EP1290189A2 - Acide nucleique codant pour un site de liaison d'une proteine kinase de la cascade de signalisation mitogene de l'enzyme catalysant la glycolyse - Google Patents

Acide nucleique codant pour un site de liaison d'une proteine kinase de la cascade de signalisation mitogene de l'enzyme catalysant la glycolyse

Info

Publication number
EP1290189A2
EP1290189A2 EP01951397A EP01951397A EP1290189A2 EP 1290189 A2 EP1290189 A2 EP 1290189A2 EP 01951397 A EP01951397 A EP 01951397A EP 01951397 A EP01951397 A EP 01951397A EP 1290189 A2 EP1290189 A2 EP 1290189A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
raf
protein
glycolysis
silent mutation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01951397A
Other languages
German (de)
English (en)
Inventor
Ulf R. Rapp
Erich Eigenbrodt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ScheBo Biotech AG
Original Assignee
ScheBo Biotech AG
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Filing date
Publication date
Application filed by ScheBo Biotech AG filed Critical ScheBo Biotech AG
Publication of EP1290189A2 publication Critical patent/EP1290189A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0104—Pyruvate kinase (2.7.1.40)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PK Pyruvate kinases
  • M2-PK is the embryonic form and replaces all other forms in proliferating cells and tumor cells (see GEJ Staal et al., Biochical and Molecular Aspects of Selected Cancers, TG Pretlow et al., Eds., Academic Press Inc., San Diego, 1, pages 313 - 337, 1991, and U. Brinck et al., Virchows Archiv 424, pages 177 - 185, 1994).
  • Rat M2-PK protein consists of 530 amino acids and differs only in one residue from human M2-PK (see T. Noguchi et al., J. Biol. Chem., 261, pages 13807-13812, 1986, and K.
  • M2-PK is a glycolytic enzyme which is present in an active tetrameric and an inactive dimeric form. The transition between the two forms ultimately regulates the glycolytic conversion in tumor cells (see W. Zwerschke et al., Proc. Natl. Acad. Sei. USA, 96, pages 1291-1296, 1999, and S. Mazurek et al., J. Bioeneg. Biomenibr., 29, pages 315v-330, 1997). The activity of M2-PK thus controls the transit of the glycolytic path and thus determines the relative proportion of glucose which is channeled into the synthesis process or used for glycolytic energy generation.
  • M2-PK allows cells to survive under conditions with low oxygen levels, since oxidative phosphorylation is not required for the production of ATP by PK.
  • an increased amount of M2-PK is found in malignant tumors and in the blood of tumor patients.
  • A-raf is known to bind to H-ras, MEK and CK2ß (see AB Vojtek et al., Cell, 74, pages 205-214, 1993, and X. Wu et al., J. Biol. Chem., 271, pages 3265 - 3271, 1996, and B. Boldyreff et al., FEBS Lett., 403, pages 197 - 199, 1997, and C.
  • the invention is based on the technical problem of finding an approach for inhibiting the anaerobic metabolism in tumor cell groups and subsequently finding active substances which at least slow down the growth of tumor cells or promote apoptosis in such tumor cell groups due to insufficient energy generation in tumor cells. Knowledge underlying the invention.
  • a PC12 cDNA library was screened using a two-hybrid system and using A-raf as bait ("bait").
  • A-raf was found as a binding partner for A-raf.
  • A-raf is therefore part of the glycolytic enzyme complex. Transformation of NIH 3T3 cells by A-raf leads to an increase in the phosphoserine content of the M2-PK protein in connection with the shift in said balance.
  • an oncogene in the mitogenic signaling cascade also has a direct effect on an enzyme that catalyzes (anaerobic) glycolysis, namely to reinforce and thus promote tumor growth.
  • the invention first relates to a nucleic acid coding for at least one partial sequence of a protein kinase of the mitogenic signaling cascade, the partial sequence coding for a binding site for an enzyme catalyzing glycolysis, or silent mutation of such a nucleic acid or nucleic acid hybridizing with such a nucleic acid or its silent mutation.
  • nucleic acid includes in particular DNA, RNA and PNA. Also subsumed under the term are double-stranded nucleic acids and single-stranded nucleic acid and consequently also nucleic acids complementary to each other.
  • Silent mutations means variants in the sequence that have no functional difference, based on the
  • silica Binding site for an enzyme catalyzing the glycolysis, the variant compared to the natural, non-mutated sequence.
  • Silent mutations can be alleles or artificial mutations.
  • Derivatives also fall under the invention. Derivatives are non-natural chemical modifications.
  • nucleic acids can be used, for example, to search for cooperation partners of the protein kinase of the mitogenic signaling cascade in the area of the enzymes catalyzing glycolysis. It is also possible to search for inhibitors for the binding sites 7
  • nucleic acids according to the invention can therefore be used in particular as a screening tool.
