JP2006519189A - Use of the sgk gene family to diagnose and treat cataracts and glaucoma - Google Patents

Abstract

The present invention relates to the use of a functional inhibitor of hsgk1 or hsgk3 protein or a negative transcriptional regulator of hsgk1 or hsgk3 gene in the manufacture of a medicament for the treatment and / or prevention of cataract, glaucoma or diabetic neuropathy. . Other aspects of the invention include the hsgk1 sequence set forth in Registry No. NM005627 or one of its fragments in the diagnosis of a predisposition to the formation of cataract, glaucoma and / or diabetic neuropathy, or described in Accession No. AF16935. A nucleic acid as described above for diagnosing a predisposition to the formation of cataract, glaucoma and / or diabetic neuropathy, in addition to the use of a single-stranded or double-stranded nucleic acid comprising one of the hsgk3 sequences or a fragment thereof Relates to a kit comprising The invention further identifies and characterizes a therapeutically effective substance for treating and / or preventing at least one disease selected from cataract, glaucoma or diabetic neuropathy from among a plurality of test substances. To various screening methods.

Description

  The present invention relates to a functional inhibitor of hsgk1 protein or hsgk3 protein, or transcription of hsgk1 gene or hsgk3 gene for producing a medicament for the treatment and / or prevention of cataract, glaucoma or diabetic neuropathy. Relates to the use of negative regulators.

  In another aspect, the present invention provides a single-stranded or double-stranded nucleic acid comprising the hsgk1 sequence set forth in Accession No. NM005627 for diagnosing a predisposition to develop cataract, glaucoma, and / or diabetic neuropathy. Or one of its fragments, or one use of a single-stranded or double-stranded nucleic acid comprising the hsgk3 sequence set forth in accession number AF16935, or a fragment thereof, as well as cataract, glaucoma, and / or The present invention relates to a kit for diagnosing a predisposition to develop diabetic neuropathy (this kit contains the above-described nucleic acid).

  Moreover, the present invention relates to various screening methods for identifying and characterizing a substance having therapeutic activity from among a large number of test substances, wherein the substance having therapeutic activity is from cataract, glaucoma and diabetic neuropathy. Used to treat and / or prevent at least one selected disease.

  Serum and glucocorticoid-inducible kinase hsgk1 was originally cloned as a glucocorticoid sensitive gene [Webster et al. Charactorization of sgk, a novel member of the serine / threonine proteinkinase family which is transcribed inco- duced and circulated. Mol Cell Biol 1993; 13: 2031-2040].

Subsequent observations reveal that hsgk1 is under the influence of numerous stimuli [Lang F, Cohen P. et al. Regulation and physical roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov. 13; 2001 (108): RE17], such stimuli include, inter alia, mineralocorticoid stimuli [Chen et al. Episodic 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; 16973-1978; 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 upgrade 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 pro-inflammatory mediators in human granulation. J Leukoc Biol. 2000: 67: 240-248].

hsgk1 is stimulated by insulin-like growth factor IGF1, by insulin and by oxidative stress by the phosphoinositol-3-kinase (PI3 kinase) and phosphoinositol-dependent kinase PDK1 via the signal cascade [Park etc. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI3-kinase-stimulated signaling pathway, EMBO J 1999; 18: 3024-3033; Characteristic of the structure and regulation of two novel forms of serum- and glucocorticoid-induced protein kinase. Biochem. J. et al. 1999; 344: 189-197]. Activation of hsgk1 by PDK1 is accompanied by phosphorylation of serine at position 422. This mutation from serine to aspartic acid ( ^S422D SGK1) results in a constitutively active kinase [Kobaashishi T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kinase acidity by 3-phosphoinoside-dependent protein kinase-1 (PDKl) and PDK2. Biochem J. et al. 1999; 339: 319-328].

