Patent Grant
10048274
The present invention involves the discovery that the efficacy of cell-based assays that screen for compounds that modulate ENaC function, preferably ENaC enhancers, is improved by the further use of a compound that at least partially inhibits ENaC function preferably an amiloride derivative such as phenamil. The present invention further relates to improved electrophysiological assays that identify human ENaC modulators using oocytes, preferably frog oocytes, that express a functional human ENaC sodium channel.
The present invention in part relates to novel cell based assays that use recombinant host cells that express amiloride-sensitive sodium channels to profile, screen for, and identify taste modulating compounds. More specifically, the invention relates to assays that utilize test cells that express a functional human epithelial sodium channel (hENaC), preferably amphibian oocytes or mammalian cells, and the use of these test cells in cells in high throughout or moderate throughput cell-based assays, preferably electrophysiological assays, to identify compounds that enhance or block hENaC function.
An amiloride-sensitive epithelial sodium channel (ENaC) mediates sodium influx across the apical membrane of taste buds cells in the tongue (Heck, et al, Science (1984) 223: 403-405). ENaC, a member of the ENaC/degenerin superfamily of ion channels involved in sodium transport, is composed of three partially homologous α, β, and γ subunits expressed at both the RNA and protein level in fungiform, foliate, and circumvallate papilla as well as the lingual epithelium in taste tissue (Li, et al, Proc. Natl. Acad. Sci. (1994) 91: 1814-1818; Kretz, et al, J. Histochem. Cytochem. (1999) 47(1): 51-64; Lin, et al, J. Comp. Neurol. (1999) 405: 406-420; Xiao-Jiang, et al, Mol. Pharmacol. (1995) 47: 1133-1140).
Complementary DNAs (cDNAs) encoding amiloride-sensitive epithelial sodium channel (ENaC) channel subunits have been isolated from kidney cells and expressed in a mammalian cell line. The channel expressed in this system has been shown to have similar properties to the distal renal sodium channel, i.e., high sodium selectivity, low conductance, and amiloride sensitivity. One form of the naturally occurring ENaC channel is comprised of three subunits of similar structure: alpha (OMIM Entry 600228), beta (OMIM Entry 600760), and gamma (OMIM Entry 600761). Each of the subunits is predicted to contain 2 transmembrane spanning domains, intracellular amino- and carboxy-termini, and a cysteine-rich extracellular domain. The three subunits share 32 to 37% identity in amino acid sequence. Alternatively spliced forms of alpha-ENaC have also been identified, indicating heterogeneity of alpha subunits of amiloride-sensitive sodium channels that may account for the multiple species of proteins observed during purification of the channel (See U.S. Pat. No. 5,693,756, which is herein incorporated by reference).
An inhibitor of ENaC sodium channel function, amiloride, is known to attenuate gustatory responses to sodium chloride in numerous non-mammalian as well as mammalian species, including humans (Halpern, Neuroscience and Behavior Reviews (1998) 23: 5-47 and all references cited within; Liu, et al, Neuron (2003) 39: 133-146; Zhao, et al, Cell (2003) 115: 255-266). In humans, amiloride reportedly reduces the intensity of sodium chloride by 15-20% when used at concentrations that specifically inhibit ENaC function (Halpern, Neurosciences and Behavior Reviews (1998) 23:5-47 and all references cited within; Feldman, et al, J. Neurophysiol. (2003) 90(3): 2060-2064). Therefore, compounds that increase the transport of sodium ions through ENaC channels may function as general salt taste enhancers and augment human salt taste perception as suggested in our previous patent application (PCT WO 02/087306 A2). Further, based on published electrophysiological data and the discovery that a human ENaC is expressed in taste bud cells, a model of salty taste transduction mediated by ENaC has been constructed. As such, the use of ENaC in the identification of substances which stimulate or block salty taste perception has been suggested (See U.S. Pat. No. 5,693,756, supra and PCT WO 02/087306 A2).
Cell-based functional expression systems commonly used for the physiological characterization of ENaC are Xenopus laevis oocytes and cultured mammalian cell lines. The oocyte system has advantages over mammalian cells in that it allows the direct injection of multiple mRNAs, provides high levels of protein expression, and can accommodate the deleterious effects inherent in the over expression of ENaC. However, the drawbacks of this system are that electrophysiological recording in Xenopus oocytes is not amenable to screening large numbers of compounds and the Xeropus oocyte is an amphibian not a mammalian system. Studies of the electrophysiological properties of rodent ENaC in mammalian cell lines (human kidney two hundred ninety three and MDCK) stably expressing the channel have been reported in the literature. While these studies used mammalian cell lines, channel function was assayed using tedious electrophysiological techniques. Such approaches do not lend themselves to high throughput screening of compounds. Thus, there remains a need in the art for identification of salt taste enhancers amenable to high throughput screening.
The development of salt taste enhancers has been the focus of numerous prior scientific publications and patents. However, direct modulation of the ENaC sodium channel involved in salt taste perception is a novel and unique approach to enhance human salty taste. Some examples of previously reported salt enhancing compounds and their properties are discussed below.
Some proteolzyed proteins, peptides, amino acids, and amino-acid esters reportedly function as salt enhancers (Tamura, et al, Argic. Biol. Chem. (1989) 53(6): 1625-1633, 1989; U.S. Pat. No. 5,711,985). However, these agents require high concentrations, between 30-60 mM, and must be supplemented with hydrochloride acid to positively modulate salty taste. In addition, the cost and difficulty in synthesizing these compounds are prohibitive for their large-scale commercial use as salt enhancers for the general population.
Choline chloride, an ammonium salt classified by the federal government as a GRAS (generally regarded as safe) compound, has been reported to function as a salt enhancer in humans and rodents. In humans, choline chloride increases the saltiness of dilute salt solutions (less than 50 mM NaCl) by a factor of two and reportedly increases the preference or hedonic ratio of both cooked peas and Campbell's low salt tomato soup (Locke, et al, Physiology & Behavior (1994) 55(6): 1039-1046; U.S. Pat. Nos. 5,260,091; 5,260,049). However, similar to peptides and amino acids described above, choline chloride requires significant concentrations (in the mM range) to enhance salty taste.
Derivatives of amiloride, which do not block ENaC function but instead block sodium-proton exchange, as well as chloride channel blockers, such as IAA-94 and anthranilic acid, reportedly increase fluid intake, an indirect measurement of salt consumption, in a rodent model system (U.S. Pat. No. 5,260,091). However, the utility of these agents as human salt enhancers has not been reported.
Cetylpyridiunium chloride (CPC) has been reported to increase amiloride-insensitive nerve responses to salt in rats and to enhance the saltiness of low salt Campbell's tomato soup by 50% in humans when used at low concentrations (high uM range) (DeSimone, et al, J. Neurphysiol. (2001) 86: 2638-2641; U.S. Pat. No. 4,997,672). However, CPC is a detergent and based on its structure likely intercalates into lipid bilayers of cells and thereby non-specifically activates salt taste cells by disrupting lipid homeostasis. Indeed, high concentrations of CPC (low mM range), above the critical micelle concentration, actually inhibit rat nerve responses to numerous salty compounds including sodium chloride, potassium chloride, and ammonium chloride, further substantiating that the reportedly observed CPC effects were likely non-specific.
Trehalose, a disaccharide composed of two glucose molecules, reportedly increases the saltiness of sodium chloride solutions by 1.2 to 2-fold (U.S. Pat. No. 6,159,529). Similar to peptides and choline chloride, high levels (1.5-12%) of this sugar are required to enhance saltiness, suggesting that the observed effects could be non-specific and attributable to taste cell volume changes (cell shrinkage) due to hyperosmolarity. In addition, the specificity of trehalose and other aforementioined salt enhancers to enhance salty taste and not modulate other tastes, including sweet, bitter, sour, and umami, was not addressed.
Alapyridaine, a derivative of the amino acid alanine that is formed as a by-product in heated sugar/amino acid mixtures, reportedly decreases the threshold for detecting sodium chloride 5-fold (Soldo, et al, Chemical Senses (2003) 28: 371-379, 2003; Ottinger, et al, J. Agric Food Chem (2003) 51: 1035-1041, 2003). Alapyridaine, however, reportedly functions as a general taste enhancer and decreases the detection thresholds for salt as well as sweet and umami tastes. In addition, the effect of alapyridaine on salt taste at higher, more physiologically-relevant, salt concentrations was not disclosed. Thus, the effects of alapyridaine may only surface when tasting low salt concentrations near threshold detection levels.
