DE10305212A1 - Use of the sgk gene family for the diagnosis and therapy of cataracts and glaucoma - Google Patents
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Abstract
Die Erfindung betrifft die Verwendung eines funktionalen Inhibitors des hsgk1- oder des hsgk3-Proteins oder eines negativen Transkriptionsregulators des hsgk1- oder hsgk3-Gens zur Herstellung eines Arzneimittels zur Therapie und/oder zur Prophylaxe eines Katarakts, eines Glaukoms oder der diabetischen Neuropathie. DOLLAR A In einem weiteren Aspekt betrifft die Erfindung die Verwendung einer einzel- oder doppelsträngigen Nukleinsäure, umfassend die hsgk1-Sequenz nach Acc. No. NM_005627 oder eines ihrer Fragmente oder umfassend die hsgk3-Sequenz nach Acc. No. AF169035 oder eines ihrer Fragmente, zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie, sowie einen Kit zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie, welcher die oben genannte Nukleinsäure umfaßt. DOLLAR A Ein weiterer Gegenstand der Erfindung sind verschiedene Screening-Verfahren zur Identifizierung und Charakterisierung von therapeutisch wirksamen Substanzen aus einer Vielzahl von Test-Substanzen zur Therapie und/oder zur Prophylaxe von mindestens einer Erkrankung, ausgewählt aus Katarakt, Glaukom und der diabetischen Neuropathie.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
Die Erfindung betrifft die Verwendung eines funktionalen Inhibitors des hsgk1- oder des hsgk3-Proteins oder eines negativen Transkriptionsregulators des hsgk1- oder hsgk3-Gens zur Herstellung eines Arzneimittels zur Therapie und/oder zur Prophylaxe eines Katarakts, eines Glaukoms oder der diabetischen Neuropathie.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 einem weiteren Aspekt betrifft die Erfindung die Verwendung einer einzel- oder doppelsträngigen Nukleinsäure umfassend die hsgk1-Sequenz nach Acc No. NM_005627 oder eines ihrer Fragmente oder umfassend die hsgk3-Sequenz nach Acc. No. AF169035 oder eines ihrer Fragmente zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie, sowie einen Kit zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie, welcher die oben genannte Nukleinsäure umfaßt.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.
Ein weiterer Gegenstand der Erfindung sind verschiedene Screening-Verfahren zur Identifizierung und Charakterisierung von therapeutisch wirksamen Substanzen aus einer Vielzahl von Test-Substanzen zur Therapie und/oder zur Prophylaxe von mindestens einer Erkrankung ausgewählt aus Katarakt, Glaukom und der diabetischen Neuropathie.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.
Die Serum- und Glucocorticoid-induzierbare Kinase hsgk1 wurde ursprünglich als Glucocorticoid-sensitives Gen cloniert [Webster et al, Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 1993; 13:2031–2040].The 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].
Folgende Untersuchungen deckten auf, daß die hsgk1 unter dem Einfluß einer Vielzahl von Stimuli steht [Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov 13;2001(108):RE17], wie unter anderem der Mineralocorticoide [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 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].
Die hsgk1 wird durch den „insulin like growth factor IGF1", durch Insulin und durch oxidativen Stress über eine Signalkaskade durch Phosphoinositol-3-kinase (PI3 kinase) und Phosphoinositol-abhängige Kinase PDK1 stimuliert [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]. Die Aktivierung der hsgk1 durch die PDK1 involviert eine Phosphorylierung am Serin der Position 422. Die Mutation dieses Serins in ein Aspartat (S422DSGK1) führt zu einer konstitutiv aktiven 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].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].
Wie frühere Untersuchungen gezeigt haben, ist die hsgk1 ein potenter Stimulator des renalen epithelialen Na+-Kanales [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]. Nachdem die hsgk1 in einer Vielzahl von Geweben gefunden wird, welche den epithelialen Na+-Kanal ENaC nicht exprimieren, sollte die Funktion der hsgk1 nicht auf die Regulation des Na+-Kanales beschränkt sein [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].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].
