WO2014195228A1

WO2014195228A1 - Screening method for a glut9 inhibitor

Info

Publication number: WO2014195228A1
Application number: PCT/EP2014/061212
Authority: WO
Inventors: Alan WISE; Andrew James MHYRE
Original assignee: Tpp Global Development Limited
Priority date: 2013-06-03
Filing date: 2014-05-29
Publication date: 2014-12-11
Also published as: GB201309876D0; WO2014195228A8

Abstract

Provided is a method for screening for a GLUT9 inhibitor, which method comprises: (a) contacting a receptor having a GLUT9 function with a test compound; (b) measuring the kinetics of transport of a species across the receptor, which species is one whose transport is facilitated by GLUT9; and (c) determining whether the test compound is a GLUT9 inhibitor from the kinetics measurements taken in step (b).

Description

SCREENING METHOD FOR A GLUT9 INHIBITOR

The present invention relates to a screening method for identifying an agent that is a GLUT9 inhibitor, and/or an agent capable if inhibiting the uptake or release of a species into or from a cell via the GLUT9 receptor. Typically the species comprises a urate anion, and the method is thus in particular a method for screening for, or identifying, a medicament for treating gout and diseases associated with hyperuricemia. The invention also relates to agents, medicaments, pharmaceutical compositions and methods of treatment containing the gout treatment agents.

The poorly soluble acid, uric acid (UA), is the end-product of purine metabolism in humans and great apes, which have lost hepatic uricase activity during hominid evolution. Loss of uricase activity leads to significantly higher serum UA (sUA) in humans (-240-360 μΜ, 4-6 mg/dl) than in other mammals (-3-120 μΜ, < l -2 mg dl). Abnormally high levels of sUA (>420 μΜ, 7 mg/dl) is known as hyperuricemia and is the precursor to the deposition of UA crystals in and around the joints, which is the causative factor in the inflammatory arthritic condition gout.

New therapeutics for the treatment of gout represent an attractive proposition for a number of reasons. The incidence of gout is around 1 -2% of adults in developed countries and is increasing rapidly in developing nations. With few new drags approved in the last 40 years, and with up to 60% of patients intolerant or unresponsive to the current first-line therapy (allopurinol), gout is a disease with significant unmet medical need.

GLUT9 has been associated with gout and identified as a high capacity urate transporter (Vitart V, Rudan I, Hayward C, Gray NK, Floyd J, Palmer CNA et al. "SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout." Nature Genetics 2008; 40(4): 437-442). About 70% of daily UA excretion occurs via the kidneys, and in 5-25% of the human population, impaired renal excretion leads to hyperuricemia. Recently, a number of genome-wide association studies led to the identification of a variety of genetic loci linked with variance of sUA levels. The major genetic variant associated with low fractional excretion of UA and susceptibility to gout was found within the SLC2A9 gene, which encodes the GLUT9 transporter. GLUT9 was originally characterised as a hexose transporter. However, following the association of various SNPs in the promoter and intron regions of SLC2A9 with hyperuricemia, a number of studies have demonstrated that GLUT9 is in fact a high capacity UA transporter expressed in the renal proximal tubule. Hereditary mutations in the SLC2A9 gene lead to impaired GLUT9 function and renal hypouricemia, which is characterised by impaired renal reabsorption of UA and subsequent low sUA levels.

In addition to the blockade of UA production through the use of xanthine oxidase inhibitors such as allopurinol, gout therapies also target the reabsorption of UA in the kidney by blocking UA transporters. Such therapeutics are termed 'uricosurics' and they increase the excretion of UA into the urine. However, existing uricosuric drags such as probenecid and benzbromarone are of limited use due to side effect issues, low potency, and lack of selectivity.

Hence, inhibition of UA reabsorption by GLUT9 represents an attractive disease-modifying opportunity for gout. The identification of novel, selective GLUT9 inhibitors may act as powerful uricosuric agents with enhanced efficacy and safety over existing therapies.

