EP1979747A1 - Procede permettant d'interferer avec la secretion d'acide gastrique induite par glucocorticoïde - Google Patents

Procede permettant d'interferer avec la secretion d'acide gastrique induite par glucocorticoïde

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Publication number
EP1979747A1
EP1979747A1 EP07702807A EP07702807A EP1979747A1 EP 1979747 A1 EP1979747 A1 EP 1979747A1 EP 07702807 A EP07702807 A EP 07702807A EP 07702807 A EP07702807 A EP 07702807A EP 1979747 A1 EP1979747 A1 EP 1979747A1
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Prior art keywords
phenyl
hydroxy
methoxy
hydrazid
benzyliden
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EP07702807A
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German (de)
English (en)
Inventor
Florian Lang
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Merck Patent GmbH
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Merck Patent GmbH
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Priority to EP07702807A priority Critical patent/EP1979747A1/fr
Publication of EP1979747A1 publication Critical patent/EP1979747A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/062—Gastritis or peptic ulcer disease

Definitions

  • Gastric acid is produced by parietal cells in the stomach. Parietal cells contain an extensive secretory network from which the gastric acid is secreted into the lumen of the stomach. These cells are part of epithelial fundic glands in the gastric muocsa. The pH of gastric acid is 2-3 in the stomach lumen, the acidity being maintained by the proton pump a H+/K+ ATPase.
  • the lining of the stomach and small intestines have protection against the irritating acids produced in the stomach.
  • the protective mechanisms may become faulty, leading to a breakdown of the lining. This results in inflammation (gastritis) or an ulcer.
  • Ulcer produces a crater-like lesion on the skin or mucous membrane of the stomach (gastric) or the upper part of the small intestine caused by an inflammatory, infectious, or malignant condition.
  • Peptic ulcer disease is a mucusal ulcer in the acid- producing zone in the distal stomach or the proximal duodenum.
  • the normal stomach produces enough mucus and alkaline juice to protect the gastric and duodenal mucosa against HCI.
  • the pancreatic bicarbonate creates a pH of 7.5 at the luminal membrane of the mucosa.
  • Risk factors which may induce peptic ulcer disease are drugs such as ASA, NSAIDs and corticoids, hyperparathyroidism where a high Ca 2+ level stimulates gastric acid secretion, gastrin-producing tumours of the pancreas and Helicobacter pylori infection of the stomach.
  • drugs such as ASA, NSAIDs and corticoids
  • hyperparathyroidism where a high Ca 2+ level stimulates gastric acid secretion, gastrin-producing tumours of the pancreas and Helicobacter pylori infection of the stomach.
  • Other contributing factors are increased pepsinogen from the chief cells, increased parietal cell mass, reduced somatostatin secretion from the antral D cells, and damage of the mucosa.
  • Acetylsalicylic acid and steroid or non-steroid anti-inflammatory drugs deplete the gastric mucosa for prostaglandins, which leads to mucosal damage.
  • the medications may include one or more of the following: Acid blockers like cimetidine, nizatidine, ranitidine, or famotidine.
  • Proton pump inhibitors such as Omeprazole or medications that protect the tissue lining like sucralfate. Bismuth protects the lining and kills the bacteria and furthermore antibiotics such as Clarithromycin are used to kill H. pylori.
  • Glucocorticoids are well known to support the development of gastric ulcer [Ahamed et al., 1983; Zamora et al., 1975], an effect considered to be due to both, enhanced H + secretion [Cooke et al., 1966; Raptis et al., 1976] and impaired defense mechanisms such as prostaglandin release [Bandyopadhyay et al., 1999; Nobuhara et al., 1985].
  • the mechanisms linking the stimulation of the glucocorticoid receptor to acid secretion are, however not known.
  • glucocorticoid signals include the serum and glucocorticoid inducible kinase SGK1, which is highly expressed in gastric tissue [Waldegger et al., 1997]. SGK1 has been shown to regulate a wide variety of transport proteins [Lang et al., 2003].
  • SGK1 regulates the Na + /H + exchanger NHE3 [Yun et al., 2002a; Yun 2003], the glutamine transporter SN1 [Boehmer et al., 2003b], the glutamate transporter EAAT1 [Boehmer et al., 2003a] the renal and intestinal glucose transporter SGLT1 [Dieter et al., 2004] and the Na + /K + -ATPase [Henke et al., 2002; Setiawan et al., 2002; Verrey et al., 2003; Zecevic et al., 2004].