As early studies have shown, hsgk1 is a synergistic stimulator of the renal epithelial Na ⁺ channel [De la Rosa et al. The serum and glucocorticoid kinase sgk increases the thebundance of epithelial sodium channels in the plasma membrane of Xenopus. J Biol Chem 1999; 274: 37834-37839; Boehmer et al. The Shrinkage-activated Na ⁺ Conductance of Rat
Hepatocytes and it's Possible Correlation to rENaC. Cell Phys Biochem. 2000; 10: 187-194; Lang et al. Derated transactional regulation of cell volume sensitive kinase hsgk in diabetic nephropathy. Proc Natl Acad Sci USA 2000; 97: 8157-8162]. Since hsgk1 is found in many tissues that do not express the epithelial Na ⁺ channel ENaC, the function of hsgk1 should not be limited to the function of modulating 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 charactarization of a putative human serine / threonine protein kinase transducedly modified anisotropy and isotonic alternations. Proc Natl Acad Sci USA 1997: 94: 4440-4445; Waldegger et al. h-sgk Serine-Threonine protein kinase gene as early transcription target of TGF-β in human intestine. Gastroenterology 1999; 116: 1081-1088].

  Because hsgk1 may regulate many other signaling pathways or components of these pathways in a way that has not yet been elucidated, hsgk1 and its human homologues are quite capable of diagnosing many diseases. It is predicted that In particular according to DE 1970173 (A1), hsgk1 is associated with many diseases (eg hypernatremia, hyponatremia, diabetes, renal dysfunction, hypermetabolism, hepatic encephalopathy and bacterial or viral infections, in this case cell volume It is clear that these changes can be used in diagnosis for important pathophysiological roles).

In WO 00/62781, hsgk1 is reported to activate endothelial Na ⁺ channels, thereby causing an increase in renal Na ⁺ reabsorption. Since this increase in renal Na ⁺ reabsorption is associated with hypertension, in this document it was expected that increased expression of hsgk1 would result in hypertension, whereas decreased expression of hsgk1 would ultimately result in hypotension. .

DE10042137 is also similarly associated with overexpression or overactivity of the human homologs hsgk2 and hsgk3, overactivation of ENaC, resulting in increased renal Na ⁺ reabsorption, and the resulting hypertension It is reported that. Moreover, this document already discusses that hsgk2 and hsgk3 kinases have diagnostic potential for arterial hypertension.

  WO 02 / 074987A2 discloses the association of the presence of two different polymorphisms of individual nucleotides (single nucleotide polymorphism (SNP)) in the hsgk1 gene with a genetically determined predisposition to hypertension. These polymorphisms are the polymorphism in intron 6 (T → C) and the polymorphism in exon 8 (C → T) in the hsgk1 gene.

  Since sgk1 is expressed in a number of tissues and sgk1 is presumed to have a number of unknown substrates, the functions of the human homologues of the sgk family, in particular the hsgk1 gene (NM005627), the hsgk2 gene and the hsgk3 gene (AF169903), It can be predicted that there is a further correlation with the onset of other diseases. By discovering the correlation of these other specific diseases in which these sgk1s are involved, it is possible to obtain nucleic acids containing polymorphic regions of the sgk family of human homologues, such regions containing the corresponding sgk It affects protein function or expression and is used to diagnose predisposition to these other diseases.

  Therefore, an object of the present invention is to discover a further correlation between the function of the human homologue of the sgk family and a new disease, whereby the polymorphic region of the gene of the human homologue of the sgk family Can provide new possibilities for the diagnostic use of nucleic acids comprising:

  This goal was achieved by the surprising discovery that hsgk1 and hsgk3 potently stimulate the glucose transporter Glut1 (see FIG. 1). In particular, the glucose transporter Glut1 mediates the uptake of glucose, particularly into 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. et al. Ultratochemical localization of the erythrocyte / HepG2-type glucose transporter (GLUT1) in the serial body of the iris of the raty. Invest Ophthalmol Vis Sc. 1991; 32: 1659-66]. Osmotic pressure causes water to be taken up with glucose, which means that an increase in Glut1 activity leads to cell swelling. As a result, increased Glut1 activity may lead to the development of cataract [Gong et al. Development of cataractous macrophthalemia in mice expressing an active MEK1 in the lens. Invest Ophthalmol Vis Sci. 2001: 42: 539-48]. In addition, overexpression of Glut1 has been shown to promote connective tissue protein formation and deposition [Ayo et al. Incremental extracellular 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 cut in normal glucose milieu mimics the diabetic phenotype. J Clin Invest. 1995; 96: 1802-1814]. Such connective tissue protein deposition interferes with the flow of the fluid in the eye, leading to an increase in intraocular pressure, resulting in damage to the retina [Fingert et al. Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced oral hypertension. Invest Ophthalmol Vis Sci. 2001: 42 (1): 145-52, Ueda et al. Distribution of myocilin and extracellular matrix components in the juxtaanalytic tissue of human eyes. Invest Ophthalmol Vis Sci. 2002: 43: 1068-76]. Indeed, glucocorticoids stimulate the expression of SGK1 (see above) and at the same time cause the development of glaucoma [Fingert et al. 2001]. However, until now, hsgk1 has never been considered to have a causative role.

  The above mentioned interference is expected to occur in any situation where hsgk1 activity is increased, ie when any of the above mentioned hormones is present in excess. A specific polymorphism of the hsgk1 gene that correlates with increased blood pressure [Busjahn et al. Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40 (3): 256-260, 2002] may simultaneously lead to an increased incidence of cataracts and glaucoma. The same genetic modification should also be associated with early appearing cataracts and / or glaucoma.

  The discovery of the present invention reveals a completely new mechanism in the regulation of the glucose transporter Glut1. Therefore, increased hsgk1 activity is expected to result in increased glucose uptake into cells. The transcription of hsgk1 is stimulated by: serum [Webster et al. 1993], glucocorticoids [Brenan and Fuller 2000, Webster et al. 1993], mineral corticoids [Chen et al. 1999, Naray-Fejes-Toth et al. 1999, Shigaev et al. 2000, Brennan and Fuller 2000, Cowling and Birnboim 2000], gonadotropins [Alliston et al. Fully stimulating home-regulated expression of serum / glucocorticoid-inducible kinase in rat ovarian fluro cer fo ro s: Mol Endocrinol. 1997; 11: 1934-1949; Alliston et al. Expression and localization of serum / glucocorticoid-induced kinase in the rat over: relation to full 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 homones, cellular signals, and regulated genes. Recent Prog Home Res. 1995: 50: 223-254] and a number of cytokines [Lang and Cohen 2001], especially TGF-β [Fillon S. et al. etc. Expression of the Serine / Threoneine kinase hsgk1 in chronic viral hepatitis. Cell Physiol Biochem 2002: 12: 47-54: Lang et al. 2000, Waldegger et al. 1999, Waerntges S et al. Executive transcription of the human serum and glucocorticoid dependent kinase hsgk1 in lug fibrosis. Cell Physiol Biochem 2002, 12: 135-142]. In addition to this, Waldeger et al. As shown by a 1997 paper (already cited above), transcription of hsgk1 is increased by contraction of cells. As occurs in diabetes, increasing glucose concentration stimulates hsgk1 expression by cell contraction and / or increased production of TGF-β [Lang et al. 2000]. Expressed hsgk1 is activated by insulin-like growth factor IGF1, insulin or oxidative stress [Kobayashi and Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].

  According to the discovery according to the invention, increased hsgk1 expression increases the activity of the glucose transporter Glut1. As a result, more glucose is taken up by the cells and subsequently water taken up by osmosis causes the cells to swell. This is the manner in which water is taken up by the cornea and lens at high levels, thereby reducing transparency and causing cataract [Gong et al. 2001].

Glaucoma can also develop in a similar manner, in addition to it can be caused by connective tissue uptake [Fingert et al. 2001].

Cell swelling is also suspected to be the cause of diabetic neuropathy [Burg et al., Sorbitol, osmorelegation, and the complications of diabetics. J Clin Invest 1988; 81: 635-40]. However, an increase in Glut1 activity is expected to occur not only in diabetes, but also under the influence of glucocorticoids or in patients exhibiting genetically determined hsgk1 overactivity [Busjahn et al., Serum. And and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40 (3): 256-260, 2002]. In fact, glucocorticoids cause glaucoma [Fingert et al. 2001]. The mechanisms involved in the development of glaucoma associated with glucocorticoid administration are unknown. In particular, it has not been found so far that hsgk1 plays a role in this mechanism and is therefore suitable for use as a target protein for diagnosing and treating glaucoma.

