary 96-well load plate used in the methods described herein.
  • FIG. 14B is is an exemplary 384-well load plate used in the methods described herein.
  • FIG.15 is an exemplary graph showing inhibition of luciferase by sodium salt. Equation 1 is used
  • FIG. 16B is an exemplary graph showing a sodium gluconate ATP response corrected with Equation 1.
  • FIG. 17 shows an exemplary ATP transmitter secretion response to sweet ligands in primary cells and a sweet clonal cell line.
  • the horizontal dashed box shows more modest responses to sweet ligands above vehicle in the high ⁇ M to low mM range. These effects are TAS1R2.TAS1R3 receptor driven.
  • the vertical dashed box shows stronger responses to sweet ligands above vehicle in the mM range. These are effects are likely driven by both TAS1R2.Tas1R3 receptors and GLUT transporters.
  • FIG. 18 shows exemplary responses in a sweet clonal cell line in the ATP transmitter detection assay to sweet ligands.
  • FIG. 19 shows concentration response curves showing stimulation by sweet ligands (sucralose and saccharin) in the hTBEC calcium assay.
  • FIG.20 shows concentration response curves showing stimulation by artificial sweeteners (advantame, neotame and alitame) in the hTBEC calcium assay.
  • FIG.21 shows concentration response curves showing stimulation by artificial sweeteners (aspartame and acesulfame K) in the hTBEC calcium assay.
  • FIG.22 shows a concentration response curve for talin, a protein sweetener, in the hTBEC calcium assay.
  • FIG. 23 shows an exemplary real-time ATP secretion detection reagent signal elicited by rebaudioside A, and blocked by known taste blockers in sweet-responsive hTBEC cells. 1 mM Reb A stimulated a robust real-time ATP secretion signal.
  • Known taste blockers, Compound A and Compound C attenuated the response and implicated taste receptors.
  • Senomyx BB68 is a bitter blocker mix that also blocks sweet taste receptors and, as such, also attenuated this Reb A assay response.
  • FIG. 24 shows the concentration-dependent increase in ATP transmitter secretion with increasing salt concentrations from a baseline of 22.5 mM sodium salt (sodium gluconate, NaGlu or sodium chloride, NaCl) in a saliva-mimicking buffer. These data also show equivalence in salt sensitivity and salt responsiveness in the primary cultures of hTBEC D.1.PFS cells versus the SV40Tt immortalized D.1.PFS cells.
  • FIG. 25 shows the concentration-dependent increase in calcium with increasing salt concentrations.
  • FIG. 26 shows that sensitivity to lower salt stimulus is present in both hTBEC D.1.PFS primary and SV40Tt immortalized cultures in the ATP transmitter secretion detection assay.
  • FIG. 27 shows amiloride inhibition of salt responses in a real-time cell calcium mobilization assay. It is possible that epithelial sodium channels (EnaCs) are involved in salt taste transduction. As little as 25 ⁇ M amiloride markedly attenuated the response.
  • EndaCs epithelial sodium channels
  • FIG. 28 shows CFTR inhibitor 172 inhibition of salt responses in a real-time cell calcium mobilization assay. It is possible that CTFR is involved in salt taste transduction. As little as 10 ⁇ M CTFR inhibitor 172 markedly attenuated the response to lower salt stimulatory concentrations and partially attenuated the response to higher salt boluses.
  • FIG. 29 shows that TRP channel inhibitors attenuate salt responses in a real-time cell calcium mobilization assay and implicate TRP channel-driven calcium entry in salt taste transduction.
  • HC 030031 is a selective TRPA1 channel inhibitor.
  • AMG-333 is a selective TRPM8 channel inhibitor.
  • FIG. 30 shows qRT-PCR studies to quantity TAS2R14 (T2R14) mRNA expression.
  • FIG. 31 shows TAS2R14 mRNA expression via qRT-PCR across all hTBEC cultures. * ⁇ High copy number based on CT values.
  • FIG. 32 shows TAS2R21 (T2R21) mRNA expression via qRT-PCR across all hTBEC cultures. * High copy number based on CT values.
  • FIG. 33 shows TAS2R39 (T2R39) mRNA expression via qRT-PCR across all hTBEC cultures. These results showed that donor H cultures are still prominent; however, a larger subset of individual cultures also showed significant expression.
  • FIG. 34 shows TAS2R38 (T2R38) mRNA expression via qRT-PCR across all hTBEC cultures. There was no amplification in most cases, but TAS2R38 was amplified to some extent in Donor H cell lines.
  • FIG. 35 shows TRPV1 mRNA expression via qRT-PCR across all hTBEC cultures.
  • FIG. 36 shows TRPML3 mRNA expression via qRT-PCR across all hTBEC cultures. TRPML3 and TRPV1 mRNA expression tracked with the TAS2R expression signals in Donor H- derived cultures. TRPML3 mRNA expression displays a large signal in H.4.TC and H.5.TC cultures.
  • FIG. 37 shows TRPA1 mRNA expression via qRT-PCR across all hTBEC cultures. TRPA1 expression did not track with previous results for Donor H-derived cultures.
  • FIG. 38 shows ATP secretion detection assay response to tenofovir alafenamide fumarate (TAF)(left panel) and quinine (right panel) in bitter-responsive primary vs. immortal H.5.TC hTBEC cultures.
  • TAF tenofovir alafenamide fumarate
  • quinine right panel
  • FIG. 39 shows ATP secretion detection assay response to tenofovir alafenamide fumarate (TAF) (right panel) and other anti-viral drugs and denatonium in bitter-responsive primary (left panel) vs. immortal H.5.TC hBEC cultures (middle panel).
  • FIG. 40 is schematic of a medium-throughput screening platform with bioassay steps to discover, validate and profile bitter blockers using the cell lines and methods provided herein.
  • FIG. 41 shows qRT-PCR amplification of phospholipase C beta 2 from bitter-responsive donor hTBEC cultures.
  • FIG. 42 shows qRT-PCR amplification of phospholipase C beta 3 from bitter-responsive donor hTBEC cultures.
  • FIG. 43 shows qRT-PCR amplification of phospholipase C beta 4 from bitter-responsive donor hTBEC cultures.
  • FIG. 44 shows qRT-PCR amplification of (T2R28) from bitter-responsive donor hTBEC cultures.
  • FIG.45 shows qRT-PCR amplification of TASR10 (T2R10) from bitter-responsive donor hTBEC cultures.
  • FIG. 46 shows qRT-PCR amplification of TRPM5 from bitter-responsive donor hTBEC cultures.
  • FIG. 47 shows qRT-PCR amplification of TRPC1 from bitter-responsive donor hTBEC cultures.
  • FIG.48 shows qRT-PCR amplification of CALHM1 from bitter-responsive donor hTBEC cultures.
  • can and “can be,” and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.
  • the terms “optional” and “optionally” mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present as well as instances where it does not occur or is not present.
  • the use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise.
  • bitter taste evolved to indicate when orally sampled substances contain chemicals that most wish to avoid. Although one can learn to enjoy low level bitterness in certain foods, such as in coffee and beer, high intensity bitterness is universally aversive and can induce nausea. Common plant alkaloids are often bitter tasting and at high levels can be toxic or even fatal. At low levels, however, many of these bitter stimuli may have medicinal properties, for example, the cardiac glycosides. Similarly, the majority of human-made active pharmaceutical ingredients (APIs) used to treat diseases also taste bitter. The bitterness of APIs leads to compliance problems for oral intake of such medicines.