  • a nucleic acid is preferably coding for a protein or peptide containing the sequence A-raf
  • nucleic acid or nucleic acid hybridizing with such a nucleic acid or its silent mutation.
  • it is preferably human A-raf.
  • Sequence 587-606, particularly 602-603, was identified as the region where the binding site for the enzymes catalyzing glycolysis is located.
  • a nucleic acid according to the invention can be shortened compared to the full sequence of A-raf, in particular at the N-terminal end.
  • sequence A-raf 255 to 587-606 or silent mutation of such a nucleic acid or nucleic acid hybridizing with such a nucleic acid or its silent mutation.
  • sequence can be in the range delimited by the sequences (255-606) and (587-606).
  • the invention further teaches a cDNA of the above structure and an isolated recombinant vector containing a nucleic acid of the above structure or an expression plasmid with this nucleic acid.
  • a DNA fragment coding for a suitable viral protein for example gag
  • gag fusion gag with, for example, A-raf or A-raf Fragment
  • a transfor ante can be formed by means of the expression plasmid, which in turn can be used to produce the protein or peptide encoded by the nucleic acid.
  • the transformant is cultivated in a suitable manner using customary methods.
  • Another aspect of the invention is an antisense nucleic acid or ribozyme which binds to, for example, an oncogenic nucleic acid, in particular RNA, coding for a protein kinase of the mitogenic signaling cascade.
  • an oncogenic nucleic acid in particular RNA
  • RNA coding for a protein kinase of the mitogenic signaling cascade.
  • A-raf can be suppressed by means of such a substance, with the result that the (anaerobic) glycolysis in the tumor tissue is inhibited.
  • a substance with a binding site for a protein or peptide encoded by a nucleic acid according to the invention selected from the group consisting of a) deactivated enzymes that catalyze glycolysis, b) inactive proteins or peptides and c) aptamers.
  • a ribozyme in the case of a ribozyme, it can be, for example, a hammerhead ribozyme which attacks within the kinase domain of a raf isoform m-RNA, for example at a GTC site.
  • the hammerhead can also attack ATG start codons that attack the kinase domain before out of frame. These make translation of an active kinase domain from the generated fragmentary RNA extremely unlikely.
  • the kinase-inactive form can be formed by a mutation in the region of the ADP binding site and / or the ATP binding site, in particular selected from the group consisting of "M2-PK K366M, R119C, T340M, Q377K, K161M, K165M and several of these mutations ".
  • the expression of the kinase-inactive form also includes only reduced forms in the kinase activity compared to normal M2-PK. Inactive mutants are generally suitable for triggering apoptosis of the tumor cells because a decrease in the ATP and ADP levels is thereby achieved.
  • ML-PK can also be considered as a natural mutant.
  • MI-PK is expressed in all non-proliferating cells.
  • M2-PK inhibition of cell proliferation occurs when Ml-PK is additionally expressed by competing reactions.
  • Ml-PK and M2-PK are different splicing products, whereby only one exon with 51 amino acids is exchanged.
  • Ml-PK and M2-PK differ in a total of only 21 amino acids.
  • Ml-PK is not phosphorylated.
  • phosphorylation sites can be derived by sequence comparison with M2-PK.
  • non-phosphorylable M2-PK mutants will act as tumor suppressor and proliferation inhibitor. Such mutants result directly from the following sequence comparison, in which potential phosphorylation sites are indicated.
  • M2-PK mutants which are mutated at least in at least one of the marked locations in accordance with the sequence comparison.
  • one or more mutations can be set up in the place of other deviations in the sequence comparison.
  • M2 Gly-Ala-Val-Glu-Ala-Ser-Phe-ys-Cys-Cys-Ser-Gly-Ala-Ile-Ile-Val-Leu Ml: Gly-Ser-Val-Glu-Ala-Ser-Tyr - ys-Cys-Leu-Ala-Ala-Ala-Leu-Ile-Val-Leu
  • proteins or peptides they are highly tailored synthetic molecules for the binding site of the protein or peptide encoded by the nucleic acid according to the invention.
  • part of the invention is also their use for blocking the cooperation or binding between a protein kinase of the mitogenic signaling cascade and an enzyme catalyzing the glycolysis, in particular the A-raf / M2-PK cooperation, and their Use for the production of a pharmaceutical preparation for the treatment of cancer, for example the urogenital tract.
  • a protein kinase of the mitogenic signaling cascade and an enzyme catalyzing the glycolysis, in particular the A-raf / M2-PK cooperation
  • the mitogenic signaling cascade is probably blocked at the same time and a synergistic effect is thus achieved.
  • Another aspect of the invention is the use of a nucleic acid according to the invention or a protein or peptide encoded thereby in a screening method for determining an enzyme which cooperates with a protein kinase of the mitogenic signaling chain and catalyzes the glycolysis.
