WO2008024902A2 - Targeting of fructokinase as therapy for cardiovascular disease, metabolic syndrome, and renal disease - Google Patents

Abstract

Disclosed herein are methods of treating and/or preventing hypertension, metabolic syndrome, and/or renal disease that involve the targeting of Fructokinase (ketohexokinase). Also disclosed herein are methods of screening for inhibitors of Fructokinase. Further disclosed herein are methods of formulating a prescriptive regimen for hypertension, metabolic syndrome, and/or renal disease that comprises evaluating the level of ketohexokinase activity in an individual.

Description

TARGETING OF FRUCTOKINASE AS THERAPY FOR CARDIOVASCULAR DISEASE,

METABOLIC SYNDROME, AND RENAL DISEASE

RELATED APPLICATIONS

[01] This application claims priority to U.S. Serial Nos. 60/862,997 filed October 26, 2006 and U. S. Serial No. 60/823,376 filed August 23, 2006, each of which are each herein incorporated by reference in their entirety.

BACKGROUND

[02] The last 100 years have seen a dramatic increase in the frequency of hypertension, metabolic syndrome, diabetes, and kidney disease. Hypertension now affects over 30% of the population (Fields, L.E., et al., The burden of adult hypertension in the United States 1999 to 2000: a rising tide. Hypertension, 2004. 44(4): p. 398-404); over two thirds of the population are overweight or obese, including 15% of our youth (Flegal, K.M. and R.P. Troiano, Changes in the distribution of body mass index of adults and children in the US population. Int J Obes Relat Metab Disord, 2000. 24(7): p. 807-18); one quarter of the population suffers from the metabolic syndrome characterized by insulin resistance, obesity and hypertriglyceridemia (Ford, E.S., W.H. Giles, and A.H. Mokdad, Increasing prevalence of the metabolic syndrome among U.S. Adults. Diabetes Care, 2004. 27(10): p. 2444-9); and chronic kidney disease has been discovered to be extremely common, affecting nearly 20 million of the population and increasing our risk for cardiovascular disease (Coresh J, Astor BC, Greene T, Eknoyan G. Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. Am J Kidney Dis. 2003 Jan;41(l):l-12).

[03] While genetics have a major influence on phenotype, the rapid rise in these disorders strongly suggests the involvement of an environmental factor. In this regard, the epidemic has been linked with industrialization and introduction of the Western diet (Forrester, T., R.S. Cooper, and D. Weatherall, Emergence of Western diseases in the tropical world: the experience with chronic cardiovascular diseases. Br Med Bull, 1998. 54(2): p. 463-73). It is critical that the potential mechanisms that underlie this great epidemic be identified and new therapies be devised to address this epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[04] FIG. 1: Biochemical reaction catalyzed by Ketohexokinase (KHK, Fructokinase), the key enzyme in the metabolism of fructose

[05] FIG. 2: A classical coupled enzymatic system to measure activity of KHK.

ADP released in KHK reaction is measured after quantitative conversion to NAD+ in two enzyme system. Decrease in absorbance of NADH at 340 nm is a readout for the amplified signal.

[06] FIG. 3: Effect of the presence of fructose in the incubation medium for 72 h on the KHK activity in cell homogenates. The typical assay of KHK activity is shown. Activity of ketohexokinase was measured in cell homogenates by coupled enzymatic assay in the reaction mixture containing 25 mM HEPES (pH 7.1), 6 mM MgCl₂, 25 mM KCl, 10 mM NaF, 5 mM ATP, 5 mM D-fructose, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 40 U/ml pyruvate kinase, 40 U/ml lactate dehydrogenase. Readout was optical density of NADH at 340 nm measured every 30 s. More significant decrease in the optical density indicates an increase in KHK activity in the homogenate of fructose-treated cells.

[07] FIG. 4: Expression of KHK and GLUT5, the fructose transporter, in human and mouse cells, (a) Expression of KHK in mouse adipocytes, GAPDH, glyceraldehyde-3 -phosphate dehydrogenase ("house-keeping" gene), NTC, non-template control. mRNA expression was detected by RT-PCR using Superscript One Step system (InVitrogen). Invereted images of amplified fragments separated in agarose gel are shown, (b) Expression of KHK inhuman cells, (c) Expression of GLUT5 in human WBC and the effect of fructose on the abundance of GLUT5 mRNA is shown. Incubation of cells in the presence of fructose (1 mM) induced dramatic increase in GLUT5 expression in comparison to cells incubated in fructose-free medium for 32 h. β-actin has been used as a "house-keeping" gene.

[08] FIG. 5: Assay for quantitative measurement of mRNA for KHK expression in human cells by real-time qRT-PCR.

[09] FIG. 6. Effect of fructose on the enzymatic activity and expression of KHK in different cell types. The values of KHK activity are presented as % of control. Absolute values of the activity in control samples varied between experiments from 0.43 to 2.34 nmol/min/mg protein. (* - P<0.05, nonparametric U-test). KHK expression at the protein level was detected by Western blotting with KHK-specific polyclonal antibody. Membranes were stripped and re-probed with GAPDH antibody to verify equal protein loading and transfer.

[010] FIG. 7. Inhibition of the KHK enzymatic activity with N-ethylmaleimide.

Cell homogenates were incubated with different concentrations of N-ethylmaleimide followed by measurement of the enzymatic activity as described in Fig. 3.

[Oil] FIG. 8. Effect of attopurinol treatment in rats with fructose-induced metabolic syndrome Hypertension develops in fructose fed (Fr) rats, which is significantly reduced by allopurinol (AP), a xanthine oxidase inhibitor that lowers uric acid. Values are means ± SD. *P

< 0.01 vs. control. #P < 0.05 vs. Fr. BP, blood pressure. Serum triglycerides (TG) are increased in Fr rats, and this is completely prevented by AP. #P < 0.01 vs. control and Fr+ AP. Fructose ingestion was associated with fasting and postprandial hyperinsulinemia. AP (150 mg/1) prevented basal hyperinsulinemia and significantly reduced postprandial hyperinsulinemia. *P

< 0.01 vs. control. #P < 0.05 vs. Fr.

[012] FIG. 9 Uric acid inhibits endothelial nitric oxide dependent vasodilation of aortic segments. Rat Aortic Rings were incubated with various concentrations of uric acid for 1 hour. Following incubation aortic rings were preconstricted with a thromboxane agonist and then the degree of vasodilation in response to acetylcholine determined. Data shown is ± standard error. * p < 0.05 compared to 0 mg/dl of uric acid.