  • APIs active pharmaceutical ingredients
  • the pediatric population is often extremely sensitive to the adverse taste profile of medicines, and one-third of children with chronic illnesses refuse to take medicines due to strong bitterness or other “bad” sensations elicited by the APIs.
  • Children have difficulty swallowing capsules and when presented with liquid formulations often reject oral medications due to their intense bitterness.
  • Effective strategies to identify methods, reagents, and tools to block bitterness remain elusive.
  • identification of the responsible bitter receptors and discovery of antagonists for those receptors can provide a method to effectively block perceived bitterness.
  • TAS2R39 and TAS2R1 are the main T2R receptors for TAF observed via heterologously expressing specific TAS2R receptors into HEK-293 cells.
  • 6-methylflavone blocked the responses of TAS2R39 to TAF.
  • the cells are human taste bud epithelial cell cultures (hTBECs) or human taste bud tissue-derived cell cultures derived from individual donors.
  • Individual donor-derived hTBEC cultures allows for analysis based on the specific genetics of the individual donor. Obtaining and using a number of unpooled, individual donor- derived hTBEC cultures captures human subject or donor diversity of heterogeneity (female and male subjects, subjects of different race and ethnicity, younger and older donors, etc.). It is also possible to isolate tissue and process it into primary cultures from subjects that have ageusia (loss of sense of taste) and other conditions relevant to taste. In some instances younger donors (aged 18-29) may be useful for these practices and methods.
  • test compounds that modulate bitter taste receptors, sweet taste receptors or salt taste receptors can be identified.
  • the cell cultures and screening assays described herein can be combined with individual human psychophysical testing to identify bitter, sweet or salt tastants as well as test compounds that inhibit or decrease the activity of a taste receptor, for example, a bitter taste receptor.
  • hTBECs Human taste bud epithelial cells
  • a method for producing a primary culture of human taste cells comprising: (a) isolating one or more human taste bud epithelial cells (hTBECs) from an individual human donor’s taste bud tissue; and (b) culturing the isolated one or more hTBECs in a culture dish or well under conditions for expanding the one or more cells to create a plurality of primary hTBECs, wherein the culture dish or well is coated with an extracellular matrix.
  • the extracellular matrix comprises human fibronectin and gelatin.
  • the taste bud tissue comprises human tongue epithelial tissue from the donor.
  • the taste cells comprise human taste receptor cells. In some embodiments, the taste cells respond to at least one taste stimulus. In some embodiments, the human taste receptors are sweet taste receptors or bitter taste receptors. In some embodiments, the method further comprises passaging the cells. In some embodiments, the cells are passaged between about two and about fifteen times.
  • an individual, or a subject is a human. The individual can be an adult subject of any age, for example, an adult at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. Pediatric subjects include subjects ranging in age from birth to eighteen years of age.
  • pediatric subjects of less than about 10 years of age, five years of age, two years of age, or one year of age are also included as individuals.
  • the individual can be male or female.
  • the individual can be an individual that has a disease, for example, cancer, HIV, diabetes, high blood pressure etc., that requires oral administration of a medication.
  • the phrase primary in the context of a primary cell, refers to a cell that has not been transformed or immortalized.
  • Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times). In some cases, the primary cells are adapted to in vitro culture conditions.
  • the primary cells are stimulated or activated.
  • the isolated one or more cells are placed on a permeable filter support, for example, on extracellular matrix-coated tissue culture plastic and expanded to create the primary cell culture.
  • the lifetime of any of the primary cultures described herein can be extended by one, two, three, four or more passages, by growing the primary hTBECs cells on a permeable filter support.
  • the cells can be grown in tissue culture plates that allow visualization of the cells during expansion.
  • the culture methods provided are used produce a culture of primary hTBECs having a viability of at least about 80%, 85%, 90%, 95% or higher, after culturing the cells for at least 3, 5, or 10 days.
  • the culture methods provided herein are used to produce a culture of primary hTBECs having a viability of at least about 80%, 85%, 90%, 95% or higher after one or more enrichment steps described herein.
  • Isolation of primary hTBECs from an individual’s taste bud tissue can comprise incubation of the taste bud tissue with proteolytic enzymes (e.g., elastase, collagenase, Pronase E etc.) for a time period such that a desired duration of cultivation and cell viability is achieved for the primary hTBECs.
  • the taste bud tissue is incubated with proteolytic enzymes for about 30 to about 90 minutes. Any method known in the art may be used to assess cell viability.
  • hTBEC cells are isolated from the taste bud tissue, the cells are seeded or placed into an appropriate growth vessel (e.g., a well, dish or plate coated with an extracellular matrix) and cultured. Cell culture is performed in an appropriate environment with appropriate CO 2 concentration and appropriate humidity, as is apparent to the skilled person. For example, depending on the chosen medium, a CO2 concentration of about 5% is appropriate. Cells are usually cultured under conditions of high humidity, for example, about 95%.
  • An appropriate temperature for culturing the primary hTBECs is any temperature that allows for cell growth and cellular processes at the desired rate, for example from about 18° C to about 37° C.
  • Culture conditions at room temperature (18-22° C) and at about 37° C e.g., from about 35 o C to 39 o C are also contemplated.
  • Culture methods at physiological temperature, (e.g., about 37° C) can also be used. It is understood that, when culturing cells for example, at about 37° C, temperature will change over time somewhat to oscillate at about 37° C, on average (e.g., ⁇ 0.5 or 1° C), depending on the cell culture system.
  • One of skill in the art can determine when medium replacement should occur during cell culture by, for example, observing cell growth and/or cell health.
  • Medium replacement can occur, for example, between about 24-72 hours after seeding.
  • Subsequent medium replacement can be performed at intervals of one, two, three, four, five, six, seven days or greater, depending on culture conditions, chosen medium, concentration of antibiotics, and growth rate, as readily determined by one of skill in the art.
  • Any of the hTBECs or primary cultures of hTBECs described herein can be transfected with a nucleic acid sequence encoding a polypeptide of interest.
  • any method known in the art for transfecting cells with a nucleic acid can be used to transfect the primary hTBECs described herein.
  • a nucleic acid for example, naked DNA, a vector or plasmid comprising a nucleic acid encoding a polypeptide, or a viral vector a nucleic acid encoding a polypeptide
  • one or more cells of the plurality of primary hTBEC cells can be immortalized using methods known in the art.
  • the cells can be transfected with a viral oncogene, for example, SV40 and/or a human telomerase (hTERT).
  • the method further comprising enriching or purifying the primary cell culture for an hTBEC subtype.
  • enriching comprises immunocapturing the hTBEC subtype and/or dilution cloning with targeted antibodies.
  • the hTBEC subtype is a sweet-responsive taste cell.
  • the immunocapturing comprises: (i) contacting the plurality of primary hTBECs with an antibody that specifically recognizes an epitope of a cell-surface receptor on the sweet-responsive taste cells; and (ii) isolating the immunocaptured cells.
  • the receptor is TAS1R2 and TAS1R3.
  • the hTBEC subtype is a bitter-responsive taste cell. See, for example, Behrens et al. “Immunohistochemical detection of TAS2R38 protein in human taste cells,” PLos One 7(7):e40304 (2012).
  • the cells for example, bitter-responsive cells, are encriched for Taste 1 Receptor Member 1 (TAS1R1), Taste 2 Receptor Member 39 (TAS2R39) and/or Taste 2 Receptor Member 14 (TAS2R14).