Aufgrund der vermutlich zahlreichen,
bisher noch unaufgeklärten
Regulationen weiterer Signaltransduktionswege bzw. ihrer Komponenten
durch die hsgk1 dürften
die hsgk1 und ihre humanen Homologen ein beträchtliches Potential zur Diagnose
zahlreicher Erkrankungen besitzen. Aus der
In der WO 00/62781 wurde beschrieben, daß die hsgk1 den endothelialen Na+-Kanal aktiviert, wodurch die renale Na+-Resorption erhöht wird. Da diese gesteigerte renale Na+-Resorption mit Hypertonie einhergeht, wurde hier vermutet, daß eine gesteigerte Expression der hsgk1 zur Hypertonie, eine verminderte Expression der hsgk1 letztlich zur Hypotonie führen sollte.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.
Auch in
In WO 02/074987 A2 wurde der Zusammenhang zwischen dem Auftreten zweier verschiedener Polymorphismen (single nucleotide polymorphism (SNP)) einzelner Nukleotide im hsgk1-Gen mit einer genetisch bedingten Prädisposition zur Hypertonie offenbart. Hierbei handelt es sich um einen Polymorphismus in Intron 6 (T→C) und um einen Polymorphismus in Exon 8 (C→T) im hsgk1-Gen.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.
Aufgrund der Expression der sGK1 in zahlreichen Geweben und aufgrund der vermutlich großen Anzahl von noch unbekannten Substraten der sgk1 ist damit zu rechnen, daß es weitere Korrelationen zwischen der Funktion der humanen Homologen der sgk-Familie, insbesondere des hsgk1-Gens (NM_005627), des hsgk2-Gens, und des hsgk3-Gens (AF169035) und der Ausbildung weiterer Erkrankungen geben wird. Die Aufdeckung solcher weiteren spezifischen Krankheits-Korrelationen der sgk1 könnten dazu führen, daß Nukleinsäuren enthaltend polymorphe Genregionen der humanen Homologen der sgk-Familie, die die Funktion oder Expression der entsprechenden sgk-Proteine beeinflussen, zur Diagnose einer Prädispostion dieser weiteren Erkrankungen verwendet werden könnten.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.
Es war daher Aufgabe der Erfindung, weitere Korrelationen zwischen der Funktion der humanen Homologen der sgk-Familie und neuen Erkrankungen aufzudecken und so neue diagnostische Einsatzmöglichkeiten für Nukleinsäuren, die polymorphe Genregionen der humanen Homologen der sgk-Familie enthalten, bereitzustellen.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.
Diese Aufgabe wurde durch die überraschende
Erkenntnis gelöst,
daß die
hsgk1 und hsgk3 den Glucosetransporter Glut1 stark stimulieren (siehe
Die oben genannten Störungen würden bei bei allen Zuständen auftreten, bei denen die hsgk1 gesteigerte Aktivität aufweist, also unter Überschuß an allen oben genannten Hormonen. Bestimmte Polymorphismen des hsgk1-Gens, die mit gesteigertem Blutdruck korrelieren [Busjahn et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 256–260, 2002], könnten gleichzeitig zu gesteigertem Auftreten von Katarakt und Glaukom führen. Die gleichen Modifikationen des Gens sollten auch mit verfrüht auftretendem Katarakt und/oder Glaukom korrelieren.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.
Die vorliegenden Befunde decken einen völlig neuen Mechanismus in der Regulation des Glucosetransporters Glut1 auf. Eine gesteigerte Aktviität der hsgk1 sollte daher zu gesteigerter Aufnahme von Glucose in die Zellen führen. Die Transcription der hsgk1 wird durch Serum [Webster et al. 1993], durch Glucocorticoide [Brenan & Fuller 2000, Webster et al. 1993], durch Mineralocorticoide [Chen et al. 1999, Naray-Fejes-Toth et al. 1999, Shigaev et al. 2000, Brennan and Fuller 2000, Cowling and Birnboim 2000], durch Gonadotropine [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. 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], und durch eine Reihe von Cytokinen [Lang & Cohen 2001], insbesondere durch 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] stimuliert. Darüberhinaus wird die hsgk1-Transcription durch Zellschrumpfung gesteigert, wie die bereits zitierte Schrift Waldegger et al. von 1997 zeigt. Eine gesteigerte Glucosekonzentration, wie sie bei Diabetes mellitus vorkommt, stimuliert die Expression der hsgk1 durch Zellschrumpfung und/oder durch gesteigerte Bildung von TGF-β [Lang et al. 2000]. Die exprimierte hsgk1 wird durch den „insulin like growth factor" IGF1, durch Insulin und durch oxidativen Stress aktiviert [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].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].