Consequently, surprisingly, the observations according to the present invention demonstrate that hsgk1 and hsgk3 increase non-epithelial glucose transport as well as epithelial Na ⁺ channels. As a result, it became clear that hsgk1 and hsgk3 have entirely novel pathophysiological significance that should be accompanied by significant diagnostic and therapeutic / preventive results.

  Thus, the present invention relates to the use of a functional inhibitor of hsgk1 protein or hsgk3 protein or a negative regulator of transcription of the hsgk1 gene or hsgk3 gene to reduce cell swelling.

  Moreover, the present invention relates to a functional inhibitor of hsgk1 protein or hsgk3 protein, or hsgk1 gene or hsgk3 gene for the manufacture of a medicament for treating and / or preventing cataract, glaucoma or diabetic neuropathy. Relates to the use of negative regulators of transcription.

  The functional inhibitor of the hsgk1 protein or hsgk3 protein can be a chemical substance having either the property of inhibiting the normal physiological activity of the hsgk1 protein or hsgk3 protein. A functional inhibitor of the hsgk1 protein or hsgk3 protein is preferably a low molecular weight chemical ("small molecule"), or a protein or peptide. In particular, functional inhibitors of hsgk1 protein or hsgk3 protein can be antagonists of these enzymes, such antagonists block the substrate binding site of hsgk1 protein or hsgk3 protein, but at the same time hsgk1 Or does not undergo any catalytic conversion by hsgk3. Suitable antagonists in such cases are preferably molecules that are structurally similar to the natural substrate of the hsgk1 protein or hsgk3 protein, i.e., in particular structurally similar to the phosphorylable amino acids serine and threonine. Molecule.

    Staurosporine and chelerythrine are two known functional inhibitors of hsgk1. Therefore, in a particularly preferred embodiment, either staurosporine or chelerythrine is a functional inhibition of hsgk1 or hsgk3 for the treatment and / or prevention of at least one disease (cataract, glaucoma and diabetic neuropathy) Used as an agent.

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

  In addition to the actual active compounds, ie functional inhibitors of hsgk1 or hsgk3, or negative regulators of transcription of hsgk1 or hsgk3, this book for the treatment and / or prevention of cataract, glaucoma or diabetic neuropathy The medicament according to the invention may also contain stabilizers and / or carrier substances, such as starch, lactose, stearic acid, fats, waxes, alcohols or other additives such as preservatives, pigments or flavoring. Agents.

  The medicament can be administered in any manner, in particular in the form of tablets, granules or capsules or orally as a solution. Other particularly suitable administration forms relate to direct administration in the form of ointments, tinctures or sprays (for example to the skin or eyes) or any type of injection (for example subcutaneously or intravenously) or infusion.

  Moreover, the present invention relates to a single strand or double chain comprising the hsgk1 sequence according to accession number NM005627, or one of its fragments, for diagnosing a predisposition to develop cataract, glaucoma and / or diabetic neuropathy. It relates to the use of double-stranded nucleic acids. In this context, the length of the hsgk1 fragment that a single-stranded or double-stranded nucleic acid can include is at least 10 nucleotides / base pairs, preferably at least 15 nucleotides / base pairs, in particular at least 20 Of nucleotides / base pairs.

  In this context, the single-stranded or double-stranded nucleic acid preferably comprises at least one polymorphic nucleotide of the hsgk1 gene, in particular a single nucleotide polymorphism (SNP) of the hsgk1 gene.

In a particularly preferred embodiment, the single-stranded or double-stranded nucleic acid in this regard comprises at least one of the following hsgkl gene SNPs:
The insertion of G at positions 732/733 in intron 2 of the hsgk1 gene,
A T / C substitution at position 2071 in intron 6 of the hsgk1 gene (WO 02 / 074987A2),
-A T / C substitution at position 2617 in exon 8 of the hsgk1 gene (WO 02 / 074987A2).

