DE10305212A1 - Use of the sgk gene family for the diagnosis and therapy of cataracts and glaucoma - Google Patents

Abstract

The invention relates to the use of a functional inhibitor of the hsgk1 or hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene for the production of a medicament for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy. DOLLAR A In a further aspect, the invention relates to the use of a single- or double-stranded nucleic acid, comprising the hsgk1 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 the diagnosis of a predisposition for the formation of cataract, glaucoma and / or diabetic neuropathy, as well as a kit for the diagnosis of a predisposition for the formation of cataract, glaucoma and / or diabetic neuropathy, which comprises the above-mentioned nucleic acid. DOLLAR A 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.

Description

The invention relates to the use a functional inhibitor of the hsgk1 or hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene for the manufacture of a medicament for therapy and / or prophylaxis a cataract, glaucoma or diabetic neuropathy.

In another aspect concerns the invention comprises the use of a single or double stranded nucleic acid the hsgk1 sequence according to Acc No. NM_005627 or one of its fragments or comprising the hsgk3 sequence according to Acc. No. AF169035 or one their fragments to diagnose a predisposition to education of cataract, glaucoma and / or diabetic neuropathy, as well as one Kit for diagnosing a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy, which comprises the above nucleic acid.

Another object of the invention are different screening methods for identification and characterization of therapeutically active substances from a variety of test substances for therapy and / or prophylaxis of at least one disease selected from cataract, glaucoma and diabetic neuropathy.

The serum and glucocorticoid inducible Kinase hsgk1 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 to 2040].

The following investigations revealed that hsgk1 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 Nat1 Acad Sci USA 1999; 96: 2514-2519; Náray-Fejes-Tóth 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].

The hsgk1 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 glucocorticoidinducible kinase ( SGK) is a target of the PI3-kinase-stimulated signaling pathway. EMBO J 1999; 18: 3024-3033; Kobayashi et al., Chaaacterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J.1999; 344: 189-197]. Activation of hsgk1 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, hsgk1 is a potent stimulator of the renal epithelial Na ⁺ channel [De 1a 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 Nat1 Acad Sci USA 2000; 97: 8157-8162]. Since the hsgk1 is found in a large number of tissues which do not express the epithelial Na ⁺ channel ENaC, the function of the hsgk1 should not be limited 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 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-β 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 hsgk1, the hsgk1 and its human homologs should have considerable potential for the diagnosis of numerous diseases. From the DE 197 08 173 A1 shows in particular that the hsgk1 in many diseases in which cell volume changes play a crucial pathophysiological role, such as, for example, hypernatremia, hyponatremia, diabetes mellitus, renal failure, hypercatabolism, hepatic encephalopathy and microbial or viral infections, can be used for diagnosis.

In WO 00/62781 it was described that the hsgk1 activates the endothelial ^{Na +} channel, whereby the renal Na ⁺ absorption is increased. Since this increased renal Na ⁺ absorption is associated with hypertension, it was assumed here that an increased expression of hsgk1 should lead to hypertension, a reduced expression of hsgk1 should ultimately lead to hypotension.

Also in DE 100 421 37 a similar relationship between overexpression and overactivity of the human homologues hsgk2 and hsgk3 with overactivation of ENaC, the resulting increased renal Na ⁺ absorption and the resulting hypertension was described. Furthermore, the diagnostic potential of the kinases hsgk2 and hsgk3 regarding arterial hypertension has already been discussed.

The relationship was described in WO 02/074987 A2 between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgk1 gene with a genetic predisposition revealed to hypertension. This is a polymorphism in intron 6 (T → C) and a polymorphism in exon 8 (C → T) in the hsgk1 gene.

Due to the expression of sGK1 in numerous fabrics and due to the presumably large number of as yet unknown substrates of the sgk1 it is to be expected that there will be more Correlations between the function of the human homologs of the sgk family, in particular the hsgk1 gene (NM_005627), the hsgk2 gene, and the hsgk3 gene (AF169035) and will give training to other diseases. The detection such further specific disease correlations of the sgk1 could cause that containing nucleic acids polymorphic gene regions of the human homologues of the sgk family, which the Influence the function or expression of the corresponding sgk proteins, for the diagnosis of a predisposition of these other diseases could be used.