[013] FIG. 10 Increasing uric acid concentrations has a stepwise effect on PPARγ and adiponectin mRNA levels. Undifferentiated cells (white bars) or fully differentiated mouse adipocytes (grey bars) were exposed to the indicated concentration of uric acid. After 12 hours exposure, total RNA was extracted and the levels of mRNA for PPARγ (A) and adiponectin (B) were determined by real-time PCR and normalized to GAPDH levels. Data is ± standard deviation.

[014] FIG. 11 Uric acid treatment of adipocytes results in an increase in reactive oxygen species, a reduction in bioavailable NO, and an increase in nitrosylated proteins.

A. Differentiated adipocytes were incubated with increasing concentrations of uric acid for 30 minutes prior to incubating with H₂DCFDA, a fiuorogenic reagent that detects reactive oxygen intermediates in cells, and quantifying fluorescence by fluorescence microscopy. B. Undifferentiating cells (open bars) or fully differentiated mouse adipocytes (black bars) were incubated with increasing concentration of uric acid for 30' prior to incubation with diaminofluorescein-FM (DAF-FM; Molecular Probes, Eugene, OR), which fluoresces in the presence of intracellular bioavailabe NO. The degree of fluorescence and thus intracellular bioavailable NO was determined by confocal microscopy. C. Fully differentiated adipocytes were incubated for 30' in the presence or absence of uric acid, 15 mg/dl, prior to lysing the cells, performing a SDS-PAGE, transferring to nitrocellulose and probing with an antibody against nitrosylated proteins. Data is ± standard deviation.

[015] FIG. 12. Patients with NAFLD have increased expression of GLUT-5 and fructokinase in their livers. Liver biopsies from 3 patients with NAFLD (black bars) and 3 patients with normal livers (open bar) were obtained and RNA isolated. Real-time PCR was used to determine the level mRNA for GLUT-5 (A) and fructokinase (B) and normalized to GAPDH. Data is shown ± standard deviation.

DETAILED DESCRIPTION

[016] Researchers associated with the subject invention have discovered that elevated serum uric acid is strongly associated with cardiovascular disease, particularly with hypertension, metabolic syndrome, and renal disease. Nevertheless, it remains controversial if the elevated uric acid has a pathogenic role in these conditions or whether it simply represents an associated 'marker'. Epidemiological studies have not resolved this issue, and experimental studies have been few. The researchers have developed an animal model of mild hyperuricemia and also performed studies in cell culture that provided the first direct evidence that uric acid plays a causal role in the pathogenesis of hypertension and renal disease. The researchers have been involved with the discovery that uric acid has a causal role in the metabolic syndrome.

[017] Not to be bound by any theory, the researchers believe that fructose intake (primarily from sugar and sweeteners) has a causal role in the obesity epidemic, and have demonstrated in animal models a potential mechanism. Namely, fructose is the only sugar that rapidly raises serum uric acid, and the researchers can demonstrate that interventions that prevent the rise in uric acid can block the development of insulin resistance, hypertriglyceridemia, and hypertension. It is believed that the mechanism by which uric acid induces the metabolic syndrome is by inhibiting endothelial NO (which accounts partly for insulin-mediated actions) and by directly activating adipocytes. The researchers have demonstrated that uric acid inhibits endothelial NO levels and can activate adipocytes to express a prediabetic phenotype. Based on these discoveries, the researchers further discovered that the metabolic syndrome and cardiovascular disease can be controlled via targeting of enzymes responsible for metabolizing fructose.

Fructose : Metabolism and kinetics

[018] Fructose (also known as fruit sugar or levulose) is a monosaccharide that is naturally present in honey and fruits. It also is present in table sugar (sucrose, which is a disaccharide consisting of one glucose and one fructose molecule) and in high fructose corn syrup (HFCS, in which the fructose content is either 42% or 55%). hi the human gut, sucrose is enzymatically hydrolyzed to fructose and glucose, the former is absorbed via the fructose transporter, Glut-5 (Hallfrisch, J., Metabolic effects of dietary fructose. Faseb J, 1990. 4(9): p. 2652-60). The fructose that is not absorbed is degraded by colonic bacteria, releasing ketoacids in the process (Davids, M.R., et al., An unusual cause for ketoacidosis. Qjm, 2004. 97(6): p. 365-76).

[019] Once absorbed, fructose is taken up primarily in the liver hepatocytes (70%) via Glut-5. However a smaller amount is metabolized by tubular cells within the kidney, in adipocytes, and to a lesser extent other organs in the body. Fructose is metabolized within the cell via two major enzyme pathways. The dominant pathway is via fructokinase (ketohexokinase or KHK), but fructose also weakly competes with glucose via the hexokinase (glucokinase) pathway. KHK catalyses phosphorylation of fructose at the expense of one molecule of ATP which is hydrolysed in this reaction to ADP, see FIG. 1. Fructokinase consists of two forms. Fructokinase C (KHK-C) is the major form present in liver and kidney, whereas Fructokinase A (KHK-A) is present throughout the body. Both KHK-A and KHK-C have polymorphisms at the 49 amino acid position (KHK-A ile and KHK-A val, respectively) but it remains unknown if these polymorphisms have different specific activities (Bonthron, D.T., et al., Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase). Hum MoI Genet, 1994. 3(9): p. 1627-31).

[020] Once inside the hepatocyte, fructose is rapidly phosphorylated at the 1 -position by KHK, and then is acted on by a series of enzymes including aldolase-B and triokinase to generate dihydroxyacetone phosphate, glyceraldehydes, and triose phosphates. Because there is no negative regulatory feedback, as is observed with the glucokinase pathway, all of the fructose is rapidly metabolized, resulting in immediate intracellular depletion of ATP and phosphate. This depletion activates several enzymes including AMP deaminase, resulting in the generation of uric acid, as well as the production of lactate (Fox, LH. and W.N. Kelley, Studies on the mechanism of fructose-induced hyperuricemia in man. Metabolism, 1972.21(8): p. 713-21). Triglycerides are also generated in the process (Havel, PJ., Dietary fructose: implications for dysregulation of energy homeostasis and lipid/carbohydrate metabolism. Nutr Rev, 2005. 63(5): p. 133-57).

The metabolic syndrome

[021] The term "metabolic syndrome" dates back to at least the late 1950's. Originally the term was used to indicate a pre-diabetic state but more recently it has become recognized as a primary risk factor for cardiovascular disease. Originally, one of the risk factors for cardiovascular disease included gout as well as upper body obesity and diabetes (Vague, J., [Obesity in the development of arteriosclerosis and diabetes.]. Sem Hop, 1954. 30(58): p. 3244-6). The ability of a hypocaloric, low carbohydrate diet to reverse the hyperglycemic state, hypercholesterolemia, and hypertriglyceridemia associated with obesity was recognized as early as 1965.