However, despite the potential benefits of a selective GLUT9 inhibitor, screening for such inhibitors has proven problematic. A typical assay to screen for inhibitors for such receptors is an uptake assay. In an uptake assay, transfected cells comprising the necessary receptors are challenged with a potential inhibitor and then the uptake of a relevant transported species is measured (Anzai N, Ichida K, Jutabha P, Kimura T, Babu E, Jin CJ et al. "Plasma Urate Level Is Directly Regulated by a Voltage-driven Urate Efflux Transporter URATv 1 (SLC2A9) in Humans." Journal of Biological Chemistry 2008; 283(40): 26834-26838). Typically radiolabeled species are employed to facilitate measurement. However, such uptake assays for GLUT9 in cell systems other than Xenopus laevis oocytes injected to express GLUT9 have not been unequivocally demonstrated. The reasons for this are not yet clearly understood. Consequently, no commercial high-throughput cell uptake assay for GLUT9 has been developed. Cell uptake assays using oocytes comprising the GLUT9 receptor have shown some effectiveness on a small scale (Anzai N, Ichida K, Jutabha P, Kimura T, Babu E, Jin CJ et al. "Plasma Urate Level Is Directly Regulated by a Voltage- driven Urate Efflux Transporter URATvl (SLC2A9) in Humans." Journal of Biological Chemistry 2008; 283(40): 26834-26838; Caulfield MJ, Munroe PB, O'Neill D, Witkowska K, Charchar FJ, Doblado M et al. "SLC2A9 is a high-capacity urate transporter in humans." PLoS Med 2008; 5(10): el 97; Vitart V, Rudan I, Hayward C, Gray NK, Floyd J, Palmer CNA et al. "SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout." Nature Genetics 2008; 40(4): 437-442; Bibert S, Hess SK, Firsov D, Thorens B, Geering K, Horisberger JD et al. "Mouse GLUT9: evidences for a urate uniporter." Am J Physiol Renal Physiol 2009; 297(3): F612-9). However, oocytes are not suitable for high-throughput screening methods. The cells are larger than regular cells and thus require more space, and are unreliable (as many as 10 cells may be required per test well before a reliable result may be expected). This is both because the cells are large, and also because viability may not be immediately apparent. Consequently tests need to be undertaken on both viable and unviable cells, which can significantly reduce efficiency. Thus, there is still a need for a high-throughput cell-based screening assay for identifying GLUT9 inhibitors.

There is a body of literature related to the imaging and use of fluorescent probes to measure activity of biological targets. In US 6,727,071 Cellomics describe an optical imaging system for analysing cells containing fluorescently labelled molecules. In US 2009/226931 Corning describe a label-free biosensor assay system for use in cell -based compound screening. However, methodologies related to changes in membrane potential, or the application of methodologies for screening for GLUT9 inhibitors are not studied, or considered, in any of these references.

There is also established literature investigating the role of GLUT9 as a uric acid transporter. Mueckler and Thorens (Molecular Aspects of Medicine, 2013, 34, 121-138) provide a review of the whole GLUT family of membrane transporters. In WO 2009/063200, Wright et al. describe the identification of GLUT9 as a uric acid transporter. JP 2009294057 from J. Pharma. describes an expression system for measuring uric acid flux across cells comprising expression of the uric acid transporters URATl and GLUT9. However, none of these applications refer to the electrogenic nature of uric acid transport by GLUT9 nor do they allude to the use of membrane potential sensitive technologies for screening for GLUT9 inhibitors.

Witkowska et al (Biochemistry and Cell Biology, 201 1, 89, 283) describe the electrogenic nature of urate uptake by GLUT9. This publication does not link the electrogenic nature of urate uptake with the use of membrane potential-sensitive dyes to provide a kinetic methodology for high-throughput screening for GLUT9 inhibitors.

Accordingly, it is an aim of the present invention to provide a high-throughput (preferably cell-based) screening assay for identifying GLUT9 inhibitors. It is a further aim of the present invention to provide a high-throughput (preferably cell-based) screening method for identifying agents suitable for treating diseases associated with hyperuricemia, which may include but are not limited to: gout, gouty attacks, hypertension, a metabolic syndrome (such as insulin resistance, obesity, dyslipidaemia), atherosclerosis, cardiovascular disease, a kidney disease (such as acute uric acid nephropathy, chronic urate nephropathy, and uric acid nephrolithiasis (urolithiasis)), sarcoidosisis, psoriasis, joint inflammation, arthritis, hyperparathyroidism, plumbism, Lesch-Nylan Syndrome and elley-Seegmiller Syndrome . It is a still further aim of the invention to provide agents, medicaments and pharmaceuticals for treating such diseases, and methods for treating such diseases using the agents, medicaments and/or pharmaceuticals.

Accordingly, the present invention provides a method for screening for a GLUT9 inhibitor, which method comprises:

(a) contacting a receptor having a GLUT9 function with a test compound;

(b) measuring the kinetics of transport of a species across the receptor, which species is one whose transport is facilitated by GLUT9; and

(c) determining whether the test compound is a GLUT9 inhibitor from the kinetics measurements taken in step (b).

