CA2514703A1 - Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma - Google Patents

Abstract

The invention relates to the use of a functional inhibitor of hsgk1or hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene in the production of a medicament for the treatment and/or prophylaxis of a cataract, glaucoma or diabetic neuropathy. Another aspect of the invention relates to the use of a single-stranded or double-stranded nucleic acid comprising the hsgk1 sequence according to Acc No. NM 005627 or one of the fragments thereof or comprising the hsgk3 sequence according to Acc. No.
AF169035 or one of the fragments thereof in the diagnosis of a predisposition to the formation of a cataract, glaucoma and/or diabetic neuropathy, in addition to a kit for diagnosis of a predisposition to the formation of a cataract, glaucoma and/or diabetic neuropathy, comprising the above-mentioned nucleic acid. The invention further relates to various screening methods for identifying and characterizing therapeutically effective substances from a plurality of test substances for the treatment and/or prophylaxis of at least one disease selected from cataracts, glaucoma or diabetic neuropathy.

Description

As originally filed Use of the sgk gene family for diagnosing and treating cataract and glaucoma 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 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 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 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.
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 therapy and/or prophylaxis of at least one disease selected from cataract, glaucoma and diabetic neuropathy.
The serum and glucocorticoid-inducible kinase hsgkl was 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].

- 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 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, Naray-Fejes-Toth et al. sgk is an 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, 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) 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 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-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 involves a phosphorylation at the serine at position 422. The mutation of this serine into an aspartate ( s4aznSGK1 ) 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].
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 in the plasma membrane of Xenopus oocytes. J Biol Chem 1999;274:37834-37839; Bohmer et a1. The Shrinkage-activated Na+ Conductance of Rat H~~patocytes and its Possible Correlation to rENaC. Cell Phys Biochem.
2000;10:187-194; Lang et al. Deranged transcriptional I5 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 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.) 20~)0;279:G998-61002;
Waldegger et al. Cloning and characterization of a 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 target of TGF-~3 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 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 A1 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 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 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.
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 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.
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 polymorphisms are a polymorphism in intron 6 (TIC) 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 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 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 further correlations between the function of the human homologs of the sgk family and new diseases and, in this way, to provide novel possibilities fox the diagnostic use of nucleic acids which contain polymorphic regions of the genes of the human homologs 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 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;
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;32:1659-6fi). Water follows the 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 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 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.
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 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 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 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):
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.
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 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 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.
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 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 [Lang & Cohen 2001), in particular by TGF-~ [Fillon S.
et al. Expression of the Serine/Threonine kinase hSGKl in chronic viral hepatitis. Cell Physiol Biochem 2002;12:47-54; Lang et al. 2000, Waldegger et al. 1999, Warntges S et al. Excessive transcription of the human serum and glucocorticoid dependent kinase hSGKl 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 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-~ [Lang et al. 2000]. The expressed hsgkl is activated by insulin-like growth factor IGF1, by insulin or by oxidative stress [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].
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 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].
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 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 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 [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 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 _ g _ 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 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 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 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.
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 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 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 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.

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 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 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 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 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, 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 injection (e.g. subcutaneous or intravenous) or infusion.
The invention furthermore relates to 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 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 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, 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, - 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).
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:
- 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 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, tissue or cells.
It is to be assumed that the activity of the expressed ' hsgkl gene depends on the version of this polymorphism 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.
The invention furthermore relates to the use of a single-stranded or double-stranded nucleic acid encompassing the hsgk3 sequence accordin<~ to Acc No.
AF169035, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy. The hsgk3 fragmen-:: which the single-stranded or double-stranded nucle~_c acid can 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 length.
In this connection, the single-stranded or double-stranded nucleic acid preferably encompa:wes at least one polymorphic nucleotide of the hsrt3 gene, in particular a single nucleotide polymorphism (SNP) of the hsgk3 gene.
In addition to the abovementioned single-stranded or 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 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 either in phosphorylated form or in unphosphorylated form.
For example, an overexpression of the hsgkl protein 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 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 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 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.
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 (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(+) 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 . . CA 02514703 2005-08-05 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.
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 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 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 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 hsgk3, - single-stranded or double-stranded nucleic acids which are able 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; 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, - 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 _ CA 02514703 2005-08-05 phosphorylated or unphosphorylated form;
in particular antibodies which are directed against the phosphorylation site of Nedd4 or Nedd4-2 in the phosphorylated or unphosphorylated form.
The invention also relates to 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 disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps:
a) Heterologously coexpressing i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in cells, b) culturing at least one cell aliquot A1 to AX in the presence of in each case at least one test substance, with the at least one test substance in each ease differing in dependence on the index 1 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 transporter Glutl in the cell aliquots Az 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 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, 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, CaP04 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 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 be expressed.
In step b), the transfected cells from a) are divided up into different cell aliquots A1 to AX and into a control cell aliquot B. The cell aliquots A1 to AX are cultured in the presence of in each case at least one test substance. The test substances) which is/are in each case added to the cell aliquots A1 to AX differ from each other (in dependence on the index 1 to X of the respective cell aliquot A1 to AX). On the other 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 A1 to AX is determined 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 Ai 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.