The antibiotic novobiocin also reportedly enhances nerve responses to sodium chloride in rats (Feigin, et al, Am. J. Physiol. (1994) 266: C1165-C1172). However, disadvantageously novobiocin reportedly forms amiloride-insensitive cation-selective ion channels in lipid bilayers suggesting that this agent pokes holes in cell membranes and, perhaps similar to CPC, non-specifically increase taste cell activity. The effect of novobiocin on human salt taste perception has not been reported.
Bretylium tosylate, an anti-fibrillary drug that modulates adrenergic and muscarinic receptors, has been reported to specifically potentiate salt taste in rodents and humans without affecting sweet, sour, or bitter taste (Schiffman, et al, Physiology & Behavior (1986) 36: 1129-1137). However, a significant disadvantage of bretylium tosylate, separate from the relatively high concentrations required to positively modulate salty taste (mM range), is that the compound is a therapeutic used to treat cardiac patients. Consequently this compound would be unsuitable for use in the general population.
Glybenclamide, an inhibitor of members of the ATP-binding cassette (ABC) protein superfamily, including the cystic fibrosis transmembrane conductance regulator and the sulfonylurea receptor, reportedly increases amiloride-sensitive ENaC sodium current by doubling the open probability of individual ENaC channels (Chraibi, et al, The Journal of Pharmacology and Experimental Therapeutics (1999) 290: 341-347, 1999; Schnizler, et al, Biochemica et Biophysica Acta (2003) 1609: 170-176). However, because Glybenclamide modulates ABC protein function, it is probable that Glybenclamide effects are due to indirect modulation of ENaC activity by ABC proteins and not attributable to direct modulation of ENaC channel function. In addition, glybenclamide has not been demonstrated to enhance human salt taste perception nor has glybenclamide been suggested as a salt taste enhancer.
Thus, based on the foregoing, it is evident that improved methods for identifying compounds that specifically modulate ENaC and salty taste are needed as are improved salty taste modulators. Preferably, such methods will comprise high or medium throughput methods and will screen for compounds having a direct effect on human ENaC function.
The present invention obviates the problems of the prior art, relating to assays for identifying compounds that modulate ENaC. Specifically, the present invention provides cell-based assays that utilize recombinant host cells, preferably mammalian cells or oocytes that express a functional human ENaC to identify compounds that modulate ENaC and consequently salty taste. More specifically, the present invention provides oocyte and mammalian cell-based assays, preferably high or medium throughput, for the profiling and screening of a sodium channel, more particularly an amiloride-sensitive epithelial sodium channel (ENaC), which assays optionally may include the addition of a compound that partially or totally inhibits ENaC function, preferably amiloride or an amiloride derivative such as phenamil. It has been found that the use of phenamil in particular enhances signal intensity during assays, preferably high or medium throughput assays for identifying compounds that modulate (enhance or inhibit) ENaC function. Such methods can be used to functionally characterize ENaC activity or to identify compounds that either enhance or block salty taste perception (herein referred to as salty taste modulators).
Accordingly, in a first aspect the invention provides recombinant host cells, preferably mammalian cells or amphibian oocytes that express a functional hENaC. In a preferred embodiment these cells will transiently or stably express all three subunits of hENaC (alpha or delta, beta and gamma), or transiently or stably express one or more subunits or functional chimeras, variants or fragments thereof. Mammalian cells suitable for use in the invention encompass any mammalian cell capable of expressing a functional hENaC, including by way of example COS, CHO, MDCK, human kidney two hundred ninety three, human kidney two hundred ninety three T, NIH3T3, Swiss3T3 and BHK cells. However, in the preferred embodiment the invention provides human kidney two hundred ninety three T cells that express a functional hENaC. Oocytes useful in the invention preferably include amphibian o ocytes, e.g., Xenopus oocytes.
In a second aspect, the invention provides cell-based assays that utilize mammalian cells or amphibian oocytes that express a functional ENaC, preferably hENaC, to identify compounds, including e.g., small organic molecules, antibodies, peptides, cyclic peptides, lipids and nucleic acids that enhance or block ENaC function. Preferably, these assays will include the addition of known ENaC inhibitor at a concentration that partially inhibits ENaC function prior to addition of use of one or more putative ENaC modulatory compounds.
Preferably the assay will comprise a mammalian or ooycte cell-based assay, preferably high or medium throughput, for the profiling and screening of putative modulators of an epithelial sodium channel (ENaC) comprising: (i) contacting a test cell expressing an ENaC loaded with a membrane potential fluorescent dye or a sodium-sensitive fluorescent dye with at least one putative modulator compound in the presence of a buffer containing sodium; (ii) prior to the addition of said at least one putative modulator compound, contacting said host cell with a compound that is known to inhibit ENaC function, at a concentration whereby ENaC function is at least partially inhibited, preferably an amiloride derivative such as phenamil; and (iii) monitoring changes in fluorescence of the membrane potential dye or sodium-sensitive dye in cells contacted with the putative modulator plus sodium after addition of the known ENaC inhibitor compound compared to the change in fluorescence of the membrane potential dye or sodium-sensitive dye for cells contacted with sodium alone to determine the extent of ENaC modulation.
In another preferred aspect of the invention, a method for monitoring the activity of an epithelial sodium channel (ENaC) is provided comprising: (i) providing test cells, e.g., mammalian cells transfected or transformed with a functional ENaC; (ii) seeding the test cell in the well of a multi-well plate and incubating for a time sufficient to reach at least about 70% confluence; (iii) dye-loading the seeded test cells with a membrane potential fluorescent dye or sodium-sensitive fluorescent dye in the well of the multi-well plate; (iv) contacting the dye-loaded test cell with at least one putative modulatory compound in the well of the multi-well plate; (v) prior to the addition of said at least one putative modulatory compound, further contacting said host cell with a compound that partially inhibits ENaC function, e.g., an amiloride derivative such as phenamil; and (vi) monitoring any changes in fluorescence using a fluorescence plate reader.
In another preferred embodiment of the invention (i) suitable cells, e.g., human kidney two hundred ninety three T cells or another mammalian cell line are transformed, tranfected with DNA sequences encoding subunits necessary to produce a functional human ENaC; (ii) the cells are seeded onto a multi-well plates, e.g., 384 well plates, preferably to about 80% confluence; (iii) the seeded test cells are loaded with a membrane potential sensitive dye such as CC2-DMPVE or DiSBAC2(3); (iv) the dye-loaded cells are then contacted with at least one putative ENaC modulatory compound; (v) the dye-loaded cells are preferably further contacted prior to contacting with said at least one ENaC modulatory compound with an amount of at least one known ENaC inhibitor at a concentration that results in at least partial ENaC inhibition; and (iv) monitoring changes in cell fluorescence using a voltage intensity plate reader e.g., VIPRII (Aurora Biosciences).
In yet another aspect of the invention, a method for identifying a salty taste modulatory compound is provided comprising: (i) providing test cells transfected, transformed with a functional human ENaC; (ii) seeding the test cell in the well of a multi-well plate and incubating for a time sufficient to reach at least about 70% confluence more preferably to about 80%, confluence; (iii) dye-loading the seeded test cells with a membrane potential dye in the well of the multi-well plate; (iv) contacting the dye-loaded test cells with at least one putative modulatory compound in the well of the multi-well plate; (v) preferably prior to the addition of said at least one putative modulatory compound further contacting the dye-loaded test cell with a known ENaC inhibitor compound at a concentration that at least partially inhibit ENaC function, e.g., an amiloride derivative such as phenamil; (vi) monitoring any changes in fluorescence of the membrane potential dye due to modulator/ENaC interactions using a fluorescence plate reader; and (vii) identifying the at least one putative modulator as a salty taste modulating compound based on the monitored changes in fluorescence.
In yet another preferred embodiment of the invention (i) suitable cells, e.g., human kidney two hundred ninety three T cells are transformed or transfected with DNA sequences encoding subunits necessary to produce a functional human ENaC; (ii) the cells are seeded onto multi-well plates, e.g., 384 well plates, preferably to about 80% confluence; (iii) the seeded test cells are loaded with a membrane potential sensitive dye such as CC2-DMPVE or DiSBAC2(3); (iv) the dye-loaded cells are then contacted with at least one putative ENaC modulatory compound; (v) preferably prior thereto the dye-loaded cells are contacted with a compound known to inhibit ENaC function, e.g., an amiloride derivative such as phenamil at a concentration that at least partially inhibits ENaC function; (vi) changes in cell fluorescence are monitored using a voltage intensity plate reader e.g., VIPRII (Aurora Biosciences); and (vii) compounds that modulate salty taste are selected based on a change in fluorescence intensity.
