WO2004069258A2

WO2004069258A2 - Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma

Info

Publication number: WO2004069258A2
Application number: PCT/EP2004/001048
Authority: WO
Inventors: Florian Lang; Andreas Busjahn
Original assignee: Florian Lang; Andreas Busjahn
Priority date: 2003-02-07
Filing date: 2004-02-05
Publication date: 2004-08-19
Also published as: PL378399A1; WO2004069258A3; JP2006519189A; CN1771039A; DE10305212A1; AU2004210416A1; BRPI0407300A; ZA200506280B; EP1663246A2; MXPA05008394A; CA2514703A1; KR20050114214A; RU2005127808A

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

Use of the sgk gene family for the diagnosis and therapy of cataracts and

glaucoma

The invention relates to the use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene for the manufacture of a medicament for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy.

In a further aspect, the invention relates to the use of a single- or double-stranded nucleic acid comprising the hsgkl sequence according to Acc No. NM_005627 or one of its fragments or comprising the hsgk3 sequence according to Acc. No. AF169035 or one of its fragments for diagnosing a predisposition to form cataract, glaucoma and / or diabetic neuropathy, and a kit for diagnosing a predisposition to form cataract, glaucoma and / or diabetic neuropathy, which comprises the above-mentioned nucleic acid.

Another object of the invention are various screening methods for the identification and characterization of therapeutically active substances from a large number of test substances 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].

The following investigations revealed that the hsgkl is under the influence of a variety 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 the mineralocorticoids [Chen et al, Epithelial sodium Channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sei USA 1999; 96: 2514-2519; Näray-Fejes-Tόth et al, sgk is an aldosterone-induced kinase in the renal collecting duet. 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, fountain pen 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].

The hsgkl is stimulated by the "insulin-like growth factor IGF1", by insulin and by oxidative stress via a signal cascade by phosphoinositol-3-kinase (PI3 kinase) and phosphoinositol-dependent kinase PDK1 [Park et al., Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated 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 phosphorylation on the serine at position 422. The mutation of this serine in an aspartate ( ^S422D SGK1) leads to a constitutively active kinase [ Kobayashi T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999; 339: 319 -328 ].

As previous studies have shown, the 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; Böhmer 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 regulation of cell volume sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sei USA 2000; 97: 8157-8162]. Since the hsgkl is found in a large number of tissues which do not express the epithelial Na ⁺ channel ENaC, the function of the hsgkl should not be restricted to the regulation of 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 putative human serme / ti-ureonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sei USA 1997; 94: 4440-4445; Waldegger et al, h-sgk Serine-Threonine protein kinase gene as early transcriptional target of TGF-ß in human intestine. Gastroenterology 1999; 116: 1081-1088]. Due to the presumably numerous, as yet unexplained regulations of further signal transduction pathways or their components by the hsgkl, the hsgkl and its human homologues should have considerable potential for the diagnosis of numerous diseases. DE 197 08 173 AI shows in particular that the hsgkl for diagnosis in many diseases in which cell volume changes play a crucial pathophysiological role, such as hypernatremia, hyponatremia, diabetes mellitus, renal failure, hypercatabolism, hepatic encephalopathy and microbial or viral infections can be used.

WO 00/62781 has described that the hsgkl activates the endothelial Na ⁺ channel, which increases renal Na absorption. Since this increased renal Na absorption is associated with hypertension, it was assumed here that an increased expression of the hsgkl should lead to hypertension, a reduced expression of the hsgkl should ultimately lead to hypotension. -

DE 100 421 37 also describes a similar relationship between the overexpression or overactivity of the human homologues hsgk2 and hsgk3 with the overactivation of the ENaC, the resulting increased renal Na absorption and the hypertension that develops from this. Furthermore, the diagnostic potential of the kinases hsgk2 and hsgk3 regarding arterial hypertension has already been discussed.

In WO02 / 074987 A2 the connection between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgkl gene with a genetically determined predisposition to hypertension was disclosed. This is a polymorphism in intron 6 (T → C) and a polymorphism in exon 8 (C → T) in the hsgkl gene.