The single-stranded or double-stranded nucleic acid described above can be preferably used to detect the SNP of the hsgk1 gene in a patient's genomic DNA or cDNA by the following method:
-Direct sequencing of genomic DNA or cDNA using the nucleic acid,
-Specifically hybridizing genomic DNA or cDNA with the nucleic acid,
-PCR oligonucleotide extension analysis or ligation analysis.

  In this connection, the patient's genomic DNA or cDNA is preferably isolated from a biological sample taken from the patient, in particular from saliva, blood, tissue or cells.

  The activity of the expressed hsgk1 gene depends on the version of this polymorphism in the patient's hsgk1 gene, and as a result, nucleic acids comprising at least one of these polymorphisms are particularly cataract, glaucoma and / or It may be well suited for diagnosing a predisposition to developing diabetic neuropathy.

  Moreover, the present invention relates to a single-stranded or double-stranded product comprising one of the hsgk3 sequences set forth in Accession No. AF169035 or a fragment thereof for diagnosing a predisposition to developing cataract, glaucoma and / or diabetic neuropathy. It relates to the use of double-stranded nucleic acids. In this context, the length of the hsgk3 fragment that a single-stranded or double-stranded nucleic acid can include is at least 10 nucleotides / base pairs, preferably at least 15 nucleotides / base pairs, in particular at least 20 Of nucleotides / base pairs.

  In this connection, the single-stranded 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-stranded or double-stranded nucleic acids described above, specific antibodies against the sgk family of human homologues, particularly the hsgk1 and hsgk3 substrates, also have at least one of the diseases of cataract, glaucoma and diabetic neuropathy. Suitable for diagnosing predisposition to onset. These diagnostic antibodies are preferably directed against epitopes of the human homologue of the sgk family (particularly of hsgk1 and hsgk3) that contain the phosphorylation site of the substrate (either phosphorylated or non-phosphorylated). is there.

  For example, overexpression of hsgk1 protein caused by the individual genetic makeup of the hsgk1 gene may result in increased hsgk substrate conversion, ie, increased enzymatic phosphorylation of the substrate by hsgk1. At the same time, overexpression of the hsgk1 protein is expected to result in stimulation of the glucose transporter Glut1, which ultimately results in high levels of glucose uptake into ocular cells, followed by high levels of water uptake due to permeability Eventually result in a predisposition to develop cataracts, glaucoma and diabetic neuropathy. Detecting more frequent phosphorylation of the hsgk1 substrate by antibodies against regions of the substrate of interest that contain the phosphorylation site of hsgk1 (phosphorylated or non-phosphorylated) results in cataracts, glaucoma and diabetics It can also be said to be a typical method for diagnosing a predisposition for developing neuropathy.

  In a preferred embodiment, the ubiquitin protein ligase Nedd4-2 (accession number BAA23711) is used as a substrate for the human homologue of the sgk family. This ubiquitin protein ligase is a protein that is specifically phosphorylated by the human homologue of the sgk family [Debonville et al., Phosphorylation of Needed4-2 by Sgk1 regulatory cells (+) channel cell surface. EMBO J.M. , 2001; 20: 7052-7059; Snyder et al., Serum and glucocorticoid-regulated kinase modulates Ned4-2-mediated inhibition of the epithelial Na (+) channel. J. et al. Biol. Chem. , 2002, 277: 5-8]. The phosphorylation site of hsgk1 has a consensus sequence (RXRXXS / T), where R is arginine, S is serine, T is threonine, and X is any arbitrary amino acid. In Nedd4-22 (accession number BAA23711), there are two possible hsgk1 phosphorylation sites that match the consensus sequence described above, namely serine at amino acid position 382 and serine at amino acid position 468.

  Therefore, an antibody for diagnosing a predisposition to develop at least one of the above-mentioned diseases of cataract, glaucoma and diabetic neuropathy is preferably the substrate Nedd4-2, particularly preferably hsgk1 It is directed to a region of the Nedd4-2 protein having a phosphorylation site sequence, ie, a consensus sequence (RXRXXS / T). In particular, these antibodies are directed to a Nedd4-2 protein region that includes at least one of two possible phosphorylation sites, serine at amino acid position 382 and / or serine at amino acid position 468.