In yet another preferred embodiment of the invention, electrophysiological assays, preferably two-electrode voltage clamp assays are provided wherein human ENaC modulatory compounds are identified based on their effect (inhibitory or enhancing) on macroscopic electrical current in oocytes, preferably amphibian oocytes (frog)), that express a functional human EnaC sodium channel. These assays are an improvement over some other cell-based assays for identifying ENaC modulators, in part because oocytes express few endogenous ion channels; consequently the ooocyte expression system advantageously allows direct measurement of ENaC sodium channel current with little or no background.
In another preferred embodiment of the invention, these electrophysiological assays will further contact said oocytes with at least a partial inhibitor of ENaC, e.g., amiloride or an amiloride derivative as a control or in order to enhance measurable changes in sodium channel cell current.
In yet another preferred embodiment of the invention, frog oocytes that functionally express a human ENaC sodium channel are provided which express human ENaC alpha, beta and gamma or delete, beta and gamma subunits.
In another preferred embodiment of the invention, the ENaC used in cell-based assays acccording to the invention can be composed of naturally occurring human ENaC subunits, one or more alternatively spliced human ENaC subunits, or a functional variant thereof. Alternatively, the ENaC can be composed of at least the alpha subunit of a naturally occurring human ENaC, or an alternatively spliced version thereof. In another embodiment, a delta subunit (such as Genebank accession U38254; see J Biol Chem, 270(46):27411-4 (1995)) or a variant thereof can substitute for the alpha subunit.
Preferably, these subunits are encoded by SEQ ID: NO.: 1, 2, 3 and 7 disclosed infra. These and other aspects of the invention will become apparent to one of skill in the art from the following detailed description, drawings, and claims.
The present invention provides assay systems that comprise test cells, preferably recombinant mammalian cells or amphibian oocytes, that express a functional hENaC as well as mammalian cell-based and amphibian oocyte cell-based assays, preferably high or medium throughput, for the profiling and screening of an epithelial sodium channel (ENaC). More specifically, the invention provides human cell lines, e.g., human kidney two hundred ninety three T cells, and amphibian oocytes, that express the α, β, and γ subunits of hENaC that can be used in cell-based assays to screen for ENaC modulators. Also the invention provides mammalian cells and amphibian oocytes that express a functional ENaC comprised of delta, beta and gamma subunits for use in functionally characterizing ENaC activity, and to identify compounds that either enhance or block salty taste perception (herein referred to as salty taste modulators). These compounds can be used as ingredients in foods, medicinals and beverages to enhance, modulate, inhibit or block salty taste.
However, prior to discussing the invention in more detail the following definitions are provided. It should be otherwise understood that the technical terms and phrases have their ordinary meaning, as they would be construed by use of ordinary skill in the art.
Definitions
The term “salty taste” or “salty taste perception” as used herein refers to a subject's perception or response to salt taste stimuli. As discussed above, it is believed that hENaC is involved in salty taste perception, in particular salts that elicit “a salty taste” in human subjects. Such stimuli include compounds such as NaCl that elicit a response in functional ENaCs, preferably hENaC.
The terms “ENaC” subunit protein or a fragment thereof, or a nucleic acid encoding one of three subunits of “ENaC” protein or a fragment thereof refer to nucleic acids and polypeptides, polymorphic variants, alleles, mutants, and interspecies homologues that: (1) have an amino acid sequence that has greater than about 80% amino acid sequence identity, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, or 500, or more amino acids, to an amino acid sequence encoded by the nucleic acid sequence contained in SEQ ID NO:1; 2 or 3; or (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by SEQ ID NO:1, 2, or 7 or immunogenic fragments thereof, and conservatively modified variants thereof; or (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding an ENaC protein, e.g., SEQ ID NO:1, 2, 3 or 7 or their complements, and conservatively modified variants thereof; or (4) have a nucleic acid sequence that has greater than about 80% sequence identity, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to SEQ ID NO:1, 2, 3 or 7 or their complements, or (5) is functionally equivalent to the hENaC described herein in a sodium conductance assay when expressed in a human kidney cell and tested by using two electrode whole cell electrophysiology or by the change in fluorescence of a membrane potential dye in response to sodium or lithium.
Functionally equivalent ENaC proteins include ENaC subunits with primary sequences different than those identified infra, but which possess an equivalent function as determined by functional assays, e.g., sodium conductance assays as described infra.
“Determining the functional effect” refers to assaying the effect of a compound that increases or decreases a parameter that is indirectly or directly under the influence of an ENaC polypeptide e.g., functional, physical and chemical effects. Such functional effects include, but are not limited to, changes in ion flux, membrane potential, current amplitude, and voltage gating, a as well as other biological effects such as changes in gene expression of any marker genes, and the like. The ion flux can include any ion that passes through the channel, e.g., sodium or lithium and analogs thereof such as radioisotopes. Such functional effects can be measured by any means known to those skilled in the art, e.g., by the use of two electrode electrophysiology or voltage-sensitive dyes, or by measuring changes in parameters such as spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties. Preferably ENaC function will be evaluated by using two electrode whole cell electrophysiology or by monitoring the change in fluorescence of a membrane potential dye in response to sodium or lithium.
“Inhibitors”, “activators”, and “modulators” of ENaC polynucleotide and polypeptide sequences are used to refer to activating, inhibitory, or modulating molecules identified using cell-based assays of ENaC polynucleotide and polypeptide sequences. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of ENaC proteins, e.g., antagonists.
“Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate ENaC protein activity. Inhibitors, activators, or modulators also include genetically modified versions of ENaC proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, peptides, cyclic peptides, nucleic acids, antibodies, antisense molecules, ribozymes, small organic molecules and the like. Such assays for inhibitors and activators include, e.g., expressing ENaC protein in cells, cell extracts, or cell membranes, applying putative modulator compounds, and optionally prior thereto contacting said ENaC protein with a known ENaC inhibitor at a concentration that results in partial ENaC inhibitors and then determining the functional effects of the rotation compound on activity, as described above.
Samples or assays comprising ENaC proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of activation, inhibition or modulation. In one embodiment of the assay, compounds are tested for their effect on the response of cells provided with a suboptimal sodium concentration. Control cells, treated with the suboptimal concentration of sodium but lacking a compound, typically exhibit a 10-20% of the maximal response. Compounds that increase the response of the suboptimal sodium concentration above the 10-20% level are putative ENaC enhancers. In contrast, compounds that reduce the response to below 10% are putative ENaC enhancers.
The term “test compound” or “test candidate” or “modulator” or grammatical equivalents thereof as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid (e.g., a sphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., to be tested for the capacity to modulate ENaC activity. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., enhancing activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Preferably, high throughput screening (HTS) methods are employed for such an analysis.
A “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
“Biological sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
“Compound that inhibits ENaC activity” refers to a compound which inhibits sodium channel activity, preferably reversibly when this compound is contacted with a functional ENaC. Preferred examples of such compounds are amiloride and amiloride derivatives such as Phenamil, benzamil, 31, 41-dichlorobenzamil, ethylisopropylamiloride; 5-(N-4-chlrobenzyl)-21,41 dimethyl-benzamil, 5-(N-methyl-N-guanidinocarbonylmethyl)amiloride; 5-(N,N-hexa-myethylene)amiloride; 5-(N-ethyl-N-isopropyl) amiloride (EIPA); 5-(N-4-chloro-benzyl) 21, 41 dimethylbenzamil, 21,41-dimethylbenzamil; 21,31-benzo-benzamil; and the like.
“Amiloride derivative” refers to a compound having a structure similar to amiloride which inhibits ENaC function. Typically such derivatives are substituted on the guanidine substituent (e.g., Phenamil) or on the 5-N position (e.g., ethylisopropylamiloride).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 80% identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequences SEQ ID NO: 1, 2, 3 or 7), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site (www.ncbi.nlm.nih.gov) or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but those functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologous, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). As noted previously, the invention embraces cells that express ENaC subunit polypeptides having primary sequences different than those disclosed in the subject application that are functionally equivalent in appropriate assays, e.g., using whole cell sodium conductance assays described in detail infra.
Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered three-dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., transmembrane domains pore domains, and cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include extracellular domains, transmembrane domains, and cytoplasmic domains. Typical domains are made up of sections of lesser organization such as stretches of α-sheet and β-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
A particular nucleic acid sequence also implicitly encompasses “splice variants.” Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternatively polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
ENaC nucleic acid sequences also include single nucleotide polymorphisms which encode ENaC subunits that are functionally equivalent to the ENaC polypeptides disclosed herein when assayed using appropriate assays, in the sodium conductance assays described herein.