[022] Haller, in 1977 used the term "metabolic syndrome" for the association of obesity, diabetes mellitus, hyperuricemia, staeatosis hepatitis (what is now termed non-alcoholic fatty liver disease), and hyperlipidemia with the increased risk of atherosclerosis (Haller, H., [Epidermiology and associated risk factors of hyperlipoproteinemia] . Z Gesamte Inn Med, 1977. 32(8): p. 124-8).

[023] The factors that go into various definitions of the metabolic syndrome are highly concordant and the diagnosis of the metabolic syndrome increases the risk for cardiovascular disease at every LDL cholesterol level. The Adult Treatment Panel (ATP) III of the National Cholesterol Education Program (NCEP) defined the metabolic syndrome as occurring when an individual has three or more of the following risk factors: 1) Abdominal Obesity determined by waist circumference (Men > 102 cm; Women > 88 cm); 2) Triglycerides > 150 mg/dL; 3) Low HDL cholesterol levels (Men < 40 mg/dL; Women < 50 mg/dL); 4) Blood pressure > 130/> 85 mmHg; 5) Fasting glucose ≥ 110 mg/dL. For purposes herein, metabolic syndrome comprises at least 3 of these 5 characteristics exhibited in an individual [024] The International Diabetes Federation has proposed a new definition for metabolic syndrome The IDF Consensus worldwide definition of the metabolic syndrome http://www.idf.org/webdata/docs/metac_syndrome_def.pdf, April 14, 2005 and more recently the American Heart Association in conjunction with the National Heart, Lung, and Blood Institute has proposed a revised version of the definition (Grundy, S. M., et al., Diagnosis and Management of the Metabolic Syndrome: An American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation, 2005. 112(17): p. 2735-2752). While all three definitions use the same five factors to make the diagnosis, the thresholds for each of the factors do differ. The IDF uses a different threshold for fasting glucose and central obesity than does the NCEP -ATPIII definition in addition obesity is required to make the diagnosis of the metabolic syndrome according to the IDF definition. The recent AHA/NHLBI statement makes only minor changes to the NCEP-ATPIII definition, most notably the lowering of the threshold for elevated fasting glucose to 100 mg/dl. Additionally, both the IDF and AHA/NHLBI definitions state that the use of pharmacologic agents to lower triglycerides, rise HDL, treat hypertension, or lower glucose would qualify as having that risk factor.

[025] According to one embodiment, the subject invention pertains to a method of screening for drugs capable of inhibiting KHK and in turn lowering UA concentration to prevent or treat hypertension; metabolic syndrome, obesity and/or diabetes.

[026] According to another embodiment, the invention pertains to a method of determining activity of KHK to determine appropriate fructose restriction diet or therapeutic regimen of therapeutic agent to down regulate or inhibit enzyme activity or expression. Thus, KHK activity can be used as a diagnostic to observe uric acid mediated conditions and to devise an appropriate prescription.

[027] According to another embodiment, the invention pertains to a therapeutic agent targeting KHK, wherein inhibition prevents or treats uric acid mediated conditions.

[028] In a further embodiment, the subject invention is directed to a method of preventing or treating hypertension; metabolic syndrome, obesity and/or diabetes comprising inhibiting KHK. KHK can be inhibited by a number of means including silencing via siRNA directed to a portion of the sedquece described at the genbank accession numbers provided below. See U. S Patent Publication 20060110440 for background on siRNA silencing. As discussed above, agents can be developed to silence KHK genes to achieve a beneficial effect on metabolic syndrome symptoms and cardiovascular disease. In certain embodiments, silencing of human KHK genes should be based on the sequences for two isoforms of the enzyme: KHK-C (predominant form of KHK, Gen Bank Accession # NM_006488 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=5670341) SEQ. ID. Nos 1 & 2 and KHK-A (Gen Bank Accession#

NM_000221(http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=4557692) SEQ. ID. Nos. 3 & 4.

1. Screening Methods

[029] The invention provides assays for screening test compounds which bind to or modulate the activity of a KHK polypeptide or bind to and inhibit or affect expression of a KHK polynucleotide. A test compound preferably binds to a KHK polypeptide. More preferably, a test compound decreases or increases KHK activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

1.1. Test Compounds

[030] Test compounds relate to agents that potentially have therapeutic activity, i.e., bind to or modulate the activity of a KHK polypeptide or bind to or affect expression of a KHK polynucleotide. Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997. [031] Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. NatL. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994).

1.2. High Throughput Screening

[032] Test compounds can be screened for the ability to bind to and inhibit KHK polypeptides or polynucleotides or to affect KHK activity or KHK gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used.

1.3. Binding Assays

[033] For binding assays, the test compound is preferably, but not necessarily, a small molecule which binds to and occupies, for example, the active site of the KHK polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

[034] In binding assays, either the test compound or the KHK polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the KHK polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

[035] Those skilled in the art equipped with teachings herein will appreciate that there are IQ multiple conventional methods of detecting binding of a test compound. For example, binding of a test compound to a KHK polypeptide can be determined without labeling either of the interactants. A microphysiometer can be used to detect binding of a test compound with a KHK polypeptide. A microphysiometer (e.g., CYTOSENSOR ^TM) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a KHK polypeptide (McConnell et al., Science 257, 19061912, 1992).

[036] In another alternative example, determining the ability of a test compound to bind to a KHK polypeptide can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal Chem. 63, 23382345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[037] In yet another aspect of the invention, a KHK polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al., J. Biol. Chem. 268, 1204612054, 1993; Bartel et al., BioTechniques 14, 920924, 1993; Iwabuchi et al., Oncogene 8, 16931696, 1993; and Brent WO94/ 10300), to identify other proteins which bind to or interact with the KHK polypeptide and modulate its activity.

[038] In many screening embodiments, it may be desirable to immobilize either the KHK polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the KHK polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the KHK polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a KHK polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

[039] In a specific embodiment, the KHK polypeptide may be a fusion protein comprising a domain that allows the KHK polypeptide to be bound to a solid support. For example, glutathione S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the nonadsorbed KHK polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

[040] Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a KHK polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated KHK polypeptides (or polynucleotides) or test compounds can be prepared from biotinNHS(Nhydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavi din-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a KHK polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the KHK polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

[041] Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the KHK polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the KHK polypeptide, and SDS gel electrophoresis under non-reducing conditions. [042] Screening for test compounds which bind to a KHK polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a KHK polypeptide or polynucleotide can be used in a cell-based assay system. A KHK polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a KHK polypeptide or polynucleotide is determined as described above.