  • the cells for example, sweet-responsive cells, are enriched for Taste 1 Receptor Member 2 (TAS1R2) and/or Taste 1 Receptor Member 3 (TAS1R3).
  • the cells for example, umami-responsive cells, are enriched for TAS1R1.
  • the cells for example, salty-responsive cells, are enriched for the epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR).
  • the cells for example, sour-responsive cells, are enriched for H+-activated channels.
  • the method further comprises cryopreserving the hTBECs.
  • a population of hTBECs enriched or purified from the primary cell culture can comprise at least about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% hTBECs comprising a taste receptor of interest, for example, a sweet taste receptor, a bitter taste receptor, a salt taste receptor, an umami taste receptor or a sour taste receptor. Any of the cells or populations of cells described herein, including enriched cells or enriched populations of cells, can be used in any of the methods described herein.
  • Cell Cultures and Cell Populations Also provided is a primary culture of hTBECs derived from an individual human donor’s taste bud tissue.
  • the primary culture of hTBECs can be made using any of the methods described herein.
  • the primary culture is produced by (a) isolating one or more human taste bud epithelial cells (hTBECs) from an individual human donor’s taste bud tissue; and (b) culturing the isolated one or more hTBECs in a culture dish or well under conditions for expanding the one or more cells to create a plurality of primary hTBECs, wherein the culture dish or well is coated with an extracellular matrix.
  • the population of hTBEC cells is a population that has been passaged one, two, three, four, five or more times.
  • the population of hTBEC cells is a population that has been passaged fewer than five, four, three or two times.
  • a population of immortalized hTBECs is also provided.
  • the population is a population derived using any of the methods for producing immortalized hTBECs as described herein.
  • the hTBECs can be sweet- responsive taste cells, bitter-responsive taste cells, umami-responsive taste cells, sour-responsive taste cells, or salt-responsive taste cells.
  • Any of the primary hTBEC cell cultures or hTBEC cell populations described herein can be cryopreserved hTBEC cell cultures or cryopreserved hTBEC cell populations.
  • any of the methods and taste cells i.e., hTBECs, described herein, can be used in assays to identify test stimuli or compounds that elicit a response in hTBECs, including tastants, taste modifiers (including stimuli that enhance, suppress, stimulate or inhibit taste, or influence a particular taste quality).
  • the assays described herein can be used to identify and evaluate the taste, taste quality, taste modifying, growth promoting, inhibiting or toxic effects of candidates. Further they may be employed to identify test compounds for treating taste loss, targets for drug development, and nutritional factors needed for maintenance of healthy taste cells.
  • such assays involve the comparison of at least one type of cellular response (for example, ATP secretion from hTBECs or hTBEC intracellular calcium) of a test compound or mixture of test compounds to a standard or control.
  • taste cell response to a test compound of unknown taste quality is compared with that of a known tastant to identify differences or similarities in taste receptor activity.
  • hBEC taste receptor activity can be determined for a known tastant, for example, as described in the Examples, or using other cellular response assays known to the skilled person.
  • the cellular response, for example, taste receptor activity, of the test compound can be determined and compared to taste receptor activity of the same hBEC cells to the known tastant.
  • any of the hTBECs, or populations of hTBECs described herein can be contacted with a test compound to identify a taste receptor modifier or modulator.
  • Such assay methods comprise contacting the hTBECs with the test compound and measuring a cellular response, for example, taste receptor activity. A measurable change in the cellular response, as compared to a control is indicative of a test compound that modulates taste receptor activity.
  • a standard or control stimulus or compound may be a known taste stimulus or tastant of a known taste quality, or a stimulus/compound that is able to elicit a particular effect on taste cells.
  • a bitter taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a bitter taste receptor; and b) measuring bitter taste receptor activity, wherein a change in bitter taste receptor activity, as compared to a control, indicates that the test compound modulates bitter taste receptor activity.
  • Bitter taster receptor activity can be modulated by increasing or decrease bitter taste receptor activity.
  • Also provided is a method for identifying a sweet taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a sweet taste receptor; b) measuring sweet taste receptor activity, wherein an increase in sweet taste receptor activity, as compared to a control, indicates that the test compound is a sweet tastant.
  • a method for identifying a salt taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a salt taste receptor; and b) measuring salt taste receptor activity, wherein an increase in salt taste receptor activity, as compared to a control, indicates that the test compound is a salt tastant.
  • the method can optionally comprise contacting the cells with an ENaC inhibitor and/or CFTR inhibitor, for example, for about 10 seconds to about 90 seconds, prior to salt stimulation (i.e., contacting the cells with a salt tastant and/or test compound).
  • an umami taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses an umami taste receptor; and b) measuring umami taste receptor activity, wherein an increase in umami taste receptor activity, as compared to a control, indicates that the test compound is an umami tastant.
  • a method for identifying a sour taste receptor modulator comprising: a) contacting a hTBEC with a test compound, wherein the hTBEC is derived from an individual donor’s human taste bud tissue, and expresses a sour taste receptor; and b) measuring sour taste receptor activity, wherein an increase in sour taste receptor activity, as compared to a control, indicates that the test compound is a sour tastant.
  • an increase in taste receptor activity can be an increase of about 5, 10, 15, 20, 25, 30, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % or greater.
  • An increase can also be a 1.5 fold, 2.0 fold, 2.5 fold, 3.0 fold, 3.5 fold, 4.0 fold, 4.5 fold, or 5.0-fold increase or greater.
  • a decrease in taste receptor activity can be a decrease of about 5, 10, 15, 20, 25, 30, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 %.
  • Screening assays for Modulators of Known Tastants any of the hTBECs, or populations of hTBECs described herein can be contacted with a test compound and a known tastant to identify a taste receptor modulator.
  • Such assay methods comprise contacting the hTBECs with the test compound and the known tastant and measuring a cellular response, for example, taste receptor activity.
  • a measurable change in taste receptor activity, as compared to taste receptor activity in the presence of the known tastant alone is indicative of a test compound that modulates taste receptor activity.
  • These methods can be used to identify a sweet taste receptor modulator, a salt taste receptor modulator, or a bitter taste receptor modulator.
  • taste cell responses to a known stimulus e.g. known tastant
  • a test compound e.g. taste modifier
  • Comparison may show that the response of the known tastant is increased, decreased, delayed, prolonged, or unaffected in the presence of the test compound.
  • a test compound producing an increase in the magnitude of the cellular response to the stimulus or an increase in the frequency of cells responding to the known stimulus shows that the test compound is useful for enhancing the intensity of the stimulus (e.g. tastant), and can be added top certain products, such as food products, to enhance the given stimulus (e.g. taste stimulus/flavor).
  • a test compound that produces a response of shorter latency or longer response duration indicates an increased taste intensity. If a test compound produces increased taste intensity, or a faster or more prolonged taste perception, the test compound can be added to food products to enhance their taste.
  • a test compound that results in inhibition of a response, decreased magnitude of response, slower latency, or shorter response duration to the target stimulus is identified as useful in masking or block a target stimulus, for example, a bitter tastant. If results show no difference in the response to the target stimulus in the presence versus absence of the test compound, the compound is not a modifier for the target stimulus.
  • a method for identifying a bitter taste receptor modulator comprising: a) contacting the hTBEC with a test compound and a bitter tastant; and b) measuring bitter taste receptor activity, wherein a change in bitter taste receptor activity, as compared to bitter taste receptor activity in the presence of the bitter tastant alone, indicates the test compound modulates the activity of the bitter tastant.