Membrane potential dyes or voltage-sensitive dyes refer to a molecule or combinations of molecules that change fluorescent properties upon membrane depolarization. These dyes can be used to detect the changes in activity of an ion channel such as ENaC expressed in a cell.
A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all. In the present invention this typically refers to cells that have been transfected with nucleic acid sequences that encode one or more ENaC subunits.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). The term “heterologous” when used with reference to cellular expression of a gene, cDNA, mRNA or protein indicates that the gene, cDNA, mRNA, or protein is not normally expressed in the cell or is from another species than the original source of the cells.
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. The wash and hybridization steps are generally carried out for ½, 1, 2, 5, 13, 15, 30, 60 or more minutes, and more typically for about 30 seconds to 2 minutes.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.
For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from a bout 50° C. to a bout 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
Particularly, such an antibody includes one which specifically binds to an ENaC disclosed herein, or a mixture of antibodies that specifically bind such ENaC polypeptides.
The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to ENaC subunit proteins, e.g., the ENaC alpha, beta, gamma or delta subunits as encoded by SEQ ID NO:1, 2, 3, or 7, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with ENaC subunit proteins i.e., ENaC alpha, beta, gamma or delta subunits, e.g., those having the amino acid sequences contained in SEQ ID NO.: 4, 5, 6 or 8, and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
Assays for Proteins that Modulate ENaC Activity
High or medium throughput functional genomics assays can be used to identify modulators of ENaC which block, inhibit, modulate or enhance salty taste. Such assays can, e.g., monitor changes in cell surface marker expression, changes in intracellular ions, or changes in membrane currents using either cell lines or primary cells or oocytes. Typically, the cells are contacted with a cDNA or a random peptide library (encoded by nucleic acids). The cDNA library can comprise sense, antisense, full length, and truncated cDNAs. The peptide library is encoded by nucleic acids. The effect of the cDNA or peptide library on the phenotype of the cells is then monitored, using an assay as described above. The effect of the cDNA or peptide can be validated and distinguished from somatic mutations, using, e.g., regulatable expression of the nucleic acid such as expression from a tetracycline promoter. cDNAs and nucleic acids encoding peptides can be rescued using techniques known to those of skill in the art, e.g., using a sequence tag.
Proteins interacting with the peptide or with the protein encoded by the cDNA (e.g., SEQ ID NO: 1, 2, or 7) can be isolated using a yeast two-hybrid system, mammalian two hybrid system, or phage display screen, etc. Targets so identified can be further used as bait in these assays to identify additional components that may interact with the ENaC channel which members are also targets for drug development (see, e.g., Fields et al., Nature 340:245 (1989); Vasavada et al., Proc. Nat'l Acad. Sci. USA 88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci. USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., Proc. Nat'l Acad. Sci. USA 9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463).
Suitable cells and cell lines that express ENaC proteins include, by way of example, kidney epithelial cells, lung epithelial cells, taste epithelial cells and other mammalian epithelial cells, preferably human, and oocytes, preferably amphibian oocytes, most preferably Xenopus oocytes.
Isolation of Nucleic Acids Encoding ENaC Proteins
This invention relies, in part, on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
Nucleic acids that encode ENaC subunits, polymorphic variants, orthologs, and alleles that are substantially identical to an amino acid sequence encoded by SEQ ID NO: 1, 2, 3 or 7 as well as other ENaC family members, can be isolated using ENaC nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening libraries. Alternatively, expression libraries can be used to clone ENaC subunit protein, polymorphic variants, orthologs, and alleles by detecting expressed homologous immunologically with antisera or purified antibodies made against human ENaC or portions thereof.
To make a cDNA library, one should choose a source that is rich in ENaC RNA. The mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel at al., supra).
For a genomic library, the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro. Recombinant phage are analyzed by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977). Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
Alternatively, ENaC cRNA encoding human ENaC subunits may be generated from α, β, γ or Δ human ENaC DNA plasmids using T7 RNA polymers to transcribe cRNA in vitro from DNA linearized with appropriate restriction enzymes and the resultant cRNA microinjected into suitable cells, e.g., oocytes, preferably frog oocytes.
An alternative method of isolating ENaC subunit nucleic acid and its orthologs, alleles, mutants, polymorphic variants, and conservatively modified variants combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of human ENaC directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Degenerate oligonucleotides can be designed to amplify ENaC homologs using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of ENaC encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
Gene expression of ENaC subunits can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, high density polynucleotide array technology, e.g., and the like.
Nucleic acids encoding ENaC subunit proteins can be used with high-density oligonucleotide array technology (e.g., GeneChip™) to identify ENaC protein, orthologs, alleles, conservatively modified variants, and polymorphic variants in this invention. In the case where the homologs being identified are linked to modulation of T cell activation and migration, they can be used with GeneChip™ as a diagnostic tool in detecting the disease in a biological sample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res. 8:435-448 (1998); and Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).
The genes encoding ENaC subunits preferably human ENaC subunits are typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression. These intermediate vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors.
1. Expression in Prokaryotes and Eukaryotes
To obtain high level expression of a cloned gene, such as those cDNAs encoding hENaC subunit, one typically subclones the hENaC subunit nucleic acid sequence into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al., and Ausubel et al, supra. Bacterial expression systems for expressing the ENaC subunit protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, Xenopus oocytes and insect cells are well known in the art and are also commercially available.
In a preferred embodiment of the invention, an oocyte expression system is used to express a functional human ENaC and to examine the effects of specific compounds on sodium transport through ENaC channels. The Xenopus oocyte expression system has previously been used for the expression of ion channels, including ENaC, and in functional studies (Dascal, CRC Crit. Rev. Biotech. (1987) 22(4):317-387; Wagner, et al., Cellular Physiology and Biochemistry (2000) 10:1-12; and Canessa et al., Nature (1994) 367:463-467). In still another embodiment retroviral expression systems may be used in the invention. In another embodiment transient expression systems may be utilized with plasmid-based vectors that are commercially available such as pcDNA 3 and derivatives thereof.
Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site, as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the ENaC subunit encoding nucleic acid in host cells. A typical expression cassette thus contains at least one promoter operably linked to a nucleic acid sequence encoding a ENaC subunit(s) and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor site.
In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Sequence tags may be included in an expression cassette for nucleic acid rescue. Markers such as fluorescent proteins, green or red fluorescent protein, β-gal, CAT, and the like can be included in the vectors as markers for vector transduction.
Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Expression of proteins from eukaryotic vectors can be also regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.
In one embodiment, the vectors of the invention may have a regulatable promoter, e.g., tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule control on the expression of the candidate target nucleic acids. This beneficial feature can be used to determine that a desired phenotype is caused by a transfected cDNA rather than a somatic mutation.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a ENaC encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
Standard transfection methods may be used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of ENaC protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983). Altternatively, in a preferred embodiment of the invention, oocytes that express human ENaC subunits are produced by microinjection of cRNA encoding said subunits therein.
Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, lipids optimized for DNA transfection, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one ENaC subunit gene into a host cell, preferably mammalian capable of expressing functional ENaC.
After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of ENaC subunit(s). In one embodiment, the cells are transiently transfected with all three hENaC genes using lipid-based transfection and cultured for 24-48 hours prior to performing the screen for ENaC modulators.
As noted previously, a preferred embodiment of the invention comprises an oocyte expression system. Those methods generally include frog surgery, oocyte isolation, cRNA preparation and oocytes microinjection. General procedures for frog surgery and oocyte isolation are conventionally known in the art. (See, Marcus-Sekur, et al., Methods in Enzymol. 152:284-288 (1987); Goldin, Methods in Enzymol. 207:266-279.) Likewise, methods for preparing cRNA are also well known and are reported, e.g., in Swansen, et al., Meth. Enzymol. 207:310-319 (1912), Golden, et al., Meth. Enzymol. 217:279-297 (1992). The resultant cRNA is then microinjected into frog oocytes by standard methods. (See, Molten, et al., Meth. Enzymol. 254:458-466 (1975); Hitchcock et al., Meth. Enzymol. 152:276-284 (1987).
A. Assays
Modulation of an ENaC protein can be assessed using a variety of assays; preferably cell-based models as described above. Such assays can be used to test for inhibitors and activators of ENaC, which modulate, block, enhance or inhibit salty taste perception.