  • the binding site of the enzyme catalysing glycolysis can also be determined and a peptide or a micris substance can be created for this which binds to the raf isoform at the same site.
  • the invention teaches the use of a nucleic acid according to the invention or a protein or peptide encoded thereby in a screening method for determining a substance which binds to a protein kinase of the mitogenic signaling chain but does not catalyze the glycolysis.
  • substances with prospective binding sites for the raf isoform binding site with an enzyme catalyzing glycolysis are subjected to a binding test, for example according to the exemplary embodiments, and those substances which bind are selected. It 12
  • Binding partners can be found in healthy or diseased tissue. In the latter use, inhibitors of the binding processes normally taking place in healthy or diseased tissue can be found.
  • the invention also relates to healing methods, for example traditionally by suitable administration of pharmaceutical preparations but also gene therapy, by means of which one or more substances according to claims 6 to 9 are introduced into a target cell or generated in the target cell.
  • Ax. 1 DNA sequence of rats Ml-PK and M2-PK,
  • Ax. 2 DNA sequence from human v-raf,
  • the two-hybrid vectors pPC86 and pPC97 were provided by D. Nathans (see also: PM Chevrey & D. Nathans, Proc. Natl. Acad. Sei. USA, 89, pages 5789-5793, 1992).
  • Full-length A-raf, B-raf and c-raf-1 cDNA were subcloned into pPC97 in fusion with the Gal4 DNA binding domain.
  • the PC12 cDNA library was subcloned into pPC97 in fusion with the Gal4 activation domain.
  • the cloning of the A-raf deletion constructs is described in C. Hagemann et al. , FEBS Lett., 403, pages 200-202, 1997.
  • A-raf (554-606) was under 14
  • M2-PK cDNA was isolated as an EcoRI fragment from pPC86 (clone 71), ligated to pGEX-2T and pcDNA3 digested with EcoRI.
  • the untranslated region was removed by introducing a BamHI site (Mutagenesis Kit Stratagene) and removing the BamHI fragment from this construct.
  • the 3 'end of the coding region of the M2-PK cDNA was isolated by PCR from the PC12 library using the primers 5'-GCC CGG TAC CGC CCA AGG GCT C-3' (sense) and 5'-CCA GGG CTG GGA ATT CTC TGG-3 '(antisense).
  • Full length M2-PK was generated by subcloning the PCR product Kpnl / EcoRI in pGEX-M2-PK ⁇ Bam and pcDNA-M2-PK ⁇ Bam, respectively, resulting in plasmids pGEX-M2-PK and pcDNA3-M2-PK.
  • pPC97-A-raf AA602 / 603RP, pcDNA3-M2-PK K366M and pGEX-2T-M2-PK K366M were made using the Stratagene Mutagenesis Kit.
  • Yeast cultures were grown at 30 ° C under standard conditions in liquid or solid media based on either YPD or minimal SD media.
  • the HF7c yeast line was transformed sequentially with first the "bait" plasmid and then the cDNA library. Transformants were drawn on SD medium in the absence of the amino acids leucine, tryptophan and histidine. After 4 days, the growing clones were tested for activation of the lacZ reporter gene in a ⁇ -Gal filter assay. Positive clones were further examined by retransforming the isolated library plasmid together with various "bait" plasmids in HF7c. Clones which only showed a ⁇ -Gal positive phenotype in the presence of raf were assessed as positive and were further investigated by sequencing and colony hybridization. For direct interaction tests, the yeast line HF7c was co-transformed with Araf deletion constructs and pPC86-M2-PK ⁇ Sal.
  • the NIH 3T3 cells were supplemented under standard conditions (37 ° C., 5% CO 2 ) in DMEM (Life Technologies, Inc.), with 10% heat-inactivated fetal bovine 16
  • Control cells NIH 3T3 and A-raf transformed NIH 3T3 cells were cultivated up to a cell density of 3.5 x 10 5 cells / dish.
  • 26 x 10 6 cells in 3 ml lysis buffer with a low salt concentration (10mM Tris, ImM NaF, ImM EDTA-Na 2 and ImM Mercaptoethanol, pH 7.4) were 17
  • Extracts of NIH 3T3 control cells and NIH 3T36A cells expressing stably gag-A-raf were prepared as described in W. Zwerschke, Proc. Natl. Acad. Be. USA, 96, pages 1291-1296, 1999.
  • the individual fractions of the focusing experiments or gel filtration experiments were diluted 1:10 with sample buffer. After separation on a 10% SDS polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane using electroblotting.
  • the following antibodies were used to detect the various antigens: M2-PK: onoclonal antibodies DF4 (ScheBo Tech, Giessen, Germany); A-raf: polyclonal antibodies which were raised against 12 C-terminal residues of A-raf representing synthetic peptides (see C. Hagemann et al., FEBS Lett. 403, pages 200-202, 1997); gag-A-raf: goat serum generated against p30 gag (see M. Huleihel, Mol. Cell.