  • the test compound modulates bitter taste receptor activity to decrease or inhibit the bitter taste receptor activity of the bitter tastant. This decrease can be a decrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90% or greater.
  • a method for identifying a sweet taste receptor modulator comprising: a) contacting the hTBEC with a test compound and a sweet tastant; and b) measuring sweet taste receptor activity, wherein a change in sweet taste receptor activity, as compared to sweet taste receptor activity in the presence of the sweet tastant alone, indicates the test compound modulates the activity of the sweet tastant.
  • the test compound modulates sweet taste receptor activity to decrease or inhibit the sweet taste receptor activity of the sweet tastant. This decrease can be a decrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90% or greater.
  • the test compound modulates sweet taste receptor activity to increase sweet taste receptor activity of the sweet tastant.
  • This increase can be an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater.
  • a method for identifying a umami taste receptor modulator comprising: a) contacting the hTBEC with a test compound and an umami tastant; and b) measuring umami taste receptor activity, wherein a change in umami taste receptor activity, as compared to umami taste receptor activity in the presence of the umami tastant alone, indicates the test compound modulates the activity of the umami tastant.
  • the test compound modulates umami taste receptor activity to decrease or inhibit the umami taste receptor activity of the umami tastant. This decrease can be a decrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90% or greater. In some methods, the test compound modulates umami taste receptor activity to increase umami taste receptor activity of the umami tastant. This increase can be an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater.
  • a method for identifying a sour taste receptor modulator comprising: a) contacting the hTBEC with a test compound and a sour tastant; and b) measuring sour taste receptor activity, wherein a change in sour taste receptor activity, as compared to sour taste receptor activity in the presence of the sour tastant alone, indicates the test compound modulates the activity of the sour tastant.
  • the test compound modulates sour taste receptor activity to decrease or inhibit the sour taste receptor activity of the sour tastant. This decrease can be a decrease of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90% or greater.
  • the test compound modulates sour taste receptor activity to increase sour taste receptor activity of the sour tastant.
  • This increase can be an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater.
  • a change in cellular response for example, a change in taste receptor activity
  • detection methods well-known in the art for example, by detecting the level of an intracellular second messenger in the hTBEC(s), for example, intracellular calcium. See, for example, Toda et al.
  • the level of ATP secreted by the hTBEC(s) into the media supernatant is measured using a luciferin-luciferase assay system. See, for example, Finger et al., “ATP signaling is crucial for communication from taste buds to gustatory nerves,” Science 310, 1495–1499 (2005).
  • Any of the methods provided herein can further comprise testing the effects of any taste receptor modulator identified herein in vivo, in a human subject.
  • the taste receptor modulator can be orally administered to the subject to determine the in vivo effects of the modulator.
  • the method can further comprise human psychophysical testing to obtain a subject’s comments on the effects of the taste modulator.
  • the taste modulator can be administered alone or in combination with a known tastant to the subject.
  • a bitter taste modulator that decreases bitter taste receptor activity in the presence of a known bitter tastant in vitro, can be orally administered to the subject with and without the bitter tastant to determine if the bitter taste modulator decreases the bitterness of the bitter tastant in vivo, as assessed, for example, via psychophysical tests on the subject.
  • Comparison of hTBEC Cells from Different Sources The response to a given stimulus (e.g. known tastant, known tastant and taste modifier) of hBECs derived from different sources, for example, from different individuals, can be compared.
  • comparisons can be used to determine whether the cellular responses to a stimulus or tastant differ between different sources of taste cells that are selected according to different cultures or individuals, or in correlation with differences in age, genetic composition, metabolic or nutritional status, disease state, medication use, and therapeutic treatment.
  • a compound that acts as a bitter taste modulator by decreasing bitter taste receptor activity in hTBECs isolated from a male individual can be compared with the effect of the bitter taste modulator on hTBECs isolated from an adult female individual. If the effects of the bitter taste modulant are similar in the hTBECs isolated from the male individual and the hTBECs isolated from the female individual, the bitter taste receptor modulator could be used as a bitter receptor modulator in males and females.
  • bitter taste modulator can be used to modify bitter taster receptor activity in males, but not in females.
  • a compound that acts as a bitter taste modulator by decreasing bitter taste receptor activity in hTBECs isolated from an elderly individual can be compared with the effect of the bitter taste modulator on hTBECs isolated from a pediatric individual. If the effects of are similar in the hTBECs isolated from the elderly individual and the hTBECs isolated from the pediatric individual, the identified bitter taste receptor could be used as a bitter receptor modulator in elderly individuals and pediatric individuals.
  • bitter taste modulator can be used to modify bitter taster receptor activity in elderly subject, but not in pediatric subjects.
  • the identified differences allow development of improved taste systems targeting flavors and flavor compositions, e.g. for food products, which are tailored to the needs of specific populations or individuals. For example, reduced responsiveness to a particular stimulus in cells derived from elderly versus young subjects would identify a necessary increase/decrease in the stimulus concentration to improve the taste quality for consumers depending on the age of the consumer group for which the product is intended.
  • TAS2R bitter taste receptors are expressed in taste receptor cells in the oral cavity and signal bitter-tasting stimuli upon entry into the mouth (Bachmanov and Beauchamp, “Taste receptor genes,” Annu Rev Nutr 27:389-414 (2007)). Although a few blockers for specific bitter receptors have been identified, the efficacy of these blockers to decrease bitter taste perception is limited. This may be partly due to the complicated interactions of bitter-taste stimuli and the TAS2Rs. A single bitter-tasting compound can activate one or several TAS2Rs.
  • the receptive field of a TAS2R may be narrowly or very broadly tuned to many compounds (Meyerhof et al. “The molecular receptive ranges of human TAS2R bitter taste receptors,” Chem Senses 35:157-170 (2010)). Moreover, there are individual polymorphic differences in bitter taste receptor genes and the receptors for which they code. Given the complexity of the interaction among the T2Rs and bitter taste stimuli, such as APIs, it was reasoned that characterization of the interaction of a target API with all 25 bitter receptors would be necessary to develop specific bitter taste blockers.
  • hTBECs human taste bud epithelial cell cultures
  • TAS2R39 was identified as a critical receptor for TAF, and the data provided herein show that the terpenoid 6-methylflavone blocks TAS2R39 responses to TAF at both the cellular and the TAS2R receptor level. It was further determined that 6-methylflavone reliably and nearly completely blocked TAF-induced bitterness in a subset of the human subjects we tested.
  • Methods Human Taste Bud Epithelial Cell (hTBEC) Platform-driven Screening of Phytochemical Small Molecules All research was conducted according to the principles expressed in the Declaration of Helsinki, and approved by an Institutional Review Board at the University of Pennsylvania. Subjects provided written, informed consent on forms approved by the Institutional Review Board prior to human taste bud tissue collection.
  • hTBECs were grown in a modified BronchiaLife media (Lifeline Cell Technology; Frederick, MD) with its additives kit, 2.5% FBS and with standard antibiotics mixed 1:1 with Advanced MEM media (Fisher Scientific/Invitrogen/Gibco-BRL) supplemented with standard antibiotics and L-glutamine supplement.
  • Advanced MEM media Fisher Scientific/Invitrogen/Gibco-BRL
  • an extracellular matrix protein coating solution of fibronectin and gelatin was also employed. This specialty media and extracellular matrix coating solution improved the growth and expansion of hTBECs and enhanced their utility in applied science experimentation.
  • a 384-well plate assay design was implemented throughout the assays.