1.4. Enzyme Assays

[043] Test compounds can be tested for the ability to increase or decrease the KHK activity of a KHK polypeptide. KHK activity can be measured such as by that described in the Examples. Enzyme assays can be carried out after contacting either a purified KHK polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases TGS activity of a KHK polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing KHK activity. A test compound which increases TGS KHK polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing TGS activity.

7.5. Gene Expression

[044] In another embodiment, test compounds which increase or decrease KHK gene expression are identified. A KHK polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the KHK polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression. [045] The level of KHK niRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a KHK polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a KHK polypeptide.

[046] Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses a KHK polynucleotide can be used in a cell-based assay system. The KHK polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

2. Pharmaceutical Compositions

[047] The invention also pertains to pharmaceutical compositions comprising one or more therapeutic agents that are identified by screening methods that utilize KHK polypeptides and/or polynucleotides. Therapeutic agent(s) can be administered to a patient to achieve a therapeutic effect, i.e. useful in modulating KHK activity and in turn, treating and/or preventing metabolic syndrome, hypertension and cardiovascular disease. Pharmaceutical compositions of the invention can comprise, for example, therapeutic agents identified by a screening method embodiment described herein, which are identified by their ability to bind to or affect activity of KHK polypeptides, or bind to and/or affect expression KHK polynucleotides. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

[048] In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[049] Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa., which is incorporated herein by reference). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

[050] This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a therapeutic agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (for example, but not limited to., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, siRNA or a KHK polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above described screening assays for treatments as described herein.

[051] Those skilled in the art will appreciate that numerous delivery mechanisms are available for delivering a therapeutic agent to an area of need. By way of example, the agent may be delivered using a liposome as the delivery vehicle. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.

[052] A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 106 cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 run, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

[053] Suitable liposomes for use in the present invention include those liposomes conventionally used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

[054] Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Pat. No. 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about

1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

[055] In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).

2.1 Determination of a Therapeutically Effective Dose

[056] The determination of a therapeutically effective dose of therapeutic agents identified by a screening method herein is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which modulates KHK activity compared to that which occurs in the absence of the therapeutically effective dose.

[057] Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 /ED50.

[058] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

[059] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

[060] Preferably, an therapeutic agent reduces expression of a KHK gene or the activity of a KHK polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a KHK gene or the activity of a KHK polypeptide can be assessed such as by hybridization of nucleotide probes to KHK-specific mRNA, quantitative RT-PCR, immunologic detection of a KHK polypeptide, or measurement of KHK activity.

[061] In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be IZ made by one of ordinary skill in the art, according to conventional pharmaceutical principles.

The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Any of the therapeutic methods described above can be applied to any subject in need of such therapy.

3. Polypeptides

[062] KHK polypeptides according to the invention comprise at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 265 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 and 4, or a biologically active variant thereof, as defined below. A KHK polypeptide of the invention therefore can be a portion of a KHK protein, a full-length KHK protein, or a fusion protein comprising all or a portion of KHK protein.

3.1 Biologically Active Variants

[063] KHK polypeptide variants which are biologically active, i.e., confer an ability to phosphorylate fructose, also are considered KHK polypeptides for purposes of this application. Preferably, naturally or non-naturally occurring KHK polypeptide variants have amino acid sequences which are at least about 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative KHK polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

[064] Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. [065] Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a KHK polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active KHK polypeptide can readily be determined by assaying for KHK activity, as described for example, in the specific Examples, below.

3.2 Fusion Proteins

[066] In some embodiments of the invention, it is useful to create fusion proteins. By way of example, fusion proteins are useful for generating antibodies against KHK polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a KHK polypeptide. Protein affinity chromatography or library-based assays for protein—protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

[067] A KHK polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. For example, the first polypeptide segment can comprise at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length KHK protein.

[068] The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include galactosidase, glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the KHK polypeptide-encoding sequence and the heterologous protein sequence, so that the KHK polypeptide can be cleaved and purified away from the heterologous moiety. [069] Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

4. Polynucleotides

[070] A KHK polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a KHK polypeptide. A coding sequence for KHK polypeptide of SEQ ID NO: 2 or 4 is shown in SEQ ID NO: 1 or 3, respectively.

[071] Degenerate nucleotide sequences encoding KHK polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO: 1 also are KHK-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affme gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of KHK polynucleotides which encode biologically active KHK polypeptides also are KHK polynucleotides.

4.1 Identification of Polynucleotide Variants and Homologs

[072] Variants and homologs of the KHK polynucleotides described above also are KHK polynucleotides. Typically, homologous KHK polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known KHK polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2 X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. [073] Species homologs of the KHK polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5° C with every 1% decrease in homology (Bonner et al, J. MoI. Biol. 81, 123 (1973). Variants of KHK polynucleotides or polynucleotides of other species can therefore be identified by hybridizing a putative homologous KHK polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

[074] Nucleotide sequences which hybridize to KHK polynucleotides or their complements following stringent hybridization and/or wash conditions also are KHK polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2^nd ed., 1989, at pages 9.50-9.51.

[075] Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C below the calculated T_mof the hybrid under study. The T_mof a hybrid between a KHK polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m=81.5° C-16.6(log₁₀ [Na⁺ ])+0.41(% G+C)-0.63(% formamide)-600/l), where l=the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4 X SSC at 65° C, or 50% formamide, 4 X SSC at 42° C, or 0.5 X SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2 X SSC at 65° C. 4.2 Preparation of Polynucleotides

[076] A naturally occurring KHK polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated KHK polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises KHK nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

[077] KHK DNA molecules can be made with standard molecular biology techniques, using KHK mRNA as a template. KHK DNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention. The inventors have successfully demonstrated this approach.

[078] Alternatively, synthetic chemistry techniques can be used to synthesize KHK polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a KHK polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.