It was therefore an object of the invention further correlations between the function of human homologues the sgk family and new diseases and thus new diagnostic applications for nucleic acids that contain polymorphic gene regions of the human homologues of the sgk family.

This task was solved by the surprising finding that the hsgk1 and hsgk3 strongly stimulate the glucose transporter Glut1 (see 1 ). The glucose transporter Glut1 mediates, among other things, glucose uptake in various cells of the eye [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 Ophthahnol Vis Sci. 1991; 32: 1659-66]. The glucose follows osmotically water, so that an increased activity of Glut1 leads to cell swelling. Thus, an increased activity of Glut1 for the development of cataracts [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, it has been shown that Glut1 overexpression promotes 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 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 that stimulate the expression of SGK1 (see above) actually lead to the development of glaucoma [Fingert et al. 2001]. However, a causal role of hsgk1 has never been assumed.

The above disorders would be at in all conditions occur in which the hsgk1 has increased activity, in excess of all hormones above. Certain polymorphisms of the hsgk1 gene, which 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 increased incidence of cataract and glaucoma to lead. The same modifications to the gene should also occur with premature Correlate cataract and / or glaucoma.

The present findings reveal a completely new mechanism in the regulation of the glucose transporter Glut1. An increased activity of hsgk1 should therefore lead to an increased uptake of glucose in the cells. The transcription of the hsgk1 is carried out 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 Sp1 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-β [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, Wärntges S et al, Excessive transcription of the human serum and glucocorticoid dependent kinase hSGK1 in lung fibrosis. Cell Physiol Biochem 2002, 12: 135-142]. In addition, the hsgk1 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 hsgk1 by cell shrinkage and / or by increased formation of TGF-β [Lang et al. 2000]. The expressed hsgk1 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].

The increased expression of hsgk1 increases according to the knowledge of the invention the activity of the glucose transporter Glut-1. This gets more glucose into the cells absorbed and the water following through osmosis causes a Cell swelling. In this way the increased water retention takes place in cornea and lens that over Decreasing transparency leads to cataracts [Gong et al. 2001].

In a similar way and additionally through Storage of connective tissue could glaucoma also develops [Fingert et al. 2001].

Even with diabetic neuropathy cell swelling is suspected as the cause [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 hsgk1 [Busjahn et al., Serum and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40 (3): 256-260, 2002] is with increased Glut1 activity to count. Lead glucocorticoids indeed on glaucoma [Fingert et al. 2001]. The mechanism responsible for glaucoma formation responsible for glucocorticoid administration was not previously known. In particular, it was not previously known that the hsgk1 used this mechanism plays a role and therefore acts as a target protein for diagnosis and therapy for glaucoma.

The observations according to the invention thus surprisingly show that the hsgk1 and the hsgk3 increase not only the epithelial Na ⁺ channel but also the non-epithelial glucose transport. Completely new pathophysiological meanings of hsgk1 and hsgk3 have been uncovered, which should have important diagnostic and therapeutic / prophylactic consequences.

An object of the invention relates thus the use of a functional inhibitor of hsgk1- or the hsgk3 protein or a negative transcription regulator the hsgk1 or hsgk3 gene to reduce cell swelling.

Another object of the invention relates to the use of a functional inhibitor of hsgk1- or the hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene for the manufacture of a medicament for Therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy.

This functional inhibitor of hsgk1 protein or the hsgk3 protein can be a chemical substance be of any kind that the normal physiological activity of the hsgk1 protein or hsgk3 protein inhibited. The functional inhibitor is preferably of the hsgk1 or hsgk3 protein is a low-molecular chemical Substance (a “small molecule ") or a protein or peptide. The functional inhibitor of the hsgk1 protein or the hsgk3 protein can in particular be an antagonist of this Enzymes, which is the substrate binding site of the hsgk1 protein or the hsgk3 protein is blocked, but at the same time no catalytic Implementation through which hsgk1 or hsgk3 is accessible. As an antagonist In this case, preference is given to those molecules which structural similarity with the natural Substrate of the hsgk1 protein or the hsgk3 protein, in particular a structural similarity with the phosphorylatable amino acids serine and threonine.