Preferably, the ENaC will be comprised of three subunits, alpha (or delta), beta and gamma and preferably the human ENaC subunit encoded by the encoded by SEQ ID NO: 1, 2, 3 or 7 or a human ortholog a conservatively modified variant thereof. Alternatively, the ENaC of the assay will be derived from a non-human epithelial cell. Generally, the amino acid sequence identity of each respective subunit will be at least 80%, preferably at least 85%, or 90%, most preferably at least 95%, e.g., 96%, 97%, 98% or 99% to the polypeptide encoded by SEQ ID NO: 1, 2, 3 or 7.
Measurement of the effect of a candidate comprised or an ENaC protein or cell expressing ENaC protein, either recombinant or naturally occurring, can be performed using a variety of assays, as described herein. Preferably to identify molecules capable of modulating ENaC, assays are performed to detect the effect of various candidate modulators on ENaC activity in an amphibian oocyte or mammalian cell that expresses a functional ENaC. Preferably, such assays will initially contact ENaC with a known ENaC inhibitor prior to the addition of at least one putative ENaC modulator, e.g., ENaC enhancer. Preferably, the inhibitor will be amiloride or an amiloride derivative such as Phenamil.
The channel activity of ENaC proteins can be assayed using a variety of assays to measure changes in ion fluxes including patch clamp techniques, measurement of whole cell currents, radiolabeled ion flux assays, and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Hoevinsky et al., J. Membrane Biol. 137:59-70 (1994)) and ion-sensitive dyes. For example, nucleic acids encoding one or more subunits of an ENaC protein or homologue thereof can be injected into Xenopus oocytes. Channel activity can then be assessed by measuring changes in membrane current. One means to obtain electrophysiological measurements is by measuring currents using patch clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595, 1997). Whole cell currents can be determined using standard methodology such as that described by Hamil et al., Pflugers. Archiv. 391:185 (1981).
Channel activity is also conveniently assessed by measuring changes in intracellular ion levels for example using ion sensitive dyes.
The activity of ENaC polypeptides can be also assessed using a variety of other assays to determine functional, chemical, and physical effects, e.g., measuring the binding of ENaC polypetides to other molecules, including peptides, small organic molecules, and lipids; measuring ENaC protein and/or RNA levels, or measuring other aspects of ENaC polypeptides, e.g., transcription levels, or physiological changes that affects ENaC activity. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as changes in cell growth or pH changes or changes in intracellular second messengers such as IP3, cGMP, or cAMP, or components or regulators of the phospholipase C signaling pathway. Such assays can be used to test for both activators and inhibitors. Modulators thus identified are useful for, e.g., as flavorants in foods, beverages and medicines.
Cell-based Assays
In another embodiment, at least one ENaC subunit protein is expressed in a cell, and functional, e.g., physical and chemical or phenotypic, changes are assayed to identify ENaC modulators. Cells expressing ENaC proteins can also be used in binding assays. Any suitable functional effect can be measured, as described herein. For example, changes in membrane potential, changes in intracellular ion levels, and ligand binding are all suitable assays to identify potential modulators using a cell based system. Suitable cells for such cell-based assays include both primary cells, e.g., taste epithelial cells that expresses an ENaC protein and cultured cell lines such as human kidney two hundred ninety three T cells that express an ENaC. Another preferred expression system will comprise amphibian oocytes. As noted, these assays will preferably initially contact the ENaC expression cell line, e.g., amphibian oocytes or human kidney two hundred ninety three with a known ENaC inhibitor, e.g., amiloride or an amiloride derivative such as Phenamil at a concentration that partially inhibits ENaC function, prior to contacting the cell line with at least one putative ENaC modulator. The ENaC protein can be naturally occurring or recombinant. Also, as described above, fragments of ENaC proteins or chimeras with ion channel activity can be used in cell based assays.
In yet another embodiment, cellular ENaC polypeptide levels are determined by measuring the level of protein or mRNA. The level of ENaC protein or proteins related to ENaC ion channel activation are measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the ENaC polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNase protection, dot blotting, is preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, ENaC expression can be measured using a reporter gene system. Such a system can be devised using an ENaC protein promoter operably linked to a reporter gene such as chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, β-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as red or green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)). The reporter construct is typically transfected into a cell. After treatment with a potential modulator, and preferably prior thereto treatment with a known ENaC inhibitor, e.g., Phenamil, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
In another embodiment, a functional effect related to signal transduction can be measured. An activated or inhibited ENaC will alter the properties of target enzymes, second messengers, channels, and other effector proteins. Assays for ENaC activity include cells that are loaded with ion or voltage sensitive dyes to report channel activity, e.g., by observing membrane depolarization or sodium influx. Assays for determining activity of such receptors can also use known antagonists for ENaC, such as amiloride or phenamil, as controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane potential will be monitored using an ion sensitive or membrane potential fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 2002 Catalog: (www.probes.com). and specific compounds disclosed infra.
A preferred assay system will use frog oocytes injected with ENaC cRNAs which are contacted with a test compound and then analyzed by the two-electrode voltage clamp electrophysiological recording technique. (See Stuhmer, Meth. Enzymol. 207:319-339 (1992); Wagner et al., Cellular Physiology and Biochemistry 10:1-12 (2000)).
Electrophysiological Assay
As noted, a preferred assay for identification of cmpounds that modulate, i.e., enhance, inhibit or block ENaC comoprises an electrophysiological assay that monitors changes in electrical current in cells that express human ENaC subunits that are contacted with at least one putative ENaC modulator (enhancer or inhibitor). These assays may use any cell that expresses a functional ENaC. In the preferred embodiment, the cells will comprise oocytes, preferably frog oocytes, mammalian cells, yeast cells or insect cells, or another expression system that is suitable for expressing a functional ENaC ion channel. Preferably, the expression system will exhibit robust and rapid human ENaC sodium channel expression and desirably will not express any or very few endogenous ion channels, thereby facilitating the identification of compounds that specifically modulate ENaC sodium channel function. Thereby, an undesirable background response is minimized or eliminated. Moreover, robust cells, such as oocytes, are desirable as this enables the cells to be reused in assays according to the invention. Oocytes have been reported previously to rapidly and robustly express other functional ion channels including ENaCs (Pascal, CRC Crit. Rev. Biotech. 22(4):317-87 (1987); Wagner et al., Cell Physiol. Biochem. 10:1-12 (2000); Canessa et al., Nature 367:463-467 (1994)).
A particularly preferred electrophysiological assay is a moderate throughput assay that measures ENaC sodium channel function in frog oocytes by the two-electrode voltage clamp technique. This robust, fast expression system provides for the expression of ˜1 million ENaC channels in an oocyte membrane after only about 18-24 hours. Moreover, because oocytes are relatively large (1 mm in diameter, relatively large compared to most mammalian cells), they are easy to handle and work with.
Based on these advantages, a single oocyte can be used to obtain multiple and repetitive electrophysiological recording. Also, an oocyte typically expresses few endogenous channels, and expression is at levels below that which cause high background relative to the background seen in some other expression systems, e.g., human kidney two hundred ninety three T cells. Thereby, oocytes allow for repeated direct measurement of the effect of target compounds on ENaC sodium channel function.
In a preferred two-electrode voltage clamp assay according to the invention (exemplified in detail in the Example 4 infra), frog oocytes that have been microinjected with ENaC α, β, and γ human ENaC cRNAs (or δ, β and γ) human ENaC cRNAs) are transferred to glass scintillation vials and incubated under appropriate conditions to facilitate ENaC protein expression.
After ENaC sodium ion channel expression is obtained, typically around 24 hours post-cRNA microinjection, ENaC function is measured according to the two-electrode voltage clamp technique using an appropriate two-electrode voltage measuring device, e.g., OpusXpress 6000A parallel oocyte voltage clamp system (Axon Instruments). The two-electrode voltage clamp technique measures the macroscopic electrical current flowing across the entire oocyte membrane through the ENaC sodium ion channels. Oocytes are punctured with a voltage-sensing electrode and a current sensing electrode; the voltage, or potential difference across the oocyte membrane, is clamped to a particular value using the voltage-sensing electrode and the current, or the flow of ions across the oocyte membrane, required to maintain the voltage is measured using the current-sensing electrode. The OpusXpresss system is one example of a commerically available two-electrode voltage measuring device which is semi-automated and which comprises a workstation that permits electrophysiological recordings to be made from 8 oocytes simultaneously. This system also provides for automated oocyte impalement and delivery of target compounds by a computer-controlled fluid handler that delivers compound into 96-well compound plates. This system can best be described as a medium or moderate-throughput system as it allows for the evaluation of about 60 compounds per week. Of course more compounds can be screened by the addition of other voltage measuring devices, as described.
