AU2004210416A1

AU2004210416A1 - Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma

Info

Publication number: AU2004210416A1
Application number: AU2004210416A
Authority: AU
Inventors: Andreas Busjahn; Florian Lang
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-02-07
Filing date: 2004-02-05
Publication date: 2004-08-19
Also published as: DE10305212A1; CA2514703A1; MXPA05008394A; WO2004069258A2; PL378399A1; CN1771039A; ZA200506280B; BRPI0407300A; WO2004069258A3; RU2005127808A; KR20050114214A; JP2006519189A; EP1663246A2

Description

IN THE MATTER of a PCT Application in the name of Prof. Dr. Florian Lang filed under PCT/EP2004/01048 and IN THE MATTER OF its transfer for an Application for a PATENT IN AUSTRALIA I, Wolfram H6rschler, Patentanwalt and European Patent Attorney in Mannheim (F.R.G.), do solemnly and sincerely declare that I am conversant with the English and German languages and am competent in translating thereof, and that the following is, to the best of my knowledge and belief, a true and correct translation of the International Patent Application filed under No. PCT/EP2004/01048 By Prof. Dr. Florian Lang for "Use of the sgk gene family for diagnosis and therepy of cataracts and glaucoma" Mannheim, August 3, 2005 Wolfram H6rschler - Patentanwalt - As originally filed Use of the sgk gene family for diagnosing and treating cataract and glaucoma 5 The invention relates to the use of a functional inhibitor of the hsgkl protein or the hsgk3 protein or of a negative regulator of the transcription of the hsgkl gene or hsgk3 gene for producing a pharmaceutical 10 for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy. In another aspect, the invention relates to the use of a single-stranded or double-stranded nucleic acid 15 encompassing the hsgkl sequence according to Acc No. NM_005627, or of one of its fragments, or encompassing the hsgk3 sequence according to Acc No. AF169035, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic 20 neuropathy, as well as to a kit for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy, which kit comprises the abovementioned nucleic acid. 25 The invention furthermore relates to different screening methods for identifying and characterizing therapeutically active substances, from among a multiplicity of test substances, with the therapeutically active substances being used for the 30 therapy and/or prophylaxis of at least one disease selected from cataract, glaucoma and diabetic neuropathy. The serum and glucocorticoid-inducible kinase hsgkl was 35 originally cloned as a glucocorticoid-sensitive gene [Webster et al. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 1993; 13:2031-2040]. 40 - 2 Subsequent investigations revealed that hsgkl is under the influence of a large number of stimuli [Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase 5 isoforms. Science STKE. 2001 Nov 13;2001(108):RE17] such as, inter alia, that of the mineralocorticoids [Chen et al. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 1999;96:2514-2519, N~ray-Fejes-T6th et al. sgk is an 10 aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na channels. J Biol Chem 1999;274:16973-16978; Shigaev et al. Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel. Am J Physiol 2000;278:F613-F619; Brenan FE, 15 Fuller PJ. Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo. Mol Cell Endocrinol. 2000;30;166:129-36; Cowling RT, Birnboim HC. Expression of serum- and glucocorticoid-regulated kinase (sgk) 20 mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes. J Leukoc Biol. 2000;67:240-248]. hsgkl is stimulated by the insulin-like growth factor 25 IGF1, by insulin and by oxidative stress by means of phosphoinositol-3-kinase (PI3 kinase) and phosphoinositol-dependent kinase PDK1 by way of a signal cascade [Park et al. Serum and glucocorticoid inducible kinase (SGK) is a target of the PI 3-kinase 30 stimulatd signaling pathway, EMBO J 1999;18:3024-3033; Kobayashi et al. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J. 1999;344:189-197]. The activation of hsgkl by PDK1 35 involves a phosphorylation at the serine at position 422. The mutation of this serine into an aspartate (s422D SGKl) results in a kinase which is constitutively active [Kobayashi T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kinase by agonists - 3 that activate phosphatidylinositide 3 kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999;339:319-328]. 5 As earlier investigations have shown, hsgkl is a potent stimulator of the renal epithelial Na channel [De la Rosa et al. The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels 10 in the plasma membrane of Xenopus oocytes. J Biol Chem 1999;274:37834-37839; B6hmer et al. The Shrinkage activated Na Conductance of Rat Hepatocytes and its Possible Correlation to rENaC. Cell Phys Biochem. 2000;10:187-194; Lang et al. Deranged transcriptional 15 regulation of cell volume sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sci USA 2000;97:8157-8162]. Since hsgkl is found in a large number of tissues which do not express the epithelial Na channel ENaC, the function of hsgkl ought not to be 20 restricted to that of regulating the Na channel [Klingel et al. Expression of the cell volume regulated kinase h-sgk in pancreatic tissue. Am J Physiol (Gastroint. Liver-Physiol.) 2000;279:G998-G1002; Waldegger et al. Cloning and characterization of a 25 putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 1997;94:4440-4445; Waldegger et al. h-sgk Serine Threonine protein kinase gene as early transcriptional 30 target of TGF-P in human intestine. Gastroenterology 1999;116:1081-1088]. Due to the fact that hsgkl probably regulates, in manners which are yet to be elucidated, a large number 35 of other signal transduction pathways or components of these pathways, hsgkl and its human homologs ought to have considerable potential for diagnosing a large number of diseases. It is evident, in particular, from DE 197 08 173 Al that hsgkl can be used for diagnosis - 4 in connection with many diseases, such as hypernatriamia, hyponatriamia, diabetes mellitus, renal insufficiency, hypercatabolism, hepatic encephalopathy and microbial or viral infections, in which changes in 5 cell volume play a crucial pathophysiological role. WO 00/62781 reported that hsgkl activates the endothelial Na channel, resulting in renal Na resorption being increased. Since this increase in 10 renal Na resorption is accompanied by hypertension, it was presumed, in this document, that an increase in the expression of hsgkl would lead to hypertension while a decrease in expression of hsgkl would ultimately lead to hypotension. 15 DE 100 421 37 also reported a similar connection between the overexpression or hyperactivity of the human homologs hsgk2 and hsgk3 and hyperactivation of the ENaC, the increase in renal Na resorption which 20 results therefrom, and the hypertension which develops from this. Furthermore, this document already discussed the diagnostic potential of the hsgk2 and hsgk3 kinases with regard to arterial hypertension. 25 WO 02/074987 A2 disclosed the connection between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgkl gene and a genetically determined predisposition for hypertension. These 30 polymorphisms are a polymorphism in intron 6 (T-*C) and a polymorphism in exon 8 (C->T) in the hsgkl gene. Because sgkl is expressed in a large number of tissues, and because sgkl presumably has a large number of yet 35 unknown substrates, it can be expected that there will be further correlations between the function of the human homologs of the sgk family, in particular of the hsgkl gene (NM_005627), of the hsgk2 gene and of the hsgk3 gene (AF169035) and the development of other - 5 diseases. The uncovering of these other specific disease correlations involving sgkl could lead to nucleic acids which contain polymorphic regions of the genes of the human homologs of the sgk family, which 5 regions influence the function or expression of the corresponding sgk proteins, being used for diagnosing a predisposition for these other diseases. The object of the invention was therefore to discover 10 further correlations between the function of the human homologs of the sgk family and new diseases and, in this way, to provide novel possibilities for the diagnostic use of nucleic acids which contain polymorphic regions of the genes of the human homologs 15 of the sgk family. This object was achieved by means of the surprising finding that hsgkl and hsgk3 powerfully stimulate the glucose transporter Glutl (see Fig. 1). Inter alia, the 20 glucose transporter Glutl mediates the uptake of glucose into various cells of the eye, inter alia [Busik et al. Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. Invest Ophthalmol Vis Sci. 2002;43:2356-63; 25 Takata K, Kasahara T, Kasahara M, Ezaki O, Hirano H. Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in the ciliary body and iris of the rat eye. Invest Ophthalmol Vis Sc. 1991;32:1659-66]. Water follows the 30 glucose osmotically, which means that an increase in the activity of Glutl leads to cell swelling. Consequently, an increase in the activity of Glutl could lead to the development of cataract [Gong et al. Development of cataractous macrophthalmia in mice 35 expressing an active MEK1 in the lens. Invest Ophthalmol Vis Sci. 2001;42:539-48]. In addition to this, it has been shown that overexpression of Glutl promotes the formation and deposition of connective tissue proteins [Ayo et al. Increased extracellular - 6 matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Am J Physiol. 1991;260:F185 191; Heilig et al. Overexpression of glucose transporters in rat mesangial cells cultured in a 5 normal glucose milieu mimics the diabetic phenotype. J Clin Invest. 1995;96:1802-1814]. Such a deposition of connective tissue proteins impedes the escape of ocular fluid and leads to pressure increases in the eye and consequently to damage of the retina [Fingert et al. 10 Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension. Invest Ophthalmol Vis Sci. 2001;42(1):145-52, Ueda et al. Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human 15 eyes. Invest Ophthalmol Vis Sci. 2002;43:1068-76]. Glucocorticoids which stimulate the expression of SGK1 (see above) do indeed at the same time lead to the development of glaucoma [Fingert et al. 2001]. However, hsgkl has never previously been suspected of having a 20 causal role. The abovementioned disturbances would occur in connection with any situations in which the activity of hsgkl was increased, that is in the presence of an 25 excess of any of the abovementioned hormones. Particular polymorphisms of the hsgkl gene which correlate with an increase in blood pressure [Busjahn et al. Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 30 256-260, 2002] could at the same time lead to an increase in the occurrence of cataract and glaucoma. The same modifications of the gene ought also to correlate with cataract and/or glaucoma which appears prematurely. 35 The present findings reveal a completely novel mechanism in the regulation of the glucose transporter Glutl. An increase in activity of hsgkl ought therefore to lead to an increase in the uptake of glucose into - 7 the cells. The transcription of hsgkl is stimulated by serum [Webster et al. 1993], by glucocorticoids [Brenan & Fuller 2000, Webster et al. 1993], by mineralocorticoids [Chen et al. 1999, Naray 5 Fejes-Toth et al. 1999, Shigaev et al. 2000, Brennan and Fuller 2000, Cowling and Birnboim 2000], by gonadotropins [Alliston et al. Follicle stimulating hormone-regulated expression of serum/glucocorticoid inducible kinase in rat ovarian granulosa cells: a 10 functional role for the Spl family in promoter activity. Mol Endocrinol. 1997;11:1934-1949; Alliston et al. Expression and localization of serum/glucocorticoid-induced kinase in the rat ovary: relation to follicular growth and differentiation. 15 Endocrinology. 2000;141:385-395; Gonzalez-Robayna et al. Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-Induced kinase (Sgk): evidence for A kinase-independent signaling by 20 FSH in granulosa cells. Mol Endocrinol. 2000;14:1283-1300, Richards et al. Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated genes. Recent Prog Horm Res. 1995;50:223-254], and by a number of cytokines 25 [Lang & Cohen 2001], in particular by TGF-P [Fillon S. et al. Expression of the Serine/Threonine kinase hSGK1 in chronic viral hepatitis. Cell Physiol Biochem 2002;12:47-54; Lang et al. 2000, Waldegger et al. 1999, Wdrntges S et al. Excessive transcription of the human 30 serum and glucocorticoid dependent kinase hSGK1 in lung fibrosis. Cell Physiol Biochem 2002,12:135-142]. In addition to this, the transcription of hsgkl is increased by cell shrinkage, as shown by the Waldegger et al. 1997 paper which has already been cited. An 35 increase in glucose concentration, as occurs in diabetes mellitus, stimulates the expression of hsgkl by cell shrinkage and/or by an increase in the formation of TGF-P [Lang et al. 2000]. The expressed hsgkl is activated by insulin-like growth factor IGF1, - 8 by insulin or by oxidative stress [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999]. 5 According to the findings in accordance with the invention, the increased expression of hsgkl increases the activity of the glucose transporter Glut-1. As a result, more glucose is taken up into the cells and the water which subsequently follows by osmosis causes the 10 cells to swell. This is the way in which water is incorporated to an increased extent into the cornea and lens, with this leading, by means of a reduction in transparency, to cataract [Gong et al. 2001]. 15 Glaucoma could also develop in a similar manner and, in addition, by the incorporation of connective tissue [Fingert et al. 2001]. Cell swelling is also suspected to be the cause in the 20 case of diabetic neuropathy [Burg et al., Sorbitol, osmoregulation, and the complications of diabetes. J Clin Invest 1988;81:635-40]. However, an increase in Glutl activity is to be expected not only in diabetes mellitus but also under the influence of 25 glucocorticoids or in patients exhibiting a genetically determined hyperactivity of hsgkl [Busjahn et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 256-260, 2002]. Glucocorticoids do indeed give rise to glaucoma 30 [Fingert et al. 2001]. The mechanism responsible for the development of glaucoma in connection with glucocorticoid administration had not previously been known. In particular, it had not previously been known that hsgkl plays a role in this mechanism and is 35 therefore suitable for use as a target protein for diagnosing and treating a glaucoma. The observations according to the invention consequently surprisingly demonstrate that hsgkl and - 9 hsgk3 increase nonepithelial glucose transport as well as increasing the epithelial Na channel. As a result, hsgkl and hsgk3 have been revealed to possess completely novel pathophysiological 5 significances which should entail important diagnostic and therapeutic/prophylactic consequences. The invention consequently relates to the use of a functional inhibitor of the hsgkl protein or the hsgk3 10 protein or of a negative regulator of the transcription of the hsgkl gene or hsgk3 gene for reducing cell swelling. The invention furthermore relates to the use of a 15 functional inhibitor of the hsgkl protein or the hsgk3 protein or of a negative regulator of the transcription of the hsgkl gene or hsgk3 gene for producing a pharmaceutical for the therapy and/or prophylaxis of a cataract, a glaucoma or diabetic neuropathy. 20 This functional inhibitor of the hsgkl protein or the hsgk3 protein can be a chemical substance of any nature which inhibits the normal physiological activity of the hsgkl protein or of the hsgk3 protein. The functional 25 inhibitor of the hsgkl protein or the hsgk3 protein is preferably a low molecular weight chemical substance (a "small molecule") or a protein or peptide. The functional inhibitor of the hsgkl protein or the hsgk3 protein can, in particular, be an antagonist of these 30 enzymes which blocks the substrate-binding site of the hsgkl protein or the hsgk3 protein but which, at the same time, is not accessible to any catalytic conversion by the hsgkl or hsgk3. Antagonists which are suitable in this case are preferably those molecules 35 which are structurally similar to the natural substrate of the hsgkl protein or of the hsgk3 protein, that is, in particular, which are structurally similar to the phosphorylatable amino acids serine and threonine.

