ZA200506280B

ZA200506280B - Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma

Info

Publication number: ZA200506280B
Application number: ZA200506280A
Authority: ZA
Inventors: Florian Lang; Andreas Busjahn
Original assignee: Florian Lang
Priority date: 2003-02-07
Filing date: 2005-08-05
Publication date: 2006-05-31
Also published as: DE10305212A1; CA2514703A1; AU2004210416A1; MXPA05008394A; WO2004069258A2; PL378399A1; CN1771039A; BRPI0407300A; WO2004069258A3; RU2005127808A; KR20050114214A; JP2006519189A; EP1663246A2

Description

- As originally filed

Use of the sgk gene family for diagnosing and treating cataract and glaucoma

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

In another aspect, the invention relates to the use of y a single-stranded or double-stranded nucleic acid encompassing the hsgkl sequence according to Acc No.

NM 005627, or of one of its fragments, or encompassing the hsgk3 sequence according to Acc No. AF169035, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy, as well as to a kit for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy, which kit comprises the abovementioned nucleic acid.

The invention furthermore relates to different screening methods for identifying and characterizing therapeutically active substances, from among a multiplicity of test substances, with the therapeutically active substances being used for the therapy and/or prophylaxis of at least one disease selected from cataract, glaucoma and diabetic neuropathy.

The serum and glucocorticoid-inducible kinase hsgkl was originally cloned as a glucocorticoid-sensitive gene [Webster et al. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 1993; 13:2031-2040].

Subsequent investigations revealed that hsgkl is under the influence of a large number of stimuli [Lang

F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov 13;2001(108):RE17] such as, inter alia, that of the mineralocorticoids [Chen et al. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 1999;96:2514-2519, Naray-Fejes-Téth et al. sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na' channels. J Biol Chem 1999;274:16973-16978; Shigaev et al. Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel. Am J Physiol 2000;278:F613-F619; Brenan FE,

Fuller PJ. Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo. Mol Cell Endocrinol. 2000;30;166:129-36; Cowling RT, Birnboim HC. Expression of serum- and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes. J

Leukoc Biol. 2000;67:240-248]. hsgkl is stimulated by the insulin-like growth factor

IGF1l, by insulin and by oxidative stress by means of phosphoinositol-3-kinase (PI3 kinase) and phosphoinositol-dependent kinase PDKl1 by way of a signal cascade [Park et al. Serum and glucocorticoid- inducible kinase (SGK) is a target of the PI 3-kinase- stimulatd signaling pathway, EMBO J 1999;18:3024-3033;

Kobayashi et al. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J. 1999:;344:189-197]. The activation of hsgkl by PDK1 involves a phosphorylation at the serine at position 422. The mutation of this serine into an aspartate (5%??’SGK1) results in a kinase which is constitutively active [Kobayashi T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kinase by agonists

- 3 = that activate phosphatidylinositide 3- kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999;339:319-328].

As earlier investigations have shown, hsgkl is a potent stimulator of the renal epithelial Na' channel [De la

Rosa et al. The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J Biol Chem 1999;274:37834-37839; Bohmer et 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 Sci USA 2000;97:8157-8162). Since hsgkl is found in a large number of tissues which do not express the epithelial

Na' channel ENaC, the function of hsgkl ought not to be restricted to that of regulating the Na’ channel [Klingel et al. Expression of the cell volume regulated kinase h-sgk in pancreatic tissue. Am J Physiol (Gastroint. Liver-Physiol.) 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-B in human intestine. Gastroenterology 1999;116:1081-1088].

Due to the fact that hsgkl probably regulates, in manners which are yet to be elucidated, a large number of other signal transduction pathways or components of these pathways, hsgkl and its human homologs ought to have considerable potential for diagnosing a large number of diseases. It is evident, in particular, from

DE 197 08 173 Al that hsgkl can be used for diagnosis in connection with many diseases, such as hypernatriamia, hyponatriamia, diabetes mellitus, renal insufficiency, hypercatabolism, hepatic encephalopathy and microbial or viral infections, in which changes in cell volume play a crucial pathophysiological role.