In an alternative embodiment, the screening method according to the invention for identifying and characterizing therapeutically active substances, from 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:
d) Heterologously coexpressing i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in at least one aliquot A1 to AX of cells, and heterologously expressing i) the glucose transporter Glutl in at least one aliquot B1 to BX of cells a ) culturing the cell aliquots A1 to AX and B1 to BX
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, f) carrying out a comparative determination of the activities of the glucose transporter Glutl in the cell aliquots A1 to AX and in the cell aliquots B1 to BX .
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.
The invention is explained in more detail by the following Fig. 1.
The uptake of 2-deoxyglucose (in pmol/1/10 min/oocyte) (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).
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 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.
The invention is explained in more detail by means of the following example.
Example 1: Expression in Xenopus laevis oocytes and 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 modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 1997;94:4440-4445]
and constitutively active SGK1 ( s4azDSGKI ) cRNA
[ Kobayashi & Cohen 1999 ] , as well as normal Glutl cRNA
[Iserovich P, Wang D, Ma L, Yang H, Zuniga FA, Pascual JM, Kuang K, De Vivo DC, Fischbarg J. Changes in glucose transport and water permeability resulting from the T310I pathogenic mutation in Glutl are consistent with two transport channels per monomer. J Biol Chem.
2002;277:30991-7j were synthesized in vitro. The 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, Broer S: The use of Xenopus laevis oocytes for the functional characterization of 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 S422DSGK1 and/or 5 ng of Xenopus Nedd4-2. Control oocytes 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 KC1, 1.8 mM CaClZ, 1 mM MgClZ and 5 mM HEPES, pH 7.4. All the substances were used at the given concentrations. The final solutions were titrated to pH 7.4 with HC1 or NaOH.
Calculations 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 for significant differences using Student's t-test.
Only results in which P < 0.05 were regarded as being statistically significant.

i. CA 02514703 2005-08-05 . SEQUENCE LISTING
<110> Prof. Dr. Lang, Florian <120> Verwendung der sgk-Genfamilie zur Diagnose and zur Therapie von Kararkt and Glaukom <130> L62135 <140> DE 103 05 212.7 <141> 2003-02-07 <160> 3 <170> PatentIn version 3.1 <210> 1 <211> 5719 <212> DNA
<213> homo sapiens, hsgkl gene, Nr4-005627 <22U>
<221> variation <222> (732)..(733) <223> Insertion of an additional G in position 732/733 in intron 2 (SNP
<220>
<221> variation <222> (2071)..(2071) <223> 'f/C- exchange in position 2071 in intron 6 (SNP) <220>
<221> variation <222> (2617)..(2617) <223> C/T- exchange in position 2617 in exon 8 (SNP) <400>