In the present context, the receptor having a GLUT9 function may be any receptor (or a receptor mimic or analogue) having the same or substantially the same function as GLUT9. It may therefore be any receptor capable of transporting the same species, substantially the same species (such as salts, ions, solvates or the like), or similar species (such as substituted versions of the species) as transported by GLUT9. It thus extends to any receptor encoded by the SCL2A9 gene, including GLUT9, UAQTL2, vURATl and URATvl . Many of these are alternative names for GLUT9, or have only minor differences that do not alter function, such as SNPs in the gene sequence. Thus, this definition extends to any such receptor (or a receptor mimic or analogue) capable of facilitating urate transport across a cell membrane.

In the present context, a test compound (the potential GLUT9 inhibitor) comprises any compound, complex, agent or the like which has potential as an inhibitor of GLUT9 (and/or an inhibitor of the receptors as defined above). Thus, the test compound may be a small molecule, such as an organic or inorganic compound, a large molecule such as an oligomer or polymer, or a biological molecule such as a peptide, polypeptide, protein, nucleic acid or the like. The compound may be present as a complex, salt, hydrate or other derivative for facilitating solubility, stability, uptake, bioavailability or the like. In this context, inhibitor means any compound capable of impairing or detrimentally affecting the GLUT9 function of the receptor in any way (such as preventing, reducing, reversing or diverting the transport of the species across the receptor), including impairing or detrimentally affecting the function of any of the receptors as defined above.

In the present context the species is any species which is capable of being transported across the GLUT9 receptor. Typically, therefore it is a species which may be transported into or out of a cell (i.e. across the cell membrane) by virtue of the GLUT9 receptor. Thus, the species is typically a urate anion, but may be a derivative thereof, such as an organic or inorganic compound, a large molecule such as an oligomer or polymer, or a biological molecule such as a peptide, polypeptide, protein, nucleic acid or the like. In particular the species may be a sugar, such as glucose or fructose, or a sugar derivative, and indeed may be any sugar capable of being transported across the GLUT9 receptor. The species may be present as a complex, salt, hydrate or other derivative for facilitating solubility, stability, uptake, bioavailability or the like.

The present inventors have discovered that measurement of kinetics provides an accurate measurement of whether a test compound is a suitable GLUT9 inhibitor. Unlike known uptake assays, which cannot be developed in a manner consistent with high-throughput screening, the kinetics measurements have surprisingly been found to provide reliable information on the throughput of species into and out of the cell, despite the cell uptake data indicating no such transport in many instances. In the present context, the measurement of kinetics of transport means the successive measurement over time of any parameter which could be related to transport. This could be as few as two measurements, each at a different point in time, but typically involves many such measurements. Typically, the kinetics measurements enable a point of comparison, or reference point, to be identified, such that the effect of different inhibitory compounds can more reliably be compared. For example, if the parameter being measured is membrane potential (which is related to the transport of species across the cell membrane) that reference point is typically the maximum change in membrane potential (see Figure 4). The change (reduction) in this maximum is indicative of an inhibitory effect on transport across the membrane, and the ability to accurately pinpoint this maximum provides reliability of comparison of inhibitory compounds that could not otherwise be achieved.

Further provided by the invention is a method for screening for a GLUT9 inhibitor that inhibits uptake or release of a species into or out of a cell, which method comprises:

(a) screening a test compound for a GLUT9 inhibitor according to any of the methods described above; and

(b) performing an assay to determine whether the test compound inhibits the uptake or release of the species into or out of the cell, which species is one whose transport is facilitated by GLUT9.

The invention also provides method for screening for an agent for treating a disease associated with hyperuricemia which method comprises a method as defined above. The disease associated with hyperuricemia is not especially limited, and may be any such disease. This may include, but is not limited to the following: gout, gouty attacks, hypertension, a metabolic syndrome (such as insulin resistance, obesity, dyslipidaemia), atherosclerosis, cardiovascular disease, a kidney disease (such as acute uric acid nephropathy, chronic urate nephropathy, and uric acid nephrolithiasis (urolithiasis)), sarcoidosisis, psoriasis, joint inflammation, arthritis, hyperparathyroidism, plumbism, Lesch-Nylan Syndrome and Kelley- Seegmiller Syndrome,

Further provided is an agent for treating a disease associated with hyperaricemia, which agent comprises an agent obtainable from a method as defined above. The disease associated with hyperaricemia is not especially limited, and may be any such disease as defined above.

Still further provided is a pharmaceutical composition for treating a disease associated with hyperaricemia, which composition comprises a compound as defined above. The disease associated with hyperaricemia is not especially limited, and may be any such disease, as defined above.

Yet further provided is a method for treating a disease associated with hyperaricemia, which method comprises administering a agent as defined above and/or a composition as defined above to a patient. The disease associated with hyperuricemia is not especially limited, and may be any such disease, as defined above.