  • a common (-5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight (Vallon et al., 2005) Jan;14(1 ):59-66.
  • SGK1 may thus contribute to metabolic syndrome.
  • SGK1 further participates in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia.
  • SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior.
  • the SGKs cover a wide variety of physiological functions and may play an active role in a multitude of pathophysiological conditions.
  • the present study delivers the unexpected finding that the regulation of gastric acid secretion is dependent on the expression and up- regulation of SGK1 activity.
  • the present invention provides evidence that SGK1 is involved in the regulation of gastric acid secretion.
  • SGK1 stimulates H + secretion by increasing the K + /H + ATPase activity and alternatively, SGK1 enhances the H + secretion by stimulating KCNQ1 channels which have been shown to be of critical importance for gastric H + secretion [Vallon et al., 2006] and are known to be upregulated by SGK1.
  • the regulation of KCNE1/KCNQ1 involves the ubiquitin ligase Nedd4-2 which decreases the affinity of the enzyme to its target proteins [Abriel et al., 2000; Debonneville et al., 2001 ;
  • a method for screening of an inhibitor of serum glucocorticoid inducible kinases (SGK) suitable for the inhibition of gastric acid secretion comprises the following steps has been described: (i) providing a recombinant pre-activated phosphorylated SGK protein, (ii) providing an SGK substrate polypeptide together with ATP or other phosphate sources, (iii) providing an inhibitor of glucocorticoid inducible kinases and (iv) evaluating SGK activity by measuring phosphorylation of the substrate.
  • a preferred embodiment of the current invention relates to the use of SGK1 for the screening of inhibitors of serum glucocorticoid inducible kinases.
  • Example 4 Examples of SGK1 inhibitors available through the claimed screening method are listed in Example 4 of the current invention and the expert recognizes that the compounds and pharmaceutical useful derivates, salts, solutions and stereoisomeres and mixtures thereof are useful drugs for the treatment of gastric acid secretion driven diseases such as peptic ulcer.
  • a preferred embodiment of the current invention is SGK1 and inhibitors directed to SGK1 and SGK1 function and diagnosis, however it is obvious to the expert that the claimed methods, use and inhibitory compounds can applied to other disease related isoforms of the protein such as SGK2 and SGK3 and selected single nucleotide polymorph variants that have been described for the three isoforms.
  • the invention delivers and claims compounds which do inhibit the function of SGK.
  • the functional inhibition is characterized by the direct inference with the activity kinase of the SGK enzyme and therefore the mechanism is unique over other known phosphorylation inhibitors.
  • the expert recognizes immediately that the determination of progression, regression or onset of gastric acid secretion driven disorders can be measured by monitoring the up-regulated expression and/or functional activity of SGK1 , SGK2 or SGK3 in organs or isolated human tissue samples and specimens taken from a patient.
  • the invention opens the possibility to diagnose a disease by the analysis of a single nucleotide polymorph variant for instance for SGK1 in order to correlate said polymorph phenotype with the presence, severity or predisposition of gastric acid secretion driven disorders.
  • Diagnosis methods for SGK are not restricted to SGK1 however may as well require the simultaneous analysis of SGK2 and or SGK3.
  • glucocorticoids on gastic H + transport has been studied in gene targeted mice lacking SGK1 (sgk ⁇ 1' ) and their wild type littermates (sgk1 +l+ ).
  • SGK1 transcript levels were determined by real-time PCR and BCECF fluorescence was utilized for determination of H + secretion ( ⁇ pH).
  • gastric SGK1 transcript levels is up-regulated significantly by a 4 day treatment with 10 ⁇ g/g BW/day dexamethasone (DEX) in gastric tissue of wild type mice.
  • DEX dexamethasone
  • Serum glucocorticoid inducible kinases of the present invention may be used as diagnostic reagents, through detecting mutations (SNP) in the associated gene.
  • SNP detecting mutations