Staurosporin and Chelerythrin are two known functional inhibitors of hsgk1. As a functional inhibitor the hsgk1 or the hsgk3 for therapy and / or prophylaxis at least one of the diseases cataract, glaucoma or diabetic neuropathy is therefore in a particularly preferred embodiment either staurosporine or chelerythrine.

A negative transcription regulator the hsgk1 gene or the hsgk3 gene is defined as a substance the expression of the hsgk1 gene or the hsgk3 gene at the transcriptional level activated.

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 ingredient, the functional ingredient bitor or the negative transcription regulator of the hsgk1 or the hsgk3, additionally contain stabilizers and / or carriers, such as starch, lactose, stearic acid, fats, waxes, alcohols or other additives such as preservatives, colorings or flavorings.

The administration of the drug can be in any way, especially orally, in the form of tablets, granules, Capsules or as a solution respectively. Other particularly suitable dosage forms concern direct applications (e.g. on skin or eyes) in the form of ointments, Tinctures, sprays or any type of injection (e.g. subcutaneous, intravenously) or the infusion.

Another object of the invention includes the use of a single or double stranded nucleic acid the hsgk1 sequence according to Acc No. NM_005627 or one of its fragments to diagnose a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy. The fragment of hsgk1 that contains the single or double stranded nucleic acid may be 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 comprises preferably at least one polymorphic nucleotide of the hsgk1 gene, especially a “single nucleotide polymorphism (SNP) "of the hsgk1 gene.

• – eine G-Insertion an Position 732/733 in Intron 2 des hsgk1-Gens,
• – der T/C-Austausch an Position 2071 in Intron 6 des hsgk1-Gens (WO 02/074987 A2),
• – der T/C-Austausch an Position 2617 in Exon 8 des hsgk1-Gens (WO 02/074987 A2).

In a particularly preferred embodiment, the single-stranded or double-stranded nucleic acid comprises at least one of the following SNPs of the hsgk1 gene:

• A G insertion at position 732/733 in intron 2 of the hsgk1 gene,
• The T / C exchange at position 2071 in intron 6 of the hsgk1 gene (WO 02/074987 A2),
• - The T / C exchange at position 2617 in exon 8 of the hsgk1 gene (WO 02/074987 A2).

The above SNPs of the hsgk1 gene in The patient's genomic DNA or cDNA can be obtained using the above single or double stranded nucleic acids preferably by the following methods:

• - by direct sequencing of genomic DNA or cDNA with the above nucleic acids,
• - by specific hybridization of the genomic DNA or cDNA with the the above nucleic acids,
• - by a PCR oligonucleotide elongation assay or by a ligation assay.

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

It is believed that the activity of the expressed hsgk1 gene from the version of this polymorphism in the hsgk1 gene of the Patient depends and that itself consequently nucleic acids, that contain at least one of these polymorphisms, especially good for diagnosing a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy suitable.

The invention further relates to comprising the use of a single or double stranded nucleic acid the hsgk3 sequence according to Acc No. AF169035 or one of its fragments to diagnose a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy. The fragment of hsgk3 that contains the single or double stranded nucleic acid may be 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 comprises preferably at least one polymorphic nucleotide of the hsgk3 gene, especially a “single nucleotide polymorphism (SNP) "of the hsgk3 gene.

In addition to the individual or double-stranded nucleic acids certain antibodies are also suitable, those against substrates of the human homologues of the sgk family, in particular against substrates of hsgk1 and hsgk3, for diagnosis a predisposition to develop at least one of the diseases cataract, glaucoma, diabetic neuropathy. These are diagnostic antibodies preferably against such an epitope of human homologues sgk family (especially the hsgk1 and the hsgk3) directed which the phosphorylation site of the substrate either in phosphorylated Contains form or in non-phosphorylated form.

For example, an overexpression of the hsgk1 protein that occurs due to the individual genetic makeup of the hsgk1 gene could lead to an increased conversion of substrates of the hsgk, ie to an increased enzymatic phosphorylation of the substrates by the hsgk1. At the same time, the overexpression of the hsgk1 protein would lead to a stimulation of the glucose transporter Glut1, which ultimately leads to a strong glucose uptake in the cells of the eye, then a strong water uptake by osmosis and thus ultimately the predisposition to the development of cataracts, glaucoma and diabetic neuropathy. The detection of the more frequent phosphorylation of substrates of the hsgk1 with the aid of an antibody which is directed against a region of the substrate in question which contains the phosphorylation site of the hsgk1 in phosphorylated form or in nonphosphorylated form could thus be a method to diagnose a predisposition to develop cataracts, glaucoma and diabetic neuropathy.