4.3 Expression of Polynucleotides

[079] To express a KHK polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding KHK polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

[080] A variety of expression vector/host systems can be utilized to contain and express sequences encoding a KHK enzyme polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

[081] The control elements or regulatory sequences are those nontranslated regions of the vector enhancers, promoters, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJoIIa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a KHK polypeptide, vectors based on S V40 or EBV can be used with an appropriate selectable marker. 5. Host Cells

[082] According to certain embodiments of the subject invention, a KHK polynucleotide will need to be inserted into a host cell, for expression, processing and/or screening. A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed KHK polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Posttranslational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

[083] Stable expression is preferred for long-term, high yield production of recombinant proteins. For example, cell lines which stably express KHK polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 12 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced KHK sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R. I. Freshney, ed., 1986.

5.7 Detecting Expression

[084] A variety of protocols for detecting and measuring the expression of a KHK polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a KHK polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A

LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158, 12111216, 1983).

5.2 Expression and Purification of Polypeptides

[085] Host cells transformed with nucleotide sequences encoding KHK polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellular^ depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode KHK polypeptides can be designed to contain signal sequences which direct secretion of soluble KHK polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound KHK polypeptide.

6. Antibodies

[086] Antibodies are referenced herein and various aspects of the subject invention utilize antibodies specific to KHK polypeptide(s). As described above, one example of an therapeutic agent may pertain to an antibody. Any type of antibody known in the art can be generated to bind specifically to an epitope of a KHK polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a KHK polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

[087] An antibody which specifically binds to an epitope of a KHK polypeptide can be used therapeutically, as mentioned, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen. Antibodies useful for embodiments of the subject invention may be polyclonal, but are preferably monoclonal antibodies.

7. Ribozymes

[088] Ribozymes may be one category of test compounds potentially useful as therapeutic agents for modulating KHK activity. Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 15321539; 1987; Cech, Ann. Rev. Biochem. 59, 543568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641 ,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

[089] Accordingly, another aspect of the invention pertains to using the coding sequence of a KHK polynucleotide to generate ribozymes which will specifically bind to mRNA transcribed from the KHK polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).

[090] Specific ribozyme cleavage sites within a KHK RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate KHK RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

[091] Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease KHK expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

[092] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

[093] Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995); Arabidopsis, Meyerowitz et al, Eds., Cold Spring Harbor Laboratory Press, New York (1994) and the various references cited therein.

8. siRNA

[094] KHK can be inhibited by a number of means including silencing via siRNA directed to a portion of the sedquece described at the genbank accession numbers provided above. siRNA molecules can be prepared against a portion of SEQ. ID. Nos 1 and 3 according to the techniques provided in U. S Patent Publication 20060110440. As discussed above, ZL agents can be developed to silence KHK genes to achieve a beneficial effect on metabolic syndrome symptoms and cardiovascular disease. In certain embodiments, silencing of human KHK genes should be based on the sequences for two isoforms of the enzyme:

ILLUSTRATIVE EXAMPLES:

Example 1: Fructose induce metabolic syndrome is mediated by uric acid in rats

[095] The researchers tested the hypothesis that fructose-induced metabolic syndrome might be partially mediated by fructose-induced hyperuricemia. First, the researchers pair fed rats either 60% glucose or 60% fructose diet in order to determine if the development of the metabolic syndrome was specific for fructose. After 4 weeks only the fructose-fed rats developed features of metabolic syndrome, such as fasting hyperinsulinemia (204±62 vs 112±43 pM), fasting hypertriglyceridemia (419±60 vs 112±28 mg/dl), and hyperuricemia (2.1±0.9 vs 1.4±0.3 mg/dl) (P<0.05 for all comparisons) (see Table 2. Nakagawa, T., et al., A causal role for uric acid in fructose-induced metabolic syndrome. Am J Physiol Renal Physiol, 2006. 290(3): p. F625-31). The observation that fructose administration raises uric acid in rats which have uncase is consistent with the hypothesis that there is a sudden and marked increase in uric acid production from the rapid phosphorylation of fructose in hepatocytes that overrides the presence of uricase.

[096] In a second experiment rats were fed fructose for 4 weeks to induce metabolic syndrome, and then were treated with either allopurinol or placebo for an additional 6 weeks. An additional control group received a normal diet (containing 46% carbohydrate). Allopurinol significantly reduced uric acid and improved features of the metabolic syndrome, including an improvement in blood pressure (FIG. 8) and triglyceride levels (FIG. 8). Insulin sensitivity was assessed with an oral glucose tolerance test using DeFronzo's formula (Matsuda, M. and R.A. DeFronzo, Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care, 1999. 22(9): p. 1462-70). While plasma glucose was not altered by fructose or with allopurinol treatment, fructose-induced hyperinsulinemia (FIG. 8) was improved with allopurinol as well as insulin sensitivity (p<0.05).

[097] Next allopurinol was administered in the drinking water with the initiation of the fructose diet. The administration of allopurinol prevented fructose induced hyperinsulinemia (272 vs.161 pmol/L, p<0.05), systolic hypertension (142 vs. 133 mmHg, p<0.05), hypertriglyceridemia (234 vs. 65 mg/dl, p<0.01) and weight gain (455 vs. 425 g, p<0.05) at 8 weeks. The mechanism by which allopurinol prevents weight gain will be evaluated in this proposal. However, evidence that it is the lowering of uric acid is important is based on the fact that a uricosuric agent, benzbromarone, also could prevent hyperuricemia (l.l±0.4 vs 2.1±0.9 mg/dl), fasting hyperinsulinemia (147±42 vs 204±62 pmol/L) and hypertriglyceridemia (293±86 vs 419±60 mg/dl) (4 week data, all p<0.05)[l]. This suggests that the mechanism of protection involves the lowering of uric acid.

[098] The effect of lowering uric acid on the features of the metabolic syndrome was investigated by treating rats with fructose-induced metabolic syndrome with allopurinol. Rats were fed fructose for 4 weeks to induce features of metabolic syndrome (Table 1) and then randomized to allopurinol or placebo for an additional 4 weeks. Allopurinol led to a significant decrease in the uric acid level of the rats, hi addition, fasting triglycerides decreased by 70% and fasting insulin levels were significantly lowered. Although there was no change in fasting blood sugars, the insulin sensitivity index (DeFronzo's formula based on fasting glucose and insulin and the mean glucose and insulin during an oral glucose tolerance test (Miyazaki, Y., M. Matsuda, and R.A. DeFronzo, Dose-response effect of pioglitazone on insulin sensitivity and insulin secretion in type 2 diabetes. Diabetes Care, 2002. 25(3): p. 517-23) significantly improved (Table 3).