In this assay system, ENaC enhancers will result in an increase in current passing through the ENaC channels in the oocyte membrane. This value is calculated by a standard formula provided infra in (Example 4). Such assays also may include appropriate negative controls, e.g., amiloride, which is a known ENaC inhibitor that blocks sodium transport through ENaC channels. Therefore, this compound functions both as an internal control to verify that oocytes express functional ENaC, and, in oocytes exhibiting amiloride inhibition, allows for the screening of putative ENaC enhancers after amiloride compound is applied (if the target compound is an ENaC enhancer it will result in an increase in current passing through ENac channels in the oocyte membrane).
Desirably, a % enhancement factor is calculated for each enhancer. For example, a 100% enhancer increases ENaC activity 100% relative to the basal control value (no compound).
Negative controls are also desirably performed to confirm that oocytes which are not injected with ENaC cRNAs do not exhibit the same effects.
As discussed in greater detail in Example 4 infra, more complex analyses are also desirably performed on compounds that exhibit maximal % enhancement valves e.g., current/voltage (I/V) curves, a miloride competitive experiments and dose-response curves to determine the concentration at which the compound exhibits half-maximal activity (EC50 value). These experiments will further confirm that the effect of the compound is ENaC-specific.
These assays will provide for the identification of ENaC modulators, preferably ENaC enhancers, which may be used as additives for foods, beverages, pharmaceuticals and the like in order to modulate the salty taste associated therewith. Desirably, an ENaC enhancer will exhibit at least 20% enhancement factor, more preferably at least 50% and even more preferably at least an 100% enhancement factor. These oocyte-based assays are discussed in further detail as well as the intrinsic advantages associated therewith in Example 4 of this application.
Animal Models
Animal models that express hENaC also find use in screening for modulators of salty taste. Similarly, transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence or increased expression of the ENaC protein. The same technology can also be applied to make knockout cells. When desired, tissue-specific expression or knockout of the ENaC protein may be necessary. Transgenic animals generated by such methods find use as animal models of responses to salty taste stimuli.
Knockout cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous ENaC gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous ENaC with a mutated version of the ENaC gene, or by mutating an endogenous gene.
A DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
B. Modulators
The compounds tested as modulators of ENaC protein can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide or a ribozyme, or a lipid. Alternatively, modulators can be genetically altered versions of an ENaC protein. Typically, test compounds will be small organic molecules, peptides, lipids, and lipid analogs. Preferably, the tested compounds are safe for human consumption.
Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including ChemDiv (San Diego, Calif.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica-Analytika (Buchs Switzerland) and the like.
In the preferred embodiment, moderate or high throughput screening methods involve providing a small organic molecule or peptide library containing a siginificant number of potential ENaC modulators (potential activator or inhibitor compounds). Such “chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual products. As noted, the preferred oocyte two-voltage clamp electrode system (a single device) permits about 60 compounds to be tested per week.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann at al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
The compounds identified using the disclosed assays, e.g., the electrophysiological (two electrode voltage-clamp technique) assays and fluorescence cell-based assay disclosed in the examples, are potentially useful as ingredients or flavorants in ingestible compositions, i.e., foods and beverages as wells as orally administered medicinals. Compounds that modulate or enhance salty taste perception can be used alone or in combination as flavorants in foods or beverages. In the preferred application, the modulator will be incorporated into a food or beverage with a reduced level of sodium and the salty taste of the resulting product will be similar to that of the high sodium product. Examples of such foods and beverages include snack foods such as pretzels, potato chips, crackers, soups, dips, soft drinks, packaged meat products, among others.
Alternatively, compounds that block or inhibit salty taste perception can be used as ingredients or flavorants in foods that naturally contain high salt concentrations in order to block or camouflage the salty taste thereof.
The amount of such compound(s) will be an amount that yields the desired degree of salty taste perception. Of course compounds used in such applications will be determined to be safe for human consumption and to be acceptable in human taste tests.
Preferred Assay Embodiment Using Phenamil or Equivalent
As disclosed supra, one of the preferred embodiments of the invention will comprise contacting a test cell expressing a functional ENaC with at least one putative modulator compound in the presence of a membrane potential dye, and preferably prior thereto contacting said test cell with at least one compound known to modulate (inhibit) ENaC function, preferably an amiloride derivative such as Phenamil and monitoring the activity of the ENaC expressed by the test cell to determine the extent of ENaC modulation. As noted, the addition of an ENaC inhibitor prior to the test compound improves assay results. This inhibitor, e.g., Phenamil, is used at a concentration that at least partially inhibits ENac function. The method can further comprise evaluating the putative modulator compound for in vivo effects on salty taste perception (e.g., performing tasting experiments to determine the in vivo effect on salty taste perception). For example, cDNAs encoding the ENaC subunits are cloned from human kidney cell cDNA, human lung cell cDNA, or human taste cell cDNA. As mentioned above, native ENaC is a multimeric protein consisting of three subunits (alpha or delta, beta, and gamma). ENaC functions as a constitutively active Na+ selective cation channel, is found in taste buds as well as other tissues, and is a candidate human salt receptor underlying the physiological perception of salty taste.
In another preferred embodiment of the invention, such a method is carried out in a high throughput assay format using multi-well plates and a fluorescence intensity plate reader (e.g., Aurora Biosciences VIPR instrument or Molecular Device's FLIPR instrument). The test cells may be seeded, dye-loaded, optionally, preferably initially contacting the test cells with a known ENaC inhibitor at a concentration whereby ENaC function is at least partially and preferably reversibly inhibited, thereafter contacting said test cell with at least one test compound, and monitoring fluorescence intensity in the same multi-well plate. Such an assay format can reliably detect both activation or inhibition of ENaC function, providing a robust screen for compounds that could either enhance or block channel activity. The assay described above has been optimized to identify ENaC enhancers. The assay described herein thus has advantages over existing assays, such as those described above, in that a human ENaC is utilized, mammalian cells are employed and the assay can be run in standard multi-well (e.g., 96, 384, or 1536 well) plates in high-throughput mode. (However, as discussed above, mammalian cells possess some disadvantageous properties, e.g., they may express endogenous ion channels at levels resulting in undesirable background levels.)
In this preferred embodiment of the invention, cells, preferably mammalian cells, will be produced that functionally express at least the alpha (or delta) subunit of ENaC. In preferred embodiments, all three subunits of hENaC (α or δ, β, and γ) are expressed either transiently or stably. The ENaC subunit(s) employed can be naturally occurring forms, variants containing SNPs, alternatively spliced forms, combinations of forms or any functional variants known in the art (see e.g., accession numbers P37088, P51168, P51170, and P51172). Preferably, the ENaC will be comprised of the human alpha, beta and gamma ENaC subunits encoded by the nucleic acid sequence in SEQ ID NO. 1, 2, 3 or the human beta, gamma and delta ENaC subunits encoded by SEQ ID NO. 2, 3 and 7. The mammalian cells can be any type known in the art such as COS, CHO, BHK, MDCK, human kidney two hundred ninety three, or human kidney two hundred ninety three T (human embryonic kidney cells expressing the large T-cell antigen). Preferably, the cell is human kidney two hundred ninety three T. The cells can be transfected using standard methods known in the art, such as but not limited to Ca2+ phosphate or lipid-based systems, or methods previously mentioned.
These transfected cells are then preferably seeded into multi-well culture plates. Functional expression is then allowed to proceed for a time sufficient to reach at least about 70% confluence, more preferably to at least about 80% confluence or to form a cell layer dense enough to withstand possible fluid perturbations caused by compound addition. Generally, an incubation time of at least 24 hours will be sufficient, but can be longer as well. The cells are then washed to remove growth media and incubated with a membrane-potential dye for a time sufficient to allow the dye to equilibrate across the plasma membranes of the seeded cells. One of skill in the art will recognize that the dye loading conditions are dependent on factors such as cell type, dye type, incubation parameters, etc. In one embodiment, the dye may be used at about 2 μM to about 5 μM of the final concentration. Further, the optimal dye loading time may range from about 30 to about 60 minutes at 37° C. for most cells. In the preferred embodiment, the membrane potential dyes are from Molecular Devices (cat# R8034). In other embodiments, suitable dyes include e.g., single wavelength-based dyes such as DiBAC, DiSBAC (Molecular Devices), and Di-4-ANEPPS (Biotium), or dual wavelength FRET-based dyes such as DiSBAC2, DiSBAC3, and CC-2-DMPE (Aurora Biosciences). [Chemical Names—Di-4-ANEPPS (Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt), DiSBAC4(2) (bis-(1,2-dibarbituric acid)-trimethine oxanol), DiSBAC4(3) (bis-(1,3-dibarbituric acid)-trimethine oxanol), CC-2-DMPE (Pacific Blue™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) and SBFI-AM (1,3-Benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)] bis-, tetra-kis[(acetyloxy)methyl]ester].