ggccgagcgcgcggcctggcgcacgatacgccgagccggtctttgagcgctaacgtcttt60 ctgtctccccycggtgytgatyacggt aactgaggctyctaagggcaccctcactta120 gaa ctccagyatyaggggcatgytggcaattctcatcggtgagtgcaggaatcttgcgygact180 tctgctccaggagacgcaaagtggaaatttttt gaaagtcccggatcagattagtgtgtg240 tggcgccgggacgttatgaagccytctaaacgtttctttatttctcctccttctatccac300 agctttcatgaagcagaggaggatgggtctgaacgactttattcagaagattgccaataa360 ctcctatgcatgcaaacagtaayttcagaccggattgaggaaataactagtatagtttga420 attt gccagcggtaaacattctcatcacggcgtttatcgygaaggcgaagacttcttctg480 gygtggygatctcatttctccttaaattctaatatatttgacacattttaaacattaaag540 ttaatttgctgatttggcttgaactggagatgtaagataaatggttcgtgttggcc,gaat6U0 tcacgctttctccatgagcaacaatcct tttctgtatttaatygggttt attattttc660 to tttaactgac taatgtattg gggtattttc agtttaaaca gtgaattatc gggtagaagt 720 cggtagagcc aggaaactca cttttgatgt tggtgtgccc cctagtggcg agctggattc 780 taaatcgtgc cctttattcc ctgcagccct gaagttcagt ccatcttgaa gatctcccaa 840 cctcaggagc ctgagcttat gaatgccaac ccttctcctc cagtaagttt ttgtatgtgc 900 cgtgcatctg tggagaactg taagggagtc agttagtatt cctacattaa tggattaaaa 960 tagcatttct agaaattagt atcaaggcag gaatgcttca ttatgcataa cagtgatata 1020 aatatttaag tattgagtca gagtattatt tttatttttt tcctgggcat attttacctc 1080 aagtggttat tttaaaaggc atatttcata aaaaggtttt atctgtctga aacaacatga 1140 ctgtgtgcag tttccatact catttgaaat gtgatgaaat gtagttttga atgtttatag 1200 atgtatggtc atttgcatca gtcatttgta gatgtaacat tttctacatc gtttatgtta 1260 tagatgtctt cctttgaagc aatggtatta aaagaaattc tttttttttt tttctagcca 1320 agtccttctc agcaaatcaa ccttggcccg tcgtccaatc ctcatgctaa accatctgac 1380 tttcacttct tgaaagtgat cggaaagggc agttttggaa aggtaatttc aaatctgaag 1440 atcttttggt acacttcctt catgtcctct tttatattct ccctggatga ggatcgaaaa 1500 atgatttttt t aaattgaaa tttcaggttc ttctagcaag acacaaggca gaagaagtgt 1560 tctatgcagt caaagtttta cagaagaaag caatcctgaa aaagaaagag gtgagatgtg 1620 cttgatgggg ctggcattgg cggtagacac tccttgaata atcttgattc tggaatgttg 1680 gtgccagttg aacatgccac taaatctgaa tcgtcatttt cctaggagaa gcatattatg 1740 tcggagcgga atgttctgtt gaagaatgtg aagcaccctt tcctggtggg ccttcacttc 1800 tctttccaga ctgctgacaa attgtacttt gtcctagact acattaatgg tggagaggtg 1860 agcagggggg atagaagtca actcttagtg tctctgcaca gcctgctttg ttttagtttg 1920 agaaaaaagt tttcaaagat ttttggtggg gagaatgtta ccagaattag catttccttc 1980 aacctgtcag gttatagtta atagattact tggggccact acct gcagtt gttcttttgc 2040 tgtgt atgtc aaaact aatt aaattacatt gtgcaaccca gaatgacttt gttctgtctc 2100 ctgcagttgt tctaccatct ccagagggaa cgctgcttcc tggaaccacg ggctcgtttc 2160 tatgctgctg aaatagccag t gcctt gggc tacctgcatt cactgaacat cgtttatagg 2220 taagcctgag agctcttcag gctaccagtt ttggtataaa ggagacgtag cactggctgt 2280 ttcatagggc cttaaaataa tttgtgttta tttgcaactt ggttcgctaa aaccagatcc 2340 cctagcacgt gagctggctt gacttaagtg ccaaggggga acagccaagt aggattgtgc 2400 ctaatccaga atagatgagc agaacaaggg ctcctttttt cttcactaca caactacagt 2460 gaacctaaat gcctctaata ccttagcaat tatctttaag aggatatctt atgaagtgaa 2520 attaacttgt gcaactactt ttctattcac ttttttacag agacttaaaa ccagagaata 2580 tttt gctaga ttcacaggga cacattgtcc ttactgactt cggactctgc aaggagaaca 2640 ttgaacacaa cagcacaaca tccaccttct gtggcacgcc ggaggtaggc gctgtcttgg 2700 tttggtgcct ggtttacccc cgccttccaa gagagagatg t acaatcatg cacttaacta 2760 ccaaaaagag taaactcctc tcagagactt cttaatacag ttcagtgcaa ataaaataca 2820 tttgctgttt gatgtagcat gagaaatccc aagtccttct gttcctttac tgaaaagtag 2880 ctgtttgtaa gtaagatctg