  • CellTiterGLO version 2.0 was employed to determine if any putative bitter blockers were cytotoxic (which would lower the signals in the assays falsely) and cell-free measurements of putative bitter blockers with ENLITEN reagent was performed to determine if any putative bitter blockers were luciferase enzyme inhibitors.
  • a single concentration validation of the 10 PM test concentration was repeated for every putative bitter blocker to further validate a putative bitter blocker.
  • multiple concentration-response curve (CRC)-based validation assays were performed to demonstrate that each putative bitter blocker had a CRC relationship in blocking the stimulation of the hTBEC platform by TAF.
  • CRC concentration-response curve
  • This final step provided potency (estimated IC 50 ) and efficacy (% inhibition of TAF stimulation) metrics for each putative bitter blocker.
  • IC 50 potency
  • efficacy % inhibition of TAF stimulation
  • a Fluo-8/AM-based real-time cell calcium assay was performed as another evidence step for validation by blocking the TAF-elicited cell calcium signals mediated by key TAS2R receptors.
  • Relative fluorescence units (excitation at 494 nm, emission at 516 nm, and auto cutoff at 515 nm) were read every 2 s for 2 min. Calcium mobilization traces were recorded. Changes in fluorescence ( ⁇ F) were calculated as the peak fluorescence minus baseline fluorescence (Lei et al. 2015). The calcium mobilization was quantified as the percentage of change ( ⁇ F) relative to baseline (F). Each data point for bar graphs and concentration-dependent responses was averaged from triplicates (mean ⁇ SD). Calcium mobilization traces and bar graphs along with concentration-dependent plots were all generated by GraphPad Prism 5. Analysis of variance with Dunnett’s multiple comparisons test was used for statistical analysis. *P ⁇ 0.05.
  • the sessions were spaced at least one hour apart to prevent carry-over effects from the long lingering sensations of the test solutions. All solutions were prepared with 1% Tween 80 (Fisher Scientific), 5% of 75.5% Everclear Grain Alcohol (Local Fine Wine and Spirits Store) and filtered water (Milli-Q Water Purification System) as 6-methylflavone is not water soluble.
  • the TAF solution was prepared by first dissolving TAF (Nanjing Bilatchem Industrial Co., Ltd.) and Tween 80 in filtered water and then adding Everclear Grain Alcohol last. The concentration of TAF was 0.5mg/ml, which is 0.84 mM TAF (within the range that was tested on cells).
  • the 6-methylflavone pre-rinse solution was prepared by first dissolving Tween 80 in filtered water and adding Everclear Grain Alcohol with 6-methylflavone (Fisher Scientific) last.
  • the admixture of TAF and 6-methylflavone was prepared by first dissolving TAF with Tween 80 in filtered water and adding Everclear Grain Alcohol with 6-methylflavone last.
  • the concentration of 6-methylflavone in the pre-rinse and admixture was 1 mM. Solutions were prepared the day of testing and stored at room temperature (about 23 °C). Each subject was instructed to refrain from smoking, eating, chewing gum, and drinking anything, except water, for one hour before participation in each session.
  • Table 1 Estimated IC 50 s from CRC of 6-methylflavone and its analogs. Counter screens were performed to insure that the putative hit bitter blockers were not cytotoxic and that they did not interfere with light-based reporters and dyes. Blockade of TAF responses was also repeated in 3 independent experiments, the last of which was a concentration-response curve (CRC)-based assay to ascertain relative potency and efficacy of the putative bitter blocker. Potency of ⁇ 10 PM and efficacy of >50% inhibition were cutoff metrics in the hTBEC screen with TAF as a bitter stimulus.
  • Literature survey indicated structurally similar compounds (e.g., 6- methoxyflavonone) are blockers of TAS2R39.
  • TAF activates a limited number of human bitter receptors TAF has been reported to be intensely bitter. However, which bitter receptor(s) mediate its bitterness is unknown.
  • a standard bitter taste receptor assay that couples the receptor activation to calcium mobilization was used to identify the bitter receptor(s) responsible for the bitter taste of TAF (Meyerhof et al. “The molecular receptive ranges of human TAS2R bitter taste receptors,” Chem Senses 35:157-170 (2010)).
  • Each TAS2R was expressed individually in heterologous HEK-293 cells, along with a coupling G protein G ⁇ 16-gust44, to examine potential TAF stimulation of each receptor(Ueda et al.
  • 6-methylflavone as bitter taste receptor blockers for hTAS2R39. PLoS One 9:e94451 (2014)).
  • HEK293 cells were transiently transfected with TAS2R39 and G ⁇ 16-gust44. The responses of HEK293 cells expressing TAS2R39 and G ⁇ 16-gust44 toward TAF were examined in the presence or absence of 6-methylflavone. In the presence of 0.1 mM 6-methylflavone, the response of TAS2R39 to 1mM TAF was completely abolished, which suggests that 6-methylflavone is an antagonist of TAS2R39 (FIG.
  • TAF also activates TAS2R1.
  • TAS2R1 HEK293 cells expressing TAS2R1 were examined for their responses toward TAF in the presence of 6-methylflavone (FIG. S1A). In these cells, no apparent differences were found between the responses of cells to TAF in the presence or absence of 6-methylflavone.
  • concentration-dependent curves were obtained. As expected, 6-methylflavone blocked the responses of TAS2R39 to TAF concentration-dependently (FIG. 3C).
  • 6-methylflavone has no effects on the responses of TAS2R1, TAS2R28 and TAS2R14 to TAF(FIG. S1B-D). Furthermore, it was shown that 6-methylflavone can block the response of TAS2R39 to denatonium (FIG.3D). 6-methylflavone analogs block TAS2R39 In addition to discovery and validation of 6-methylflavone as a TAS2R39 antagonist, several other flavonoids were identified as putative bitter blockers from the hTBEC screen of phytochemicals and phytochemical derivatives.
  • Table 2 The TimTac catalog numbers of 6-methylflavone and its analogs and other phytochemical derivatives tested in receptor-based assays 6-methylflavone reduces bitterness of TAF in human subjects
  • 6-methylflavone reduces the bitterness of TAF
  • sixteen subjects rated the perceived bitterness of TAF with and without 6-methylflavone.6-methylflavone itself is tasteless on average at the concentration employed, as tested in these sixteen subjects (Fig 5A and B (first bar)); this was not true for some of the other putative flavonoid bitter blockers that emerged from the screen.
  • the perceived bitterness of TAF was nearly completely blocked reliably by 6-methylflavone (FIG. 4).
  • the need for consistent medical compliance is so vital that surgical procedures are undertaken to enable medicines to be placed directly in the stomach, thus bypassing the bitter taste system.
  • the TAF-inhibition screen described herein using novel, native hTBEC culture platforms that use primary hTBEC derived from individual human donors, led to the discovery of 6- methylflavone, which blocks the activation of cultured human taste cells' response to TAF.
  • Subsequent receptor-based activity assays in heterologous human cells demonstrated that TAF activated a limited number of bitter taste receptors, including TAS2R39, TAS2R1, TAS2R8, TAS2R14.
  • limitations include cultured cells: i) not representing native taste cells precisely, ii) not fully reflecting individual human differences in bitter taste, iii) not targeting specific receptors, since native cells tend to express multiple T2Rs, and iv) not delineating whether inhibition is TAS2R-based or downstream signaling elements- based. Therefore, to address limitations iii) and iv), initial human taste cell-based screening with receptor-based assays in heterologous human cells for identification of compounds that specifically target hTAS2Rs for further evaluation were performed. To address limitations i) and ii), 6-methylflavone was validated as a bitter inhibitor of TAF in human psychophysical tests with a small population sample.