- 10 Staurosporine and chelerythrine are two known functional inhibitors of hsgkl. In a particularly preferred embodiment, either staurosporine or chelerythrine is therefore used, as a functional 5 inhibitor of hsgkl or hsgk3, for the therapy and/or prophylaxis of at least one of the diseases cataract, glaucoma and diabetic neuropathy. A negative regulator of the transcription of the hsgkl 10 gene or the hsgk3 gene is defined as a substance which activates the expression of the hsgkl gene or the hsgk3 gene at the transcriptional level. In addition to the actual active compound, i.e. the 15 functional inhibitor of hsgkl or hsgk3 or the negative regulator of the transcription of hsgkl or hsgk3, the pharmaceutical according to the invention for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy can also comprise stabilizers 20 and/or carrier substances, such as starch, lactose, stearic acid, fats, waxes, alcohols or other additives such as preservatives, dyes or flavorings. The pharmaceutical can be administered in any manner, 25 in particular orally in the form of tablets, granules or capsules or as a solution. Other particularly suitable administration forms concern direct administrations (e.g. on the skin or the eye) in the form of ointments, tinctures or sprays or any type of 30 injection (e.g. subcutaneous or intravenous) or infusion. The invention furthermore relates to the use of a single-stranded or double-stranded nucleic acid 35 encompassing the hsgkl sequence according to Acc No. NM_005627, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy. The hsgkl fragment which the single-stranded or double-stranded nucleic acid can - 11 encompass in this connection is at least 10 nucleotides/base pairs in length, preferably at least 15 nucleotides/base pairs in length, and, in particular, at least 20 nucleotides/base pairs in 5 length. In this connection, the single-stranded or double stranded nucleic acid preferably encompasses at least one polymorphic nucleotide of the hsgkl gene, in 10 particular a single nucleotide polymorphism (SNP) of the hsgkl gene. In a particularly preferred embodiment, the single stranded or double-stranded nucleic acid encompasses, 15 in this connection, at least one of the following SNPs of the hsgkl gene: a G insertion at position 732/733 in intron 2 of the hsgkl gene, 20 - the T/C substitution at position 2071 in intron 6 of the hsgkl gene (WO 02/074987 A2), - the T/C substitution at position 2617 in exon 8 of the hsgkl gene (WO 02/074987 A2). 25 The abovementioned single-stranded or double-stranded nucleic acids can preferably be used to detect the above SNPs of the hsgkl gene in the genomic DNA or cDNA of the patient by means of the following methods: 30 - by means of directly sequencing the genomic DNA or cDNA using the above nucleic acids, - by means of specifically hybridizing the genomic DNA or cDNA with the above nucleic acids, - by means of a PCR oligonucleotide elongation assay 35 or by means of a ligation assay. In this connection, the genomic DNA or cDNA of the patient is preferably isolated from a body sample taken from the patient, in particular from saliva, blood, - 12 tissue or cells. It is to be assumed that the activity of the expressed hsgkl gene depends on the version of this polymorphism 5 in the hsgkl gene of the patient and that, consequently, nucleic acids which contain at least one of these polymorphisms are particularly well suited for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy. 10 The invention furthermore relates to the use of a single-stranded or double-stranded nucleic acid encompassing the hsgk3 sequence according to Acc No. AF169035, or of one of its fragments, for diagnosing a 15 predisposition for developing cataract, glaucoma and/or diabetic neuropathy. The hsgk3 fragment which the single-stranded or double-stranded nucleic acid can encompass in this connection is at least 10 nucleotides/base pairs in length, preferably at least 20 15 nucleotides/base pairs in length and, in particular, at least 20 nucleotides/base pairs in length. In this connection, the single-stranded or double stranded nucleic acid preferably encompasses at least 25 one polymorphic nucleotide of the hsgk3 gene, in particular a single nucleotide polymorphism (SNP) of the hsgk3 gene. In addition to the abovementioned single-stranded or 30 double-stranded nucleic acids, particular antibodies which are directed against substrates of the human homologs of the sgk family, in particular against substrates of hsgkl and hsgk3, are also suitable for diagnosing a predisposition for developing at least one 35 of the diseases cataract, glaucoma and diabetic neuropathy. These diagnostic antibodies are preferably directed against an epitope of the human homologs of the sgk family (in particular of hsgkl and hsgk3) which contains the phosphorylation site of the substrate - 13 either in phosphorylated form or in unphosphorylated form. For example, an overexpression of the hsgkl protein 5 arising due to the individual genetic makeup of the hsgkl gene could lead to an increase in the conversion of substrates of the hsgk, i.e. to an increase in the enzymatic phosphorylation of the substrates by the hsgkl. At the same time, the overexpression of the 10 hsgkl protein would lead to stimulation of the glucose transporter Glutl, with this ultimately bringing about a high level of glucose uptake into the cells of the eye and, subsequently, a high level of water uptake by osmosis and, as a result, ultimately bringing about 15 predisposition for developing cataract, glaucoma and diabetic neuropathy. Detecting the more frequent phosphorylation of hsgkl substrates by means of an antibody which is directed against a region of the substrate in question which contains the 20 phosphorylation site of the hsgkl in phosphorylated form or unphosphorylated form could consequently represent a method for diagnosing a predisposition for developing cataract, glaucoma and diabetic neuropathy. 