Die gesteigerte Expression der hsgk1 steigert nach den erfindungsgemäßen Erkenntnissen die Aktivität des Glucosetransporters Glut-1. Damit wird mehr Glucose in die Zellen aufgenommen und das durch Osmose nachfolgende Wasser bewirkt eine Zellschwellung. Auf diese Weise erfolgt die gesteigerte Wassereinlagerung in Hornhaut und Linse, die über Minderung der Transparenz zum Katarakt führt [Gong et al. 2001].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 ähnlicher Weise und zusätzlich durch Einlagerung von Bindegewebe könnte auch ein Glaukom entstehen [Fingert et al. 2001].In a similar way and additionally through Storage of connective tissue could glaucoma also develops [Fingert et al. 2001].
Auch bei der diabetischen Neuropathie wird als Ursache eine Zellschwellung vermutet [Burg et al., Sorbitol, osmoregulation, and the complications of diabetes. J Clin Invest 1988;81:635–40]. Doch nicht nur bei Diabetes mellitus, sondern auch unter dem Einfluß von Glucocorticoiden oder bei Patienten mit einer genetisch bedingten Überaktivität der hsgk1 [Busjahn et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 256–260, 2002] ist mit gesteigerter Glut1-Aktivität zu rechnen. Glucocorticoide führen tatsächlich zum Glaukom [Fingert et al. 2001]. Der Mechanismus, der für die Glaukom-Bildung bei Glucocorticoid-Gabe verantwortlich ist, war bislang nicht bekannt. Insbesondere war bislang nicht bekannt, daß die hsgk1 bei diesem Mechanismus eine Rolle spielt und sich daher als Target-Protein für die Diagnose und Therapie eines Glaukoms eignet.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.
Die erfindungsgemäßen Beobachtungen zeigen somit überraschenderweise, daß die hsgk1 und die hsgk3 nicht nur den epithelialen Na+-Kanal, sondern auch den nicht-epithelialen Glucosetransport steigern. Damit sind völlig neue pathophysiologische Bedeutungen der hsgk1 und der hsgk3 aufgedeckt worden, welche wichtige diagnostische und therapeutische/prophylaktische Konsequenzen nach sich ziehen sollten.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.
Ein Gegenstand der Erfindung betrifft somit die Verwendung eines funktionalen Inhibitors des hsgk1- oder des hsgk3-Proteins oder eines negativen Transkriptionsregulators des hsgk1- oder hsgk3-Gens zur Verringerung der Zellchwellung.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.
Ein weiterer Gegenstand der Erfindung betrifft die Verwendung eines funktionalen Inhibitors des hsgk1- oder des hsgk3-Proteins oder eines negativen Transkriptionsregulators des hsgk1- oder hsgk3-Gens zur Herstellung eines Arzneimittels zur Therapie und/oder zur Prophylaxe eines Katarakts, eines Glaukoms oder der diabetischen Neuropathie.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.