  • TAF can activate hTAS2R39 and hTAS2R1, and to a lesser extent hTAS2R8 and hTAS2R14.
  • TAS2R14 when expressed in HEK cells has shown broad tuning; it responds to multiple structurally diverse compounds (Meyerhof et al., 2010). The other hTAS2Rs are more narrowly tuned (Slack et al. “Modulation of bitter taste perception by a small molecule hTAS2R antagonist. Curr. Biol. 20:1104-1109 (2010)).
  • TAS2R39, TAS2R1, and TAS2R8 could be critical receptors for transducing TAF bitterness.
  • 6-methylflavone was an effective blocker of TAS2R39. Blocking one TAF receptor (TAS2R39), however, may not be sufficient to suppress bitter taste across a large heterogeneous human population (Meyerhof et al., 2010).
  • TAS2R39 Blocking one TAF receptor (TAS2R39)
  • 6-methylflavone may not be sufficient to suppress bitter taste across a large heterogeneous human population.
  • 6-methylflavone six other subjects also reliably reported reduction of TAF bitterness in the presence of 6-methylflavone. 6-methylflavone was not, however, effective at blocking TAF bitterness for all subjects.
  • TAF was utilized at approximately 1 mM, a standard patient formulation, which is within the same range that was blocked in cellular assays by 6-methylflavone. Note that other candidate flavone bitter blockers had no impact on TAF bitterness for any subjects.
  • sensitivity to 6-methylflavone as an inhibitor of TAF is a physiological and perceptual trait for some but not all people. These individual differences in response to pharmacological agents comprises the basis for individualized (personal) medicine. Sensitivity to 6-methylflavone could be a multi-genic individual trait, based on several T2R responses to treatment. For some subjects, 6-methylflavone significantly and almost completely reduces bitterness, in others it suppresses bitterness moderately, and in still others it makes bitterness stronger.
  • TAF bitterness of TAF
  • TAS2R1 or TAS2R8 receptors
  • identification of antagonists for these other bitter receptors using the tripartite approach described herein would generate a cocktail of inhibitors that when administered would have potent bitterness inhibitory effects for the majority of subjects.
  • a cocktail containing 6- methylflavone plus antagonists for TAS2R1 and TAS2R8 could be tested to see if it would decrease the medicine’s bitterness for the majority of patients. If so, it would be possible to increase compliance in taking medicines and ultimately be more successful in treating diseases.
  • hTBEC cultures originated from human taste bud tissue donated by multiple individuals (3-6 subjects), were initially used. This practice allowed the amount of cells isolated into primary culture to be maximized. However, this resulted in a genetic mixture within the cultures. To overcome this issue, individual donor-derived hTBEC cultures were established. These individual, donor-derived hTBEC cultures capture human subject or donor diversity of heterogeneity (female and male subjects, subjects of different race and ethnicity, younger and older donors, etc.). Using the methods described herein, it is also possible to isolate tissue and process it into primary cultures from subjects that have ageusia (loss of sense of taste) and other conditions relevant to taste.
  • STI Kill Media 1 mg/ml Soybean Trypsin Inhibitor (STI) in 10 mls Ca2+ Mg2+-free DMEM/F12 media
  • STI Soybean Trypsin Inhibitor
  • This material was transferred to the same 15 ml conical tube as above, so as to have all cells and tissue fragments together. Also, the well where the mincing occurred was washed multiple times to recover all minced tissue and dissociated cells. Maximum recovery is critical because the tissues are tiny and precious. When processing tissues from multiple individual donors it is essential to keep these preps separate in their tubes and wells (10) The tubes were centrifuged for 5 minutes at 2,000 RPM and looked at carefully for a pellet in the bottom of the tube. The supernatant was decanted and a little liquid was left in the bottom of the tube to suspend the cell pellet by finger vortexing.
  • the coated 48-well plate and the coated permeable filter supports in the 24-well plate were incubated in a cell culture incubator for at least 3 days without disturbing the plates.
  • the cells were inspected and imaged on Day 3.
  • the cells were fed every 48 hours by first add- feeding media (standard hTBEC media) to the wells on top of existing media. When the well was full, the media was carefully aspirated and fresh media was gently added.
  • Institutional Review Board (IRB) approvals secured by Monell from the University of Pennsylvania IRB human taste bud tissues were harvested from 10 different, individual donors that varied in sex, ethnicity and age.
  • the demographics of the individual donors 00A through 00J is shown in Table 3.
  • the human sensory testing data for 9 of these donors is shown in FIG. 7.
  • FIG. 8 A schematic of how these primary cultures were initiated is shown below in FIG. 8. Simply put, the goal was to integrate three important parameters: (a) human taste genetics; (b) human taste sensory properties; and (c) the hTBEC cell assay responses from the cultures derived from individual donors that were also examined for points (a) and (b).
  • Donor 00B yielded a B.1.TC primary culture that maintained typical primary hTBEC cell morphology and growth characteristics for a record 15 passages before growth arrest. It is a standard for hTBEC growth characteristics and for the qRT-PCR results below. It responds modestly to different taste stimuli such as sweet ligands, bitter ligands and to salt.
  • Donor 00H was the most successful donor tissue, in that eleven, different primary cultures were established, from 12 possible wells. This donor’s demographics and human sensory testing results are provided in Table 3, and FIG.7, respectively. Examples of Donor 00H primary cultures growing out from digested tissue fragments are shown in FIG.8.
  • Donor 00H was as close to a ‘super taster’ profile in the human sensory testing. This subject had the strongest responses to denatonium benzoate, quinine, and phenylthiocarbamide (PTC or phenylthiourea) and also rated water as modestly bitter during the panel of tastants tested. Donor 00H is highlighted because of qRT-PCR and functional assay profiling results (see below) that led to selection of Donor 00H primary cultures isolated, expanded and grown on ECM-coated TC plastic, as an optimal primary hTBEC culture for the bitter blocker MTS campaign.
  • a magnetic immunocapture method was performed using antibodies to predicted external epitopes of the sweet taste receptor proteins TAS1R2 and TAS1R3 to enrich the cultures in sweet- responsive cells (FIG.9). Also, for adequate doubling time, growth and expansion of either sweet hTBEC clonal cell lines or magnetically immunocaptured cells, lactisole was included in the hTBEC specialty media as an additive so as to enhance cell growth at a concentration of 0.5 to 1 mM (the IC50 for binding to the TAS1R2/TAS1R3 sweet taste receptor heterodimer complex).
  • FIG.10 shows the results of quantitative RT-PCR (qRT-PCR) for TAS1R3.
  • Staining for membrane proteins involved in glucose sensing and glucose transport are also positive validation for a sweet-responsive cell. There is evidence for their involvement in sweet taste transduction. Transport of glucose (and other sugars) by the GLUT transporters may also play a role in sweet taste transduction, in addition to the classical TAS1R2/TAS1R3 heterodimer.
  • the pooled hTBEC 56 lineage (the primary culture, the SV40Tt immortalized cell culture as well as the magnetically immunocaptured “enriched” culture and the clonal cell lines derived from this immortalized culture) are used as the sweet-responsive hTBEC platforms. Assay data shown herein utilized these platforms to show responses to a variety of sweet ligands.
