WO2005017196A2 - A method for the identification of irs protein kinase inhibitors or agonists - Google Patents
A method for the identification of irs protein kinase inhibitors or agonists Download PDFInfo
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- WO2005017196A2 WO2005017196A2 PCT/EP2004/007900 EP2004007900W WO2005017196A2 WO 2005017196 A2 WO2005017196 A2 WO 2005017196A2 EP 2004007900 W EP2004007900 W EP 2004007900W WO 2005017196 A2 WO2005017196 A2 WO 2005017196A2
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- 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
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
Definitions
- the present invention relates to a method for the identification of IRS protein kinase inhibitors or agonists as well as to selected IRS peptides comprising phosphorylation sites for PKC- ⁇ and other IRS serine kinases and to the use of these peptides for the identification of a pharmaceutical composition for the treatment of diabetes type 2.
- Non-insulin dependent diabetes mellitus occurs predominantly in adults and is characterized by a reduced sensitivity of tissues being capable of clearing glucose from the blood.
- IDDM insulin dependent diabetes mellitus
- diabetes type II is not characterized by an impaired insulin secretion from the pancreatic beta cells.
- insulin receptor substrate (IRS) proteins are phosphorylated on multiple tyrosine residues by the activated insulin receptor, the insulin-like-growth factor receptor and JAK1/2 and play a pivotal role in the process of downstream insulin signaling (1 , 2, 3).
- PI-3 kinase phosphatidylinositol-3 kinase
- PI-3 kinase is composed of a catalytic 110-kDa subunit (p110) and a regulatory 85-kDa subunit (p85) containing two SH2 domains that bind to tyrosine-phosphorylated pYMXM and pYXXM motifs in IRS proteins and induce PI-3 kinase activation (7).
- PKB/Akt serine/threonine kinase
- PKC- ⁇ / ⁇ atypical protein kinase C isoforms- ⁇ and - ⁇ (PKC- ⁇ / ⁇ ) (10, 11) by phosphoinositide- dependent kinase 1.
- Activation of PKB and PKC- ⁇ / ⁇ and its downstream signals have been shown to play a critical role in mediating the metabolic actions of insulin such as GLUT4 translocation and glucose transport (10, 11 ), GSK3 serine phosphorylation and glycogen synthesis (12), PDE serine phosphorylation and anti-lipolysis (13, 14), and mTOR activation and protein synthesis (15, 16).
- IRS-1 serine/threonine phosphorylation of IRS-1 occurs constitutively in the cell (21) and it is further promoted by cytokines and metabolites that inhibit signal transduction like tumor necrosis factor (TNF) ⁇ (22), free fatty acids, glucose or ceramide (23). Furthermore, hyperphosphorylation of IRS-1 on serine/threonine residues is a common finding during insulin resistance and type 2 diabetes (24).
- IRS-1 serine phosphorylation protein kinases
- JNK c-Jun N-terminal kinase
- IkappaB kinase- ⁇ 26
- MAP kinase 27
- Casein kinase 28
- glycogensynthase kinase 29
- phosphoinositol-3-kinase 30
- protein kinase A 31
- protein kinase C 32
- PBB protein kinase B
- AMPK AMP-activated protein kinase
- Ser 789 is targeted by AMPK and positively modulates insulin action (34), however, Ser 789 phosphorylation by unidentified kinases was also found to attenuate insulin signaling (37).
- Ser 612 , Ser 632 , Ser 662 and Ser 731 are located within or near the PI 3-kinase interaction domain, however, the functional implications of these sites has remained elusive (38-40).
- PKC protein kinase C
- IRS-1 insulin signal and in the negative feedback control of IRS-1 function (11 , 41-44).
- PKC- ⁇ was found to colocalize with GLUT4 and to be essential for insulin-regulated GLUT4 translocation and glucose transport in skeletal muscle (41) and adipocytes (11 ). Further, defective activation of PKC- ⁇ may contribute to obesity-dependent development of skeletal muscle insulin resistance (42).
- IRS-1 represents a novel substrate for PKC- ⁇ and in a parallel study Zick and co- workers (44) found that this process inhibits PI 3-kinase activation, suggesting that PKC- ⁇ represents a key element in the negative feedback control of insulin action.
- IRS especially IRS-1 interacts with PKC- ⁇
- PKC- ⁇ the nature of this interaction is not known. Consequently, there are no agents known in the art interacting with IRS-PKC- ⁇ interaction. However, such agents would be highly needed since it is very likely that an inhibition of the interaction of IRS and PKC- ⁇ would result in down-regulation of the inhibition of IRS and, more downstream, PI 3-kinase which in turn would result in an improvement in GLUT4 translokation and glucose transport.