25 In a preferred embodiment, the ubiquitin protein ligase Nedd4-2 (Acc No. BAA23711) is employed as the substrate of the human homolog of the sgk family. This ubiquitin protein ligase is a protein which is specifically phosphorylated by the human homologs of the sgk family 30 [Debonneville et al., Phosphorylation of Nedd4-2 by Sgkl regulates epithelial Na(+) channel cell surface expression. EMBO J., 2001;20:7052-7059; Snyder et al., Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na(+) 35 channel. J. Biol. Chem., 2002, 277: 5-8]. Phosphorylation sites for hsgkl possess the consensus sequence (R X R X X S/T), where R is arginine, S is serine, T is threonine and X is any arbitrary amino acid. In Nedd4-2 2 (Acc No. BAA23711) there are two - 14 potential phosphorylation sites for hsgkl with which the abovementioned consensus sequence matches, i.e. the serine at amino acid position 382 and the serine at amino acid position 468. 5 The abovementioned antibodies for diagnosing a predisposition for developing at least one of the diseases cataract, glaucoma and diabetic neuropathy are therefore preferably directed against the substrate 10 Nedd4-2 and, particularly preferably, against a region of the Nedd4-2 protein having the sequence of the potential phosphorylation site for hsgkl, i.e. the consensus sequence (R X R X X S/T). In particular, these antibodies are directed against Nedd4-2 protein 15 regions which encompass at least one of the two potential phosphorylation sites serine at amino acid position 382 and/or serine at amino acid position 468. The invention furthermore relates to a kit for 20 diagnosing one of the diseases cataract, glaucoma and diabetic neuropathy, which kit comprises at least one of the following components: - antibodies which are directed against hsgkl or 25 hsgk3, - single-stranded or double-stranded nucleic acids which are able to hybridize, under stringent conditions, with the hsgkl gene according to Acc 30 No. NM 005627 or with the hsgk3 gene according to Acc No. AF169035; in particular those single stranded or double-stranded nucleic acids which encompass polymorphic nucleotides, in particular "SNPs" of the hsgkl gene or of the hsgk3 gene, 35 - antibodies which are directed against a substrate of a human homolog of the sgk family; preferably antibodies which are directed against the phosphorylation site of this substrate in the - 15 phosphorylated or unphosphorylated form; in particular antibodies which are directed against the phosphorylation site of Nedd4 or Nedd4-2 in the phosphorylated or unphosphorylated 5 form. The invention also relates to a screening method for identifying and characterizing therapeutically active substances, from among a multiplicity of test 10 substances, with the therapeutically active substances being used for the therapy and/or prophylaxis of at least one disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps: 15 a) Heterologously coexpressing i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in cells, 20 b) culturing at least one cell aliquot A, to Ax in the presence of in each case at least one test substance, with the at least one test substance in each case differing in dependence on the index 1 25 to X of the cell aliquot, and culturing a control cell aliquot B in the absence of any test substance, c) determining the activity of the glucose 30 transporter Glutl in the cell aliquots A, to Ax as compared with the activity of the glucose transporter Glutl in the control cell aliquot B. A substance library, preferably a small molecular 35 library, or else a protein library or the like, can be employed as the "multiplicity of test substances". In step a), suitable cells, preferably mammalian cells or cell lines, in particular human cells or cell lines, - 16 are transfected with suitable expression vectors, which contain suitable expression cassettes for expressing Glutl and hsgkl and/or hsgk3, using standard methods such as electroporation, CaPO4 5 precipitation, lipofection or the like. The expression cassettes contain the genomic DNA or the cDNA of the relevant target gene (Glutl, hsgkl or hsgk3) under the control of suitable promoters which are active in the cell type in question and which are able to express the 10 target gene in a suitable quantity. The expression vectors can additionally contain selection markers. The transfected cells are then cultured under conditions which enable the target genes i) and ii) to 15 be expressed. In step b), the transfected cells from a) are divided up into different cell aliquots A, to Ax and into a control cell aliquot B. The cell aliquots A, to Ax are 20 cultured in the presence of in each case at least one test substance. The test substance(s) which is/are in each case added to the cell aliquots A, to Ax differ from each other (in dependence on the index 1 to X of the respective cell aliquot A, to Ax). On the other 25 hand, the control cell aliquot B is cultured in the absence of any test substance. In step c), the activity of the glucose transporter Glutl in the cell aliquots A 1 to Ax is determined 30 quantitatively in comparison with the activity of the glucose transporter Glutl in the control cell aliquot B. A test substance which is able to functionally inhibit hsgkl or hskg3, or which reduces their expression, must have been added to the cell aliquots 35 A, to Ax in which a markedly lower value is measured for the Glutl activity than that which is measured in the control cell aliquot B. Such a substance could be suitable for treating at least one of the diseases cataract, glaucoma and diabetic neuropathy.