In a preferred embodiment is the substrate of the human homologue of the sgk family, the ubiquitin protein ligase Nedd4-2 (Acc No. BAA23711) used. This ubiquitin protein ligase represents a protein which is derived from the human homologues of the sgk family is specifically phosphorylated [Debonneville et al., Phosphorylation of Nedd4-2 by SGK1 regulates epithelial Na (+) channel cell surface expression. FMBO 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 hsgk1 have the consensus sequence (R X R X X S / T), where R for arginine, S for serine, T for threonine and X for any amino acid stands. There are two potentials in Nedd4-2 2 (Acc No. BAA23711) Phosphorylation sites for the hsgk1 on which the above consensus sequence fits: the Serine at the amino acid position 382 and the serine at the amino acid position 468th

The above antibodies for Diagnosis of a predisposition to develop at least one of the diseases cataract, glaucoma, diabetic neuropathy is therefore preferred against the substrate Nedd4-2 directed and particularly preferred against a protein region of Nedd4-2 with the sequence of the potential phosphorylation site for the hsgk1, the consensus sequence (R X R X X S / T). In particular are these antibodies directed against those Nedd4-2 protein regions that at least one of the two potential phosphorylation sites serine at the amino acid position 382 and / or serine at the amino acid position Include 468.

• – Antikörper, die gegen hsgk1 oder hsgk3 gerichtet sind,
• – einzel- oder doppelsträngige Nukleinsäuren, die mit dem hsgk1-Gen nach Acc No. NM_005627 oder mit dem hsgk3-Gen nach Acc No. AF169035 unter stringenten Bedingungen hybridisieren können; insbesondere solche einzel- oder doppelsträngigen Nukleinsäuren, die polymorphe Nukleotide, insbesondere „SNPs" des hsgk1-Gens oder des hsgk3-Gens umfassen,
• – Antikörper, die gegen ein Substrat eines humanen Homologen der sgk-Familie gerichtet sind; vorzugsweise Antikörper, die gegen die Phosphorylierungsstelle dieses Substrates in der phosphorylierten oder nicht phosphorylierten Form gerichtet sind; insbesondere Antikörper, die gegen die Phosphorylierungsstelle von Nedd4 oder Nedd4-2 in der phosphorylierten oder nicht phosphorylierten Form gerichtet sind.

The invention further relates to a kit for diagnosing one of the diseases of cataract, glaucoma and diabetic neuropathy, comprising at least one of the following components:

• - antibodies directed against hsgk1 or hsgk3,
• - Single- or double-stranded nucleic acids, which with the hsgk1 gene acc. NM_005627 or with the hsgk3 gene acc. Can hybridize AF169035 under stringent conditions; in particular those single-stranded or double-stranded nucleic acids which comprise polymorphic nucleotides, in particular “SNPs” of the hsgk1 gene or of 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.

• a) Heterologe Koexpression von i) dem Glucosetransporter Glut1 und ii) der hsgk1 und/oder der hsgk3 in Zellen,
• b) Kultivierung mindestens eines Zell-Anteils A₁ bis A_X in Gegenwart von jeweils mindestens einer Test-Substanz, wobei sich die mindestens eine Test-Substanz in Abhängigkeit vom Index 1 bis X des Zell-Anteils jeweils unterscheidet und Kultivierung von einem Kontroll-Zell-Anteil B in Abwesenheit jeglicher Test-Substanz,
• c) Bestimmung der Aktivität des Glucosetransporters Glut1 in den Zell-Anteilen A₁ bis A_X im Vergleich zur Aktivität des Glucosetransporters Glut1 im Kontroll-Zell-Anteil B.