catcataaaa actttctaat cctaagtaag agatatcaag 2940 tgccagcagt ttcctaaatg tcagtacaca taggtagcca gtcaccctca aaaagtccag 3000 cagttttatc aggaaggaat ctaaagatat ctatcttcca agctggctct gggtctctca 3060 gctttttcaa actaaatgtg tggtcgtggg attgcttgc-t ttcgcaggtt ctaaacgctg 3120 tttccctggt ctgtttttca gt atctcgca cctgaggtgc ttcataagca gccttatgac 3180 aggactgtgg actggtggtg cctgggagct gtcttgtatg agatgctgta tggcctggtg 3240 agt ggcacat tgggaaccac tggaacactg cctgctccct acaatattgc cttcacacag 3300 caaaagcagc taagaggcat attggttatt ttatagttca t aagaataat cacttacctg 3360 gttcttttgt gcatttcaca ttttactaga taggaccaca ttgaacctgt gtggtggtga 3420 aaaactacca cttattaaca tctaccccct accctccaca cacacacaca caaacacaca 3480 cacgggttgc aaagtagaca cttaaatagc aagggaaaag aaagcattga ggtggggaga 3540 gtttctcaaa tcgagcctaa tatttattgc cgtttatatc tttttctcta ctggtaatgt 3600 gtgccatatg aaacttccaa ttaagtctaa agtaattttc cccttctttc agccgccttt 3660 tt atagccga aacacagctg aaatgtacga caacattctg aacaagcctc tccagctgaa 3720 accaaatatt acaaattccg caagacacct cctggagggc ctcctgcaga aggacaggac 3780 aaagcggctc ggggccaagg atgacttcgt gagtgatgtt ttcctgtcct cctgggccgg 3840 ccgggacgt g cactagacct ccctgccctt attgaatgca cctgtclaaa ttaatcttgg 3900 gtttcttatc aacagatgga gattaagagt catgtcttct tctccttaat taact gggat 3960 gatctcatta ataagaagat tactccccct tttaacccaa atgtggtgag tatctgtctc 407.0 tcttctaagt atagagaagc caagcgattt attttaattc agaattgtct gggggagggt 4080 tggaaggaat acattggcag atgttttctc cataaacctg ttattttacc tacatagaca 4140 catttatcaa ttcgaagcac caaaaggcaa caagtgaaca ttattc-ttat gtttaactgt 4200 gtgtagcctt ttgagatttt gtgcttgaag t gggtgatta tggaagttga tataagactt 4260 aaacttggta tttaaagcct ggtcaagatt tccctytcct gtgtctagtg tgagttcttg 4320 acaagagtgt ttttcccttc ccgtcacaga gtgggcccaa cgagctacgg cacttt gacc 4380 ccyagtttac cgaagagcct gtccccaact ccattggcaa gtcccctgac agcgtcctcg 4440 tcacagccag cgtcaaggaa gctgccgagg ctttcctagg cttttcctat gcgcctccca 4500 cggactcttt cctctgaacc ctgttagggc ttggttttaa aggattttat gtgtgtttcc 4560 gaatgtttta gttagccttt tggt ggagcc gccagctgac aggacatctt acaagagaat 4620 ttgcacatct ctggaagctt agcaatctta ttgcacactg ttcgctggaa ttttttgaag 4680 agcacattct cctcagtgag ctcatgaggt tttcattttt attcttcctt ccaacgtggt 4740 gctatctctg aaacgagcgt tagagtgccg ccttagacgg aggcaggagt ttcgttagaa 4800 agcggacctg ttctaaaaaa ggtctcctgc agatctgtct gggctgtgat gacgaatatt 4860 atgaaatgtg cctttLctga agagattgtg ttagctccaa agcttttcct atcgcagtgt 4920 ttcagttctt tattttccct t gtggatatg ctgtgtgaac cgtcgtgtga gtgtggtatg 4980 cctgatcaca gatggatttt gttataagca tcaatgtgac acttgcagga cactacaacg 5040 tgggacattg tttgtttctt ccatatttgg aagataaatt tatgtgtaga cttttttgta 5100 agatacggtt aataactaaa atttattgaa atggtcttgc aatgactcgt attcagatgc 5160 ct aaagaaag cattgctgct acaaatattt ctatttttag aaagggtttt tatggaccaa 5220 tgccccagtt gtcagtcaga gccgttggtg tttttcattg tttaaaatgt cacctgtaaa 5280 atgggcatta tttatgtttt tttttttgca ttcctgataa ttgtatgtat tgtataaaga 5340 acgtctgtac attgggttat aacactagta tatttaaact t acaggctta tttgtaatgt 5400 aaaccaccat tttaatgtac tgtaattaac atggttataa tacgtacaat ccttccctca 5460 tcccatcaca caactttttt tgtgtgtgat aaactgattt tggtttgcaa taaaaccttg 5520 aaaaatattt acatatattg tgtcatgtgt tatttt gtat attttggtta agggggtaat 5580 catgggttag tttaaaattg aaaaccatga aaatcctgct gtaatttcct gcttagtggt 5640 ttgctccaac agcagtggt t tctgactcca gggagtatag gatggcttaa gccaccacgt 5700 ccaggccttt agcagcatt 5719 <210>