  • FIG. 13 Before discussing the data using assays to detect ATP transmitter secretion and to measure mobilization of intracellular calcium for sweet, salty and bitter taste modalities, a schematic of taste signaling, in the context of an hTBEC cell, is provided in FIG. 13, as well as the methods used to perform these dual assays measuring cell signaling at different points in the taste signal transduction cascade.
  • the first is the last step in taste signaling: the secretion of ATP as the transmitter that stimulates sensory nerves.
  • ATP Assay Determine ENLITEN inhibition by salt (Biological Assay) x Make set of salt solutions that covers the range of the assay x Put 50 ⁇ l of salt solutions containing 300 nM ATP in a 384 plate o Consider putting blanks between the wells because the signal can be high ⁇ Signal can bleed over into neighboring wells x Run assay on luminescence reader o For example, use a Biotek Neo 2 o Run the assay in well mode ⁇ Inject 50 ⁇ l of ENLITEN assay buffer ⁇ Read the luminescence ⁇ Plot the luminescence signal vs the Na + concentration (FIG.15) x Curve fit the data using "one phase decay" model Determine ATP Secretion by hTBEC cells (Cellular Assay) x Wash cells in Saliva Buffer o Wash each well 6 times with 100 ⁇ l Saliva Buffer
  • An intermediate step in the taste signal transduction cascade is real-time measurement of cell calcium mobilization.
  • increases in cell calcium can be triggered by release from intracellular ER stores and/or from calcium entry from extracellular stores via transient receptor potential (TRP) channels or other types of plasma membrane calcium channels.
  • TRP transient receptor potential
  • the methods of measurement of cell calcium mobilization in real- time with the Fluo-8 AM dye (Abcam, Cambridge, United Kingdom) using the hTBEC platform technology are included below. Example data follows within the sections for sweet-responsive hTBEC platforms, salt-responsive hTBEC platforms, and bitter-responsive hTBEC platforms.
  • 96-well plate well A1 loads into 384-well plate wells A1-B2 (FIG.14B) . Same for 96-well plate A2-A12 x
  • the numbers in each well represent the injected NaCl concentration in mM (Table 1) Make 10X Sodium Solutions x Make Input Buffer o (Important Note) Additives can be introduced to the assay using the Input Buffer . For example, adding the small molecule amiloride to the assay .
  • FIG.17 shows the ATP transmitter secretion response to sweet ligands in primary cells (left panel) and in the sweet clonal cell line (right panel).
  • the hTBEC 56.SV.18 sweet clonal cell line has a higher ATP signal to all sweet ligands than the primary hTBEC 56 cells (note different scales on the Y-axis of 20,000 max vs. 8,000 max).
  • FIG. 17 includes a heat map of assay results that show signal-to-noise at both micromolar and millimolar concentrations above the vehicle signal-to-noise in bright blue. It is not meant to show quantitative data.
  • FIG. 18 shows the responses in the sweet clonal cell line in the ATP transmitter secretion detection assay to sweet ligands.
  • FIG.18 focuses on the micromolar range that is driven solely by sweet taste receptor activation; with the exception of AceK, the other sweet stimuli triggered a 1.5 to 3 fold increase in ATP secretion.
  • FIG.19 shows a comparison between sucralose and saccharin. It may be necessary to push the saccharin concentrations higher, because saccharin elicited a stronger signal than the other sweet ligands in the cell calcium assay.
  • the hTBEC platform provided herein can also detect their activity at the sweet taste receptors. These sweet proteins as well as the best known one, miraculin, isolated from the miracle fruit berry stimulate a response.
  • the response in both cell calcium and ATP secretion assays is typically more sustained because sweet proteins bind to and interact with sweet taste receptors in a manner that seems to attenuate their desensitization. Their binding is also appears to be pH-dependent.
  • a real-time ATP secretion detection reagent was used to examine stimulation by rebaudoside A (Reb A).
  • the rebaudosides are another family of sweet ligands. Like many artificial sweeteners, they can also have a bitter taste or bitter “after taste” along with a sweet taste.
  • Reb A rebaudoside A
  • the real- time ATP secretion detection reagent allows for the time kinetics of the ATP assay to be similar to the Fluo-8/AM-based real-time cell calcium mobilization assay.
  • the use of both, in concert, in the sweet-responsive hTBEC panel of cultures is provided herein.
  • ECM-coated Permeable Filter Support (PFS) Culture Yielded the Best Salt-responsive hTBEC Cultures or Platforms – Bioassay Data in Support Upon examining the individual donor-derived hTBEC cultures to determine if any of them are enriched or more responsive to salt as a stimulus, D.1.PFS cells emerged as a salt-responsive primary culture. These were immortalized successfully with SV40T to establish a salt-responsive D.1.PFS SV40Tt immortalized cell line. Critically, this culture was initiated and established on ECM-coated permeable filter supports.
  • hTBEC cultures initiated and grown in this manner on a permeable growth substrate might exhibit a more polarized phenotype which is critical for ion channel proteins such as the epithelial sodium channels (the ENaCs) and other membrane proteins. Since the specific membrane proteins (channels, transporters, receptors) involved in human salt taste transduction are not defined, functional assays were used to identify D.1.PFS hTBEC cultures as the best salt-sensitive platform at this time. Shown herein is the salt responsiveness and salt sensitivity of D.1.PFS primary and D.1.PFS SV40Tt immortalized hTBEC cultures.
  • FIG. 25 shows the concentration-dependent increase in ATP transmitter secretion with increasing salt concentrations from a baseline of 22.5 mM sodium salt (sodium gluconate, NaGlu or sodium chloride, NaCl) in our saliva-mimicking buffer.
  • FIG. 27 shows ATP transmitter secretion detection data that confirms a similar salt sensitivity in both the primary and immortal D.1.
  • PFS salt-responsive hTBEC platforms when one examines the overall salt titration as well as a modest 82.5 mM salt stimulus. Identification of a distinct salt-responsive hTBEC platform allowed interrogation of this platform as to potential cellular and molecular mechanisms of action.
  • FIG. 27 shows results using the ENaC inhibitor, amiloride.
  • this blocker failed to inhibit the salt responses in assays or inhibited only modestly.
  • the cells were pre- treated with amiloride for 1 minute prior to salt stimulation, there was marked inhibition of the salt-responsive hTBEC assay responses. As little as 25 micromolar amiloride attenuated the response markedly. This result made sense, because amiloride binding to the ENaC extracellular domains is known to be charge-dependent.
  • cystic fibrosis transmembrane conductance regulator CFTR
  • ENaC cystic fibrosis
  • CFTR cystic fibrosis transmembrane conductance regulator
  • FIG.28 shows an interesting and novel result that the selective CFTR inhibitor, CFTR inhibitor 172, also attenuates the response to lower salt stimulus and to the concentration-dependent stimulation with salt.
  • CTFR inhibitor 172 attenuated the response markedly to lower salt stimulatory concentrations and partially to higher salt boluses.
  • Cellular and molecular mechanism experiments with pharmacological inhibitors were performed using the Fluo-8/AM real-time cell calcium assays; however, similar data is accrued in the ATP transmitter secretion detection assay.
  • a fundamental question emerges with regard to where the cellular calcium comes from.
  • CFTR and ENaC may interact functionally with transient receptor potential (TRP) channels in specialized epithelial cells.
  • TRP transient receptor potential
  • TRP channels are calcium-permeable non-selective cation channels that contribute to calcium and sodium entry across the plasma membrane. As such, a pair of commercially available TRP channel inhibitors were employed, and partial inhibition of the salt-driven cell calcium responses was observed.