Moreover, the present invention relates to a kit for diagnosing one of the diseases of cataract, glaucoma and diabetic neuropathy, the kit comprising at least one of the following components:
An antibody directed against hsgk1 or hsgk3,
A single-stranded or double-stranded nucleic acid (which can hybridize under stringent conditions to the hsgk1 gene described in accession number NM005627 or the hsgk3 gene described in accession number AF16935); in particular, of the hsgk1 gene or the hsgk3 gene Single-stranded or double-stranded nucleic acid comprising a polymorphic nucleotide, in particular "SNP",
An antibody against a substrate of the human homologue of the sgk family; preferably an antibody against the phosphorylation site (phosphorylated or non-phosphorylated) of this substrate; in particular the phosphorylation site of Nedd4 or Nedd4-2 (phosphorylated or Non-phosphorylated) antibody.

The present invention also relates to a screening method for identifying and characterizing a substance having therapeutic activity from among a large number of test substances, wherein the substance having therapeutic activity is selected from the group consisting of cataract, glaucoma and diabetic neuropathy Used to treat and / or prevent at least one of the diseases to be treated, the method comprises the following steps:
a) heterogeneously co-expressing the following in the cell:
i) the glucose transporter Glut1, and
ii) hsgk1 and / or hsgk3,
b) culturing at least one of the aliquots A _{1 to} A _x of the cells in each case in the presence of at least one test substance (the at least one test substance is in each case a cell Culturing a control cell aliquot B in the absence of the test substance,
c) Measuring the activity of the glucose transporter Glut1 in the aliquots A ₁ -A _x of the cells compared to the activity of the glucose transporter Glut1 in the aliquot B of the control cells.

  As “various test substances”, substance libraries, preferably small molecule libraries, protein libraries, and the like can be used.

In step a), suitable cells, preferably mammalian cells or cell lines, in particular human cells or cell lines, are prepared using standard methods (eg electroporation, CaPO ₄ precipitation, lipofection etc.) using Glut1 and hsgk1. And / or transfected with an appropriate expression vector containing an expression cassette suitable for expressing hsgk3. Such an expression cassette is active in the cell type of interest and under the control of an appropriate promoter capable of expressing the target gene in an appropriate amount, the genome of the relevant target gene (Glut1, hsgk1 or hsgk3) Includes DNA or cDNA. The expression vector may further comprise a selection marker.

  The transfected cells are then cultured under conditions that allow expression of the target genes i) and ii).

In step b), the transfected cells of a) are distributed into different cell aliquots A ₁ -A _x and control cell aliquot B. Cell aliquots A ₁ -A _x are in each case cultured in the presence of at least one test substance. The test substances added to the aliquots A _{1 to} A _x of the cells in each case are different from each other (corresponding to the indices _{1 to X} of the aliquots A _{1 to} A _x of the respective cells). Meanwhile, an aliquot B of control cells is cultured in the absence of the test substance.

In step c), the activity of the glucose transporter Glut1 in the aliquots A _{1 to} A _x of the cells is measured quantitatively compared to the activity of the glucose transporter Glut1 in the aliquot B of the control cells. Cell aliquots A ₁ -A _x that are significantly lower in terms of Glut1 activity than those measured in control cell aliquot B include test substances capable of functionally inhibiting hsgk1 or hsgk3, or Test substances that can reduce their expression will be added. Such substances may be suitable for treating at least one of the diseases of cataract, glaucoma and diabetic neuropathy.

An alternative embodiment is a screening method according to the present invention for identifying and characterizing a substance having therapeutic activity from among a large number of test substances, wherein the substance having therapeutic activity is cataract, glaucoma and diabetes Used to treat and / or prevent at least one disease selected from the group consisting of sexual neuropathy, the method comprises the following steps:
d) heterogeneously co-expressing in at least one of the aliquots A ₁ -A _x of the cells:
i) the glucose transporter Glut1, and
ii) hsgk1 and / or hsgk3,
and,
Heterologously expressing in at least one of aliquots B ₁ -B _x of cells:
i) Glucose transporter Glut1,
In the case of e), respectively, in the presence of at least one test substance, culturing aliquots A ₁ to A _x and B ₁ .about.B _x cell (the at least one test substance, in each case , Corresponding to the index 1 to X of the aliquot of cells),
f) A step of comparing and measuring the activity of the glucose transporter Glut1 in the cell aliquots A _{1 to} A _x and the cell aliquots B _{1 to} B _x .