The invention will now be explained in more detail, by way of example only, with reference to the following Figures.

Figure 1 shows an overview of FLIPR™ membrane potential dye kit technology (Molecular Devices LLC).

Figure 2 shows a Western blot of crude cell lysates from H4 cells transfected to express hGLUT9. High levels of expression are confirmed in clone #10.

Figure 3 shows immunocytochemistry demonstrating cell surface expression of hGLUT9 in H4 cells.

Figure 4 shows that membrane potential changes are increased by replacement of NaCl with choline chloride in both the dye loading and urate stimulation buffers. Figure 5 shows representative concentration-response curve data for pharmacological reference compounds in the membrane potential assay.

Figure 6 shows representative inhibition data for two hit molecules in an orthogonal oocyte uptake assay.

The invention will now be described in more detail.

As has been described, the invention relates to a method for screening for a GLUT9 inhibitor, which method comprises:

(a) contacting a receptor having a GLUT9 function with a test compound;

In the present context, the receptor having a GLUT9 function may be any receptor (or a receptor mimic or analogue) having the same or substantially the same function as GLUT9. It may therefore be any receptor capable of transporting the same species, substantially the same species (such as salts, ions, solvates or the like), or similar species (such as substituted versions of the species) as transported by GLUT9. It thus extends to any receptor encoded by the SCL2A9 gene, including GLUT9, UAQTL2, vURATl and URATvl . Many of these are alternative names for GLUT9, or have only minor differences that do not alter function, such as SNPs in the gene sequence. Thus, this definition extends to any such receptor capable of facilitating urate transport across a cell membrane.

The test compound (the GLUT9 inhibitor) is not especially limited, and may comprise any compound, complex, agent or the like which has potential as an inhibitor of GLUT9 (and/or an inhibitor of the receptor as defined above). Thus, the test compound may be a small molecule, such as an organic or inorganic compound, a large molecule such as an oligomer or polymer, or a biological molecule such as a peptide, polypeptide, protein, nucleic acid or the like. The compound may be present as a complex, salt, hydrate or other derivative for facilitating solubility, stability, uptake, bioavailability or the like. In this context, inhibitor means any compound capable of impairing or detrimentally affecting the GLUT9 function of the receptor in any way (such as preventing, reducing, reversing or diverting the transport of the species across the receptor), including impairing or detrimentally affecting the function of any of the receptors as defined above.

The species is not especially limited, and may be any species which is capable of being transported across the GLUT9 receptor. Typically, therefore it is a species which may be transported into or out of a cell (i.e. across the cell membrane) by virtue of the GLUT9 receptor. Thus, the species is typically a urate anion, but may be a derivative thereof, such as an organic or inorganic compound, a large molecule such as an oligomer or polymer, or a biological molecule such as a peptide, polypeptide, protein, nucleic acid or the like. In particular the species may be a sugar, such as glucose or fructose, or a sugar derivative, and indeed may be any sugar capable of being transported across the GLUT9 receptor. The species may be present as a complex, salt, hydrate or other derivative for facilitating solubility, stability, uptake, bioavailability or the like.

In a typical embodiment, the method preferably comprises the following:

(a) contacting a cell comprising a GLUT9 receptor with a test compound;

(b) measuring the kinetics of transport of a species across a membrane of the cell, which species is one whose transport across the membrane is facilitated by GLUT9; and

Thus, in these typical embodiments, the receptor having the GLUT9 function is a GLUT9 receptor. In other embodiments, the receptor may be an artificially modified receptor, a receptor mimic or the like, provided that it retains the GLUT9 receptor function, and in particular its solute carrying function. In addition, the kinetics are typically measured across a cell membrane so as to representative of the transport of the species into and out of the cell itself. However, in other embodiments, the system may be an artificial system modelling transport across a cell membrane, and need (in that case) not involve the cell itself. The species is not especially limited, provided that its transport into and out of a cell is capable of being facilitated by the GLUT9 receptor. The species is typically selected from a substituted or unsubstituted uric acid compound, a substituted or unsubstituted urate compound, and a mixture of two or more of the above. In the more preferred embodiments, the species comprises a substituted or unsubstituted urate anion.

Thus, typically the species is uric acid or an anion of uric acid, preferably being a substituted or unsubstituted species with the following structure:

In all of the embodiments mentioned in connection with this invention, both above and in the following, the term 'substituent' is not especially limited and may be any functional group or any atom, especially any functional group or atom common in organic chemistry, provided that the ability of the species to be transported by the GLUT9 receptor is not prevented.

The substituent may be present at any one or more of the C or N atoms in the uric acid ring system above, provided that at least one N atom is attached to H such that the uric acid retains its ability to form an anion.