WO 00/62781 reported that hsgkl activates the endothelial Na' channel, resulting in renal Na’ resorption being increased. Since this increase in renal Na' resorption is accompanied by hypertension, it was presumed, in this document, that an increase in the expression of hsgkl would lead to hypertension while a decrease in expression of hsgkl would ultimately lead to hypotension.

DE 100 421 37 also reported a similar connection between the overexpression or hyperactivity of the human homologs hsgk2 and hsgk3 and hyperactivation of the ENaC, the increase in renal Na’ resorption which results therefrom, and the hypertension which develops from this. Furthermore, this document already discussed the diagnostic potential of the hsgk2 and hsgk3 kinases with regard to arterial hypertension.

WO 02/074987 A2 disclosed the connection between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgkl gene and a genetically determined predisposition for hypertension. These polymorphisms are a polymorphism in intron 6 (T—C) and a polymorphism in exon 8 (C—T) in the hsgkl gene.

Because sgkl is expressed in a large number of tissues, and because sgkl presumably has a large number of yet unknown substrates, it can be expected that there will be further correlations between the function of the human homologs of the sgk family, in particular of the hsgkl gene (NM 005627), of the hsgk2 gene and of the hsgk3 gene (AF169035) and the development of other diseases. The uncovering of these other specific disease correlations involving sgkl could lead to nucleic acids which contain polymorphic regions of the genes of the human homologs of the sgk family, which regions influence the function or expression of the corresponding sgk proteins, being used for diagnosing a predisposition for these other diseases.

The need exists to discover further correlations between the function of the human homologs of the sgk family and new diseases and, in this way, to provide novel possibilities for the diagnostic use of nucleic acids which contain polymorphic regions of the genes of the human homologs of the sgk family.

This need is fulfilled by means of the surprising finding that hsgkl and hsgk3 powerfully stimulate the glucose transporter Glutl (see Fig.l). Inter alia, the glucose transporter Glutl mediates the uptake of glucose into various cells of the eye, inter alia [Busik et al. Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. Invest Ophthalmol Vis Sci. 2002;43:2356-63;

Takata K, Kasahara T, Kasahara M, Ezaki O, Hirano H.

Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1l) in the ciliary body and iris of the rat eye. Invest

Ophthalmol Vis Sc. 1991;32:1659-66]. Water follows the glucose osmotically, which means that an increase in the activity of Glutl leads to cell swelling.

Consequently, an increase in the activity of Glutl could lead to the development of cataract [Gong et al.

Development of cataractous macrophthalmia in mice expressing an active MEK1 in the lens. Invest

Ophthalmol Vis Sci. 2001;42:539-48]. In addition to this, it has been shown that overexpression of Glutl promotes the formation and deposition of connective tissue protein [Ayo et al. Increased extracellular

AMENDED SHEET matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Am J Physiol. 1991;260:F185- 191; Heilig et al. Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype. J

Clin Invest. 1995;96:1802-1814]. Such a deposition of connective tissue proteins impedes the escape of ocular fluid and leads to pressure increases in the eye and consequently to damage of the retina [Fingert et al.

Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension.

Invest Ophthalmol Vis Sci. 2001;42(1):145-52, Ueda et al. Distribution of myocilin and extracellular matrix components in the Jjuxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci. 2002;43:1068-76].

Glucocorticoids which stimulate the expression of SGKl1 (see above) do indeed at the same time lead to the development of glaucoma [Fingert et al. 2001]. However, hsgkl has never previously been suspected of having a causal role. :

The abovementioned disturbances would occur in connection with any situations in which the activity of hsgkl was increased, that is in the presence of an excess of any of the abovementioned hormones.

Particular polymorphisms of the hsgkl gene which correlate with an increase in blood pressure [Busjahn et al. Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 256-260, 2002] could at the same time lead to an increase in the occurrence of cataract and glaucoma.

The same modifications of the gene ought also to correlate with cataract and/or glaucoma which appears prematurely.