- the present invention is based on the surprising identification of specific serine phosphorylation sites in the sequence of IRS, which are specifically recognized by PKC- ⁇ . Consequently, in the context of the present invention, the molecular mechanism providing the IRS-PKC- ⁇ interaction were identified. Moreover its likely that other protein kinases like c-Jun N-terminal kinase (JNK), IkappaB kinase- ⁇ , MAP kinase, Casein kinase, glycogensynthase kinase, phosphoinositol-3-kinase, protein kinase A, protein kinase C, protein kinase B 8PKB) and AMP-activated protein kinase (AMPK) might be able to recognize these phosphorylation sites. Therefore the determination of the serine sites, which are phosphorylated by protein kinases, especially PKC- ⁇ .enables the identification of molecules interfering with this interaction, either in an antagonistic or
- IRS protein kinase inhibitor relates to a substance which interferes with the interaction between IRS and a protein kinase, exemplified by PKC- ⁇ .
- IRS peptide relates to a peptide comprising a stretch of at least 5, preferably 7, preferably at least 10 amino acids of IRS.
- IRS peptide includes that the IRS-peptide may comprise, in addition to the stretch of amino acids derived from IRS, further amino acids which are not IRS derived.
- PKC- ⁇ is commercially available, e.g. from CalBiochem (San Diego, CA, USA). Furthermore, methods for the isolation of PKC- ⁇ are described in the literature cited in the present application.
- IRS-1 The sequence of IRS, especially IRS-1 and IRS-2 from different species are known in the art.
- the rat IRS-1 sequence is herein provided as SEQ ID NO: 16.
- mutated proteins e.g. by exchanging one or more amino acids or by deleting stretches of amino acids, are known in the art (see above). These methods include site directed mutagenesis of the IRS gene and expressing the modified gene in appropriate cells.
- a reduced phosphorylation of the PKC- ⁇ -Ser- phosphorylation site in comparison to the phosphorylation in the absence of the at least one putative inhibitor is indicative for the inhibitory properties of the putative inhibitor.
- PKC- ⁇ is of mammalian, preferably, of rodent or human origin, more preferably of rat or human origin.
- the IRS peptide is derived from an. IRS, preferably IRS-1 , of mammalian, preferably of human or rodent, more preferably of rat origin.
- the IRS-1 is of rat origin and the at least one PKC- ⁇ -Ser-phosphorylation site is selected from the group consisting of Ser 458, 469, 481 , 498, 522, 526, 530, 536, 538, 539, 542, 560, 570, 577, 599, 600, 612, 620, 632, 635, 662, and 664 wherein the sequence numbers correspond to rat IRS-1 as depicted in SEQ ID NOJ 6.
- the IRS-1 may be of human origin and the at least one PKC- ⁇ -Ser- phosphorylation site is selected from Ser-residues corresponding to the above Ser- residues of the rat IRS-1 ,
- the PKC- ⁇ -Ser-phosphorylation site in the context of the present invention may be selected from the group consisting of Ser 498 , Ser 570 and Ser 612 , more preferably Ser 570 .
- the peptide is rlRS 449 - 664 (SEQ ID NO: 17).
- the inhibitor is selected from the group consisting of binding peptides, antibodies, and Low molecular weight compounds (LMWs).
- LMWs Low molecular weight compounds
- binding protein or "binding peptide” refers to a class of proteins or peptides which bind and inhibit IRS including, without limitation, polyclonal or monoclonal antibodies, antibody fragments and protein scaffolds directed against IRS, e.g. anticalins which are directed against IRS
- the procedure for preparing an antibody or antibody fragment is effected in accordance with methods which are well known to the skilled person, e.g. by immunizing a mammal, for example a rabbit, with IRS, where appropriate in the presence of, for example, Freund's adjuvant and/or aluminum hydroxide gels (see, for example, Diamond, B.A. et al. (1981 ) The New England Journal of Medicine: 1344- 1349).
- the polyclonal antibodies which are formed in the animal as a result of an immunological reaction can subsequently be isolated from the blood using well known methods and, for example, purified by means of column chromatography.
- Monoclonal antibodies can, for example, be prepared in accordance with the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299).
- antibody or antibody fragment is also understood as meaning antibodies or antigen-binding parts thereof, which have been prepared recombinantly and, where appropriate, modified, such as chimaeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab) or F(ab) 2 fragments (see, for example, EP-B1-0 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884).