Due to the expression of the sgkl in numerous tissues and due to the presumably large number of still unknown substrates of the sgkl, it can be expected that there will be further correlations between the function of the human homologues of the sgk family, in particular the hsgkl gene (NM_005627), des hsgk2 gene, and the hsgk3 gene (AF 169035) and the formation of further explanations. The discovery of such further specific disease correlations of the sgk could lead to the fact that nucleic acids containing polymorphic gene regions of the human homologues of the sgk family, which influence the function or expression of the corresponding sgk proteins, could be used to diagnose a predisposition for these further diseases , It was therefore an object of the invention to uncover further correlations between the function of the human homologs of the sgk family and new diseases and thus to provide new diagnostic uses for nucleic acids which contain polymorphic gene regions of the human homologues of the sgk family.

This task was solved by the surprising finding that the hsgkl and hsgk3 strongly stimulate the glucose transporter Glutl (see Fig. 1). The glucose transporter Glutl provides, among other things, glucose uptake into different cells of the eye [Busik et al., Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. luvest Ophthalmol Vis Be. 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 Sei. 1991; 32: 1659-66]. The glucose follows osmotically, so that an increased activity of glutl leads to cell swelling. Thus, an increased activity of Glutl for the development of cataracts [Gong et al., Development of cataractous macrophthalmia in mice expressing an active MEK1 in the lens. Invest Ophthalmol Vis Sei. 2001; 42: 539-48]. In addition, it was shown that Glutl overexpression requires the formation and deposition of connective tissue proteins £ Ayo et al., Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. At the 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 to 1814]. Such deposition of connective tissue proteins prevents the drainage of eye fluid and leads to pressure increases in the eye and thus damage to the retina [Fingert et al., Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension. Invest Ophthalmol Vis Sei. 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 Sei. 2002; 43: 1068-76]. Glucocorticoids that stimulate the expression of SGK1 (see above) actually lead to the development of glaucoma [Fingert et al. 2001]. However, a causal role of hsgkl has never been suspected.

The above-mentioned disorders would occur in all conditions in which the hsgkl has increased activity, that is to say in excess of all the above-mentioned hormones. Certain polymorphisms of the hsgkl gene that correlate with increased 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 increase Lead to occurrence of cataract and glaucoma. The same modifications of the gene should also correlate with premature cataract and / or glaucoma.

The present findings reveal a completely new mechanism in the regulation of the glucose transporter Glutl. An increased activity of the hsgkl should therefore lead to an increased uptake of glucose in the cells. The transcription of the hsgkl is carried out by serum [Webster et al. 1993], by glucocorticoids [Brenan & Füller 2000, Webster et al. 1993], by mineralocorticoids [Chen et al. 1999, Naray-Fejes-Toth et al. 1999, Shigaev et al. 2000, Brennan and Füller 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. Mole of endocrinol. 1997; 11: 1934 to 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. Mole of 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-ß [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, Wärntges S et al, Excessive transcription of the human serum and glucocorticoid dependent kinase hSGKl in hing fibrosis. Cell Physiol Biochem 2002, 12: 135-142]. In addition, the hsgkl transcription is increased by cell shrinkage, as the Waldegger et al. from 1997 shows. An increased glucose concentration, as occurs in diabetes mellitus, stimulates the expression of hsgkl by cell shrinkage and / or by increased formation of TGF-ß [Lang et al. 2000]. The expressed hsgkl is activated by the "insulin like growth factor" IGF1, by insulin and by oxidative stress [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].

According to the findings of the invention, the increased expression of the hsgkl increases the activity of the glucose transporter Glut-1. This means that more glucose is absorbed into the cells and the water that follows through osmosis causes cell swelling. In this way, there is increased water retention in the cornea and lens, which leads to cataracts by reducing transparency [Gong et al. 2001]. Glaucoma could also develop in a similar way and additionally through the incorporation of connective tissue [Fingert et al. 2001].

Cell swelling is also suspected to be the cause of diabetic neuropathy [Burg et al., Sorbitol, osmoregulation, and the complications of diabetes. J Clin Invest 1988; 81: 635-40]. Not only in diabetes mellitus, but also under the influence of glucocorticoids or in patients with a genetic overactivity of hsgkl [Busjahn et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40 (3): 256-260, 2002], increased glutl activity can be expected. Glucocorticoids actually lead to glaucoma [Fingert et al. 2001]. The mechanism responsible for glaucoma formation when glucocorticoid is administered has not yet been known. In particular, it was not previously known that hsgkl plays a role in this mechanism and is therefore suitable as a target protein for the diagnosis and therapy of glaucoma.