  • Detection of a mutated form of the gene characterized by the polynucleotide polymorphism disclosed in Example 5 of the current application in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene.
  • Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art.
  • the molecular diagnosis should always be confirmed either by an upper intestinal endoscopy, which allows direct examination of the ulcer.
  • endoscopy a biopsy specimen can be removed from the body for examination.
  • the tissue will be examined under a microscope using immuno staining methods and molecular diagnosis serum of glucocorticoid inducible kinases expression to assist in diagnosis. Therefore, only very small samples are needed.
  • the nucleic acids for diagnosis may as well be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled SGK1 , SGK2, or SGK3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401 ).
  • An array of oligonucleotides probes comprising SGK1 , SGK2 or SGK3 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Such arrays are preferably high density arrays or grids.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M. Chee et al., Science, 274, 610-613 (1996) and other references cited therein.
  • Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantification of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them.
  • the techniques used are well known in the art and include in situ hydridization techniques to clones arrayed on a grid, such as cDNA microarray hybridization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
  • a preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.
  • Antibody based assay technique is another possibility that can be used to determine levels of SGK1 , SGK2 or SGK3 of the present invention.
  • SGK antibodies from commercial suppliers such as Sigma or Cell Signalling Technologies are available or have been published by others. Additional antibodies against selected polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G.
  • the antibodies are useful in assays which include radio-immunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagnostic kit comprising:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1 , or a fragment or an RNA transcript thereof;
  • b a nucleotide sequence complementary to that of (a);
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
  • kits may comprise a substantial component.
  • Such a kit will be of use in diagnosing a disease or ⁇ Q susceptibility to a disease, particularly diseases of the invention, amongst others.
  • antibodies may be employed to diagnose isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • -I C invention may also be employed to treat diseases of the invention, amongst others.
  • mice lacking SGK1 have been generated as described previously [Wulff et al., 2002].
  • a conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons.
  • 25 neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4-11 , which code for the Sgk1 kinase domain, were "floxed” by inserting a third loxP site into intron 3. Targeted R1 ES cells were transiently transfected with Cre recombinase. A clone with a recombination between the first and third loxP site (type I recombination) was injected into
  • mice 30 C57BL/6 blastocytes.
  • Male chimeras were bred to 129/SvJ females.
  • Heterozygous sg/ct-deficient mice were backcrossed to 129/SvJ wild-type mice for two generations and then intercrossed to generate homozygous sgkr ⁇ and Sgk1 +/+ littermates.
  • the animals were genotyped by PCR using standard methods.
  • mice For analysis of dexamethasone effects, sgk1 +l+ and sgk1 ' ' ⁇ mice were injected with dexamethasonephosphate disodiumsalt (Sigma, Taufkirchen, Germany; dissolved in 0.9% saline) at a dose of 10 ⁇ g/g BW for four consecutive days at 8 pm. sgk1 ⁇ ' ⁇ and sgk1 +l+ mice injected with 0.9% saline alone served as controls. Mice had free access to a standard mouse diet (Altromin diet 1310, Heidenau, Germany) and tap water. On the 4 th day of dexamethasone treatment, the animals were fasted 16 hours on wire grids prior to the experiments with free access to tap water.
  • Example 2 Quantitative real-time PCR was used to determine the effect of glucocorticoids on SGK1 transcript levels, gastric tissue was quickly removed and frozen in liquid nitrogen. Automated disruption and homogenization of frozen tissue was performed using the MagNa Lyser Instrument TM (Roche Diagnostics, Mannheim, Germany). For each sample one-way special tubes were filled with ceramic beads, 20-30 mg of frozen tissue and 600 ⁇ l of RLT-buffer (Qiagen, Hilden, Germany). Cleared cell lysate was transferred for further RNA purification process (RNAeasy Mini Kit, Qiagen, Hilden, Germany).