<211>

<212>
DNA

<213> hsgk3 AF169035 hocno mRNA, Sapiens, <400>

ggtgtgctcttgagggattaaatgcaaagagatcacaccatggactacaaggaaagctgc60 ccaagtgtaagcattcccagctccgatgaacacagagagaaaaagaagaggtttactgtt120 tataaagttctggtttcagtgggaagaagtgaatggtttgtcttcaggagatatgcagag180 tttgataaactt-tataacactttaaaaaaacagtttcctgctatggccctgaagattcct240 gccaagagaatatttggtgataattttgatccagattttattaaacaaagacgagcagga300 ctaaacgaattcattcagaacct agttaggtatccagaactttataaccatccagatgtc360 aggctaaggc aggagaatcg cttgaacccg ggaggeggag gttgcagtga gccgagatcg 2340 caccattgca ctcctgcctg ggcaacaaga gtgaaactcc atctccaaaa a 2391 <210> 3 <211> 995 <212> PRT
<213> homo sapiens, Nedd 4-2 protein, BAA23711 <400> 3 Pro Gly Gly Trp Leu Arg Arg Ala Leu Pro Gly Arg Glu Arg Leu Gln 1 5 lU 15 Ser Pro Val His Ala Val Pro Pro Gln His Gly Thr Ser His Ser Arg Leu Leu Val Thr 'lrp Pro Gly Ala Gly Arg Asp Gln Asp Phe Ser Ser Pro Pro Leu Leu Leu Leu Gly G1u '1'hr Asp His Leu His Leu Asp Leu Pro Leu Ser Pro Leu Pro Thr Ser Asp Glu Leu Plte Leu Pro G1y Ile Cys Asp Pro Tyr Val Lys Leu Ser Leu Tyr Val Ala Asp Glu Asn Arg Glu Leu Ala Leu Va1 Gln i'hr Lys Thr I1e Lys Lys Thr Leu Asn Pro Lys 'rrp Asn Glu Glu Phe '1'yr Phe Arg Val Asn Pro Ser Asn His Arg Leu Leu Plue Glu Val Phe Asp Glu Asn Arg Leu Thr Arg Asp Asp Phe Leu Gly Gln Val Asp Val Pro Leu Ser His Leu Pro 1'hr Glu Asp Pro T'hr Met Glu Arg Pro Tyr i'hr Plne Lys Asp Phe Leu Leu Arg Pro Arg Ser His Lys Ser Arg Val Lys Gly Phe Leu Arg Leu Lys Met A1a '1'yr Met Pro Lys Asn Gly Gly Gln Asp Glu Glu Asn Ser Asp Gln Arg Asp agagcattcc ttcaaatgga cagtccaaaa caccagtcag atccatctga agatgaggat 42U
gaaagaagtt ctcagaagct acactctacc tcacagaaca tcaacctggg accgtctgga 480 aatcctcatgccaaaccaactgactttgatttcttaaaagttattggaaaaggcagcttt540 ggcaaggttcttcttgcaaaacggaaactggatggaaaattttatgct caaagtgtta600 gt cagaaaaaaatagttctcaacagaaaagagcaaaaacatatt atggctgaacgtaatgt 660 g ctcttgaaaaatgtgaaacatccgtttttggttggattgcattattccttccaaacaact720 gaaaagctttattttgttctggattttgttaatggaggggagctttttttccacttacaa780 agagaacggtcctttcctgagcacagagctaggttttacgctgctgaaattgctagtgca840 ttgggttacttacattccatcaaaatagtatacagagacttgaaaccagaaaatattctt900 ttggattcagtaggacatgttgtcttaacagattttgggctttgtaaagaaggaattgct960 atttctgacaccact attttgtgggacaccagagtatcttgcacctgaagtaatt1020 accac agaaaacagccctatgacaatactgtagattggtggtgccttggggctgttctgtatgaa1080 