The present findings reveal a completely novel mechanism in the regulation of the glucose transporter

Glutl. An increase in activity of hsgkl ought therefore to lead to an increase in the uptake of glucose into the cells. The transcription of hsgkl is stimulated by serum [Webster et al. 1993], by glucocorticoids [Brenan & Fuller 2000, Webster et al. 1993], by mineralocorticoids [Chen et al. 1999, Naray-

Fejes-Toth et al. 1999, Shigaev et al. 2000, Brennan and Fuller 2000, Cowling and Birnboim 2000}, by gonadotropins [Alliston et al. Follicle stimulating hormone-regulated expression of serum/glucocorticoid- inducible kinase in rat ovarian granulosa cells: a functional role for the Spl family in promoter activity. Mol Endocrinol. 1997;11:1934-1949; Alliston et al. Expression and localization of serum/glucocorticoid-induced kinase in the rat ovary: relation to follicular growth and differentiation.

Endocrinology. 2000;141:385-395; Gonzalez-Robayna et al. Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-Induced kinase (Sgk): evidence for A kinase-independent signaling by

FSH in granulosa cells. Mol Endocrinol. 2000;14:1283-1300, Richards et al. Ovarian cell differentiation: a cascade of multiple hormones, - cellular signals, and regulated genes. Recent Prog Horm

Res. 1995;50:223-254], and by a number of cytokines [Lang & Cohen 2001], in particular by TGF-p [Fillon S. et al. Expression of the Serine/Threonine kinase hSGK1 in chronic viral hepatitis. Cell Physiol Biochem 2002;12:47-54; Lang et al. 2000, Waldegger et al. 1999,

Warntges S et al. Excessive transcription of the human serum and glucocorticoid dependent kinase hSGK1l in lung fibrosis. Cell Physiol Biochem 2002,12:135-142]. In addition to this, the transcription of hsgkl is increased by cell shrinkage, as shown by the Waldegger et al. 1997 paper which has already been cited. An increase in glucose concentration, as occurs in diabetes mellitus, stimulates the expression of hsgkl by cell shrinkage and/or by an increase in the formation of TGF-R [Lang et al. 2000]. The expressed hsgkl is activated by insulin-like growth factor IGF1,

by insulin or by oxidative stress [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].

According to the findings in accordance with the invention, the increased expression of hsgkl increases the activity of the glucose transporter Glut-1l. As a result, more glucose is taken up into the cells and the water which subsequently follows by osmosis causes the cells to swell. This is the way in which water is incorporated to an increased extent into the cornea and lens, with this leading, by means of a reduction in transparency, to cataract [Gong et al. 2001].

Glaucoma could also develop in a similar manner and, in addition, by the incorporation of connective tissue [Fingert et al. 2001].

Cell swelling is also suspected to be the cause in the case of diabetic neuropathy [Burg et al., Sorbitol, osmoregulation, and the complications of diabetes. J

Clin Invest 1988;81:635-40]. However, an increase in

Glutl activity is to be expected not only in diabetes mellitus but also under the influence of glucocorticoids or in patients exhibiting a genetically determined hyperactivity of hsgkl [Busjahn et al.,

Serum- and glucocorticoid-regulated kinase (SGK1l) gene and blood pressure. Hypertension 40(3): 256-260, 2002].

Glucocorticoids do indeed give rise to glaucoma [Fingert et al. 2001]. The mechanism responsible for the development of glaucoma in connection with glucocorticoid administration had not previously been known. In particular, it had not previously been known that hsgkl plays a role in this mechanism and is therefore suitable for use as a target protein for diagnosing and treating a glaucoma.

The observations according to the invention consequently surprisingly demonstrate that hsgkl and hsgk3 increase nonepithelial glucose transport as well as increasing the epithelial Na* channel. As a result, hsgkl and hsgk3 have been revealed to possess completely novel pathophysiological significances which should entail important diagnostic and therapeutic/prophylactic consequences.

The invention consequently relates to the use of a _ functional inhibitor of the hsgkl protein or the hsgk3 protein or of a negative regulator of the transcription of the hsgkl gene or hsgk3 gene for reducing cell swelling.