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

  The present invention is described in more detail with reference to FIG. 1 below.

  The uptake of 2-deoxyglucose (picomoles / l / 10 min / oocyte) (arithmetic mean ± SEM) is plotted on the ordinate A of FIG. Xenopus laevis oocytes were injected with cRNA of Glut1 with or without SGK1, SGK2, SGK3 or protein kinase B (PKB) cRNA (see Example 1).

  FIG. 1 shows the increased 2-deoxy-glucose uptake that occurs in oocytes that express hsgk1 or hsgk3 in addition to Glut1, compared to oocytes that express only Glut1. This demonstrates that the functions of hsgk1 and hsgk3 efficiently stimulate the activity of the glucose transporter Glut1. Similar effects are not observed in oocytes expressing hsgk2 or PKBmut instead of hsgk1 or hsgk3.

  The following examples illustrate the invention in more detail.

〔Example〕
Example 1: Expression in Xenopus oocytes, and two-electrode voltage clamp normal SGK1 cRNA [Waldegger S, Barth P, Laber G, Lang F: Cloning and charactarization of a human serotype modified during anisotonic and isotonic alternations of cell volume. Proc Natl Acad Sci USA 1997; 94: 4440-4445], and constitutively active SGK1 ( ^S422D SGK1) cRNA [Kobayashi and Cohen 1999], as well as normal Glut1 cRNA [Iserovic P, Wang D Ma L, Yang H, Zuniga FA, Pasqual JM, Kuang K, De Vivo DC, Fischburg J. et al. Changes in glucose transport and water permeability resetting form the T310I pathogenetic mutation in Glut1 are consistent with two transporters. J Biol Chem. 2002; 277: 30991-7] was synthesized in vitro. Xenopus ovarian incision and oocyte collection and processing have already been described in detail [Wagner CA, Friedrich B, Setiawan I, Lang F, Breeer S: The use of Xenopus laevis oocites characterisation of heterologously expressed membrane proteins. Cell Physiol Biochem 2000; 10: 1-12]. Oocytes were injected with human Glut1 (5 ng), human ^S422D SGK1 (7.5 ng), and / or Xenopus Nedd4-2 (5 ng). Water was injected into control oocytes. The uptake of radiolabeled glucose was measured for 2 days at room temperature after each cRNA injection. The control bath solution contains 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 at the prescribed concentrations. The final solution was titrated to pH 7.4 with HCl or NaOH.

Calculated data is shown as arithmetic mean ± SEM; n is the number of oocytes tested. All experiments were performed with at least three different oocyte groups. The results were tested for significant differences using Student's t test. Only results with P <0.05 were considered statistically significant.

2 shows increased 2-deoxy-glucose uptake occurring in oocytes expressing hsgk1 or hsgk3 in addition to Glut1 compared to oocytes expressing only Glut1.

Claims (15)