atgctgtatggattgcctcctttttattgccgagatgttgctgaaatgtatgacaatatc1140 cttcacaaacccctaagtttgaggccaggagtgagtcttacagcctggtccattctggaa1200 gaactcctagaaaaagacaggcaaaatcgacttggtgccaaggaagactttcttgaaatt1260 cagaatcatcctttttttgaatcactcagctgggctgaccttgt acaaaagaagattcca1320 ccaccatttaatcctaat ggctggaccagatgatatcagaaactttgacacagcattt1380 gt acagaagaaacagttccatattctgtgtgtgtatcttctgactattctatagtgaatgcc1440 agtgtattggaggcagatgatgcattcgttggtttctcttatgcacctccttcagaagac1500 ttatttttgtgagcagtttgccattcagaaaccattgagcaaaataagtctatagatggg1560 actgaaacttctatttgtgtgaatatattcaaatatgtataactagtgcctcatttttat1620 atgtaatgatgaaaactatgaaaaaatgtattttcttctatgtgcaagaaaaatagggca1680 tttcaaagagctgttttgattaaaatttatattcttgtttaataagctttttttaaaca1740 a atttaaaagctattattcttagcattaacctatttttaaagaaaccttttttgctattga1800 ctgtt-ttttccctctaagtttacactaacatctacccaagatagact ttttaacagt1860 gtt caatttcagttcagctaacatatattaatacctttgtaactctttgctatggcttttgtt1920 atcacaccaaaactatgcaattggtacatggttgttt aagaaaccgtatttttccat1980 aag gataaatcaCtgtttgaaatatttggttcatggtatgatcgaaatgtaaaagcataatta2040 acacattggctgctagttaacaattggaataactttattctgcagatcatttaagaagta2100 acaggccgggcgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggc2160 agatcacctaggtcaggagttggagacca.gcctgaccaacatggacaaaccccgtctct2220 g actaaaaatacaaaattggcagggtgtggtggcacatgcctataatcccagctacttggg2280 Asp Met Glu His Gly 'Prp Glu Val Val Asp Ser Asn Asp Ser Ala Ser Gln His Gln Glu Glu Leu Pro Pro Pro Pro Leu Pro Pro Gly Trp Glu Glu Lys Val Asp Asn Leu Gly Arg i'hr Tyr Tyr Val Asn His Asn Asn Arg '1'hr '1'hr Gln Trp His Arg Pro Ser Leu Met Asp Val Sex Ser Glu Ser Asp Asn Asn Ile Arg Gln Ile Asn Gln Glu Ala Ala His Axg Arg Phe Arg Ser Arg Arg His Ile Ser Glu Asp Leu Glu Pro G1u Pro Ser Glu Gly Gly Asp Val Pro Glu Pro Trp Glu Thr Ile Ser Glu Glu Val Asn Ile Ala Gly Asp Ser Leu Gly Leu Ala Leu Pro Pro Pro Pro Ala Ser Pro Gly Ser Arg Thr Ser Pro Gln Glu Leu Ser Glu Glu Leu Ser Arg Arg Leu Gln Ile Thr Pro Asp Ser Asn Gly Glu Gln Phe Ser Ser Leu Ile Gln Arg Glu Pro Ser Ser Arg Leu Arg Ser Cys Ser Val Thr Asp Ala Val Ala Glu Gln Gly His Leu Pro Pro Pro Ser Val Ala Tyr Val His '1'hr 'lhr Pro Gly Z,eu Pro Ser Gly 'lrp Glu Glu Arg Lys Asp Ala Lys Gly Arg '1'hr l'yr '1'yr Val Asn His Asn Asn Arg Thr '1'hr Thr Trp Thr Arg Pro Ile