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

This functional inhibitor of the hsgkl protein or the hsgk3 protein can be a chemical substance of any nature which inhibits the normal physiological activity of the hsgkl protein or of the hsgk3 protein. The functional inhibitor of the hsgkl protein or the hsgk3 protein is preferably a low molecular weight chemical substance (a “small molecule”) or a protein or peptide. The functional inhibitor of the hsgkl protein or the hsgk3 protein can, in particular, be an antagonist of these enzymes which blocks the substrate-binding site of the hsgkl protein or the hsgk3 protein but which, at the same time, is not accessible to any catalytic conversion by the hsgkl or hsgk3. Antagonists which are suitable in this case are preferably those molecules which are structurally similar to the natural substrate of the hsgkl protein or of the hsgk3 protein, that is, in particular, which are structurally similar to the phosphorylatable amino acids serine and threonine.

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

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

In addition to the actual active compound, i.e. the functional inhibitor of hsgkl or hsgk3 or the negative regulator of the transcription of hsgkl or hsgk3, the pharmaceutical according to the invention for the therapy and/or prophylaxis of a cataract, of a glaucoma or of diabetic neuropathy can also comprise stabilizers and/or carrier substances, such as starch, lactose, stearic acid, fats, waxes, alcohols or other additives such as preservatives, dyes or flavorings.

The pharmaceutical can be administered in any manner, in particular orally in the form of tablets, granules or capsules or as a solution. Other particularly suitable administration forms concern direct administrations (e.g. on the skin or the eye) in the form of ointments, tinctures or sprays or any type of injection (e.g. subcutaneous or intravenous) or infusion.

The invention furthermore relates to the use of a single-stranded or double-stranded nucleic acid encompassing the hsgkl sequence according to Acc No.

NM 005627, or of one of its fragments, for diagnosing a predisposition for developing cataract, glaucoma and/or diabetic neuropathy. The hsgkl fragment which the single-stranded or double-stranded nucleic acid can encompass in this connection is at least 10 nucleotides/base pairs in length, preferably at least 15 nucleotides/base pairs in length, and, in particular, at least 20 nucleotides/base pairs in length.

In this connection, the single-stranded or double- stranded nucleic acid preferably encompasses 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- stranded or double-stranded nucleic acid encompasses, in this connection, at least one of the following SNPs of the hsgkl gene: - a G insertion at position 732/733 in intron 2 of the hsgkl gene, - the T/C substitution at position 2071 in intron 6 of the hsgkl gene (WO 02/074987 A2), - the T/C substitution at position 2617 in exon 8 of the hsgkl gene (WO 02/074987 A2).

The abovementioned single-stranded or double-stranded nucleic acids can preferably be used to detect the above SNPs of the hsgkl gene in the genomic DNA or cDNA of the patient by means of the following methods: - by means of directly sequencing the genomic DNA or cDNA using the above nucleic acids, - by means of specifically hybridizing the genomic

DNA or cDNA with the above nucleic acids, - by means of a PCR oligonucleotide elongation assay or by means of a ligation assay.

In this connection, the genomic DNA or cDNA of the patient is preferably isolated from a body sample taken from the patient, in particular from saliva, blood,

Claims

Patent Claims

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

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

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

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

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

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

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

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

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

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

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

12. A kit for diagnosing one of the diseases cataract, glaucoma and diabetic neuropathy, comprising antibodies which are directed against hsgkl or hsgk3 or comprising nucleic acids which are able to hybridize, under stringent conditions, with the hsgkl gene according to Acc No. NM 005627 or with the hsgk3 gene according to Acc No. AF169035, or comprising these antibodies and nucleic acids jointly.

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

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

Ay as compared with the activity of the glucose transporter Glutl in the control cell aliquot B.

15. A screening method for identifying and characterizing therapeutically active substances, from among a multiplicity of test substances, with the therapeutically active substances being used for the therapy and/or prophylaxis of at least one disease selected from the group comprising cataract, glaucoma and diabetic neuropathy,

CL . comprising the following steps: Ce a. d) Heterologously coexpressing - 15 i) the glucose transporter Glutl and ii) hsgkl and/or hsgk3 in at leest one aliquot A; to Ax of cells, and heterologously expressing i) the glucose transporter Glutl in at leest one aliquot B; to By of cells e) culturing the cell aliquots A; to Ax and B; to Bx in the presence of in each case at least one test substance, with the at least one test substance in each case differing in dependence on the index 1 to X of the cell aliquots, f) carrying out a comparative determination of the activities of the glucose transporter Glutl in the cell aliquots A; to Ay and in the cell aliquots B; to By.