Dieser funktionale Inhibitor des hsgk1-Proteins oder des hsgk3-Proteins kann eine chemische Substanz jeglicher Art sein, die die normale physiologische Aktivität des hsgk1-Proteins oder des hsgk3-Proteins inhibiert. Vorzugsweise ist der funktionale Inhibitor des hsgk1- oder des hsgk3-Proteins eine niedermolekulare chemische Substanz (ein „small molecule") oder ein Protein oder Peptid. Der funktionale Inhibitor des hsgk1-Proteins oder des hsgk3-Proteins kann insbesondere ein Antagonist dieser Enzyme sein, welcher die Substratbindungsstelle des hsgk1-Proteins oder des hsgk3-Proteins blockiert, aber gleichzeitig keinerlei katalytischen Umsetzung durch die hsgk1 oder hsgk3 zugänglich ist. Als Antagonist eignen sich in diesem Fall vorzugweise solche Moleküle, die strukturelle Ähnlichkeit mit dem natürlichen Substrat des hsgk1-Proteins oder des hsgk3-Proteins, also insbesondere eine strukturelle Ähnlichkeit mit den phosphorylierbaren Aminosäuren Serin und Threonin, haben.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 und Chelerythrin sind zwei bekannte funktionale Inhibitoren der hsgk1. Als funktionaler Inhibitor der hsgk1 oder der hsgk3 zur Therapie und/oder zur Prophylaxe mindestens einer der Erkrankungen Katarakt, Glaukom oder diabetische Neuropathie wird daher in einer besonders bevorzugten Ausführungsform entweder Staurosporin oder Chelerythrin eingesetzt.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.
Ein negativer Transkriptionsregulator des hsgk1-Gens oder des hsgk3-Gens ist als eine Substanz definiert, die die Expression des hsgk1-Gens bzw. des hsgk3-Gens auf Transkriptionsebene aktiviert.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.
Das erfindungsgemäße Arzneimittel zur Therapie und/oder zur Prophylaxe eines Katarakts, eines Glaukoms oder der diabetischen Neuropathie kann neben dem eigentlichen Wirkstoff, dem funktionalen Inhibitor oder dem negativen Transkriptionsregulator der hsgk1 oder der hsgk3, zusätzlich Stabilisatoren und/oder Trägersubstanzen, wie beispielsweise Stärke, Laktose, Stearinsäure, Fette, Wachse, Alkohole oder andere Additiva wie Konservierungsstoffe, Farbstoffe oder Geschmacksstoffe enthalten.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.
Die Verabreichung des Arzneimittels kann auf jede Art, insbesondere oral, in Form von Tabletten, Granulaten, Kapseln oder als Lösung erfolgen. Andere besonders geeignete Darreichungsformen betreffen direkte Applizierungen (z.B. auf Haut oder Auge) in Form von Salben, Tinkturen, Sprays oder jegliche Art von Injektion (z.B. subkutan, intravenös) oder die Infusion.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.
Ein weiterer Gegenstand der Erfindung ist die Verwendung einer einzel- oder doppelsträngigen Nukleinsäure umfassend die hsgk1-Sequenz nach Acc No. NM_005627 oder eines ihrer Fragmente zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie. Das Fragment der hsgk1, das die einzel- oder doppelsträngige Nukleinsäure hierbei umfassen kann, ist mindestens 10 Nukleotide/Basenpaare lang, vorzugsweise mindestens 15 Nukleotide/Basenpaare lang und insbesondere mindestens 20 Nukleotide/Basenpaare lang.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.
Die einzel- oder doppelsträngige Nukleinsäure umfaßt hierbei vorzugsweise mindestens ein polymorphes Nukleotid des hsgk1-Gens, insbesondere einen „Single nucleotide polymorphism (SNP)" des hsgk1-Gens.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.
In einer besonders bevorzugten Ausführungsform umfaßt die einzel- oder doppelsträngige Nukleinsäure hierbei mindestens einen der folgenden SNPs des hsgk1-Gens:
- – 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).
- 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).
Die obigen SNPs des hsgk1-Gens in der genomischen DNA oder cDNA des Patienten können mit Hilfe der oben genannten einzel- oder doppelsträngigen Nukleinsäuren vorzugsweise durch die nachfolgenden Verfahren nachgewiesen werden: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:
- – durch direkte Sequenzierung der genomischen DNA oder cDNA mit den obigen Nukleinsäuren,- by direct sequencing of genomic DNA or cDNA with the above nucleic acids,
- – durch spezifische Hybridisierung der genomischen DNA oder cDNA mit den obigen Nukleinsäuren,- by specific hybridization of the genomic DNA or cDNA with the the above nucleic acids,
- – durch einen PCR-Oligonukleotid-Elongationsassay oder durch einen Ligations-Assay.- by a PCR oligonucleotide elongation assay or by a ligation assay.