- protein scaffolds against IRS e.g. anticalins which are ' based on lipocalin
- the natural ligand-binding sites of the lipocalins for example the retinol-binding protein or the bilin-binding protein, can be altered, for example by means of a "combinatorial protein design" approach, in such a way that they bind to selected haptens, here to IRS (Skerra, 2000, Biochim. Biophys. Acta, 1482, 337-50).
- Other known protein scaffolds are known as being alternatives to antibodies for molecular recognition (Skerra (2000) J. Mol. Recognit, 13, 167-187).
- LMWs are molecules which are not proteins, peptides antibodies or nucleic acids, and which exhibit a molecular weight of less than 5000 Da, preferably less than 2000 Da, more preferably less than 2000 Da, most preferably less than 500 Da. Such LMWs may be identified in High-Through-Put procedures starting from libraries.
- the inhibitor can be in the form of a natural product extract, either in crude or in purified form.
- the extract can be produced according to standard procedures, such as water and/or alcohol and/or organic solvent extraction and/or column chromatography and/or precipitation from an animal, plant or microbial source, like snake poison, leaves or microbial fermentation broths.
- IRS and PKC- ⁇ are provided e.g. in an assay system and brought directly or indirectly into contact with a test compound, in particular a biochemical or chemical test compound, e.g. in the form of a chemical compound library. Then, the influence of the test compound on the phosphorylation of IRS is measured or detected. Thereafter, suitable inhibitors can be analyzed and/or isolated.
- the use of high-throughput assays are preferred which are known to the skilled person or which are commercially available.
- chemical compound library refers to a plurality of chemical compounds that have been assembled from any of multiple sources, including chemically synthesized molecules and natural products, or that have been generated by combinatorial chemistry techniques.
- the influence of the test compound on the interaction is measured or detected by determining the degree of phosphorylation of the IRS peptide. This can be done by using phosphor-specific antibodies. Such antibodies are known in the art and available e.g: from Clonetech, Santa Cruz and Cellsignal.
- the degree of phosphorylation may be measured by using radio labeled ATP in the assay.
- the ATP can be labeled with 32-P or 33-P, and the amount of radioactive phosphate incorporated into the IRS can be measured by methods known in the art (see Example 2, 9.).
- the intensity of a signal measured by auto radiography may be indicative for the degree of phosphorylation.
- the method of the present invention is carried out in a robotics system e.g. including robotic plating and a robotic liquid transfer system, e.g. using micro fluidics, i.e. channeled structured.
- the method is carried out in form of a high-through put screening system.
- the screening method is automated and miniaturized, in particular it uses miniaturized wells and micro fluidics controlled by a roboter.
- the invention further relates to a method for the identification of an IRS agonist, comprising the steps of a) bringing into contact PKC- ⁇ with at least one IRS peptide comprising at least one PKC- ⁇ -Ser-phosphorylation site in the presence of at least one putative agonist comprising at least one PKC- ⁇ -Ser-phosphorylation site, and b) measuring the phosphorylation of the PKC- ⁇ -Ser-phosphorylation site of the putative agonist.
- the agonist is a peptide.
- Peptide libraries which could be used in the context of the present invention are known in the art.
- an increased phosphorylation of the PKC- ⁇ -Ser-phosphorylation site of the agonist in comparison to the phosphorylation of the PKC- ⁇ -Ser-phosphorylation site of the IRS peptide is indicative for the agonistic properties of the putative agonist.
- the invention further relates to a method for the determination of PKC- ⁇ activity, comprising the steps of a) bringing into contact PKC- ⁇ with at least one IRS peptide comprising at least one PKC- ⁇ -Ser-phosphorylation site in the presence of at least one putative inhibitor, and b) measuring the phosphorylation of the PKC- ⁇ -Ser-phosphorylation site. Consequently, the present invention provides a method to measure the activity of PKC- ⁇ . Such a method is especially useful when the activity of PKC- ⁇ from different patients has to measured in order to gain more information about the signal transduction system in patients, especially diabetic patients.
- PKC- ⁇ is preferably of mammalian, more preferably of human origin.
- the invention further relates to an IRS-1 peptide comprising Ser 570 , preferably IRS- ,
- this peptide of the invention is especially useful for the identification of IRS analogues or inhibitors.
- the invention further provides a kit comprising a) at least one IRS peptide, b) a PKC- ⁇ preparation, and c) at least one putative IRS protein kinase inhibitor or agonist.
- the present invention provides an IRS-1 peptide, wherein Ser 570 and/or Ser 612 are mutated, preferably to alanine.