  Use of a functional inhibitor of hsgk1 protein or hsgk3 protein or a negative regulator of transcription of the hsgk1 gene or hsgk3 gene to reduce cell swelling.   Use of a functional inhibitor of hsgk1 protein or hsgk3 protein or a negative regulator of transcription of hsgk1 gene or hsgk3 gene to produce a medicament for the treatment and / or prevention of cataract, glaucoma or diabetic neuropathy .   Use according to claim 1 or 2, characterized in that the functional inhibitor of hsgk1 protein or hsgk3 protein is staurosporine or chelerythrine.   A medicament comprising a functional inhibitor of hsgk1 protein or hsgk3 protein or a negative regulator of transcription of hsgk1 gene or hsgk3 gene for the treatment and / or prevention of cataract, glaucoma or diabetic neuropathy.   Use of a single-stranded or double-stranded nucleic acid comprising the hsgk1 sequence according to accession number NM005627, or one of its fragments, for diagnosing a predisposition to develop cataract, glaucoma and / or diabetic neuropathy.   Use according to claim 5, characterized in that the single-stranded or double-stranded nucleic acid comprises at least one polymorphic nucleotide of the hsgk1 gene, in particular a "SNP" of the hsgk1 gene.   The SNP of the hsgk1 gene is a G insertion at position 732/733 in intron 2 of the hsgk1 gene, a T / C substitution at position 2071 in intron 6 of the hsgk1 gene, and a T at position 2617 in exon 8 of the hsgk1 gene. Use according to claim 6, characterized in that it is selected from the group of SNPs consisting of / C substitutions.   Use of a single-stranded or double-stranded nucleic acid comprising a hsgk3 sequence according to accession number AF169035, or one of its fragments, for diagnosing a predisposition to develop cataract, glaucoma and / or diabetic neuropathy.   Use according to claim 8, characterized in that the single-stranded or double-stranded nucleic acid comprises at least one polymorphic nucleotide of the "SNP" of the hsgk3 gene, in particular the hsgk1 gene.   Use of an antibody against a substrate of a human homologue of the sgk family for diagnosing a predisposition to developing at least one of the diseases (cataract, glaucoma and diabetic neuropathy), said antibody being a phosphorylated site (phosphorylated or To human homologous epitopes, including any of the non-phosphorylated forms).   Use according to claim 10, characterized in that the substrate of the human homologue of the sgk family is Nedd4-2 (accession number BAA23711).   A kit for diagnosing one of diseases (cataract, glaucoma and diabetic neuropathy), comprising an antibody against hsgk1 or hsgk3, or under stringent conditions, the hsgk1 gene described in Accession No. NM005627, Alternatively, the above kit comprising a nucleic acid capable of hybridizing with the hsgk3 gene described in Accession No. AF169035, or a combination of these antibodies and nucleic acid.   The nucleic acid, under stringent conditions, includes a polymorphic nucleotide of the hsgk1 gene or the hsgk3 gene, particularly the “sNP”, the hsgk1 gene described in Accession No. NM005627, or the DNA region of the hsgk3 gene described in Accession No. AF16935 The kit according to claim 12, which can hybridize with the kit. A screening method for identifying and characterizing a substance having a therapeutic activity from among a large number of test substances, wherein the substance having a therapeutic activity is selected from the group consisting of cataract, glaucoma and diabetic neuropathy Is used to treat and / or prevent at least one of the following steps:
a) heterogeneously co-expressing the following in the cell:
i) the glucose transporter Glut1, and
ii) hsgk1 and / or hsgk3,
b) culturing at least one of the aliquots A _{1 to} A _x of the cells in the presence of at least one test substance in each case (the at least one test substance is in each case a cell Culturing a aliquot B of control cells in the absence of any test substance,
c) measuring the activity of the glucose transporter Glut1 in the aliquots A _{1 to} A _x of the cells compared to the activity of the glucose transporter Glut1 in the aliquot B of the control cells;
Including the above method. A screening method for identifying and characterizing a substance having a therapeutic activity from among a large number of test substances, wherein the substance having a therapeutic activity is selected from the group consisting of cataract, glaucoma and diabetic neuropathy Is used to treat and / or prevent at least one of the following steps:
d) heterogeneously co-expressing in at least one of the aliquots A ₁ -A _x of the cells:
i) the glucose transporter Glut1, and
ii) hsgk1 and / or hsgk3,
and,
Heterologously expressing in at least one of aliquots B ₁ -B _x of cells:
i) Glucose transporter Glut1,
In the case of e), respectively, in the presence of at least one test substance, one of the test substance the at step (said at culturing aliquots A ₁ to A _x and B ₁ .about.B _x cells, in each case , Corresponding to the index 1 to X of the aliquot of cells),
f) comparing and measuring the activity of the glucose transporter Glut1 in cell aliquots A ₁ -A _x and cell aliquots B ₁ -B _x ,
Including the above method.

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