- 17 In an alternative embodiment, the screening method according to the invention for identifying and characterizing therapeutically active substances, from 5 among a multiplicity of test substances, with the therapeutically active substances being used for therapy and/or prophylaxis of at least one disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprises the following steps: 10 d) Heterologously coexpressing i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in at least one aliquot A, to Ax of cells, and 15 heterologously expressing i) the glucose transporter Glutl in at least one aliquot B 1 to Bx of cells e) culturing the cell aliquots A, to Ax and B 1 to Bx 20 in the presence of in each case at least one test substance, with the at least one test substance in each case differing in dependence on the index 1 to X of the cell aliquots, 25 f) carrying out a comparative determination of the activities of the glucose transporter Glutl in the cell aliquots A, to Ax and in the cell aliquots B 1 to Bx. 30 The explanations which are given above with regard to the individual procedural steps a) to c) apply in a corresponding manner to the procedural steps d) to f) of the alternative screening method according to the invention. 35 The invention is explained in more detail by the following Fig. 1. The uptake of 2-deoxyglucose (in pmol/1/10 min/oocyte) - 18 (arithmetic means ± SEM) is plotted on the ordinate A of Fig. 1. Xenopus laevis oocytes were injected with Glut-1 cRNA with or without SGK1, SGK2, SGK3 or protein kinase B (PKB) cRNA (see Example 1). 5 Fig. 1 shows the increase in the uptake of 2-deoxy glucose which occurs in oocytes which are expressing hsgkl or hsgk3 in addition to Glutl as compared with oocytes which are expressing Glutl on its own. This 10 thereby demonstrates that the functions of hsgkl and hsgk3 efficiently stimulate the activity of the glucose transporter Glutl. A similar effect is not seen in the case of oocytes which are expressing hsgk2 or PKBmut instead of hsgkl or hsgk3. 15 The invention is explained in more detail by means of the following example. Example 1: Expression in Xenopus laevis oocytes and 20 two-electrode voltage clamp Normal SGK1 cRNA [Waldegger S, Barth P, Raber G, Lang F: Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally 25 modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 1997;94:4440-4445] and constitutively active SGK1 (s 422 DSGK1) cRNA [Kobayashi & Cohen 1999], as well as normal Glut1 cRNA [Iserovich P, Wang D, Ma L, Yang H, Zuniga FA, Pascual 30 JM, Kuang K, De Vivo DC, Fischbarg J. Changes in glucose transport and water permeability resulting from the T310I pathogenic mutation in Glut1 are consistent with two transport channels per monomer. J Biol Chem. 2002;277:30991-7] were synthesized in vitro. The 35 dissection of the Xenopus laevis ovaries and the collection and treatment of the oocytes have already been described in detail [Wagner CA, Friedrich B, Setiawan I, Lang F, Br6er S: The use of Xenopus laevis oocytes for the functional characterization of - 19 heterologously expressed membrane proteins. Cell Physiol Biochem 2000;10:1-12]. The oocytes were injected with 5 ng of human Glutl, 7.5 ng of human s 422 DSGK1 and/or 5 ng of Xenopus Nedd4-2. Control oocytes 5 were injected with water. The uptake of radioactively labeled glucose was measured at room temperature for 2 days after the injection of the respective cRNAs. The control bath solution contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgC1 2 and 5 mM HEPES, pH 7.4. All the 10 substances were used at the given concentrations. The final solutions were titrated to pH 7.4 with HCl or NaOH. Calculations 15 The data are given as arithmetic means ± SEM; n is the number of oocytes which were examined. All the experiments were carried out in at least three different groups of oocytes. The results were tested 20 for significant differences using Student's t-test. Only results in which P < 0.05 were regarded as being statistically significant.