  • RNA was reverse transcribed to cDNA utilizing the reverse transcription system (Bioscience, USA) with oligo(dT) primers according to the manufacturer's protocol.
  • reverse transcription system Bioscience, USA
  • oligo(dT) primers according to the manufacturer's protocol.
  • quantitative real-time PCR with the LightCycler System TM (Roche Diagnostics, Mannheim, Germany) was established.
  • PCR reactions for mSGK1 were performed in a final volume of 20 ⁇ l containing 2 ⁇ l cDNA, 2.4 ⁇ l MgCI 2 (3 ⁇ M), 1 ⁇ l primermix (0.5 ⁇ M of both primers), 2 ⁇ l cDNA Master SybrGreen I mix (Roche Molecular Biochemicals, Mannheim, Germany) and 12.6 ⁇ l DEPC treated water.
  • the transcript levels of the housekeeping gene mGAPDH were determined in each sample using a commercial primer kit (Search LC, Heidelberg, Germany).
  • PCR reactions for GAPDH were performed in a final volume of 20 ⁇ l containing 2 ⁇ l cDNA, 2 ⁇ l primer mix (Search LC, Heidelberg, Germany), 2 ⁇ l cDNA Master Sybr Green I mix (Roche Molecular Biochemicals, Mannheim, Germany) and 14 ⁇ l DEPC treated water.
  • Amplification of the target DNA was performed during 35 cycles of 95°C for 10s, 68°C for 10s and 72 0 C for 16s, each with a temperature transition rate 5 of 20°C/s and a secondary target temperature of 58°C with a step size of
  • mSGK1 sense 5' TGT CTT GGG GCT GTC CTG TAT G 3' mSGK1 antisense: 5' GCT TCT GCT GCT TCC TTC ACA C 3' 15
  • BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm using an intensified charge-coupled device camera (Proxitronic, Germany) and specialized computer software (Metafluor, USA).
  • Intensity ratio data (490/440) were converted into pH values using the high-K + /nigericin calibration technique [Ganz et al., 1989]. Where indicated 50 ⁇ M omeprazole (Astra-Zeneca Sweden) was added to the BCECF incubation medium and standard Hepes solution.
  • ⁇ pHj is the decrease of cytosolic pH (pHj) following ammonia removal and A[NH 4 + Ii the decrease of cytosolic NH 4 + concentration, which is identical to the concentration of NH 4 + immediately before the removal of ammonia.
  • pHo extracellular pH
  • NH 4 + ] O extracellular fluid
  • the solutions were composed of (in mM): standard Hepes 115 NaCI, 5 KCI, 1 CaCI 2 , 1.2 MgSO 4 , 2 NaH 2 PO 4 10 glucose, 32.2 Hepes; sodium free Hepes 132.8 NMDG, 3 KCI, 1 CaCI 2 , 1.2 MgSO 4 , 2 KH 2 PO 4 , 32 Hepes, 10 Mannitol, 10 Glucose; sodium free ammonium chloride 10 mM NMDG and mannitol was replaced with 20 mM NH 4 CI; High K + for calibration 105 KCI, 1 CaCI 2 , 1.2 MgSO 4 , 32.2 Hepes, 10 mannitol 5 ⁇ M nigericin. The pH of the solutions was titrated to 7.4 or 7.0 with HCI/NaOH, HCI/NMDG and HCI/KOH, respectively, at 37°C.
  • R 1 , R 5 is either H, OH, OA, OAc or Methyl
  • R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 is either
  • R 4 , R 5 is either H, A, OH, OA, Alkenyl, Alkinyl, NO 2 , NH 2 , NHA, NA 2 , Hal,
  • CONR 8 R 9 two groups selected from R 1 , R 2 , R 3 , R 4 , R 5 or as well -0-CH 2 -CH 2 -, -0-CH 2 -O- or
  • R 6 , R 7 is either H, A, Hal, OH, OA or CN
  • R 8 , R 9 is either H or A
  • X, X 1 is either NH or is missing
  • the SGK1 assay was performed with activated/phosphorylated SGK1.
  • 20 Activated SGK1 was generated by incubating truncated SGK1 peptide ( ⁇ (1- 60)hSGK1 S422D) together with a PDK1 -derivative and ATP.
  • the preactivated/phosphorylated SGK1 was incubated for 60 min at room temperature together with biotinylated Crosstide peptide (biotin- KGSGSGRPRTSSFAEG) and 5 ⁇ M [ 33 P-ATP] (50-1000 cpm/pmole) in the 25 presence or absence of compounds to be tested.
  • the assay buffer was 20 mM MOPS pH 7.2, 5 mM EGTA, 0.5 ⁇ M substrate peptide, 15 mM MgCb, 25 mM glycerophosphate, 1 mM Na 3 VO 4 , 1 mM DTT, 0.01 % Brij-35.
  • the reaction was stopped by addition of 200 ⁇ l of the assay buffer without ATP and aliquots of the reaction mixture have been transferred into a 96- well streptavidin coated FLASH-plate, and incubated for 20 min. Thereafter, the solution is removed and the plate washed three times with PBS buffer. For measuring the bound radioactivity the FLASH-plates have been placed in a TOPCOUNT microtiter plate scintillation counter.
  • 96-wells microtiter-plates have been coated by adding 50 ⁇ l of a 0.5 ⁇ M solution of the GSK-3 fusion protein (Cell Signalling; No. 9278) in 0.2 M carbonate puffer. The MTP was incubated for 60 min at 37 0 C. Thereafter, the MTP-wells have been washed 3 x with 100 ⁇ l/well of 140 mM NaCI, 10 mM NaH 2 PO 4 (wash buffer).