Thus, substituent may have any of the following meanings. The substituent may comprise any organic group and/or one or more atoms from any of groups III A, IV A, VA, VIA or VIIA of the Periodic Table, such as a B, Si, N, P, O, or S atom (e.g. OH, OR, NH₂, NHR, NR₂, SH, SR, S0₃H, PO4H2 etc.) or a halogen atom (e.g. F, CI, Br or I) where R is a lower hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or more, e.g. 7-40 C atoms).

When the substiruent comprises an organic group, the organic group preferably comprises a hydrocarbon group. The hydrocarbon group may comprise a straight chain, a branched chain or a cyclic group. Independently, the hydrocarbon group may comprise an aliphatic or an aromatic group. Also independently, the hydrocarbon group may comprise a saturated or unsaturated group.

When the hydrocarbon comprises an unsaturated group, it may comprise one or more alkene functionalities and/or one or more alkyne functionalities. When the hydrocarbon comprises a straight or branched chain group, it may comprise one or more primary, secondary and/or tertiary alkyl groups. When the hydrocarbon comprises a cyclic group it may comprise an aromatic ring, an aliphatic ring, a heterocyclic group, and/or fused ring derivatives of these groups. The cyclic group may thus comprise a benzene, naphthalene, anthracene, indene, fluorene, pyridine, quinoline, thiophene, benzothiophene, furan, benzofuran, pyrrole, indole, imidazole, thiazole, and/or an oxazole group, as well as regioisomers of the above groups.

The number of carbon atoms in the hydrocarbon group is not especially limited, but preferably the hydrocarbon group comprises from 1-40 C atoms. The hydrocarbon group may thus be a lower hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or more, e.g. 7-40 C atoms). The lower hydrocarbon group may be a methyl, ethyl, propyl, butyl, pentyl or hexyl group or regioisomers of these, such as isopropyl, isobutyl, tert-butyl, etc. The number of atoms in the ring of the cyclic group is not especially limited, but preferably the ring of the cyclic group comprises from 3-10 atoms, such as 3, 4, 5, 6 or 7 atoms.

The groups comprising heteroatoms described above, as well as any of the other groups defined above, may comprise one or more heteroatoms from any of groups 1IIA, IVA, VA, VIA or V1IA of the Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen atom (e.g. F, CI, Br or I). Thus the substiruent may comprise one or more of any of the common functional groups in organic chemistry, such as hydroxy groups, carboxylic acid groups, ester groups, ether groups, aldehyde groups, ketone groups, amine groups, amide groups, imine groups, thiol groups, thioether groups, sulphate groups, sulphonic acid groups, and phosphate groups etc. The substituent may also comprise derivatives of these groups, such as carboxylic acid anhydrydes and carboxylic acid halides.

In addition, any substituent may comprise a combination of two or more of the substituents and/or functional groups defined above.

The cell is not especially limited, provided that it comprises a receptor with a GLUT9 function. However, in typical embodiments it is a cell capable of being used in a high-throughput cell-based assay or method, such as HEK-293 or CHO cells. It is further typical that the cell is a cell endogenously expressing GLUT9 (such as RPTEC (Renal Proximal Tubule Epithelial Cells)), or is a cell that has been transfected such that it comprises a GLUT9 receptor at its surface. In some embodiments the cell comprises an H4 cell. When transfection has been employed, typically the cell is one that has been transfected with a plasmid containing hGLUT9. Transfection is beneficial, since it may give rise to a cell overexpressing GLUT9, which may improve the efficiency of the assay.

The kinetics measured in accordance with the present method are not especially limited, provided that they provide a measure of uptake of the species. Typically, however, the measurement of kinetics of transport comprises a measurement of flux. In some embodiments, when the species is ionic, such as a urate anion, the ionic nature of the species is advantageous allowing the measurement of kinetics via changes in membrane potential. In such embodiments, it is especially beneficial to employ a dye to facilitate the measurement. Typically the dye comprises a membrane permeable dye. It is further typical that the dye comprises a fluorescent dye. In especially favourable embodiments, the measurement of kinetics is performed using a Fluorescent Imaging Plate Reader (FLIPR ) technique.