[099] Thus, in rats, allopurinol treatment significantly improves features of the metabolic syndrome once the metabolic syndrome is established. To determine if allopurinol treatment can prevent the development of the metabolic syndrome, rats were treated simultaneously with allopurinol at the time of initiation of a 60% fructose diet (Table 4). Treatment of allopurinol while the rats were ingesting a 60% fructose diet led to a significant reduction in uric acid, fasting insulin, and fasting triglyceride levels. Notably, in the fructose and allopurinol animals whose intake was a 4% less (not statistically significantly different) than the fructose fed animals their weight gain was 75% less (an average of 6 grams vs. an average of 26 grams), hi addition, there is no statistical difference in the final weight of the control animals and the weight of the fructose animals treated with allopurinol even though their caloric consumption was 10% greater per day [2]. These studies demonstrate that uric acid has an important role in the development of the fructose-induced metabolic syndrome and blocking the rise of uric acid may be able to slow down the gain of weight from fructose intake. Example 2: Proposed mechanism by which uric acid causes metabolic syndrome — effect on endothelial cell function.

[0100] It has been reported that uric acid inhibits endothelial nitric oxide (NO) in human umbilical vein endothelial cells, porcine aortic endothelial cells, and bovine aortic endothelial cells (Khosla, U.M., et al., Hyperuricemia induces endothelial dysfunction. Kidney Int, 2005. 67(5): p. 1739-42; and Kang, D.H., et al., Uric acid-induced C-reactive protein expression: implication on cell proliferation and nitric oxide production of human vascular cells. J Am Soc Nephrol, 2005. 16(12): p. 3553-62). More recently, the researchers have demonstrated that uric acid also inhibits acetylcholine dependent vasodilation in rat aortic rings (FIG. 9).

[0101] The mechanism for this inhibition appears to be driven by several pathways. First, uric acid stimulates C-Reactive Protein which can be shown to partly inhibit endothelial NO release (Kang, D. H., et al., Uric acid-induced C-reactive protein expression: implication on cell proliferation and nitric oxide production of human vascular cells. J Am Soc Nephrol, 2005. 16(12): p. 3553-62). Second, uric acid stimulates arginase in endothelial cells (S. Zharikov, unpublished data) diminishing the concentration of arginine available for NO synthase. Third, and perhaps most exciting, uric acid can react with peroxynitrite to generate radicals (Gersch, CJ., RJ. Imaran, W. Angerhofer, A. Palii, S. Henderson, G.N., Uric acid's reaction with peroxynitrite: Formation of labile intermediates. Journal of American Society of Nephrology, 2006) and can also react directly with NO itself. Regardless of mechanism, the observation that uric acid can inhibit endothelial NO provides a mechanism for the development of insulin resistance. Insulin requires NO for its action. It is well known that eNOS knockout mice develop metabolic syndrome; similarly blocking NO production with L-NAME results in the same phenotype.

[0102] Additionally, there is now excellent evidence in humans that uric acid also inhibits endothelial NO levels. Thus, uric acid and plasma NO vary inversely during the day (Kanabrocki, EX., et al., Circadian relationship of serum uric acid and nitric oxide. Jama, 2000. 283(17): p. 2240-1), an elevated uric acid is associated with impaired endothelial function (Zoccali, C, et al., Uric acid and endothelial dysfunction in essential hypertension. J Am Soc Nephrol, 2006. 17(5): p. 1466-71), and lowering uric acid results in improved endothelial function in 6 studies (Mercuro, G., et al., Effect of hyperuricemia upon endothelial function in patients at increased cardiovascular risk. Am J Cardiol, 2004. 94(7): p. 932-5).

While one study reported that the acute infusion of uric acid into humans does not cause an immediate fall in endothelial NO (as measured by brachial artery reactivity) (Waring, W.S., et al., Hyperuricaemia does not impair cardiovascular function in healthy adults. Heart, 2004. 90(2): p. 155-9) the researchers have also found that the decrease in endothelial NO is not immediate.

Example 3: Uric acid leads to adipocyte activation

[0103] The prediabetic phenotype of adipocytes is marked by a reduction in PPARγ and a reduction in adiponectin release. To determine if uric acid has a direct effect on adipocytes, the researchers evaluated the effect of incubating well differentiated mouse adipocytes with increasing concentrations of uric acid (Sautin, Y.Y., Nakagawa., T. Zharikov, S. Johnson, RJ. , Uric Acid as a stimulator of NADPH oxidase-dependent redox signaling and oxidate/nitrosative stress in adipocytes). Submitted Incubating adipocytes with uric acid led to a reduction of PPARγ expression and a reduction in adiponectin mRNA expression (FIG. 10).

[0104] To determine if uric acid's effect on adiponectin is associated with an increase in reactive oxygen species, the oxidant generation within adipocytes was determined after incubation with increasing concentrations of uric acid. Not only did uric acid exposure result in a marked increase in oxidant generation (FIG. HA), but as has previously been shown with endothelial cells, uric acid exposure also resulted in a marked decrease in intracellular, bioavailable NO (FIG. HB). Increase intracellular peroxynitrate were demonstrated indirectly by demonstrating increased levels of nitrosylated proteins with uric acid exposure (FIG. HC).

[0105] To investigate whether fructose could have a direct effect on adipocytes, the researchers first determined whether adipocytes express the fructose transporter (Glut 5) and fructokmase. The researchers found that Adipocytes express Glut 5 and fructokinase (data not shown), and that incubation of adipocytes with fructose, as with uric acid, results in oxidant generation (data not shown). Thus, uric acid and fructose have prodiabetic effects on the adipocyte.

Example 4: Studies in Humans with Metabolic Syndrome-Fatty Liver Disease.