  • Fructose 1, 6-bisphosphate, pyruvate and phosphoenol pyruvate concentrations were determined in perchloric acid extracts of the cells, as described in S. Mazurek et al. , J. Biol. Chem. 272, pages 4941-4952, 1997. According to this reference, the concentrations of glucose, glutamine, glutamate and lactate in the cell supernatants were also determined in order to determine the flow rates.
  • M2-PK the above interactions are known and thus serve as evidence of the functionality of the system used (expression and folding).
  • M2-PK an enzyme that catalyzes glycolysis
  • the isolated clone represents a partial sequence 10 which contains a part of the untranslated region in front of the N-terminus and which lacks 29 amino acids at the C. terminus (M2-PK (1-501)).
  • FIG. 2 shows in detail the determination of the M2-PK activity in the presence of 2 mM and 0.2 mM PEP in the different fractions (upper part) and the determination of M2-PK, p-serine and p-threonine by direct immunoblotting after SDS gel electrophoresis of the different fractions.
  • Transformation of NIH 3T3 cells by stable expression of a fusion between the A-raf kinase domain with viral gag protein led to a selective increase in the tetrameric form (amount of tetramers in control cells: 69%; after A-raf transformation: 76%), for which reference is made to FIG. 2.
  • FIG. 3 shows in the upper part the determination of the activities of pyruvate kinase (circles) and phosphoglyceromutase in the different fractions, pI values for some fractions are given as a reference.
  • the lower part shows the detection of A-raf, gag-A-raf, M2-PK and MEK 1 by direct immunoblotting after SDS gel electrophoresis of the different fractions. It is first seen that the tetrameric form of M2-PK co-focuses with enolase, glyceraldehyde 3-phosphate dehydrogenase (not shown) and part of phosphoglyceromutase type B.
  • A-raf and c-raf focus in the glycolytic enzyme complex in fractions 35-37 and gag-A-raf in fractions 40-44.
  • MEK 1 and MEK 2 the substrates of the raf kinases, both focused in the same fractions 34-45 of the glycolytic enzyme complex.
  • the majority of ERK 1 and ERK 2 the substrates of MEK, focused outside of the glycolytic enzyme complex in fractions 32-35 (not shown). 22
  • M2-PK, A-raf, gag-A-raf, c-raf, MEK and ERK were detected with the antibodies specified under Methods.
  • the immunologically detectable amount of M2-PK protein and the amounts of phosphoserine and phosphothronone on the M2-PK protein were determined densitrometrically using the Scion Image Program (Beta 3B version, Scion Corporation).
  • Scion Image Program Beta 3B version, Scion Corporation.
  • the total content of M2-PK protein increased 1.3-fold in A-raf transformed cells, while the phosphoserine content of M2-PK protein increased 2.6-fold and that of phosphothreonine 1.2-fold.
  • the ratio between phosphoserine and M2-PK protein increased from 0.7 in control cells to 1.3 cells transformed in Araf. The ratio for phosphothreonine, however, was unchanged.
  • the glycolytic complex was extracted by extracting the cells with high phosphate concentrations and the ratio between the tetrameric and the dimeric form of M2-PK was determined directly by means of gel permeation.
  • gel permeation also showed a shift from the dimeric form to the tetrameric form in A-raf transformed cells 23
  • NIH 3T3 cells were transfected with gag-A-raf and M2-PK cDNA and the focus formation after 10 days of cultivation was examined (Table 2). While A-raf transfection only led to the formation of 2 foci per 1 ⁇ g of transfected DNA, cotransfection resulted in an increase in the number to 6 foci per 1 ⁇ g DNA, which is a cooperative effect of A-raf and M2-PK in the Cell transformation speaks.
  • M2-PK K366M did not show this, but actually antagonized the morphological transformation by A-raf.
  • the cells showed a rather flat, less refractile phenotype, as can be seen in FIG. 4 (empty vector: pcDNA3).
  • Protein expression was controlled by Western blots in all experiments (data not shown).
  • x-.DF 11 is the delogarithmic form of the arithmetic mean and the standard deviation of the previously logarithmically transformed data.
  • a one-way analysis of the covariance with the cell density as a covariable was carried out for the statistical analysis. In the case of glutamate, positive values mean medium production and negative values consumption.
  • NIH 3T3 cells were transfected with A-raf, M2-PK and M2-PK K366M in the specified combinations. Focus formation was determined after 10 days of growth (left column).
  • NIH 6A leuk cells expressing Stbail gag-A-raf were transfected with M2-PK and M2-PK K366M. Colonies of surviving cells were counted after 10 days of G418 selection (right column).
  • Vector empty vector pcDNA3.
  • FIG. 5 shows a first GTC for an attack by the hammerhead shown on the corresponding mRNA.
  • a different target sequence for the hammerhead attack is also possible, as are other positions of the same target sequences.
  • the hammerhead can easily be adapted to this by a specialist. It is only essential that the translation of an active kinase domain is reduced or suppressed.