In typical embodiments, especially those employing cells, the measurement of kinetics is carried out in a buffer medium. The buffer medium is not especially limited, provided that it is not detrimental to the assay. Typically the buffer medium comprises choline chloride. In such embodiments, the choline chloride is present in the buffer in a concentration of from 50 mM to 200 mM, preferably from 75 mM to 175 mM, preferably from 100 mM to 150 mM and most preferably about 125 mM. In further preferred embodiments, the buffer medium does not comprise NaCI. In other embodiments, the buffer medium further comprises one or more of: KC1, MgS0₄, and KHP0₄. Typically, the KC1 is present in a concentration of from 2-8 mM, preferably from 3-7 mM, preferably from 4-6 mM and most preferably about 4.8 mM. Typically the MgS0₄ is present in a concentration of from 0.2-2.2 mM, preferably from 0.5-2 mM, preferably from 1-1.5 mM and most preferably about 1.2 mM. Typically the KHPO₄ is present in a concentration of from 0.2-2.2 mM, preferably from 0.5-2 mM, preferably from 1-1.5 mM and most preferably about 1.2 mM. The pH of the buffer is not especially limited, provided that it is not detrimental to the assay. Advantageously, the buffer medium pH is from 6.5 to 8.5, preferably from 7.0 to 7.7, preferably from 7.2-7.5, preferably from 7.3-7.5 and most preferably about 7.4.

It is not especially limited how the test compound may be determined to be effective. However, typically the test compound is determined to be a GLUT9 inhibitor by comparing the kinetics measurements obtained in step (b) with kinetics measurements obtained for one or more known GLUT9 inhibitors. Any such GLUT9 inhibitors may be employed, but typical GLUT9 inhibitors are selected from one or more of benzbromarone, indomethacin and oxypurinol.

As has been mentioned, the present invention further provides a method for screening for a GLUT9 inhibitor that inhibits uptake or release of a species into or from a cell, which method comprises:

(b) performing an assay to determine whether the test compound inhibits the uptake or release of the species into or from the cell, which species is one whose transport is facilitated by GLUT9.

In this method, the species is typically one as already defined above. However, in this method it is advantageous for the uptake assay (step b) if the species is radiolabelled, preferably ¹⁴C labelled. In typical embodiments, the cell for the uptake assay comprises an oocyte. Oocytes have been found to be useful in a cell-based assay to confirm cell uptake, although are not suitable for a high-throughput cell-based assay. Therefore, in the present methods, if a small number of inhibitors are identified in a high-throughput method of the invention, this small number may be tested in a cross-check for cell uptake using the oocyte system.

Typically the cell, such as the oocyte, is a cell that has been injected with GLUT9 mRNA transcript such that it comprises a GLUT9 receptor at its surface.

As has been mentioned, the present screening methods are suitable for screening for an agent for treating a disease associated with hyperuricemia. The disease associated with hyperuricemia may be any disease as defined above, such as gout, gouty attacks, hypertension, a metabolic syndrome (such as insulin resistance, obesity, dyslipidaemia), atherosclerosis, cardiovascular disease, a kidney disease (such as acute uric acid nephropathy, chronic urate nephropathy, and uric acid nephrolithiasis (urolithiasis)), sarcoidosis, psoriasis, joint inflammation, arthritis, hyperparathyroidism, plumbism, Lesch-Nylan Syndrome and Kelley-Seegmiller Syndrome. Any test compound identified as a GLUT9 inhibitor is considered a potential agent for treating the disease in accordance with the present invention. Thus the invention provides such agents, pharmaceutical compositions for treating such a disease comprising such agents, and methods for treating such a disease, comprising administering such agents as and/or compositions to a patient. The patient is not especially limited, and may be a human or an animal, although is typically a human.

The invention will now be described in more detail, by way of example only, with reference to the following specific embodiments.

EXAMPLES

In order to identify inhibitors of GLUT9, a functional cell-based assay was developed that measures changes in membrane potential in response to transport of urate anions across the plasma membrane. For this purpose the Fluorescent Imaging Plate Reader (FLIPR™) is a routinely used platform for real-time measurement of ion flux across cell membranes with appropriate fluorescent dyes. A subset of such membrane-permeable dyes rapidly redistribute across the membrane in response to changing membrane potential. Hence, a fluorescent read-out provides a measure of changes to membrane potential following addition of a suitable electrogenic agent such as an anion. Consequently, this assay provides the opportunity to measure the effects of test agents which modulate ion flux across the membrane. An overview of this technology is depicted in Figure 1.

Methods

Stable cell line generation

To support assay development, a cell line stably expressing human GLUT9 was first generated. Human H4 cells were transfected with the pReceiver-Lvl 05 plasmid containing hGLUT9 with Lipofectamine 2000. hGLUT9-overexpressing polyclonal populations were isolated under purinomycin selective pressure (1 μ§/ηι!) and maintained in Dulbecco's modified Eagle's medium supplemented with 1 p- /ml purinomycin + 10% foetal bovine serum (FBS). Monoclonal cell lines were then isolated and high hGLUT9-expressing clones selected following confirmation of protein expression by Western blotting (see Figure 2) and cell-surface expression by immunocytochemistry (see Figure 3).