[0106] The researchers have examined the role of fructose in patients suffering from fatty liver disease. NAFLD can lead to progressive liver disease and has become the most common cause of chronic liver disease and the second most common reason for liver transplantation (Neuschwander-Tetri, B. A., Nonalcoholic steatohepatitis and the metabolic syndrome. Am J Med Sci, 2005. 330(6): p. 326-35). The researchers have found that patients with NAFLD have a history of markedly excessive soft drink intake compared to the general population. Patients with NAFLD consumed an average of 356 Kcal/day from soft drinks. That is nearly 2.5 times the average mean caloric intake from soft-drinks, 144 kcal/day in the general population (Nielsen, S.J. and B.M. Popkin, Changes in beverage intake between 1977 and 2001. Am J Prev Med, 2004. 27(3): p. 205-10). Since soft drinks are the major source of HFCS, these subjects are ingesting a vast excess of fructose compared to the general population. Consistent with this observation was evidence in liver biopsies that these patients had a marked elevation of the fructose associated enzymes, Glut-5 the fructose transporter and fructokinase (FIG. 12). It is known that high fructose (or sucrose) intake will upregulate fructokinase levels in both the intestine and liver of rats (Weiser, M.M., H. Quill, and KJ. Isselbacher, Effects of diet on rat intestinal soluble hexokinase and fructokinase activities. Am J Physiol, 1971. 221(3): p. 844-9; Korieh, A. and G. Crouzoulon, Dietary regulation of fructose metabolism in the intestine and in the liver of the rat. Duration of the effects of a high fructose diet after the return to the standard diet. Arch Int Physiol Biochim Biophys, 1991. 99(6): p. 455-60; Grand, RJ., M.I. Schay, and S. Jaksina, Development and control of intestinal and hepatic fructokinase. Pediatr Res, 1974. 8(8): p. 765-70). The researchers have also demonstrated that fructose increases both Glut-5 and fructokinase in a human hepatocyte cell line (data not shown). These studies suggest that excessive fructose may lead to an upregulation of these key proteins involved in the fructose metabolism. The researchers have not ruled out the possibility that liver disease itself leads to an upregulation of these proteins. However, if Glut-5 is upregulated fructose is transported into cells more rapidly, with the concominant upregulation of fructokinase, uric acid levels would increase more rapidly for a given concentration of fructose. Thus, if individuals continue to consume the same amount of fructose there may be a greater rise in uric acid, hi addition, even if fructose ingestion is reduced uric acid may still be increased above the uric acid levels of individuals who have not upregulated the fructose enzymes.

[0107] Accordingly, another embodiment of the subject invention pertains to evaluating the level of Glut5 in an individual. The level of Glut5 can serve as a basis to devise an appropriate treatment regimen or to diagnose the state of a uric-acid related disease including hypertension, metabolic syndrome and/or renal disease. Values relating to a state of disease or presence of a disease can be obtained by empirical studies. Thus, when a data value for an individual is obtained, this data value can indicate the state of a disease or presence of disease by comparison to the predetermined values. In a specific embodiment, the level of Glut5 is obtained from leukocytes.

[0108] The conventional fructose tolerance test is difficult to conduct in a clinical setting. The researchers have found that fructokinase is produced in peripheral blood mononuclear cells and that this fructokinase is upregulated in response to fructose and correlates with the conventional fructose tolerance test. Thus, based on the level of fructokinase in response to a dosage of fructose can be obtain via the procurement of a small blood sample. This can serve as the basis for prescribing an appropriate diet and/or drug regimen.

Example 5: KHK assay.

[0109] To measure enzymatic activity of KHK, we used a coupled enzymatic assay based on existing methods (Asipu, A., Hayward, B.E., O'Reilly, J. & Bonthron, D.T. Properties of normal and mutant recombinant human ketohexokinases and implications for the pathogenesis of essential fructosuria. Diabetes 52, 2426-32 (2003); Davies, D.R., Detheux, M. & Van Schaftingen, E. Fructose 1 -phosphate and the regulation of glucokinase activity in isolated hepatocytes. Eur J Biochem 192, 283-9 (1990); and Weiser, M.M. & Quill, H. Estimation of fructokinase in crude tissue preparations. Anal Biochem 43, 275-81 (1971). The researchers modified these methods and optimized them for 96-well format, convenient for screening (and note that one skilled in the art will appreciate that the method can be optimized for 384- well format, in view of the teachings herein). The assay measures KHK activity in the reaction mixture containing HEPES buffer (pH 7.1), MgCl₂, KCl, NaF, ATP, D-fructose, NADH, phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase, and crude preparations of cells and tissues by measuring ADP released in KHK reaction after quantitative conversion to NAD⁺(FIG. 2). Decrease in absorbance of NADH at 340 run is a readout for the amplified signal. This signal can be conveniently measured by many plate readers. The typical experiment showing increase in KHK activity in human renal epithelial cells in response to stimulation with fructose is shown in FIG. 3. Example 6: KHK expression.

[0110] The researchers measured expression of KHK in different mouse and human cells and tissues by RT-PCR with pairs of primers specific for mouse and human KHK, respectively (FIG. 4). The data obtained confirm that KHK is expressed in all tested cell types including human white blood cells, which can be important for the purposes of detection of KHK as a marker of the metabolic syndrome. In addition, treatment of human white blood cells with fructose stimulated expression of mRNA for GLUT5 (FIG. 4), the fructose transporter immediately upstream from KHK in the pathway of the intracellular fructose metabolism.

[0111] The researchers designed and validated an assay for quantitative measurement of mRNA for KHK expression in human cells by real-time qRT-PCR (FIG. 5). KHK mRNA can now be measured quantitatively in small samples of human white blood cells for diagnostic purposes.

Using all these methods, one can now measure expression and enzymatic activity of KHK in all samples of white blood cells. The enzymatic assay can also be used for screening for KHK inhibitors.

Example 7: The effect of fructose on KHK activity and expression.

[0112] The researchers demonstrated that human renal epithelial cells and mouse adipocytes respond to elevating the fructose level in the medium with significant increase in KHK activity (FIG. 4). Fructose induced also increase in the expression of KHK protein detected by Western blotting with specific antibody recognizing both human and mouse KHK (FIG. 6).

[0113] The researchers also demonstrated that we can detect an inhibition of KHK. To inhibit KHK, we used SH-groups agent N-ethylmaleimide. KHK function depends on intact SH groups (Ponz, F. & Llinas, J.M. Essential -SH groups in liver ketohexokinase. Nature 197, 696 (1963)). As expected, we observed dose-dependent decrease in KHK activity in response to N-ethylmaleimide (FIG. 7). This experiment demonstrates potential applicability of our method for screening for specific pharmacological inhibitors of KHK. Example 8: Labeling of Food Items based on fructose levels and regulation of fructose intake

[0114] Discussed herein are the various mechanisms that the researchers have elucidated that might be responsible for the adverse effects of a high fructose diet. The researchers are not aware of a heretofore identified method or standard of determining the amount of fructose in foods versus carbohydrates or sugars as a whole. Thus, according to another embodiment, the invention pertains to an article of manufacture containing a food item. The article of manufacture comprises a label on an external surface of the article which annotates the amount of fructose present in the contained food item. The fructose is described as its load (total amount) and/or as a percentage of sugars, carbohydrates and/or total calories. In a related embodiment, the invention pertains to a method comprising determining the fructose content of an item in a container and labeling the container with an annotation of the fructose content. The fructose content is described by load and/or percentage, hi another related embodiment, the invention pertains to a method of regulating fructose intake comprising providing a first food product comprising a first fructose content score; providing a second food product comprising a second fructose score; and instructing a dieter to restrict diet to no more than a first predetermined total score for a first predetermined time frame. The method may also comprise a first phase relating, e.g., a first total score per day for a set time frame such as 2 weeks the followed by a second phase relating, e.g., a second total score per day for a set time frame. Thus, the method may further comprise instructing a dieter to restrict diet to no more than a second predetermined fructose content score for a second predetermined time frame.