Met Gln Leu Ala Glu Asp Gly Ala Ser Gly Ser Ala 'i'hr Asn Ser Asn Asn His Leu Ile G1u Pro Gln Ile Arg Arg Pro ' Arg Ser Leu Ser Ser Pro '1'hr Val Thr Leu Ser Ala Pro Leu Glu Gly Ala Lys Asp Ser Pro Val Arg Arg Ala Val Lys Asp Thr Leu Ser Asn Pro Gln Ser Pro Gln Pro Ser Pro Tyr Asn Ser Pro Lys Pro Gln His Lys Val Thr G1n Ser Phe Leu Pro Pro Gly Trp Glu Met Arg Ile Ala Pro Asn Gly Arg Pro Phe Phe Ile Asp His Asn Thr Lys 'lhr Thr 'Ch r Trp Glu Asp Pro Arg Leu Lys Phe Pro Val His Met Arg Ser Lys 'I'hr Ser Leu Asn Pro Asn Asp Leu Gly Pro Leu Pro Pro Gly Trp Glu Glu Arg Ile His Leu Asp Gly Arg Thr Phe Tyr Ile Asp His Asn Ser Lys Ile 'lhr Gln Trp G1u Asp Pro Arg Leu Gln Asn Pro Ala Ile Thr Gly Pro Ala Val Pro 1'yr Ser Arg Glu Phe Lys Gln Lys Tyr Asp Tyr Phe Arg Lys Lys Leu Lys Lys Pro Ala Asp Ile Pro Asn Arg Phe Glu Met Lys Leu His Arg Asn Asn Ile Phe Glu Glu Ser Tyr Arg Arg Ile Met Ser Va1 Lys Arg Pro Asp Val Leu Lys Ala Arg Leu Trp Ile Glu Phe Glu Ser Glu Lys Gly Leu Asp Tyr Gly Gly Val Ala Arg Glu 1'rp Phe Phe Leu Leu Ser Lys Glu Met Pyre Asn Pro Tyr Tyr Gly Leu Phe Glu Tyr Ser Ala Thr Asp Asn 'i'yr Thr Leu Gln Ile Asrr Pro Asn Ser Gly Leu Cys Asn Glu Asp His Leu Ser 'lyr Phe Thr Phe Tle G1y Arg Val Ala Gly Leu Ala Val Phe His Gly Lys Leu Leu Asp Gly Phe Phe Ile Arg Pro Phe Tyr Lys Met Met Leu Gly Lys Gln Ile Thr Leu Asn Asp Met Glu Ser Val Asp Ser Glu Tyr Tyr Asn Ser Leu Lys Trp Ile Leu Glu Asn Asp Pro Thr Glu Leu Asp Leu Met Phe Cys Tle Asp Glu Glu Asn Phe Gly G1n Thr Tyr Gln Val Asp Leu Lys Pro Asn Gly Ser Glu Ile Met Val 'lhr Asn Glu Asn Lys Arg Glu 7.'yr Ile Asp Leu Val Ile Gln Trp Arg Plue Val Asn Arg Val Gln Lys Gln Met Asn Ala Phe Leu Glu Gly Phe 'lhr Glu Leu Leu Pro Ile Asp Leu Ile Lys Ile Phe Asp Glu Asn Glu Leu Glu Leu Leu P4et Cys Gly Leu Gly Asp Val Asp Val Asn Asp 'I'rp Arg Gln His Ser Ile '1'yr Lys Asn Gly Tyr Cys Pro Asn His Pro Val Ile Gln 1'rp Phe 'i'rp Lys Ala Val Leu Leu Met Asp Ala Glu Lys Arg Ile Arg Leu Leu G1n Plre Val Thr Gly 'Phr Ser Arg Val Pro Met Asn Gly Phe Ala Glu Leu 'Iyr Gly Ser Asn Gly Pro Gln Leu Phe Thr Ile Glu Gln Trp Gly Ser Pro Glu Lys Leu Pro Arg Ala His 'Phr Cys Phe Asn Arg Leu Asp Leu Pro Pro '.Cyr GIu 2'hr Phe Glu Asp Leu Arg Glu Lys Leu Leu Met Ala Val Glu Asn Ala Gln Gly Phe Glu 9f30 985 990 Gly Val Asp