Claims

1. The use of a functional inhibitor of the hsgkl protein or the hsgk3 protein or of a negative 5 regulator of the transcription of the hsgkl gene or hsgk3 gene for reducing cell swelling.

2. The use of a functional inhibitor of the hsgkl protein or the hsgk3 protein or of a negative 10 regulator of the transcription of the hsgkl gene or hsgk3 gene for producing a pharmaceutical for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy. 15

3. The use as claimed in claim 1 or 2, characterized in that the functional inhibitor of the hsgkl protein or of hsgk3 protein is staurosporine or chelerythrine. 20

4. A pharmaceutical comprising a functional inhibitor of the hsgkl protein or the hsgk3 protein, or a negative regulator of the transcription of the hsgkl gene or hsgk3 gene, for the therapy and/or prophylaxis of a cataract, of a glaucoma or of 25 diabetic neuropathy.

5. The use of a single-stranded or double-stranded nucleic acid encompassing the hsgkl sequence according to Acc No. NM 005627, or of one of its 30 fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy.

6. The use as claimed in claim 5, characterized in 35 that the single-stranded or double-stranded nucleic acid encompasses at least one polymorphic nucleotide of the hsgkl gene, in particular an "SNP" of the hsgkl gene. - 21

7. The use as claimed in claim 6, characterized in that the SNP of the hsgkl gene is selected from the group of SNPs comprising the G insertion at position 732/733 in intron 2 of the 5 hsgkl gene, the T/C substitution at position 2071 in intron 6 of the hsgkl gene and the T/C substitution at position 2617 in exon 8 of the hsgkl gene. 10

8. The use of a single-stranded or double-stranded nucleic acid encompassing the hsgk3 sequence according to Acc No. AF169035, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic 15 neuropathy.

9. The use as claimed in claim 8, characterized in that the single-stranded or double-stranded nucleic acid encompasses at least one polymorphic 20 nucleotide of the hsgk3 gene, in particular an "SNP" of the hsgkl gene.

10. The use of an antibody directed against a substrate of a human homolog of the sgk family for 25 diagnosing a predisposition for developing at least one of the diseases cataract, glaucoma and diabetic neuropathy, with the antibody being directed against an epitope of the human homolog which contains the phosphorylation site either in 30 phosphorylated form or in unphosphorylated form.

11. The use as claimed in claim 10, characterized in that the substrate of the human homolog of the sgk family is Nedd4-2 having the Acc No. BAA23711. 35

12. A kit for diagnosing one of the diseases cataract, glaucoma and diabetic neuropathy, comprising antibodies which are directed against hsgkl or hsgk3 or comprising nucleic acids which are able - 22 to hybridize, under stringent conditions, with the hsgkl gene according to Acc No. NM_005627 or with the hsgk3 gene according to Acc No. AF169035, or comprising these antibodies and 5 nucleic acids jointly.

13. The kit as claimed in claim 12, characterized in that the nucleic acids are able to hybridize, under stringent conditions, with the DNA regions 10 of the hsgkl gene according to Acc No. NM 005627 or of the hsgk3 gene according to Acc No. AF169035 which encompass polymorphic nucleotides, in particular "SNPs" of the hsgkl gene or of the hsgk3 gene. 15

14. A screening method for identifying and characterizing therapeutically active substances, from among a multiplicity of test substances, with the therapeutically active substances being used 20 for the therapy and/or prophylaxis of at least one disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps: 25 a) Heterologously coexpressing i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in cells, 30 b) culturing at least one cell aliquot A, to Ax in the presence of in each case at least one test substance, with the at least one test substance in each case differing in dependence on the index 1 to X of the cell 35 aliquot, and culturing a control cell aliquot B in the absence of any test substance, c) determining the activity of the glucose transporter Glutl in the cell aliquots A 1 to - 23 Ax as compared with the activity of the glucose transporter Glutl in the control cell aliquot B. 5

15. A screening method for identifying and characterizing therapeutically active substances, from among a multiplicity of test substances, with the therapeutically active substances being used for the therapy and/or prophylaxis of at least one 10 disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps: d) Heterologously coexpressing 15 i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in at least one aliquot A, to Ax of cells, and heterologously expressing i) the glucose transporter Glutl 20 in at least one aliquot B 1 to Bx of cells e) culturing the cell aliquots A, to Ax and B 1 to Bx in the presence of in each case at least one test substance, with the at least one 25 test substance in each case differing in dependence on the index 1 to X of the cell aliquots, f) carrying out a comparative determination of 30 the activities of the glucose transporter Glutl in the cell aliquots A to Ax and in the cell aliquots B 1 to Bx.