[0115] The fructose content score may be described, in some specific embodiments, according to a predetermined formula. Illustrative, but non-limiting examples of formulas include one or both of the following:

1) Number of calories from fructose divided by the total number of calories) x 2

So if bread has 1 gram of fructose and 80 kcal. The index would be 4 kcal fructose x 2 divided by 80 which is 8/80 = 0.1

2) Grams of fructose divided by percent of total calories of an average 2000 kcal diet.

So a piece of bread with 1 gram of fructose in 80 kcal would get an index of 25 (1/ (80/2000)). This would indicate to the dieter that if they were to intake 2000kcal of bread they would take in 25 grams of fructose. Accordingly, if the dieter ate all foods below an index of 25, this would be a specific example of a predetermined fructose content score. Furthermore, 100 grams of a protein food, such as steak, this would get an index of 0 implying that if the dieter ate 2000 kcal of steak they would not take in fructose.

[0116] According to another specific embodiment, the method of regulating fructose in diet might also take the form of fructose point program, which might make it easier for the dieter to regulate fructose intake.

A specific, non-limiting example of the formula for the "fructose points" might be: --Grams of fructose multiplied by 5.

So a piece of bread with 1 gram of fructose would be 5 points. The target for a dieter who is obese would be to eat less than a total of 100 fructose points per day. If the dieter is healthy, they should eat less than 200 points per day, indefinitely. If the dieter has the metabolic syndrome, they should eat 0 points for 2 weeks and than eat less than 100 points total a day.

[0117] According to one embodiment, the first predetermined fructose content score would be 100 points per day. In an additional embodiment, the first predetermined fructose content score would be 200 points per day for a continued period of time. In another embodiment the first fructose content score would be 0 points per for a first predetermined time frame of 2 weeks; the second predetermined fructose content score would be 100 points per day for a second predetermined time frame of 2 weeks or longer, including for the remainder of life.

[0118] While the invention has been illustrated and described in reference to various illustrative embodiments, accordingly it is not limited to the details shown or discussed, since it will be understood that various omissions, modifications, substitutions and changes in the forms and details of the embodiments illustrated and discussed can be made by those skilled in the art without departing in any way from the spirit of the present invention.

[0119] The disclosures of the cited patent documents, publications and references are incorporated herein in their entirety to the extent not inconsistent with the teachings herein. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims

1. A method of screening for compounds capable of inhibiting BCHK comprising: contacting at least one KHK inhibitor test compound with a KHK polypeptide; and detecting binding of said at least one KHK inhibitor test compound to said KHK polypeptide, wherein a test compound which binds to said KHK polypeptide is identified as potential KHK inhibitor agent.

2. A method of screening for compounds capable of inhibiting KHK comprising: i) determining the activity of a KHK polypeptide without contact with a test compound, ii) determining the activity of said KHK polypeptide upon contact of said test compound, wherein a test compound that modulates activity of said KHK polypeptide is identified as potential KHK inhibitor agent.

3. The method of claim 1, wherein the step of contacting is in or at the surface of a cell.

4. The method of claim 1, wherein the cell is in vitro.

5. The method of claim 1, wherein the step of contacting is in a cell-free system.

6. The method of claim I₅ wherein the polypeptide is coupled to a detectable label.

7. The method of claim 1, wherein the test compound displaces a ligand which is first bound to the polypeptide.

8. The method of claim 1, wherein the polypeptide is attached to a solid support

9. The method of claim 1, wherein the compound is attached to a solid support.

10. A method of screening for compounds capable of inhibiting BCHK comprising the steps of i) contacting a test compound with a KHK polynucleotide, ii) detecting binding of said test compound to said KHK polynucleotide, wherein a test compound that binds to a KHK polynucleotide is identified as potential KHK inhibitor agent.

11. The method of claim 10 wherein the nucleic acid molecule is RNA.

12. The method of claim 10 wherein the contacting step is in or at the surface of a cell.

13. The method of claim 10 wherein the contacting step is in a cell-free system.

14. The method of claim 10 wherein polynucleotide is coupled to a detectable label.

15. The method of claim 10 wherein the test compound is coupled to a detectable label.

16. A pharmaceutical composition for the prevention and/or treatment of hypertension, metabolic syndrome and/or kidney disease comprising a therapeutic agent which binds to a KHK polypeptide.

17. A pharmaceutical composition for the prevention and/or treatment of hypertension, metabolic syndrome and/or kidney disease comprising a therapeutic agent which regulates the activity of a KHK polypeptide.

18. A pharmaceutical composition for the prevention and/or treatment of hypertension, metabolic syndrome and/or kidney disease comprising a therapeutic agent which regulates the activity of a KHK polypeptide, wherein said therapeutic agent is i) a small molecule, ii) an RNA molecule, including siRNA, iii) an antisense oligonucleotide, iv) a polypeptide, v) an antibody, or vi) a ribozyme.

19. Method for the preparation of a pharmaceutical composition useful for the prevention and/or treatment of hypertension, metabolic syndrome, and/or kidney disease comprising the steps of i) identifying a KHK inhibitor in accord with the method of claim 1 ; ii) determining whether said KHK inhibitor ameliorates the hypertension, metabolic syndrome, and/or kidney disease in a human or nonhuman mammal; and iii) combining of said KHK inhibitor with an acceptable pharmaceutical carrier.

22. A method of diagnosing a uric acid mediated health condition in a human or nonhuman animal comprising determining activity of KHK in a human or nonhuman animal to obtain a KHK activity value and determining a state of said uric acid mediated health condition by comparing said KHK activity value to a predetermined value.

23. A method of prescribing an appropriate fructose restriction diet and/or a therapeutic regimen of a KHK inhibitor comprising determining activity of KHK in a human or nonhuman animal to obtain a KHK activity value and formulating a fructose restriction diet and/or therapeutic regimen based on said KHK activity value.

24. A method of diagnosing a disease or state thereof, comprising determining activity and/or concentration of KHK and/or Glut5 in an individual to obtain a data value; and comparing said data value with a predetermined value.