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Abstract

L'invention concerne un acide nucléique codant pour au moins une séquence partielle d'une protéine kinase de la cascade de signalisation mitogène, la séquence partielle étant codante pour un site de liaison d'une enzyme catalysant la glycolyse, ou bien une mutation silencieuse d'un tel acide nucléique ou des acides nucléiques d'hybridation par un tel acide nucléique ou sa mutation silencieuse.
EP01951397A 2000-06-14 2001-06-14 Acide nucleique codant pour un site de liaison d'une proteine kinase de la cascade de signalisation mitogene de l'enzyme catalysant la glycolyse Withdrawn EP1290189A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10029131 2000-06-14
DE10029131A DE10029131A1 (de) 2000-06-14 2000-06-14 Nukleinsäure codierend eine Bindungsstelle einer Protein Kinase der mitogenen Signalisierungskaskade für ein die Glykolyse katalysierendes Enzym
PCT/DE2001/002246 WO2001096535A2 (fr) 2000-06-14 2001-06-14 Acide nucleique codant pour un site de liaison d'une proteine kinase de la cascade de signalisation mitogene de l'enzyme catalysant la glycolyse

Publications (1)

Publication Number Publication Date
EP1290189A2 true EP1290189A2 (fr) 2003-03-12

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EP01951397A Withdrawn EP1290189A2 (fr) 2000-06-14 2001-06-14 Acide nucleique codant pour un site de liaison d'une proteine kinase de la cascade de signalisation mitogene de l'enzyme catalysant la glycolyse

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US (1) US20040115631A1 (fr)
EP (1) EP1290189A2 (fr)
AU (1) AU7234301A (fr)
DE (1) DE10029131A1 (fr)
WO (1) WO2001096535A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2002026246A2 (fr) * 2000-09-29 2002-04-04 Gsf-Forschungszentrum Für Umwelt Und Gesundheit, Gmbh Compositions pharmaceutiques contenant des polynucleotides codant une proteine raf
CA2478924A1 (fr) * 2002-03-14 2003-09-18 Qlt Inc. Proteine kinase araf1 associee au cancer et ses utilisations
WO2004016646A2 (fr) * 2002-08-12 2004-02-26 Amynon Bio Tech Gmbh Modulateurs peptidiques d'un sous-type de pyruvate kinase m2 (m2-pk) specifique de tumeurs
US10213493B2 (en) * 2012-03-15 2019-02-26 Georgia State University Research Foundation, Inc. Protein to promote blood vessel growth and uses thereof
EP2877214B1 (fr) * 2012-07-26 2019-04-24 Joslin Diabetes Center, Inc. Prédiction de complications diabétiques

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Publication number Priority date Publication date Assignee Title
US5656612A (en) * 1994-05-31 1997-08-12 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of raf gene expression

Non-Patent Citations (1)

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Title
See references of WO0196535A3 *

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US20040115631A1 (en) 2004-06-17
AU7234301A (en) 2001-12-24
WO2001096535A3 (fr) 2002-07-04
WO2001096535A2 (fr) 2001-12-20
DE10029131A1 (de) 2002-01-03

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