Die genomische DNA oder cDNA des Patienten wird hierbei vorzugsweise aus einer Körperprobe des Patienten, insbesondere aus Speichel, Blut, Gewebe oder Zellen, isoliert.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.
Es ist anzunehmen, daß die Aktivität des exprimierten hsgk1-Gens von der Version dieses Polymorphismus im hsgk1-Gen des Patienten abhängt und daß sich folglich Nukleinsäuren, die mindestens einen dieser Polymorphismen enthalten, besonders gut zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie eignen.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.
Die Erfindung betrifft weiterhin die Verwendung einer einzel- oder doppelsträngigen Nukleinsäure umfassend die hsgk3-Sequenz nach Acc No. AF169035 oder eines ihrer Fragmente zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und/oder diabetischer Neuropathie. Das Fragment der hsgk3, das die einzel- oder doppelsträngige Nukleinsäure hierbei umfassen kann, ist mindestens 10 Nukleotide/Basenpaare lang, vorzugsweise mindestens 15 Nukleotide/Basenpaare lang und insbesondere mindestens 20 Nukleotide/Basenpaare lang.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.
Die einzel- oder doppelsträngige Nukleinsäure umfaßt hierbei vorzugsweise mindestens ein polymorphes Nukleotid des hsgk3-Gens, insbesondere einen „Single nucleotide polymorphism (SNP)" des hsgk3-Gens.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.
Neben den oben genannten einzel- oder doppelsträngigen Nukleinsäuren eignen sich auch bestimmte Antikörper, die gegen Substrate der humanen Homologen der sgk-Familie, insbesondere gegen Substrate der hsgk1 und der hsgk3, gerichtet sind, zur Diagnose einer Prädisposition zur Ausbildung mindestens einer der Erkrankungen Katarakt, Glaukom, diabetische Neuropathie. Diese diagnostischen Antikörper sind vorzugsweise gegen ein solches Epitop der humanen Homologen der sgk-Familie (insbesondere der hsgk1 und der hsgk3) gerichtet, welches die Phosphorylierungsstelle des Substrates entweder in phosphorylierter Form oder in nicht phosphorylierter Form enthält.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.
Beispielsweise könnte eine aufgrund der individuellen genetischen Ausstattung des hsgk1-Gens auftretende Überexpression des hsgk1-Proteins zu einer verstärkten Umsetzung von Substraten der hsgk, d.h. zu einer verstärkten enzymatischen Phosphorylierung der Substrate durch die hsgk1 fuhren. Gleichzeitig würde die Überexpression des hsgk1-Proteins zu einer Stimulierung des Glucosetransporters Glut1 fuhren, welche letztlich eine starke Glucoseaufnahme in die Zellen des Auges, anschließend eine starke Wasseraufnahme durch Osmose und dadurch letztlich die Prädisposition zur Ausbildung von Katarakt, Glaukom und diabetischer Neuropathie bewirkt. Der Nachweis der häufigeren Phosphorylierung von Substraten der hsgk1 mit Hilfe eines Antikörpers, der gegen eine Region des betreffenden Substrates gerichtet ist, welche die Phosphorylierungsstelle der hsgk1 in phosphorylierter Form oder in nicht phosphorylierter Form enthält, könnte somit ein Verfahren zur Diagnose einer Prädisposition zur Ausbildung von Katarakt, Glaukom und diabetischer Neuropathie darstellen.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 einer bevorzugten Ausführungsform wird als Substrat des humanen Homologen der sgk-Familie die Ubiquitin-Protein-Ligase Nedd4-2 (Acc No. BAA23711) eingesetzt. Diese Ubiquitin-Protein-Ligase stellt ein Protein dar, welches von den humanen Homologen der sgk-Familie spezifisch phophoryliert wird [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]. Phosphorylierungsstellen für die hsgk1 besitzen die Konsensus-Sequenz (R X R X X S/T), wobei R für Arginin, S für Serin, T für Threonin und X für eine beliebige Aminosäure steht. In Nedd4-2 2 (Acc No. BAA23711) gibt es zwei potentielle Phosphorylierungsstellen für die hsgk1, auf die die oben genannte Konsensus-Sequenz paßt: das Serin an Aminosäure-Position 382 und das Serin an Aminosäure-Position 468.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
Die oben genannten Antikörper zur Diagnose einer Prädisposition zur Ausbildung mindestens einer der Erkrankungen Katarakt, Glaukom, diabetische Neuropathie sind daher vorzugsweise gegen das Substrat Nedd4-2 gerichtet und besonders bevorzugt gegen eine Proteinregion von Nedd4-2 mit der Sequenz der potentiellen Phosphorylierungsstelle für die hsgk1, der Konsensus-Sequenz (R X R X X S/T), gerichtet. Insbesondere sind diese Antikörper gegen solche Nedd4-2-Protein-Regionen gerichtet, die mindestens eine der beiden potentiellen Phosphorylierungsstellen Serin an Aminosäure-Position 382 und/oder Serin an Aminosäure-Position 468 umfassen.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.