The invention further relates to a screening method for identifying and characterizing 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 Glut1 and ii) the hsgk1 and / or the hsgk3 in cells,
• b) cultivation of at least one cell component A ₁ to A _X in the presence of at least one test substance, the at least one test substance depending on the index 1 to X of the cell component differs in each case and cultivation by a control Cell portion B in the absence of any test substance,
• c) Determination of the activity of the glucose transporter Glut1 in the cell fractions A ₁ to A _X in comparison to the activity of the glucose transporter Glut1 in the control cell fraction B.

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

In step a), suitable cells, preferably mammalian cells or cell lines, especially human cells or cell lines with suitable ones Expression vectors containing suitable expression cassettes for Expression of Glut1 and of hsgk1 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 (Glut1, hsgk1, hsgk3) under the control of suitable promoters in the questionable Cell type are active and express the target gene in an appropriate amount can. The expression vectors can continue to contain selection markers.

Then the transfected Cells are cultivated under such conditions that the expression of Allow target genes i) and ii).

In step b), the transfected cells from a) are divided into different cell portions (aliquots) A ₁ to A _X and a control cell portion B. The cell portions A ₁ to A _X are each in the presence of cultivated at least one test substance. The test substances added to the cell portions A ₁ to A _X differ from one another (depending on the index 1 to X of the respective cell portion A ₁ to A _X ). 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 Glut1 in the cell portions A ₁ to A _{X is} quantitatively determined in comparison to the activity of the glucose transporter Glut1 in the control cell portion B. A test substance that functionally functions the hsgk1 or the hsgk3 should have been added to the cell portions A ₁ to A _X , in which a significantly lower value of the Glut1 activity is measured compared to the control cell portion B. can inhibit or reduce their expression. Such a substance could be suitable for the therapy of at least one of the diseases cataract, glaucoma or diabetic neuropathy.

• d) Heterologe Koexpression von i) dem Glucosetransporter Glut1 und ii) der hsgk1 und/oder der hsgk3 in mindestens einem Anteil A₁ bis A_X von Zellen und heterologe Expression von
• i) dem Glucosetransporter Glut1 in mindestens einem Anteil B₁ bis B_X von Zellen
• e) Kultivierung der Zell-Anteile A₁ bis A_X und B₁ bis B_X in Gegenwart von jeweils mindestens einer Test-Substanz, wobei die mindestens eine Test-Substanz sich in Abhängigkeit von dem Index 1 bis X der Zell-Anteile jeweils unterscheidet,
• f) vergleichende Bestimmung der Aktivität des Glucosetransporters Glut1 in den Zell-Anteilen A₁ bis A_X und in den Zell-Anteilen B₁ bis B_X.

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 Glut1 and ii) the hsgk1 and / or the hsgk3 in at least a proportion A ₁ to A _X of cells and heterologous expression of
• i) the glucose transporter Glut1 in at least a portion B ₁ to B _X of cells
• e) Culturing the cell fractions A ₁ to A _X and B ₁ to B _X in the presence of at least one test substance, the at least one test substance differing depending on the index 1 to X of the cell fractions .
• f) Comparative determination of the activity of the glucose transporter Glut1 in the cell parts A ₁ to A _X and in the cell parts B ₁ to B _X.

The explanations above of the individual process steps a) to c) apply to the process steps d) to f) corresponding to the alternative screening method according to the invention.

The invention is illustrated by the following 1 explained in more detail.

On ordinate A the 1 the uptake of 2-deoxyglucose (in pmol / 1/10 min / oocyte) is plotted (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).

1 shows the increased uptake of 2-deoxyglucose in those oocytes which, in addition to glut 1, express hsgk1 or hsgk3 in comparison to those oocytes which express glut 1 alone. This shows that the function of the hsgk1 and the hsgk3 efficiently stimulate the activity of the glucose transporter Glut1. A similar effect is not seen for those oocytes which express the hsgk2 or PKBmut instead of hsgk1 or hsgk3.

The invention is illustrated by the following Example closer explained.

Example 1: Expression in Xenopus laevis oocytes and two-electrode voltage clamp

cRNA of normal SGK1 [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 the constitutively active SGK1 ( ^S422D SGK1) [Kobayashi & Cohen 1999], and normal glow1 [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 Glut1 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 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 glut1, 7.5 ng of human ^S422D SGK1 and / or with 5 ng of Xenopus Nedd4-2. Control oocytes 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 KCl, 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 titrated to pH 7.4 with HCl or NaOH.

calculations

The data is called arithmetic Mean values ± SEM indicated, n is the number of oocytes examined. All experiments were carried out in at least three different groups of oocytes. The Results were tested using the Student's t-test for significant differences tested. Only results with P <0.05 were viewed as statistically significant.