- a peptide is useful for blocking PKC- ⁇ activity in vitro or in vivo.
- the invention further relates to the use of an IRS peptide as defined above for the production of antibodies, preferably against a PKC- ⁇ -Ser-phosphorylation site, preferably against Ser 498 , Ser 570 and Ser 612 , more preferably against Ser 570 . Consequently, with the help of the IRS peptides as defined in the present invention, it is possible to produce IRS specific antibodies, especially antibodies which are directed against PKC- ⁇ phosphorylation sites. Such antibodies can serve both in in vitro diagnostics as well as in pharmaceutical compositions.
- the present invention relates to the use of an IRS peptide as defined above or of an IRS-1 peptide with mutated Ser-sites as defined above for the preparation of a pharmaceutical composition for the treatment of diabetes type 2.
- Such peptides may inhibit the interaction between IRS and PKC- ⁇ and may, therefore, serve as antagonists of IRS phosphorylation.
- the IRS protein kinase inhibitors or agonists as identified in the present invention or the peptides of the present invention are usually formulated with one or more pharmaceutically acceptable additives or auxiliary substances, such as physiological buffer solution, e.g. sodium chloride solution, demineralized water, stabilizers, such as protease or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA, gel formulations, such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
- physiological buffer solution e.g. sodium chloride solution
- demineralized water demineralized water
- stabilizers such as protease or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA
- gel formulations such as white vaseline, low-visco
- Suitable further additives are, for example, detergents, such as, for example, Triton X-100 or sodium deoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred.
- the physiological buffer solution preferably has a pH of approx. 6.0-8.0, expecially a pH of approx. 6.8-7.8, in particular a pH of approx. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter, preferably of approx. 290-310 milliosmol/liter.
- the pH of the pharmaceutical composition is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3-morpholino- 1-propanesulphonic acid).
- a suitable organic or inorganic buffer such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino
- the pharmaceutical composition can be administered in a conventional manner, e.g. by means of oral dosage forms, such as, for example, tablets or capsules, by means of the mucous membranes, for example the nose or the oral cavity, in the form of depositories implanted under the skin, by means of injections, infusions or gels which contain the pharmaceutical compositions according to the invention. It is further . possible to administer the pharmaceutical composition topically and locally, if appropriate, in the form of liposome complexes. Furthermore, the treatment can be carried out by means of a transdermal therapeutic system (TTS), which makes possible a temporally controlled release of the pharmaceutical compositions.
- TTS transdermal therapeutic system
- TTS are known for example, from EP 0 944 398 A1 , EP 0 916 336 A1 , EP 0 889 723 A1 or EP 0 852 493 A1.
- Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body.
- Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more liters, are to be administered. Since, in contrast to the infusion solution, only a few milliliters are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or the tissue fluid in the injection do not make themselves noticeable or only make themselves noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according to the invention before use is therefore in general not necessary.
- the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained.
- An example of an isotonic solution is a 0.9% strength sodium chloride solution.
- the dilution can be carried out, for example, using sterile water while the administration can be carried out, for example, via a so-called bypass.
- the invention further relates to a method for the preparation of a pharmaceutical composition, comprising the steps: a) identifying an IRS protein kinase inhibitor or agonist as defined above, b) providing adequate amounts of the IRS protein kinase inhibitor, and c) formulating the IRS protein kinase inhibitor into a pharmaceutical composition, optionally in combination with a pharmaceutical acceptable carrier.
- Example 1 MATERIALS Oligonucleotide primers were obtained from MWG-Biotech (Ebersberg, Germany). BL21 Codon Plus and QuikChangeTM Site-Directed Mutagenesis Kit were purchased from Stratagene (La Jolla, CA, USA). One Shot TOP 10 Competent Cells were from Invitrogen (Karlsruhe, Germany). A plasmid miniprep kit was obtained from Qiagen (Hilden, Germany). A polyclonal anti-IRS-1 antiserum was a gift from Dr. J. A. Maassen (Leiden, The Netherlands). Anti-phosphotyrosine antibody (RC20) coupled to horseradish peroxidase and anti-p85 antibody were obtained from Transduction Laboratories, Inc.
- RC20 Anti-phosphotyrosine antibody
- Alpha Thrombin was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Enzymes for molecular biology, Complete protease inhibitor cocktail, and modified trypsin sequencing grade were obtained from Roche (Mannheim, Germany). Okadaic acid, phosphatidylserine, and wheat germ agglutinin (Triticum vulgaris) were purchased from SIGMA (M ⁇ nchen, Germany). IRS-1 peptides were synthesized by Dr. Hoffmann (BMFZ, University of Dusseldorf, Dusseldorf, Germany).