  • Blocking of free sides was performed adding 100 ⁇ l of the blocking buffer (wash buffer + 3 % (w/v) bovine serum albumin) to each well and incubation for 60 min at 37 °C. After washing, the kinase reaction was carried out in a total volume of 50 ⁇ l by incubation of 10 ng of hSGK ( ⁇ (1-60)hSGK1 S422D-derivative) in the presence of increasing concentrations of the test compounds at 30 - 300 ⁇ M ATP in the ADBI-buffer (ADBI-buffer: 20 mM MOPS pH 7.2, 5 mM EGTA, 15 mM MgCI 2 , 25 mM glycerophosphate, 1 mM Na 3 VO 4 , 1 mM DTT 1 0.01 % Brij-35). The incubation is carried out for 120 min at room temperature.
  • ADBI-buffer 20 mM MOPS pH 7.2, 5 mM EGTA, 15 mM MgCI 2
  • the MTP was incubated for 30 min at 37 °C and then three times washed. Next, 50 ⁇ l of a goat-anti-rabbit horse-radish-peroxidase (POD) conjugated antibody solution (1 :15,000 dilution in blocking buffer; Sigma #A-0545) was added to each well. After further incubation and washing the assay was developed with ABTS (2,2'-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid), (Calbiochem # 194434) and H 2 O 2 . The read-out was performed with a goat-anti-rabbit horse-radish-peroxidase (POD) conjugated antibody solution (1 :15,000 dilution in blocking buffer; Sigma #A-0545) was added to each well. After further incubation and washing the assay was developed with ABTS (2,2'-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid), (Calbiochem # 194434)
  • Inhibitors SGK1 as claimed in this application have been identified by a decrease in the OD in compared to the control incubations.
  • NDRG1 -protein has be described as a specific substrate of the SGK1 expressed in HeLa-cells (see Murray JT, et al. Biochem. J. 2004, 384:477- 88). In the current application HeLa-cells have been used as a cellular test system for the characterization of SGK1 -inhibitors.
  • HeLa cells are plated in 6-wells MTPs (Costar Corning, # 3506) at a density of 10 - 20 x 10 3 cells / cm 2 in DMEM medium, supplemented with 10 % foetal calf serum (FCS), 2 mM glutamine and 1 mM sodium pyruvate have been incubated for 24 hrs at 37 0 C at 5 % CO 2 . Then serial dilutions of the compound have been added resulting in the anticipated SGK1 -inhibitor concentration at a 1 % DMSO concentration. The cells have been incubated for another 24 hrs.
  • FCS foetal calf serum
  • 16 ⁇ l aliquots of the cell lysates are transferred to 6 ⁇ l of the 4 X NuPage ® LDS-sample buffer plus 1 ⁇ l ⁇ -mercaptoethanol and heated.
  • the samples have been further investigated by SDS-PAGE and Western blot analysis, using NDRG1- and P-NDRG1-antisera.
  • the decrease in the intensity of the band on the Western blot, using the P-NDRG1- antibody was determined and used to assess the intracellular inhibitory potency of the SGK1- inhibitorsclaimed in the current application.
  • RK Dexamethasone makes the gastric mucosa susceptible to ulceration by inhibiting prostaglandin synthetase and peroxidase-two important gastroprotective enzymes. MoI Cell Biochem 1999;202:31-36.
  • Boehmer C Okur F 1 Setiawan I, Broer S, Lang F: Properties and regulation of glutamine transporter SN1 by protein kinases SGK and PKB. Biochem Biophys Res Commun 2003b;306:156-162.
  • FSH Follicle-Stimulating hormone
  • ROMK1 by the serum- and glucocorticoid-inducible kinase SGK1.
  • SGK Serum and glucocorticoid-inducible kinase
  • Ovarian cell differentiation a cascade of multiple hormones, cellular signals, and regulated genes. Recent Prog Horm Res 1995;50:223-254.
  • Warntges S Klingel K, Weigert C, Fillon S, Buck M, Schleicher E, Rodemann HP, Knabbe C 1 Kandolf R, Lang F: Excessive transcription of the human serum and glucocorticoid dependent kinase hSGK1 in lung fibrosis. Cell Physiol Biochem 2002;12:135-142.
  • NHERF2 NHERF2
  • Fig. 1 SGK1 Transcript levels in gastric tissue
  • Fig. 2 pH recovery in parietal cells following an ammonium pulse
  • cytosolic pH in ileum following an ammonium pulse.
  • 20 mM NH 4 CI were added and Na + removed (replaced by NMDG) in a first step (see bars below each original tracing), NH 4 CI removed in a second step, Na + added in a third step and nigericin (Nig.) applied in a fourth step to calibrate each individual experiment.
  • B Original tracings illustrating alterations of pH in typical experiments in sgk1 +/+ (left panels) and sgk1 v ⁇ (right panels) in dexamethasone treated mice