Ein weiterer Gegenstand der Erfindung ist ein Kit zur Diagnose einer der Erkrankungen Katarakt, Glaukom und diabetische Neuropathie, enthaltend mindestens eine der folgenden Komponenten:
- – 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.
- - 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.
Ein weiterer Gegenstand der Erfindung betrifft ein Screening-Verfahren zur Identifizierung und Charakterisierung von therapeutisch wirksamen Substanzen aus einer Vielzahl von Test-Substanzen zur Therapie und/oder zur Prophylaxe von mindestens einer Erkrankung ausgewählt aus der Gruppe bestehend aus Katarakt, Glaukom und der diabetischen Neuropathie, umfassend die folgenden Schritte:
- 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 AX 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 AX im Vergleich zur Aktivität des Glucosetransporters Glut1 im Kontroll-Zell-Anteil B.
- 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.
Als „Vielzahl von Test-Substanzen" kann eine Substanz-Bibliothek, vorzugsweise eine „small molecule-library", aber auch eine Protein-Bibliothek oder ähnliches eingesetzt werden.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 Schritt a) werden geeignete Zellen, vorzugsweise Säugetier-Zellen oder Zellinien, insbesondere humane Zellen oder Zellinien mit geeigneten Expressionsvektoren, enthaltend geeignete Expressionskassetten zur Expression von Glut1 und von hsgk1 und/oder hsgk3 nach Standardmethoden, wie beispielsweise Elektroporation, CaPO4-Präzipitation, Lipofektion oder ähnliches, transfiziert. Die Expressionskassetten enthalten die genomische DNA oder die cDNA des fraglichen Zielgens (Glut1, hsgk1, hsgk3) unter der Kontrolle geeigneter Promotoren, die in dem fraglichen Zelltyp aktiv sind und das Zielgen in einer geeigneten Menge exprimieren können. Die Expressionsvektoren können weiterhin Selektionsmarker enthalten.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.
Anschließend werden die transfizierten Zellen unter solchen Bedingungen kultiviert, die die Expression der Zielgene i) und ii) erlauben.Then the transfected Cells are cultivated under such conditions that the expression of Allow target genes i) and ii).
In Schritt b) erfolgt die Aufteilung der transfizierten Zellen aus a) in verschiedene Zell-Anteile (Aliquots) A1 bis AX und in einen Kontroll-Zell-Anteil B. Die Zell-Anteile A1 bis AX werden in Gegenwart von jeweils mindestens einer Test-Substanz kultiviert. Die jeweils in die Zell-Anteile A1 bis AX zugegebene(n) Test-Substanzen) unterscheiden sich untereinander (in Abhängigkeit von dem Index 1 bis X des jeweiligen Zell-Anteils A1 bis AX). Der Kontroll-Zell-Anteil B hingegen wird in Abwesenheit jeglicher Test-Substanz kultiviert.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 Schritt c) wird die Aktivität des Glucosetransporters Glut1 in den Zell-Anteilen A1 bis AX im Vergleich zur Aktivität des Glucosetransporters Glut1 im Kontroll-Zell-Anteil B quantitativ bestimmt. In den Zell-Anteilen A1 bis AX, in denen im Vergleich zum Kontroll-Zell-Anteil B ein deutlich geringerer Wert der Glut1-Aktivität gemessen wird, sollte eine Test-Substanz zugegeben worden sein, die die hsgk1 bzw. die hsgk3 funktional inhibieren kann oder deren Expression mindert. Eine solche Substanz könnte sich zur Therapie mindestens einer der Erkrankungen Katarakt, Glaukom oder diabetische Neuropathie eignen.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.