- Example 2 METHODS 1.) Construction and Expression of Fusion Proteins
- the regulatory p85 ⁇ subunit of bovine PI 3-kinase cloned into the expression vector pGEX-2T was a kind gift of Dr. P. Shepherd (London, UK).
- a glutathione S-transferase (GST) fusion protein containing the amino acids 449-664 of rat IRS-1 (rlRS-1 449"664 , M w of 51.2 kDa) was prepared based on the method described by Smith and Johnson (40) using the pGEX-5X-3 vector.
- Corresponding rat cDNA was generated from RNA r isolated from rat heart by reverse transcription using avian myeloblastosis virus reverse transcriptase and subsequent amplification by polymerase chain reaction using Pwo.DNA Polymerase and the following oligonucleotide primers: 5 ' -primer, ATATTGTCGACCAC-ACCCCACCAGCCAGG, 3 ' -primer, ATGTACTACTACAGAGGGTC-ACGCCGGCGTAAGAATA (SEQ ID NO: 1 and 2). The PCR products were isolated, digested with appropriate restriction enzymes and subcloned into pGEX-5X-3.
- rat IRS-1 clone was verified by restriction endonuclease analysis and nucleotide sequencing.
- This vector and the p85 ⁇ -pGEX-2T construct were used to transform Escherichia coli BL21.
- Transformed cells were grown to an A 6 oo nm of 0.6 - 0.8 in 2x YTA medium (16 g/l tryptone, 10 g/l yeast, NaCl 5 g/l) supplemented with 0.1 mg/ml ampicillin and induced for 2 h with 0.1 mM isopropyl- ⁇ -D- thiogalactoside (IPTG).
- IPTG isopropyl- ⁇ -D- thiogalactoside
- Fusion proteins were purified by affinity chromatography on glutathione-sepharose columns and eluted by 10 mM glutathione in 50 mM Tris-HCI (pH 8.0).
- the GST part of the p85 GST-fusion protein was proteolytically removed using bovine thrombin in PBS.
- the protease was added to the fusion protein bound to the glutathione sepharose column, incubated for 2 h at room temperature and then the eluate was collected. Protein was determined using a modification of the Bio-Rad protein assay. All GST fusion proteins had the expected molecular weight when analyzed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE).
- SDS sodium dodecyl sulphate
- PAGE polyacrylamide gel electrophoresis
- the supernatant was then applied to an agarose-bound wheat germ agglutinin (WGA) column.
- WGA wheat germ agglutinin
- the column was washed with 50 mM Hepes (pH 7.4), 0.1% Triton X-100, and bound glycoproteins were eluted from the WGA column with this buffer containing 0.3 M N-acetylglucoseamine.
- rlRS-1 449"664 phosphorylation by insulin receptor 5 ⁇ g of the WGA-purified glycoprotein fraction was preincubated for 30 min at 30°C with 100 nM of insulin in a phosphorylation buffer containing 20 mM Hepes (pH 7.4), 1 mM DTT, 10 mM MgC ⁇ , 100 ⁇ g/ml bovine serum albumin, 0.2 mM Na 3 V0 , 1.7 mM CaCI 2 , 0.6 mg/ml phosphatidylserine, and 0.5 ⁇ g/ml okadaic acid. Autophosphorylation was initiated by the addition of ATP at a concentration of 50 ⁇ M and continued for 10 min at 30 °C.
- Substrate phosphorylations were initiated by addition of equal volumes of rlRS-1 449"669 (1 ⁇ g) with or without pre-treatment (30 min) by the PKC isoforms in the same buffer in the presence of 50 ⁇ M ATP and was allowed to proceed for 10 min at 30°C in a final volume of 50 ⁇ l.
- the reaction was terminated by the addition of 6x sample buffer (0.35 M Tris-HCI (pH 6.8), 10.28 % (w/v) SDS, 36 % (v/v) glycerol; 0.6 M DTT, 0.012 % (w/v) bromphenol blue) and boiling for 5 min.
- Proteins were separated by SDS-PAGE and analyzed by immunodetection with an anti-phosphotyrosine antibody after transfer to nitrocellulose. Serine/threonine phosphorylation of rlRS-1 449"664 by different PKC isoforms was assessed by incubating 1 ⁇ g rlRS-1 449"664 with 0.5 ⁇ g PKC-rb or PKC- ⁇ in phosphorylation buffer for 30 min at 30°C in the presence of 50 ⁇ M ATP plus 2 ⁇ Ci [ ⁇ - 32 P]ATP in a volume of 20 ⁇ l. Proteins were resolved by SDS-PAGE and the stained and dried gels were subjected to autoradiography.