The observations according to the invention thus surprisingly show that the hsgkl and the hsgk3 increase not only the epithelial Na ⁺ channel, but also the non-epithelial glucose transport. Completely new pathophysiological meanings of the hsgkl and hsgk3 have been uncovered, which should have important diagnostic and therapeutic / prophylactic consequences.

One object of the invention thus relates to the use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene to reduce cell swelling.

Another object of the invention relates to the use of a 'functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene for the production of a medicament for the therapy and / or prophylaxis of a cataract, a glaucoma or the diabetic neuropathy.

This functional inhibitor of the hsgkl protein or the hsgk3 protein can be a chemical substance of any kind which inhibits the normal physiological activity of the hsgkl protein or the hsgk3 protein. The functional inhibitor of the hsgkl or hsgk3 protein is preferably a low-molecular 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 at the same time, no catalytic conversion is accessible through the hsgkl or hsgk3. In this case, suitable antagonists are preferably those molecules which have structural similarity to the natural substrate of the hsgkl protein or the hsgk3 protein, that is to say in particular a structural similarity to the phosphorylatable amino acids serine and threonine.

Staurosporin and chele-rythrin are two known functional inhibitors of hsgkl. In a particularly preferred embodiment, either staurosporin or chelerythrine is therefore used as the functional inhibitor of the hsgkl or the hsgk3 for the therapy and / or prophylaxis of at least one of the diseases cataract, glaucoma or diabetic neuropathy.

A negative transcription regulator 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 transcription level.

The medicament according to the invention for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy can, in addition to the actual active substance, the functional inhibitor or the negative transcription regulator of the hsgkl or the hsgk3, additionally stabilizers and / or carrier substances, such as starch, lactose , Stearic acid, fats, waxes, alcohols or other additives such as preservatives, colors or flavors. The drug can be administered in any way, in particular orally, in the form of tablets, granules, capsules or as a solution. Other particularly suitable dosage forms relate to direct applications (e.g. on the skin or eye) in the form of ointments, tinctures, sprays or any type of injection (e.g. subcutaneous, intravenous) or infusion.

Another object of the invention is the use of a single or double-stranded nucleic acid comprising the hsgkl sequence according to Acc No. NM_005627 or one of its fragments to diagnose a predisposition to develop cataracts, glaucoma and / or diabetic neuropathy. The fragment of hsgkl, which the single or double-stranded nucleic acid can comprise, is at least 10 nucleotides / base pairs long, preferably at least 15 nucleotides / base pairs long and in particular at least 20 nucleotides / base pairs long.

The single- or double-stranded nucleic acid here preferably comprises at least one polymorphic nucleotide of the hsgkl gene, in particular a “single nucleotide polymorphism (SNP)” of the hsgkl gene. In a particularly preferred embodiment, the single- or double-stranded nucleic acid comprises 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 exchange at position 2071 in intron 6 of the hsgkl gene (WO02 / 074987 A2), ^• the T / C exchange at position 2617 in exon 8 of the hsgkl gene (WO02 / 074987 A2).

The above SNPs of the hsgkl gene in the patient's genomic DNA or cDNA can preferably be detected using the abovementioned single or double-stranded nucleic acids by the following methods: by direct sequencing of the genomic DNA or cDNA with the above nucleic acids, by specific hybridization the genomic DNA or cDNA with the above nucleic acids, by a PCR oligonucleotide elongation assay or by a ligation assay.

The patient's genomic DNA or cDNA is preferably isolated from a patient's body sample, in particular from saliva, blood, tissue or cells.

It can be assumed that the activity of the expressed hsgkl gene depends on the version of this polymorphism in the patient's hsgkl gene and that nucleic acids which contain at least one of these polymorphisms are therefore particularly good for diagnosing a predisposition to the development of cataracts, glaucoma and / or diabetic neuropathy.

The invention further relates to the use of a single- or double-stranded nucleic acid comprising the hsgk3 sequence according to Acc No. AF169035 or one of its fragments to diagnose a predisposition to develop cataract, glaucoma and / or diabetic neuropathy. The fragment of hsgk3, which the single or double-stranded nucleic acid can comprise, is at least 10 nucleotides / base pairs long, preferably at least 15 nucleotides / base pairs long and in particular at least 20 nucleotides / base pairs long.