Claims (15)

Use of a functional inhibitor of the hsgk1 or hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene to reduce cell swelling. Use of a functional inhibitor of the hsgk1- or the hsgk3 protein or a negative transcription regulator of the hsgk1 or hsgk3 gene for the manufacture of a medicament for Therapy and / or prophylaxis of cataracts, glaucoma or diabetic neuropathy. Use according to claim 1 or 2, characterized in that that the functional inhibitor of the hsgk1 protein or the hsgk3 protein Is staurosporine or chelerythrine. Medicament containing a functional inhibitor of the hsgk1 or hsgk3 protein or a negative transcriptional regulator of the hsgk1 or hsgk3 gene for the therapy and / or prophylaxis of cataracts, glaucoma or diabetic neuropathy. Comprehensive use of a single or double stranded nucleic acid the hsgk1 sequence acc. to Acc No. NM_005627 or one of its fragments, for diagnosis a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy. Use according to claim 5, characterized in that the single or double stranded nucleic acid at least one polymorphic nucleotide of the hsgk1 gene, in particular an "SNP" of the hsgk1 gene. Use according to claim 6, characterized in that that the SNP of the hsgk1 gene selected is from the group of SNPs consisting of the G insertion at position 732/733 in intron 2 of the hsgk1 gene, the T / C exchange at position 2071 in intron 6 of the hsgk1 gene and the T / C exchange in position 2617 in exon 8 of the hsgk1 gene. Comprehensive use of a single or double stranded nucleic acid the hsgk3 sequence acc. to Acc No. AF169035 or one of its fragments used to diagnose a predisposition for the development of cataracts, glaucoma and / or diabetic neuropathy. Use according to claim 8, characterized in that the single or double stranded nucleic acid at least one polymorphic nucleotide of the hsgk3 gene, in particular an "SNP" of the hsgk1 gene. Use of an antibody against a substrate human homologues of the sgk family to diagnose a predisposition to develop at least one of the diseases cataract, glaucoma, diabetic neuropathy, the antibody against such an epitope of the human homologue which is the phosphorylation site either in phosphorylated form or in non-phosphorylated Contains form. Use according to claim 10, characterized in that that this Substrate of the human homologue of the sgk family Nedd4-2 with the Acc No. BAA23711 is. Kit for diagnosing one of the diseases of cataract, Glaucoma and diabetic neuropathy containing antibodies that directed against hsgk1 or hsgk3 or containing nucleic acids which with the hsgk1 gene acc. NM_005627 or with the hsgk3 gene acc. to Acc No. Hybridize AF169035 under stringent conditions can, or these antibodies and nucleic acids together. Kit according to claim 12, characterized in that the nucleic acids with such DNA regions of the hsgk1 gene according to Acc No. NM_005627 or of the hsgk3 gene acc. AF169035 under stringent conditions can hybridize the polymorphic nucleotides, in particular "SNPs" of the hsgk1 gene or the hsgk3 gene include. 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 Glut1 and ii) the hsgk1 and / or the hsgk3 in cells, b) cultivation of at least one cell component A ₁ to A _X in the presence of at least one test substance, the at least one test being -Substance differentiated depending on the index 1 to X of the cell portion and cultivation of a control cell portion B in the absence of any test substance, c) determination of the activity of the glucose transporter Glut1 in the cell portions A ₁ to A _X compared to the activity of the glucose transporter Glut1 in the control cell part B. 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 Glut1 and ii) the hsgk1 and / or the hsgk3 in at least a portion A ₁ to A _X of cells and heterologous expression of i) the glucose transporter Glut1 in at least a portion B ₁ to B _X of cells e ) Culturing the cell fractions A ₁ to A _X and B ₁ to B _X in the presence of at least one test substance, the at least one test substance differing depending on the index 1 to X of the cell fractions, f) Comparative determination of the activity of the glucose transporter Glut1 in the cell parts A ₁ to A _X and in the cell parts B ₁ to B _X.