In one embodiment, the dye-loaded cells are preferably contacted with a known ENaC inhibitor, e.g., Phenamil, then contacted with test compounds (or controls), and the cell cultures are monitored using standard fluorescence analysis instrumentation such as or VIPR or FLIPR®. The addition of NaCl or other test compounds which pharmacologically act on ENaC elicit a change in membrane potential which is then detected as a change in the resting fluorescence in a standard fluorescence intensity plate reader (e.g., FLIPR) or voltage intensity plate reader (e.g. VIPR). As such, the method of the present invention can be used to identify salty taste modulating compounds by monitoring the activity of ENaC in the test cells through fluorescence. For instance, a decrease in fluorescence may indicate a taste (salty) blocker, while an increase in fluorescence may indicate a taste (salty) enhancer.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting. It is understood that various modifications and changes can be made to the herein disclosed exemplary embodiments without departing from the spirit and scope of the invention.
DNA sequences encoding the alpha, beta and gamma subunit of a human ENaC expressed in human taste cells were cloned from human kidney cells by RT-PCR.
Methods for Cloning Human Epithelium Sodium Channel Subunit DNA Sequences (ENaCs)
Human ENaC cDNAs for α, β and γ ENaC were amplified from human kidney cDNA (Origene Technologies Inc.) by PCR using the following primer pairs, respectively: 5′ CGC GGA TCC GCC CAT ACC AGG TCT CAT G 3′ and 5′ CCG GAA TTC CTG CAC ATC CTT CAA TCT TGC 3′; 5′ CGC GGA TCC AGC AGG TGC CAC TAT GCA C 3′ and CCG CTC GAG GTC TTG GCT GCT CAG TGA G 3′; 5′ CGC GGA TCC CCT CAA AGT CCC ATC CTC G 3′ and 5′ CCG GAA TTC GAC TAG ATC TGT CTT CTC AAC 3′. The primers were designed to be complementary to 5′ and 3′-untranslated region sequence in order to retain the endogenous translation initiation signal, and they introduced terminal restriction endonuclease sites that were used to clone amplified ENaC cDNAs into the mammalian expression vector pcDNA3 (Invitrogen) for functional expression experiments. The cloned ENaC cDNAs were sequenced and compared to ENaC sequences in public DNA databanks. Each cloned subunit is a composite of polymorphisms present in different databank alleles; that is, every polymorphism in each cloned subunit identified by pairwise comparison of the cloned subunit to a databank allele could be found in another databank allele. In addition, polymorphisms in cloned ENaC subunits were verified by sequencing of cloned cDNAs amplified in independent PCR experiments.
The nucleic acid sequences encoding cloned sequences alpha, beta and gamma hENaC subunits are respectively contained in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and the corresponding amino acid sequences in SEQ ID NO: 4, 5 and 6. Each of these DNA sequences was inserted into the expression vector pcDNA3 to produce alpha, beta and gamma subunit plasmids that express human ENaC subunit polypeptides. Also, the nucleic acid sequence for the human amiloride sensitive sodium channel delta subunit (ΔNaCh) is contained in SEQ ID NO: 7, which functions equivalently to the ENaC alpha subunit. The amino acid sequence for the delta subunit is contained in SEQ ID NO: 8. human kidney two hundred ninety three T cells were transiently transfected via Ca2+ phosphate with 1:1:1 weight ratios of αβ, and γsubunit plasmids expressing human ENaC. Such transfection resulted in a Na+ dependent amiloride sensitive fluorescence change, as compared to mock-transfected cells. With reference to
As depicted in
DNA sequences encoding the alpha, beta and gamma subunits of a human ENaC, SEQ ID 1, 2, and 3, respectively, were each cloned into the expression vector pcDNA3 to produce alpha, beta and gamma subunit plasmids that express human ENaC subunit polypeptides. human kidney two hundred ninety three T cells were transiently transfected via lipofection with 1:1:1 weight ratios of αβ, and γ subunit plasmids expressing human ENaC (2 μg of each subunit/20 million cells). Transfected cells were plated into 384-well plates and analyzed on a VIPRII Instrument (Aurora Biosciences) using voltage-sensitive fluorescent dyes. Cells expressing ENaC exhibited a Na+ dependent fluorescence change, as compared to mock-transfected cells (
Methods and Materials for Example 2:
[The procedures herein are conditions for transfection of human kidney two hundred ninety three T cells equivalent to ten screening 384-well plates (200,000,000 cells). These conditions can be altered e.g., by increasing or decreasing cell confluency by use of different size multi-well plates etc.]
Materials
2. Cell Loading
Preparation of CC2-DMPE Loading Buffer
Loading of Cells with CC2-DMPE
Preparation of DiSBAC2(3) Loading Buffer
(can be done during CC2 incubation)
Loading of Cells with DiSBAC2(3) Loading Buffer
Modulation of ENaC functions is monitored in human embryonic kidney human kidney two hundred ninety three cells expressing the three different ENaC subunits. human kidney two hundred ninety three cells are transiently transfected with the ENaC subunit plasmids using lipid-based systems. Transfected human kidney two hundred ninety three cells are seeded into 384-well screening plates and functional expression of ENaC is allowed to proceed for a total of 24 hours. Cells are then labeled with specific dyes (such as DisBac2(3) and CC2-DMPE, Panvera) allowing the detection in subtle changes in membrane potential. Changes in dye fluorescence properties, upon modulation of ENaC functions, are monitored using a Voltage-Intensity-Plate-Reader (VIPR, Panvera). Using this technology, we can detect inhibition of ENaC function (Na+-induced change in membrane potential dye fluorescence) with increasing concentration of a known ENaC inhibitor, Phenamil (
These results indicate that the use of an ENaC inhibitor such as Phenamil, prior to screening with one or more potential ENaC modulators, e.g., ENaC enhancers, enhances signal intensity, thereby significantly improving the likelihood of identifying molecules enhancing ENaC activity in throughput cell-based assays.
The oocyte expression system has intrinsic advantages (expression levels, robust, low endogenous ion channel expression, et al.) that render it useful to examine the effects of compounds on sodium transport through ENaC channels. These compounds are candidates for enhancing salt taste perception. The oocyte expression system has been used earlier for the rapid and robust expression of ion channels, including ENaC, in functional studies (Dascal, CRC Crit. Rev. Biochem. (1987) 22(4): 317-387; Wagner, et al, Cellular Physiology and Biochemistry (2000) 10:1-12; Canessa, et al, Nature (1994) 367: 463-467). Therefore, this system was selected for use in a two-electrode voltage clamp assay using methods and materials as described below.
The oocyte expression system is comprised of the following steps and methodologies, which collectively comprise the screen for ENaC enhancers: frog surgery and oocyte isolation, cRNA preparation, oocyte microinjection, and measurement of ENaC currents in oocytes using two-electrode voltage clamp electrophysiological recordings. The following references describe general practices for frog surgery and oocyte isolation (Marcus-Sekura, et al, Methods in Enzymology (1987) 152: 284-288; Goldin, Methods in Enzymology (1992) 207: 266-279), cRNA preparation (Swanson, et al, Methods in Enzymology (1992) 207: 310-319; Goldin, et al, Methods in Enzymology (1992) 207: 279-297), oocyte microinjection (Matten, et al, Methods in Enzymology (1995) 254: 458-466; Hitchcock, et al, Methods in Enzymology (1987) 152: 276-284), and two-electrode voltage clamp electrophysiological recording (Stuhmer, Methods in Enzymology (1992) 207: 319-339; Wagner, et al, Cellular Physiology and Biochemistry (2000) 10: 1-12). Each of these methodologies, as they pertain to the screen for ENaC enhancers, is described in further detail below.