The single- or double-stranded nucleic acid preferably comprises at least one polymorphic nucleotide of the hsgk3 gene, in particular a “single nucleotide polymorphism (SNP)” of the hsgk3 gene. In addition to the single or double-stranded nucleic acids mentioned above, certain antibodies which are directed against substrates of the human homologues of the sgk family, in particular against substrates of the hsgkl and hsgk3, are also suitable for diagnosing a predisposition to the formation of at least one of the diseases cataract, Glaucoma, diabetic neuropathy. These diagnostic antibodies are preferably directed against such an epitope of the human homologues of the sgk family (in particular the hsgkl and the hsgk3) which contains the phosphorylation site of the substrate either in phosphorylated form or in nonphosphorylated form.

For example, an overexpression of the hsgkl protein that occurs due to the individual genetic makeup of the hsgkl gene could lead to an increased implementation of

Hsgk substrates, i.e. to an increased enzymatic phosphorylation of the

Guide substrates through the hsgkl. At the same time, the overexpression of the hsgkl -

Proteins lead to a stimulation of the glucose transporter Glutl, which ultimately leads to a strong glucose uptake in the cells of the eye, then a strong water uptake through osmosis and thus ultimately the predisposition to the development of cataracts, glaucoma and diabetic neuropathy. Detection of the more frequent phosphorylation of substrates of the hsgkl with the help of an antibody which is directed against a region of the substrate in question, which the

Contains phosphorylation site of hsgkl in phosphorylated form or in non-phosphorylated form, could therefore be a method for diagnosing a predisposition

Represent formation of cataracts, glaucoma and diabetic neuropathy.

In a preferred embodiment, the ubiquitin protein ligase Nedd4-2 (Acc No. BAA23711) is used as the substrate of the human homologue of the sgk family. This ubiquitin protein ligase is a protein which is specifically phosphorylated by the human homologues 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 the hsgkl have the consensus sequence (R X R X X S / T), where R is arginine, S is serine, T is threonine and X is any amino acid. In Nedd4-2 2 (Acc No. BAA23711) there are two potential phosphorylation sites for the hsgkl that the above consensus sequence fits: the serine at amino acid position 382 and the serine at amino acid position 468.

The abovementioned antibodies for diagnosing a predisposition to develop at least one of the diseases cataract, glaucoma, diabetic neuropathy are therefore preferably directed against the substrate Nedd4-2 and particularly preferably against directed a protein region of Nedd4-2 with the sequence of the potential phosphorylation site for the hsgkl, the consensus sequence (RXRXXS / T). In particular, these antibodies are directed against those Nedd4-2 protein regions which comprise at least one of the two potential phosphorylation sites serine at amino acid position 382 and / or serine at amino acid position 468.

Another object of the invention is a kit for the diagnosis of one of the diseases cataract, glaucoma and diabetic neuropathy, comprising at least one of the following components: ^" - Antibodies which are directed against hsgkl or hsgk3, single or double-stranded nucleic acids which with the hsgkl - Gene according to Acc No. NM_005627 or with the hsgk3 gene according to Acc No. AF169035 can hybridize under stringent conditions; in particular those single- or double-stranded nucleic acids which comprise polymorphic nucleotides, in particular “SNPs” of the hsgkl gene or the hsgk3 gene,

Antibodies directed against a substrate of a human homologue of the sgk family; preferably antibodies directed against the phosphorylation site of this substrate in the phosphorylated or non-phosphorylated form; especially antibodies directed against the phosphorylation site of Nedd4 or Nedd4-2 in the phosphorylated or non-phosphorylated form.

The invention further relates to a screening method for identifying and characterizing therapeutically active substances from a large number of test substances for therapy and / or prophylaxis of at least one disease selected from the group consisting of cataract, glaucoma and diabetic neuropathy, comprising the following steps: a) heterologous coexpression of i) the glucose transporter Glutl and ii) the hsgkl and / or the hsgk3 in cells, b) cultivation of at least one cell portion Ai to A in the presence of at least one test substance, wherein the at least one test substance differs depending on the index 1 to X of the Zeil portion and Cultivation of a control cell part B in the absence of any test substance, c) determination of the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax compared to the activity of the glucose transporter Glutl in the control cell part B.