H4/hGLUT9 cells (clone #10) were seeded in black, clear bottom tissue culture treated 384- well plates at l xlO⁴ cells/well in DMEM containing 10% FBS and grown for 18-22 hrs in a tissue culture incubator maintained at 37°C with 5% C0₂. In order to measure membrane potential, the media was removed from the cell assay plates and the cells loaded with 20 μΐ/well membrane potential-sensitive blue dye (Molecular Devices LLC) dissolved at 1.2x in Assay Buffer (25 mM HEPES buffer containing 125 mM choline chloride, 4.8 mM KC1, 1.2 mM MgS0₄, and 1.2 mM KHP0₄ at pH 7.4). After a 30 min pre-incubation at room temperature, the assay plates were delivered by robotic arm to the FLIPR™ tetra for real-time imaging of the stimulation with 2.5 mM urate (final) prepared in assay buffer. During assay development, it was found that replacement of NaCl in the assay buffer during both dye loading and urate stimulation phases significantly improved the assay window and also that of urate solubility (see Figure 4). In order to pharmacologically validate the assay a number of small molecule agents previously shown to inhibit urate-mediated uptake via GLUT9 were tested. In these instances, compounds were added to the cells simultaneously with the blue dye and incubated for 30 min prior to the addition of urate. Example data using reference compounds is shown in Figure 5. In all cases, IC50 values generated were in agreement and rank order with literature reports.

Expression of GLUT9 and [14CJ -urate uptake in Xenop s laevis oocytes

Adult female X. laevis oocytes were injected with 50 ng of human GLUT9 mRNA transcript or with an equivalent volume of ¾0 and then maintained in Barth's buffer for 2 days at 18°C before use. To determine urate uptake, oocytes were incubated at 18°C in uptake buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM KC1, 1 mM CaCl₂, MgCl₂) containing 30-52 μΜ [¹⁴C] -urate for 1 h. Oocytes were extensively washed with ice-cold buffer. After washing, oocytes were individually transferred to scintillation tubes and solubilized with 200 μΐ of 10% (w/v) SDS. Samples were counted after addition of 2 ml scintillation fluid. Data are expressed as rate of urate uptake in picomoles per oocyte per hour calculated from the counted dpm. For urate uptake inhibition studies, compounds (at 10, 30 and 80 μΜ final test concentrations) and oocytes were pre-incubated in uptake buffer for 30 min prior to the addition of [^! C] -urate.

High-throughput Screening and Hit Identification

Small molecule inhibitors of hGLUT9 were identified by screening a diverse library of 121 ,000 samples at a final testing concentration of 10 μΜ solubilized in 100% DMSO. 'Hits' were selected based on a triaging strategy. The control inhibitors, benzbromarone, indomethacin and oxypurinol, were routinely tested on quality control plates located within each batch process in a 10-point concentration response format to monitor the overall performance of the assay. In order to remove 'false positive' or 'technology artefact' hits, hit molecules were also screened in a counter-screen assay which measured the flux of chloride anions across the plasma membrane. Table 1 shows representative Κ¾ο data for inhibition of urate uptake in the membrane potential assay by 2 hit compounds. Table 1 - Representative pICSO data for hit compound inhibition of urate uptake in H4 GLUT9 cells using the membrane potential assay.

Hit confirmation

Selected hit compounds were then profiled in the orthogonal in vitro assay system measuring the inhibition of [ ^l4C] -urate uptake in X. laevis oocytes injected to express human GLUT9. Figure 7 shows representative inhibition data for 2 hit molecules.

The data clearly show that not only does the assay provide a suitable assay for a GLUT9 inhibitor, which had not been possible previously, but that the assay is predictive for inhibiting urate uptake in cells, and hence a viable screening method for gout treatments.

Claims

CLAIMS:

1. A method for screening for a GLUT9 inhibitor, which method comprises:

(a) contacting a receptor having a GLUT9 function with a test compound;

2. A method according to claim 1, which method comprises:

(a) contacting a cell comprising a GLUT9 receptor with a test compound;

3. A method according to claim 1 or claim 2, wherein the species is selected from a substituted or unsubstituted uric acid compound, a substituted or unsubstituted urate compound, and a mixture of two or more of the above.

4. A method according to claim 3, wherein the species comprises a substituted or unsubstituted urate anion.

5. A method according to any of claims 2-4, wherein the cell is a cell endogenously expressing GLUT9, or is a cell that has been transfected such that it comprises a GLUT9 receptor at its surface.