Claims (18)

Claims

1. The use of an antagonist of the hsgk1 protein or the hsgk3 protein, which antagonist is structurally similar to a natural substrate of the hsgk1 protein or the hsgk3 protein, or the use of a negative regulator of the transcription of the hsgk1 gene or hsgk3 gene, for producing a pharmaceutical for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy.

2. The use of an antagonist of the hsgk1 protein or the hsgk3 protein as claimed in claim 2, wherein this antagonist is structurally similar to the phosphorylatable amino acid serine or threonine.

3. A pharmaceutical comprising an antagonist of the hsgk1 protein or the hsgk3 protein, which antagonist is structurally similar to a natural substrate of the hsgk1 protein or the hsgk3 protein, or comprising a negative regulator of the transcription of the hsgk1 gene or the hsgk3 gene, for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy.

4. The pharmaceutical comprising an antagonist of the hsgk1 protein or the hsgk3 protein as claimed in claim 3, wherein this antagonist is structurally similar to the phosphorylatable amino acid serine or threonine.

5. The use of a single-stranded or double-stranded nucleic acid encompassing the sequence of a human homology of the hsgk family, or of one of its fragment, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy.

6. The use as claimed in claim 5, characterized in that the single-stranded or double-stranded nucleic acid encompasses the sequence of the hsgk1 gene according to Acc No. NM_005627, or of one of its fragments.

7. The use as claimed in claim 6, characterized in that the hsgk1 gene encompasses at least one polymorphic nucleotide, in particular an "SNP" of the hsgk1 gene.

8. The use as claimed in claim 7, characterized in that the polymorphic nucleotide of the hsgk1 gene is selected from the group comprising the G
insertion at position 732/733 in intron 2 of the hsgk1 gene, the T/C substitution at position 2071 in intron 6 of the hsgk1 gene and the T/C
substitution at position 2617 in exon 8 of the hsgk1 gene.

9. The use as claimed in claim 5, characterized in that the single-stranded or double-stranded nucleic acid encompasses the sequence of the hsgk3 gene according to Acc No. AF169035, or of one of its fragments.

10. The use as claimed in claim 9, characterized in that the hsgk1 gene encompasses at least one polymorphic nucleotide, in particular an "SNP" of the hsgk3 gene.

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

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

13. A kit for diagnosing one of the diseases cataract, glaucoma and diabetic neuropathy, comprising antibodies which are directed against hsgk1 or hsgk3 or comprising nucleic acids which are able to hybridize, under stringent conditions, with the hsgk1 gene according to Acc No. NM_005627 or with the hsgk3 gene according to Acc No. AF169035, or comprising these antibodies and nucleic acids jointly.

14. The kit as claimed in claim 13, characterized in that the nucleic acids are able to hybridize, under stringent conditions, with the DNA regions of the hsgk1 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 hsgk1 gene or of the hsgk3 gene.

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 disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps:
a) Heterologously coexpressing i) the glucose transporter Glut1 and ii) hsgk1 and/or hsgk3 in cells, b) culturing at least one cell aliquot A1 to A X
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, and culturing a control cell aliquot B in the absence of any test substance, c) determining the activity of the glucose transporter Glut1 in the cell aliquots A1 to A X as compared with the activity of the glucose transporter Glut1 in the control cell aliquot B.

6. The screening method as claimed in claim 15, wherein, in step a), the hsgk1 substrate Nedd4-2 is concomitantly coexpressed in addition to, or instead of, the glucose transporter Glut1 and wherein, in step c), the degree of phosphorylation of the hsgk1 substrate Nedd4-2 (Acc No. BAA23711) is determined in the cell aliquots A1 to A X in comparison with the control cell aliquot B.

7. 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 disease selected from the group comprising cataract, glaucoma and diabetic neuropathy, comprising the following steps:
a) Heterologously coexpressing i) the glucose transporter Glut1 and ii) hsgk1 and/or hsgk3 in at least one aliquot A1 to A X of cells, and heterologously expressing i) the glucose transporter Glut1 in at least one aliquot B1 to B X of cells b) culturing the cell aliquots A1 to A X and B1 to B X 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, c) carrying out a comparative determination of the activities of the glucose transporter Glut1 in the cell aliquots A1 to A X and in the cell aliquots B1 to B X.

18. The screening method as claimed in claim 17, wherein, in step a), the hsgk1 substrate Nedd4-2 is concomitantly coexpressed, both in the cell aliquots A1 to A X and in the cell aliquots B1 to B X, in addition to, or instead of, the glucose transporter Glut1 and wherein, in step c), the degree of phosphorylation of the hsgk1 substrate Nedd4-2 (Acc No. BAA23711) is determined in the cell aliquots A1 to A X in comparison with the cell aliquots B1 to B X.