In einer alternativen Ausführungsform umfaßt das erfindungsgemäße Screening-Verfahren zur Identifizierung und Charakterisierung von therapeutisch wirksamen Substanzen aus einer Vielzahl von Test-Substanzen zur Therapie und/oder zur Prophylaxe von mindestens einer Erkrankung ausgewählt aus der Gruppe bestehend aus Katarakt, Glaukom und der diabetischen Neuropathie die folgenden Schritte:
- d) Heterologe Koexpression von i) dem Glucosetransporter Glut1 und ii) der hsgk1 und/oder der hsgk3 in mindestens einem Anteil A1 bis AX von Zellen und heterologe Expression von
- i) dem Glucosetransporter Glut1 in mindestens einem Anteil B1 bis BX von Zellen
- e) Kultivierung der Zell-Anteile A1 bis AX und B1 bis BX 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 AX und in den Zell-Anteilen B1 bis BX.
- 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.
Die oben genannten Erläuterungen der einzelnen Verfahrensschritte a) bis c) gelten für die Verfahrensschritte d) bis f) der alternativen erfindungsgemäßen Screening-Verfahrens entsprechend.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.
Die Erfindung wird durch die nachfolgende
Auf der Ordinate A der
Die Erfindung wird durch das nachfolgende Beispiel näher erläutert.The invention is illustrated by the following Example closer explained.
Beispiel 1: Expression in Xenopus laevis Oocyten und Zwei-Elektroden-SpannungsklemmeExample 1: Expression in Xenopus laevis oocytes and two-electrode voltage clamp
cRNA der normalen 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] und der konstitutiv aktiven SGK1 (S422DSGK1) [Kobayashi & Cohen 1999], sowie von normalem Glut1 [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] wurden in vitro synthetisiert. Die Dissektion der Xenopus laevis Ovarien und die Kollektion und Behandlung der Oocyten wurden bereits detailliert beschrieben [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]. Die Oocyten wurden mit 5 ng von humanem Glut1, 7.5 ng von humaner S422DSGK1 und/oder mit 5 ng von Xenopus Nedd4-2 injeziert. Kontrolloocyten wurden mit Wasser injeziert. Die Aufnahme radioaktiv markierter Glucose wurde bei Raumtemperatur 2 Tage nach Injektion der jeweiligen cRNA's gemessen. Die Kontrollbadlösung enthielt 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.4. Alle Substanzen wurden in den angegebenen Konzentrationen eingesetzt. Die endgültigen Lösungen wurden mit HCl bzw. NaOH auf pH 7.4 tritriert.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.
Berechnungencalculations
Die Daten werden als arithmetische Mittelwerte ± SEM angegeben, n ist die Zahl der untersuchten Oocyten. Alle Experimente wurden in mindestens drei verschiedenen Gruppen von Oocyten durchgeführt. Die Ergebnisse wurden mit dem Student t-test auf significante Unterschiede getestet. Nur Ergebnisse mit P < 0.05 wurden als statistisch significant angesehen.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.