- Proteins were separated by SDS-PAGE using 8-18 % gradient gels followed by transfer to nitrocellulose in a semi-dry blotting apparatus. The membrane was then blocked 60 min in Tris-buffered saline containing 0.05% Tween 20 and 1 % BSA or 5% non fat dry milk and probed with appropriate antibodies (anti-IRS-1 , anti-pTyr, anti- p85 ⁇ ). After extensive washing, the membranes were incubated with horseradish
- the HPLC flow rate was 0.5 ml/min. After injection of sample, the peptides were eluted beginning at 100% buffer A (20 mM NH 4 CH 3 COOH, pH 7.0) and 0% of buffer B (1 M KH 2 P0 4 , pH 4.0). The amount of buffer B was increased to 10% within 40 min and from 10 to 50% during the following 75 min. Fractions of 0.5 ml were collected, and radioactivity was measured by Cerenkov counting. Radioactive fractions were subjected to reversed phase HPLC.
- Mass spectra were recorded on an electrospray quadrupole time-of-flight mass spectrometer (QSTAR Pulsar I, Applied Biosystems, Foster City, CA, USA) using a nanospray source (Protana, Odense, Denmark). Selected peptides were analyzed in tandem mass spectrometry mode and the sequence and posttranslational modifications were retrieved by manual interpretation.
- the serine 570 to alanine and serine 612 to alanine mutants of rlRS-1 449"664 were generated by site-directed mutagenesis using the QuikChangeTM Site-Directed Mutagenesis Kit according to the manufacturer's instructions using pGEX-5X-3/rlRS- 1 449"664 as a template.
- the following primers were used: S570A, 5 ' -CCCGGCTACCGGCATGCCGCCTTCGTGCCCACC (SEQ ID NO:3) and 3 ' -GGGCCGATGGCCGTACGGCGGAAGCACGGGTGG (SEQ ID NO:4); S612A, 5 ' -GGCTACATGCCCATGGCTCCCGGAGTGGCTCC (SEQ ID NO:5) and 3 ' -CCGATGTACGGGTACCGAGGGCCTCACCGAGG (SEQ ID NO:6). Presence of the desired mutations was confirmed by sequencing the recombinant molecules by Qiagen Sequencing Services (Hilden, Germany).
- the mixture was adjusted with water to an end volume of 20 ⁇ l minus the necessary ATP volume. Then the phosphorylation reaction was started by addition of ATP from a stock solution to an end concentration of 50 ⁇ M ATP plus 2 ⁇ Ci [ ⁇ - 32 P]ATP and incubated for 30 min at 30°C. The reaction was stopped by addition of 4 ⁇ l 6x sample buffer (0.35 M Tris-HCI (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol; 0.6 M 30 DTT, 0.012% (w/v) bromphenol blue) and boiling for 5 min. The proteins were then resolved by SDS-Page and the stained and dried gels were subjected for analysis to autoradiography.
- Example 3 An IRS-1 domain is phosphorylated by the insulin receptor and interacts with PI 3- kinase
- a selected part of the rat IRS-1 protein was cloned, expressed as a GST-fusion protein and purified from E. coli.
- This GST-fusion protein (rlRS-1 449"664 ) covers a domain of 216 amino acids (449-664) of the rat IRS-1 protein
- rlRS-1 449"664 incubated with the same amount of PKC in the presence of PKC inhibitors exhibited no significant incorporation of phosphate (Fig. 3 A).
- a dose-response curve with increasing amounts of PKC was then determined to establish conditions of max. phosphorylation, which was observed with 0.5 ⁇ g PKC rat brain or PKC- ⁇ (data not shown).
- rlRS-1 449"664 was treated with or without PKC and then incubated with WGA-purified IR.
- p85 ⁇ association was subsequently determined by coupling rlRS-1 449"664 to glutathione sepharose beads via its GST part and incubation with 0.5 ⁇ g p85 ⁇ , as outlined in Figure 2 A.
- Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against phosphotyrosine (pTyr), p85 ⁇ , and IRS-1.
- pTyr phosphotyrosine
- IRS-1 IRS-1
- rlRS-1 449"664 was incubated with PKC- ⁇ and separated by SDS-PAGE.
- Phosphorylated rlRS-1 449"664 was digested with trypsin and extracted from the gel.