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne un procédé permettant de modifier la sécrétion d'acide gastrique induite par un glucocorticoïde qui consiste à mettre en contact des cellules exprimant une kinase inductible par sérum et glucocorticoïde (SGK) avec une substance qui module cette kinase inductible par glucocorticoïde. Cette invention concerne aussi le diagnostic et l'identification de composés qui peuvent être des agonistes, des antagonistes potentiellement utiles dans une thérapie de sécrétion acide gastrique pathologique.
EP07702807A 2006-01-31 2007-01-17 Procede permettant d'interferer avec la secretion d'acide gastrique induite par glucocorticoïde Withdrawn EP1979747A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07702807A EP1979747A1 (fr) 2006-01-31 2007-01-17 Procede permettant d'interferer avec la secretion d'acide gastrique induite par glucocorticoïde

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP06001934 2006-01-31
PCT/EP2007/000350 WO2007087985A1 (fr) 2006-01-31 2007-01-17 Procédé permettant d'interférer avec la sécrétion d'acide gastrique induite par glucocorticoïde
EP07702807A EP1979747A1 (fr) 2006-01-31 2007-01-17 Procede permettant d'interferer avec la secretion d'acide gastrique induite par glucocorticoïde

Publications (1)

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EP1979747A1 true EP1979747A1 (fr) 2008-10-15

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EP (1) EP1979747A1 (fr)
JP (1) JP2009531020A (fr)
AR (1) AR059258A1 (fr)
AU (1) AU2007211655A1 (fr)
CA (1) CA2641325A1 (fr)
IL (1) IL193081A0 (fr)
WO (1) WO2007087985A1 (fr)

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KR20210008193A (ko) * 2019-07-10 2021-01-21 한양대학교 산학협력단 염증성 신경 질환의 치료제로서 Sgk1 저해제의 용도
WO2022093780A1 (fr) * 2020-10-26 2022-05-05 University Of North Dakota Utilisation d'activateurs de fak à petites molécules pour favoriser la cicatrisation des muqueuses

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DE10346913A1 (de) * 2003-10-09 2005-05-04 Merck Patent Gmbh Acylhydrazonderivate
CN1929831A (zh) * 2004-03-08 2007-03-14 默克专利有限公司 改变胰岛素分泌的方法
CN1929846A (zh) * 2004-03-11 2007-03-14 默克专利有限公司 包括采用血清和糖皮质激素诱导的激酶的调节物来调节谷氨酸受体以治疗神经精神障碍的方法
JP2007527875A (ja) * 2004-03-11 2007-10-04 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 線維症を抑制するための方法
WO2005106491A2 (fr) * 2004-04-30 2005-11-10 Bayer Healthcare Ag Diagnostic et traitement therapeutique de maladies associees a la kinase 1 regulee par serum/glucocorticoide (sgk1)

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Also Published As

Publication number Publication date
WO2007087985A1 (fr) 2007-08-09
JP2009531020A (ja) 2009-09-03
AU2007211655A1 (en) 2007-08-09
CA2641325A1 (fr) 2007-08-09
IL193081A0 (en) 2009-02-11
AR059258A1 (es) 2008-03-19

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