A "substance library, preferably a" small molecule library ", but also a protein library or the like can be used as the" multitude of test substances ".

In step a) suitable cells, preferably mammalian cells or cell lines, in particular human cells or cell lines with suitable expression vectors, contain suitable expression cassettes for the expression of Glutl and of hsgkl and / or hsgk3 according to standard methods, such as electroporation, CaPO4 precipitation, lipofection or the like, transfected. The expression cassettes contain the genomic DNA or the cDNA of the target gene in question (Glutl, hsgkl, hsgk3) under the control of suitable promoters which are active in the cell type in question and can express the target gene in a suitable amount. The expression vectors can also contain selection markers.

The transfected cells are then cultivated under conditions which allow expression of the target genes i) and ii). In step b), the transfected cells from a) are divided into different Zeil parts (aliquots) Ai to Ax and a control cell part B. The Zeil parts Ai to Ax are each in the presence of at least one test Cultivated substance. The test substance (s) added to the Zeil fractions Ai to Ax differ from one another (depending on the index 1 to X of the respective Zeil fractions A _\ to Ax). The control cell portion B, on the other hand, is cultivated in the absence of any test substance.

In step c) the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax is quantitatively determined in comparison to the activity of the glucose transporter Glutl in the control cell fraction B. A test substance that can functionally inhibit the hsgkl or the hsgk3 should have been added to the Zeil portions Ai to Ax, in which a significantly lower value of the Glutl activity is measured compared to the control Zeil portion B. or reduces their expression. Such a substance could be suitable for the therapy of at least one of the diseases cataract, glaucoma or diabetic neuropathy. In an alternative embodiment, the screening method according to the invention for the identification and characterization of therapeutically active substances from a large number of test substances for the therapy and / or prophylaxis of at least one disease selected from the group consisting of cataract, glaucoma and diabetic neuropathy following steps: d) heterologous coexpression of i) the glucose transporter Glutl and ii) the hsgkl and / or the hsgk3 in at least a portion of Ai to Ax of cells and heterologous expression of i) the glucose transporter Glutl in at least a portion B _t to Bx of Cells e) cultivation of the Zeil fractions A _\ to Ax and Bi to Bx in the presence of at least one test substance, the at least one test substance depending on the index 1 to X of the Zeil fractions, f ) Comparative determination of the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax and in the line proportions Bi to B.

The above explanations of the individual process steps a) to c) apply correspondingly to process steps d) to fj of the alternative screening process according to the invention.

The invention is illustrated by the following Fig. 1.

The ordinate A in Fig. 1 shows the uptake of 2-deoxyglucose (in pmol 1/10 min / oocyte) (arithmetic mean ± SEM). Xenopus laevis oocytes were injected with cRNA from Glut-1 with or without cRNA from SGK1, SGK2, SGK3 or protein kinase B (PKB) (see Example 1).

Fig. 1 shows the increased uptake of 2-deoxyglucose in those oocytes which, in addition to glut 1, express the hsgkl or the hsgk3 in comparison to those oocytes which express glut 1 alone. This shows that the function of the hsgkl and the hsgk3 efficiently stimulate the activity of the glucose transporter Glutl. A similar effect is not found for those oocytes which express the hsgk2 or PKBmut instead of hsgkl or hsgk3. The invention is illustrated by the following example.

Example 1: Expression in Xenopus laevis oocytes and two-electrode voltage clamp cRNA of normal SGKl [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 Sei USA 1997; 94: 4440-4445] and the constitutively active SGKl ( ^S422D SGK1) [Kobayashi & Cohen 1999], and of normal Glutl [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: 30991-7] were synthesized in vitro. The dissection of Xenopus laevis ovaries and the collection and treatment of oocytes have already been described in detail [Wagner CA, Friedrich B, Setiawan I, Lang F, Bröer S: The use of Xenopus laevis ooeytes 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 SGKl and / or with 5 ng of Xenopus Nedd4-2. ControUocytes were injected with water. The uptake of radioactively labeled glucose was measured at room temperature 2 days after injection of the respective cRNAs. The control bath solution contained 96 mM NaCl, 2 mM KC1, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ and 5 mM HEPES, pH 7.4. All substances were used in the specified concentrations. The final solutions were nitrated to pH 7.4 with HC1 or NaOH.

calculations

The data are given as arithmetic mean ± SEM, n is the number of oocytes examined. All experiments were carried out in at least three different groups of oocytes. The results were tested for significant differences using the Student t-test. Only results with P <0.05 were considered to be statistically significant.