- the peptides generated by digestion were resolved by two dimensional HPLC and the content of radioactivity in the fractions was monitored by Cerenkov counting.
- the HPLC profile of the first separation using an anion exchange column showed 6 reproducible major peaks (Fig. 7 A).
- Phosphoserine 358 was identified by ESI-MS/MS.
- the fragment ion with a mass difference of 97.9 Da to the parent ion indicates a phosphopeptide (Fig. 8 A).
- a mass difference 97.9 Da correlates to the loss of phosphoric acid.
- the site of phosphorylation was identified by the loss of the phosphate group (HPO 3 ) and phosphoric acid (H 3 P0 ) from the fragment ions bg and b-io. This dephosphorylation of the fragment ions indicates that the phosphorylation could only take place by the Y355 or S358.
- S358 is the phosphoaminoacid, because there was no dephosphorylation detected from the b 4 ion, indicating a phosphorylation at Y355 (data not shown).
- Phosphoserine 612 could not be detected by mass spectrometry despite being detected by a phosphosite specific antibody (Fig. 6 A), but a peptide including this site was found in peak 2 together with three additional peptides. Peak 1 and peak 3 contained only one peptide covering 13% and 21% of the radioactivity, respectively (THSAGTSPTISHQK and TPSQSSVVSIEEY-TEMMPAAYPPGGGSGGR). (SEQ ID NO:10 and 11 ) To further confirm that the phosphorylation sites found were serine 570 and 612, two additional GST fusion proteins with mutation of serine to alanine were generated.
- the abbreviations used are: GST, glutathione S-transferase; HPLC, high performance liquid chromatography; IR, insulin receptor; IRS, insulin receptor substrate; ESI-MS, electrospray ionization mass spectrometry; p85, regulatory subunit of phosphatidylinositol (PI)-3 kinase; PAGE, polyacrylamide gel electrophoresis; PI 3- kinase, phosphatidylinositol-3-kinase; RP, reversed phase; PKC, protein kinase C; PKB, protein kinase B; RTKs, receptor tyrosine kinases; SH2, src-homology domain 2; WGA, wheat germ agglutinine; SDS, sodium dodecyl sulphate; ECL, enhanced chemiluminescence.
- PI phosphatidylinositol
- FIG. 1 Schematic overview of IRS-1 with known interaction partners and known serine/threonine phosphorylation sites.
- Upper panel The relative positions of the pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domain are indicated followed by a C-terminal tail that contains numerous tyrosine phosphorylation sites. Potential binding partners including PI 3-kinase, Grb2 and SHP-2 are also shown.
- Middle panel Known (S307, 612, 632, 789) and potential serine phosphorylation sites are highlighted.
- Bottom panel Construction of a GST-fusion protein containing aa 449-664 of rat IRS-1 including the major binding site of the PI 3-kinase.
- FIG. 2 Tyrosine phosphorylation of rlRS-1 449"664 and interaction with p85 ⁇ subunit of . PI 3-kinase.
- A Schematic diagram of the experimental procedure. 5 ⁇ g IR was autophosphorylated for 10 min at 30°C in phosphorylation buffer after a 30 min preincubation with 100 nM insulin. Substrate phosphorylation was subsequently initiated by addition of autophosphorylated IR to aliquots of 1 ⁇ g rlRS-1 449"664 . The reaction proceeded for 10 min and then glutathione sepharose beads were added and samples were incubated at 4 °C on a rotator for 1 h.
- FIG. 3 Effect of PKC from rat brain on tyrosine phosphorylation of rlRS-1 449"664 and interaction with p85 .
- A 1 ⁇ g of rlRS-1 449 - 664 was incubated with 0.5 ⁇ g PKC from rat brain (PKC-rb) or 0.5 ⁇ g PKC- ⁇ in the presence of 2 ⁇ Ci ( 32 P)-ATP (final cone. 50 ⁇ M), as detailed in Methods. The reaction was inhibited by bisindolylmaleimide I (BIM) or pseudosubstrate peptide for PKC-rb or PKC- ⁇ , respectively. Proteins were resolved by SDS-PAGE and subjected to autoradiography.
- BIM bisindolylmaleimide I
- BIM pseudosubstrate peptide for PKC-rb or PKC- ⁇
- FIG. 4 Effect of PKC isoforms on IR autophosphorylation.
- A Autophosphorylation of IR was conducted as outlined in Fig. 2. Either PKC-rb or PKC- ⁇ was added after 10 min of autophosphorylation and incubation was continued for another 10 min. Tyrosine phosphorylation of IR ⁇ -subunit was then analyzed by immunoblotting.