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DE10305212A DE10305212A1 (en) | 2003-02-07 | 2003-02-07 | Use of the sgk gene family for the diagnosis and therapy of cataracts and glaucoma |
EP04708350A EP1663246A2 (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
KR1020057014582A KR20050114214A (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
CNA2004800070348A CN1771039A (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
PCT/EP2004/001048 WO2004069258A2 (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
RU2005127808/15A RU2005127808A (en) | 2003-02-07 | 2004-02-05 | APPLICATION OF SGK FAMILY GENES FOR DIAGNOSTICS AND THERAPY CATARACTS AND GLAUKOMA |
MXPA05008394A MXPA05008394A (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma. |
BR0407300-2A BRPI0407300A (en) | 2003-02-07 | 2004-02-05 | Uses of a functional hsgk1 protein or hsgk3 protein inhibitor or negative transcription regulator of the hsgk1 gene or hsgk3 gene, a single stranded or double stranded nucleic acid, and an antibody, drug, diagnostic kit, and, screening method for identifying and characterizing therapeutically active substances |
JP2006501737A JP2006519189A (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family to diagnose and treat cataracts and glaucoma |
CA002514703A CA2514703A1 (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
PL378399A PL378399A1 (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
AU2004210416A AU2004210416A1 (en) | 2003-02-07 | 2004-02-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
ZA200506280A ZA200506280B (en) | 2003-02-07 | 2005-08-05 | Use of the sgk gene family for diagnosis and therapy of cataracts and glaucoma |
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DE102008029072A1 (en) * | 2008-06-10 | 2009-12-17 | Lang, Florian, Prof. Dr.med. | Substance, which inhibits serum and glucocorticoid dependent kinase 3, useful for the prophylaxis and/or treatment or diagnosis of age-related diseases e.g. arteriosclerosis, skin atrophy, myasthenia, infertility, stroke and kyphosis |
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JP2007529423A (en) * | 2004-03-11 | 2007-10-25 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Methods for modulating glutamate receptors for the treatment of neuropsychiatric disorders, including the use of modulators of serum and glucocorticoid-inducible kinases |
DE102004059781A1 (en) * | 2004-12-10 | 2006-06-22 | Sanofi-Aventis Deutschland Gmbh | Use of serum / glucocorticoid-regulated kinase |
WO2007002670A2 (en) * | 2005-06-28 | 2007-01-04 | Bausch & Lomb Incorporated | Method of lowering intraocular pressure |
JPWO2007037560A1 (en) * | 2005-09-30 | 2009-04-16 | リンク・ジェノミクス株式会社 | Therapeutic or diagnostic use of SGK2 gene |
CN101360999B (en) * | 2005-11-22 | 2013-07-17 | 麦吉尔大学 | Intraocular pressure-regulated early genes and uses thereof |
WO2008079980A1 (en) * | 2006-12-22 | 2008-07-03 | Alcon Research, Ltd. | Inhibitors of protein kinase c-delta for the treatment of glaucoma |
BR112013018374A2 (en) * | 2011-01-25 | 2016-10-11 | Monell Chemical Senses Centre | compositions and methods for providing or modulating sweet taste and methods for tracking them |
KR102357260B1 (en) * | 2020-12-10 | 2022-02-08 | 주식회사 레피겐엠디 | Method for diagnosis and predict of diabetic neuropathy using micro-rna and kit therefor |
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EP0832295A1 (en) * | 1995-06-07 | 1998-04-01 | Ligand Pharmaceuticals Incorporated | Method for screening for receptor agonists and antagonists |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
DE19708173A1 (en) * | 1997-02-28 | 1998-09-03 | Dade Behring Marburg Gmbh | Cell volume regulated human kinase h-sgk |
AU3991499A (en) * | 1998-05-15 | 1999-12-06 | Joslin Diabetes Center | Independent regulation of basal and insulin-stimulated glucose transport |
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US6399655B1 (en) * | 1998-12-22 | 2002-06-04 | Johns Hopkins University, School Of Medicine | Method for the prophylactic treatment of cataracts |
DE19917990A1 (en) * | 1999-04-20 | 2000-11-02 | Florian Lang | Medicament containing inhibitors of cell volume regulated human kinase h-sgk |
AU5840300A (en) * | 1999-07-14 | 2001-01-30 | University Of Lausanne | Glutx polypeptide family and nucleic acids encoding same |
US6416759B1 (en) * | 1999-09-30 | 2002-07-09 | The Regents Of The University Of California | Antiproliferative Sgk reagents and methods |
US20030236246A1 (en) * | 2002-04-30 | 2003-12-25 | Brazzell Romulus Kimbro | Method for decreasing capillary permeability in the retina |
DE10225844A1 (en) * | 2002-06-04 | 2003-12-18 | Lang Florian | sgk and nedd as diagnostic and therapeutic targets |
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AU2004210416A1 (en) | 2004-08-19 |
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