6. A method according to claim 5, wherein the cell comprises an H4 cell.

7. A method according to claim 5 or claim 6 wherein the cell is one that has been transfected with a plasmid containing hGLUT9.

8. A method according to any of claims 5-7 wherein the cell is a cell overexpressing GLUT9.

9. A method according to any preceding claim, wherein the measurement of kinetics of transport comprises a measurement of flux.

10. A method according to any preceding claim, wherein the measurement of kinetics comprises measurement of changes in membrane potential.

1 1. A method according to claim 8 or claim 9, wherein a dye is employed to facilitate the measurement of kinetics.

12. A method according to claim 1 1 , wherein the dye comprises a membrane permeable dye.

13. A method according to claim 1 1 or claim 12, wherein the dye comprises a fluorescent dye.

14. A method according to any of claims 11-13, wherein the measurement of kinetics is performed using a Fluorescent Imaging Plate Reader technique.

15. A method according to any preceding claim, wherein the measurement of kinetics is carried out in a buffer medium.

16. A method according to claim 15, wherein the buffer medium comprises choline chloride.

17. A method according claim 16, wherein the choline chloride is present in the buffer in a concentration of from 50 mM to 200 mM, preferably from 75 mM to 175 mM, preferably from 100 mM to 150 mM and most preferably about 125 mM.

18. A method according to any of claims 15-17 wherein the buffer medium does not comprise NaCL

19. A method according to any of claims 15-18 wherein the buffer medium further comprises one or more of: KC1, MgS0 , and KHPO₄.

20. A method according to claim 19, wherein the KC1 is present in a concentration of from 2-8 mM, preferably from 3-7 mM, preferably from 4-6 mM and most preferably about 4.8 mM.

21. A method according to claim 19 or claim 20, wherein the MgS0₄ is present in a concentration of from 0.2-2.2 mM, preferably from 0.5-2 mM, preferably from 1 -1.5 mM and most preferably about 1.2 mM.

22. A method according to any of claims 19-21 , wherein the HPO₄ is present in a concentration of from 0.2-2.2 mM, preferably from 0.5-2 mM, preferably from 1-1.5 mM and most preferably about 1.2 mM.

23. A method according to any of claims 15-22, wherein the buffer medium pH is from 6.5 to 8.5, preferably from 7.0 to 7.7, preferably from 7.2-7.5, preferably from 7.3-7.5 and most preferably about 7.4.

24. A method according to any preceding claim, wherein the test compound is determined to be a GLUT9 inhibitor by comparing the kinetics measurements obtained in step (b) with kinetics measurements obtained for one or more known GLUT9 inhibitors.

25. A method according to claim 24, wherein the one or more known GLUT9 inhibitors are selected from one or more of benzbromarone, indomethacin and oxypurinol.

26. A method for screening for a GLUT9 inhibitor that inhibits uptake or release of a species into or from a cell, which method comprises:

(a) screening a test compound for a GLUT9 inhibitor according to any of claims 1 -25; and

27. A method according to claim 26, wherein the species is selected from a substituted or unsubstituted uric acid compound, a substituted or unsubstituted urate compound, and a mixture of two or more of the above.

28. A method according to claim 27, wherein the species comprises a substituted or unsubstituted urate anion.

29. A method according to any of claims 26-28, wherein the species is radiolabeled, preferably ¹⁴C labelled.

30. A method according to any of claims 26-29, wherein the cell comprises an oocyte.

31. A method according to any of claims 26-30, wherein the cell is a cell that has been injected with GLUT9 mRNA transcript such that it comprises a GLUT9 receptor at its surface.

32. A method for screening for an agent for treating a disease associated with hyperuricemia, which method comprises a method as defined in any of claims 1-31.

33. A method according to claim 32, wherein the disease comprises one or more diseases selected from: gout, gouty attacks, hypertension, a metabolic syndrome (such as insulin resistance, obesity, dyslipidaemia), atherosclerosis, cardiovascular disease, a kidney disease (such as acute uric acid nephropathy, chronic urate nephropathy, and uric acid nephrolithiasis (urolithiasis)), sarcoidosis, psoriasis, joint inflammation, arthritis, hyperparathyroidism, plumbism, Lesch-Nylan Syndrome and Kelley-Seegmiller Syndrome.

34. An agent for treating a disease associated with hyperuricemia, which agent comprises an agent obtainable from a method as defined in claim 32 or claim 33.

35. A pharmaceutical composition for treating a disease associated with hyperuricemia, which composition comprises a compound as defined in claim 34.

36. A method for treating a disease associated with hyperuricemia, which method comprises administering an agent as defined in claim 34 and/or a composition as defined in claim 35 to a patient.

37. A method according to claim 36, wherein the patient is human.