  • FIG. 29 shows that TRP channel inhibitors attenuate salt responses in the real-time cell calcium-mobilization assay and implicate TRP channel-driven calcium entry in salt tast transduction.
  • HC030031 is a selective TRPA1 channel inhibitor.
  • AMG- 333 is a selective TRPM8 channel inhibitor.
  • the example data below show how more bitter-responsive cultures were characterized and selected from the hTBEC culture cryorepository, how bitter ligands were profiled, and how this applied science platform can be used in medium-throughput screening campaigns to discover and validate bitter blockers.
  • Humans have selected for more and more TAS2R bitter receptors throughout evolution to the present. It is thought to protect us from ingesting toxins or poisons.
  • TAS2R bitter taste receptor genes There have been 29 TAS2R bitter taste receptor genes and characterizing the functionality of each TAS2R is still being characterized.
  • scRNASeq single cell RNA Sequencing
  • TAS2R family are functional important in binding/responding to bitter medicines (small molecule biopharmaceutical drugs.
  • the key functional TAS2Rs include TAS2R1, TAS2R7, TAS2R8, TAS2R14, TAS2R38, and TAS2R39.
  • TAS2R1, TAS2R7, TAS2R8, TAS2R14, TAS2R38, and TAS2R39 To compare mRNA expression of these key functional TAS2Rs to cell calcium mobilization and ATP transmitter secretion bioassay responses in our hTBEC cell cultures, we performed qRT-PCR across our arsenal of hTBEC cultures so as to identify the best candidate cultures for a bitter blocker discovery platform.
  • FIGS.30-37 summarize qRT-PCR results that led to focus on the Donor 00H TC cultures.
  • TAS2R14 is expressed significantly in all of the hTBEC primary cultures examined.
  • TAS2R14 is an important target to block or antagonize, because many bitter medicines are ligands for this particular bitter taste receptor.
  • TAS2R14 is also expressed in human cells and tissues outside of the taste bud: so-called extraoral tissues.
  • TAS2R14 is expressed most abundantly in a subset of the Donor 00H TC cultures, especially H.2.TC, H.4.TC and H.5.TC (highlighted by the arrows inside the hatched box in FIG.30). TAS2R14 mRNA was also easier to amplify than other mRNA targets; however, the inset plot in FIG. 3 shows that TAS2R14 is not as abundant as ⁇ - actin and that amplification occurs late in the 40 cycles.
  • FIG.31 assesses the entire repository of individual donor-derived cultures for TAS2R14 mRNA expression.
  • Donor 00H There were a few additional cultures outside of Donor 00H such as Donor 00G (G.1.PFS), Donor 00I (I.3.PFS) and Donor 00J (also known as Donor 00Na1, Na1.1.PFS) that showed higher expression of TAS2R14 relative to many of the other cultures. They are marked by the additional arrows and will be useful for confirming bitter blocker effects on cultures from other donors. Interestingly, however, none of the Donor 00H PFS cultures were remarkable for TAS2R14 expression, hence the hatched box focusing on the H TC cultures.
  • TAS2R1 (FIG. 32) and TAS2R39 (FIG. 33) were also assessed; because, like TAS2R14, they are typically stimulated by many bitter medicines such as the anti- virals (tenofovir, etc.) and anti-malarials (praziquantel, piperaquine, chloroquine, ferroquine).
  • TAS2R38 mRNA expression was also quantified because of its interesting genetics (FIG. 35); it is also the specific TAS2R for PTC, a bitter taste industry standard.
  • TAS2R and TRP channel targets that must be expressed in the specific hTBEC screening platform to be used in bitter blocker discovery.
  • This analysis with human sensory testing in advance of tissue harvest and primary culture creation, links the human genetics, human taste behavior, and human taste bud tissue-derived cell assay responses in the appropriate integrated way. This integrated and human-focused effort has not been undertaken before, with the vast majority of knowledge coming from rodent models. It is also possible that multiple transient receptor potential (TRP) channels are expressed by hTBEC cultures and may act as signal amplifiers for bitter taste perception.
  • TRP transient receptor potential
  • TRPV1 (FIG. 35) and TRPML3 (FIG. 36) mRNA expression cluster well with the TAS2R subtype mRNA expression data.
  • TRPV1 has been associated with taste sensory mechanisms in past published work; however, TRPML3 has not yet been linked to taste transduction. Its heightened signal in H.4.TC and H.5.TC cultures is consistent with the TRPV1 result.
  • TRPA1 (FIG.37) data do not agree with the results of the other two TRP channels. This finding is a positive result, because TRPA1 is thought to be an irritant receptor not expressed by taste cells or taste buds.
  • TAS2Rs 30, 31, and 4 TAS2Rs 30, 31, and 4.
  • TAS2R variant analysis was performed across the 10 individual donors from which our individual donor-derived hTBEC cultures 00A through 00J were isolated. Table 7 shows these results on the subsequent page. Importantly, we need to do deeper investigation to determine if any of these TAS2R variants are functional and how they contribute to bitter taste transduction.
  • Table 6 Results of RNA sequencing analysis
  • MTS was performed on primary hTBEC cultures because they maintained a robust signal-to-noise in the dual functional assays and primary cultures could be expanded and passaged enough to support MTS. While concentration-response curve- based relationships were observed for all of the anti-viral drugs and significant ‘bitter’ signal from remdesivir and valpatasvir (FIG. 39), tenofovir alafenamide fumarate (TAF) was selected as the bitter stimulus for our MTS campaign to discover and validate bitter blockers.
  • TAF tenofovir alafenamide fumarate
  • TAS2R bitter taste receptors TAS2R14, TAS2R1, and TAS2R39
  • TRP transient receptor potential channels that are calcium-permeable and promote calcium entry from extracellular stores may be signaling amplifiers for TAS2R bitter receptors.
  • TRPV1 and TRPML3 were amplified initially. These data are shown on Figs.35 and 36, respectively.
  • TRPA1 was not as well expressed in hTBEC cultures. In fact, it is an irritant receptor and should not be expressed in human taste cells. These data are shown on FIG.37. Subsequent to this analysis, additional targets were analyzed to confirm the hTBEC cultures were suitable for bitter taste receptor analysis.
  • PLC beta isoforms 2, 3 and 4 different TRP channel targets (TRPM5, in particular), additional TAS2Rs (TAS2R8 and TAS2R10), and CALHM1 (a candidate ATP release channel) were analyzed by qRT-PCR.
  • TRPM5 phospholipase C
  • TAS2R8 and TAS2R10 additional TAS2Rs
  • CALHM1 a candidate ATP release channel
  • PLC beta isoforms 2, 3 and 4 were amplified from the most bitter responsive donor H cultures and from a culture for a different donor by using more targeted qRT-PCR assays. Additional bitter receptors, TAS2R8 and TAS2R10, were also amplified (FIGS.44 and 45). Additional TRP channel family subtypes, TRPM5 and TRPC1, were also amplified (FIGS. 46 and 47). The candidate ATP release channel, CALHM1, was also amplified easily (FIG.48).

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Abstract

La présente invention concerne des cellules épithéliales de bourgeons gustatifs humains (hTBEC) dérivées d'un donneur humain individuel et des procédés d'utilisation de celles-ci pour identifier des substances gustatives amères, sucrées, umami, acides et salées. Les composés qui inhibent l'activité des agents gustatifs amers peuvent également être identifiés en utilisant les hTBEC et les procédés décrits dans la présente invention.
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