- Figure 5 Effect of PKC- ⁇ on tyrosine phosphorylation of rlRS-1 449"664 and interaction with p85 ⁇ .
- rlRS-1 449"664 was preincubated with PKC- ⁇ (0.5 ⁇ g) for 30 min. Tyrosine phosphorylation by IR and interaction with p85 ⁇ was determined by immunoblotting, as detailed in Fig. 2.
- FIG. 6 Identification of IRS-1 serine 612 as a target of PKC and interaction analysis with p85 ⁇ using surface plasmon resonance.
- Figure 7 HPLC analysis of tryptic phosphopeptides derived from rlRS-1 449"664 phosphorylated by PKC- ⁇ .
- Fig. 8 ESI-MS/MS spectra from the phosphopeptid 352-378.
- Fig. 9 HPLC analysis of tryptic phosphopeptides of rlRS-1 449"664 and mutants S570A and S612A.
- FIG. 10 Functional analysis of serine 570 and serine 612.
- riRS-1 449"664 and the indicated mutants were preincubated with PKC- ⁇ and subjected to tyrosine phosphorylation by IR and interaction with p85 ⁇ , as outlined in Fig. 5. Representative Western, blots are shown.
- NAHTPPARGEEELSNYICMGGK SEQ TD 233-254 4 & 2 NOJ2
- GASTLTAPNGHYILSR SEQ LO NOJ3
- 225555--227700 2 20.7
- YTPGATMGTSPALTGDEAAGAADLDNR 277-303 S286 5 & 6 10.6 (SEQ ID NO:9)
- THSAGTSPTISHQK (SEQ ID NO: 10) 308-321 1 12.9 TPSQSSVNSffiEYTEMMPAAYPPGGGSGGR 322-351 3 21.2 (SEQ LD ⁇ OJ1)
- NDPNGYMMMSPSAAAS (SEQ LD NO:15) 441-456 2 20.7 a Tryptic peptides of rIRS-1 " were analyzed after anion exchange HPLC and reversed phase _-_ _ H
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Application Number | Priority Date | Filing Date | Title |
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EP04763258A EP1658501A2 (en) | 2003-08-16 | 2004-07-16 | A method for the identification of irs protein kinase inhibitors or agonists |
BRPI0413610-1A BRPI0413610A (en) | 2003-08-16 | 2004-07-16 | method for the identification of protein kinase irs agonists or inhibitors |
MXPA06001618A MXPA06001618A (en) | 2003-08-16 | 2004-07-16 | A method for the identification of irs protein kinase inhibitors or agonists. |
CA002535649A CA2535649A1 (en) | 2003-08-16 | 2004-07-16 | A method for the identification of irs protein kinase inhibitors or agonists |
AU2004265390A AU2004265390B2 (en) | 2003-08-16 | 2004-07-16 | A method for the identification of IRS protein kinase inhibitors or agonists |
JP2006523543A JP4796490B2 (en) | 2003-08-16 | 2004-07-16 | Method for identifying an IRS protein kinase inhibitor or agonist |
IL173545A IL173545A (en) | 2003-08-16 | 2006-02-05 | Method for the identification of irs protein kinase inhibitors |
NO20061215A NO20061215L (en) | 2003-08-16 | 2006-03-15 | Method for Identifying IRS Protein Kinase Inhibitors or Agonists |
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EP03018517A EP1508806A1 (en) | 2003-08-16 | 2003-08-16 | A method for the identification of IRS protein kinase inhibitors or agonists |
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US20100239586A1 (en) * | 2009-03-21 | 2010-09-23 | The Regents Of The University Of California | Procalin polypeptides, antigens, and methods of use |
RU2694964C2 (en) * | 2017-12-19 | 2019-07-19 | ФЕДЕРАЛЬНОЕ АГЕНТСТВО ПО ТЕХНИЧЕСКОМУ РЕГУЛИРОВАНИЮ И МЕТРОЛОГИИ (Росстандарт) | Method of measuring catalytic activity (catalytic concentration) of enzymes using methods of mass spectrometry and spectrophotometry with preliminary identification of target analyte and determination of purity of substances |
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US6511811B1 (en) * | 1995-06-07 | 2003-01-28 | The Regents Of The University Of California | Protein kinase C antagonist related to insulin receptor |
JPWO2002083730A1 (en) * | 2001-04-10 | 2004-08-05 | 大正製薬株式会社 | Novel protein IRSAL and gene irsal encoding the protein |
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