Frog Surgery and Oocyte Isolation
Female Xenopus laevis South African clawed frogs greater than or equal to 9 cm in length are obtained from NASCO (Fort Atkinson, Wis.). Frogs are anesthetized in 0.15% MS-222 (tricaine or ethyl-3-aminobenzoate methanesulfonate; Sigma) in distilled water and placed on ice. Using sterile surgical tools, sequential 1-2 cm incisions are made in the abdomen through both the outer skin layer and the inner peritoneal layer. Excised ovarian lobes (containing 1000-2000 oocytes) are placed in OR-2 calcium-free media (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES pH 7.5 with NaOH) and sequentially digested with 2 mg/ml collagenase type IA (Sigma), prepared immediately before use, for 45 min followed by 1 mg/ml collagenase type IA for 15 min on a rocking platform at room temperature. After enzymatic digestion, at which point the majority of oocytes are released from the ovarian lobes, oocytes are rinsed in OR-2 without collagenase and transferred to a Petri dish containing Barth's saline (88 mM NaCl, 2 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 2.4 mM NaHCO3, and 5 mM HEPES pH7.5; Specialty Media) supplemented with 2.5 mM sodium pyruvate. Mature stage V or VI oocytes (˜1 mm diameter) containing distinct animal poles, corresponding to the dark side of the egg containing melanin pigment granules, and vegetal poles, corresponding to the light side of the egg containing yolk proteins, are selected for microinjection Frogs are sutured using a C6 needle with a 3-0 black braid suture (Harvard Apparatus) and resused for oocyte isolation following a 2-3 month recovery period.
cRNA Preparation
ENaC cRNA is generated using the mMESSAGE mMACHINE kit according to the manufacturer's instructions (Ambion) from α, β, and γ human ENaC DNA plasmids described in our previous patent (PCT WO 02/087306 A2) using T7 RNA polymerase to transcribe cRNA in vitro from DNA linearized with Eco RI for α and γ ENaC and linearized with Xho I for β ENaC. cRNA quality is checked by denaturing agarose gel electrophoresis and spectrophometric absorbance readings at 260 and 280 nm to ensure that full-length, non-degraded cRNA is generated.
Microinjection
Microinjection needles are pulled on a Model P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co.) using borosilicate glass capillaries (World Precision Instruments Inc.), back-filled with mineral oil (Sigma), and then front-filled with ENaC cRNA using a Nanoliter 2000 injector with a Micro4 MicroSyringe Pump Controller (World Precision Instruments). Oocytes are microinjected in the animal pole with 10-15 nl containing 1-3 ng of each α, β, and γ human ENaC cRNA. Following microinjection, oocytes are transferred to glass scintillation vials containing Barth's solution supplemented with 2.5 mM sodium pyruvate and incubated at 18-19° C. overnight under normal atmospheric conditions. During this time, the oocytes translate injected ENaC cRNA into protein.
Measurement of ENaC Currents in Oocytes Using Two-Electrode Voltage Clamp Electrophysiological Recordings
Twenty-four hours post-microinjection, ENaC function is measured in oocytes using the two-electrode voltage clamp technique on an OpusXpress 6000A parallel oocyte voltage clamp system (Axon Instruments). The two-electrode voltage clamp technique is an electrophysiology method that measures the macroscopic electrical current flowing across the entire oocyte membrane though protein channels such as ENaC (Stuhmer, Methods in Enzymology (1992) 207: 319-339). Oocytes are impaled with a voltage-sensing electrode and a current-sensing electrode; the voltage, or potential difference across the oocyte membrane, is clamped to a particular value using the voltage-sensing electrode and the current, or the flow of ions across the oocyte membrane, required to maintain that voltage is measured using the current-sensing electrode. The OpusXpress system is a semi-automated two-electrode voltage clamp workstation that allows recordings to be made from 8 oocytes simultaneously. Oocyte impalement is automated and compound delivery is performed by computer-controlled fluid handlers from 96-well compound plates. This medium-throughput system dramatically increases the number of compounds we can examine from ˜1 compound per week using a conventional single oocyte voltage clamp system to ˜60 compounds per week using the OpusXpress system.
Oocytes are placed in the OpusXpress system and bathed in ND-96 solution (96 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES pH 7.5 with NaOH). Oocytes are impaled with voltage-sensing and current-sensing electrodes, pulled on a Model P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co.) using borosilicate glass capillaries (World Precision Instruments Inc.) and back-filled with 3M KCl, containing silver chloride wires. Electrodes exhibit resistances between 2-10 Mohm for voltage-sensing electrodes and between 0.5-2 Mohm for current-sensing electrodes. Following impalement, oocytes are voltage clamped to −60 mV and experimental recordings are initiated. Data are acquired at 50 Hz and low-pass filtered at 5 Hz using a 4-pole Bessel filter.
A preferred procedure used to screen a compound for ENaC enhancement in an oocyte assay according to the invention is as follows (
Negative control experiments are performed in oocytes not injected with ENaC cRNA to demonstrate that effects observed with compounds in ENaC expressing oocytes are due to currents flowing through ENaC channels and not due currents flowing through channels endogenously expressed in the oocyte membrane. (Dascal, CRC Crit. Rev. Biochem. (1987) 22(4): 317-387). Compounds specifically enhancing ENaC should not affect currents in uninjected oocytes and should exhibit % enhancement factors of 0 (
More complex analyses are performed on compounds displaying large % enhancement factors and having no effect on oocytes not injected with ENaC cRNA. The assays include current/voltage (I/V) curves, amiloride competition experiments, and dose-response curves. For I/V curves, currents are measured in voltage steps from −100 to +60 mV, in 20 mV increments, in the presence and absence of amiloride, to verify ENaC expression as above, and in the presence and absence of compound, to investigate the magnitude of compound enhancement (
Amiloride competition experiments are desirably performed to demonstrate that compound effects are ENaC dependent (
Dose-response curves are performed to determine the concentration at which the compound exhibits half-maximal activity (EC50) (
A flowchart which schematically illustrates the sequence of experiments performed to examine the effect of a compound on ENaC function is depicted in
In our previous patent application (PCT WO 02/087306 A2), we utilized a high-throughput mammalian cell-based assay in human kidney two hundred ninety three T human embryonic kidney cells that indirectly measured ENaC activity by assaying membrane voltage in ENaC-transfected cells loaded with voltage-sensitive fluorescent probes. Although this approach was high-throughput, and identified some compounds that modulated ENaC, unfortunately, it was not specific, and ˜90% of identified compounds did not directly modulate ENaC function but instead likely modulated the activity of other ion channels endogenously expressed in human kidney two hundred ninety three T cells. The efficacy of such high throughput assays is improved herein by the use of phenamil and similar ENaC inhibitors as described supra. In addition, the subject application also provides a more direct (specific), but lower throughput assay methodology to measure ENaC sodium channel function in oocytes using the two-electrode voltage clamp technique. This system allows rapid and robust expression of ion channels (˜1 million ENaC channels can be expressed in the oocyte membrane after only about 18-24 hours). Other advantages of the oocyte expression system include: oocytes are large (˜1 mm in diameter) and robust making them easy to handle and work with; multiple and repetitive recordings can be obtained from the same oocyte to the same or different compounds of interest; oocytes express few endogenous channels at levels sufficient to cause high background current in comparison to the current stemming from an exogenously expressed ion channel; and oocytes allow direct measurement of ion channel function. Thus, in contrast to assays that indirectly measure ENaC function in human kidney two hundred ninety three T cells using voltage-sensitive probes, the oocyte expression system allows direct measurement of ENaC sodium channel current with virtually no background.
Nucleic Acid and Amino Acid Sequences of ENaC Subunits Used in Examples
While the invention has been described by way of examples and preferred embodiments, it is understood that the words which have been used herein are words of description, rather than words of limitation. Changes may be made, within the purview of the appended claims, without departing from the scope and spirit of the invention in its broader aspects. Although the invention has been described herein with reference to particular means, materials, and embodiments, it is understood that the invention is not limited to the particulars disclosed. The invention extends to all equivalent structures, means, and uses which are within the scope of the appended claims.
This application is a Divisional Patent Application of U.S. application Ser. No. 10/887,233 filed on Jul. 9, 2004, which claims priority from U.S. Provisional Application No. 60/485,745 filed on Jul. 10, 2003. This application further is a continuation-in-part of U.S. Ser. No. 10/133,573 filed on Apr. 29, 2002, all of which are incorporated herein by reference in their entireties.
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| 20120070857 A1 | Mar 2012 | US |
| Number | Date | Country | |
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| 60485745 | Jul 2003 | US |
| Number | Date | Country | |
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| Parent | 10887233 | Jul 2004 | US |
| Child | 13206656 | US |