Claims

claims

1. Use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene to reduce cell swelling.

2. Use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene for the manufacture of a medicament for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy.

3. Use according to claim 1 or 2, characterized in that the functional inhibitor of the hsgkl protein or the hsgk3 protein is staurosporine or chelerythrine.

4. Medicament containing a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene

Therapy and / or prophylaxis of cataracts, glaucoma or diabetic neuropathy.

5. Use of a single- or double-stranded nucleic acid comprising the hsgkl sequence according to Acc No. NM_005627 or one of its fragments to diagnose a predisposition to develop cataracts, glaucoma and / or diabetic

Neuropathy.

6. Use according to claim 5, characterized in that the single- or double-stranded nucleic acid comprises at least one polymorphic nucleotide of the hsgkl gene, in particular an "SNP" of the hsgkl gene. 7. Use according to claim 6, characterized in that the SNP of the hsgkl gene is selected from the group of SNPs consisting of the G insertion at position 732/733 in intron 2 of the hsgkl gene, the T / C exchange at position 2071 in intron 6 of the hsgkl gene and the T / C exchange at position 2617 in exon 8 of the hsgkl gene.

8. Use of a single- or double-stranded nucleic acid comprising the hsgk3 sequence according to Acc No. AF169035 or one of its fragments used to diagnose a

Predisposition to develop cataracts, glaucoma and / or diabetic neuropathy.

, Use according to claim 8, characterized in that the single- or double-stranded nucleic acid comprises at least one polymorphic nucleotide of the hsgk3 gene, in particular an "SNP" of the hsgkl gene.

10. Use of an antibody against a substrate of a human homologue of the sgk family for the diagnosis of a predisposition to the formation of at least one of the

Diseases cataract, glaucoma, diabetic neuropathy, the antibody being directed against such an epitope of the human homologue which contains the phosphorylation site either in phosphorylated form or in nonphosphorylated form. 11. Use according to claim 10, characterized in that the substrate of the human homologue of the sgk family Nedd4-2 with the Acc No. BAA23711 is.

12. Kit for diagnosing one of the diseases cataract, glaucoma and diabetic neuropathy, containing antibodies which are directed against hsgkl or hsgk3 or containing nucleic acids which are linked to the hsgkl gene according to Acc No. NM_005627 or with the hsgk3 gene acc. AF 169035 can hybridize under stringent conditions, or these antibodies and nucleic acids together.

13. Kit according to claim 12, characterized in that the nucleic acids with such DNA regions of the hsgkl gene according to Acc No. NM_005627 or the hsgk3 gene acc. AF169035 can hybridize under stringent conditions that comprise polymorphic nucleotides, in particular “SNPs” of the hsgkl gene or the hsgk3 gene.

14. Screening method for the identification and characterization of therapeutically active substances from a large number of test substances for the therapy and / or prophylaxis of at least one disease selected from the group consisting of cataract, glaucoma and diabetic neuropathy, comprising the following steps: a) heterologous coexpression of i) the glucose transporter Glutl and ii) the hsgkl and / or the hsgk3 in cells, b) cultivation of at least one cell portion A _t to Ax in the presence of at least one test substance, the at least one Test substance differs depending on the index 1 to X of the Zeil portion and Cultivation of a control cell part B in the absence of any test substance, c) determination of the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax compared to the activity of the glucose transporter Glutl in the control cell part B.

15. Screening method for the identification and characterization of therapeutically active substances from a large number of test substances for the therapy and / or prophylaxis of at least one disease selected from the group consisting of cataract, glaucoma and diabetic neuropathy, comprising the following steps: d) heterologous coexpression of i) the glucose transporter Glutl and ii) the hsgkl and / or the hsgk3 in at least a portion A to Ax of cells and heterologous expression of i) the glucose transporter Glutl in at least a portion Bi to Bx of cells e) cultivation the Zeil fractions Ai to A and Bi to Bx in the presence of at least one test substance, the at least one test substance depending on the index 1 to X of the Zeil fractions, f) comparative determination of the activity of the glucose transporter Glutl in the Zeil parts A _t to Ax and in the Zeil parts Bi to Bx.