WO1994016069A2 - Proteines gap associees aux ras - Google Patents

Proteines gap associees aux ras Download PDF

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WO1994016069A2
WO1994016069A2 PCT/US1994/000198 US9400198W WO9416069A2 WO 1994016069 A2 WO1994016069 A2 WO 1994016069A2 US 9400198 W US9400198 W US 9400198W WO 9416069 A2 WO9416069 A2 WO 9416069A2
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WO1994016069A3 (fr
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Masato Nakafuku
Yoshito Kaziro
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Schering Corporation
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Priority to JP6516210A priority Critical patent/JPH08507204A/ja
Priority to EP94920844A priority patent/EP0679185A1/fr
Priority to AU60838/94A priority patent/AU6083894A/en
Publication of WO1994016069A2 publication Critical patent/WO1994016069A2/fr
Publication of WO1994016069A3 publication Critical patent/WO1994016069A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C07K14/4706—Guanosine triphosphatase activating protein, GAP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the Ras family includes three functional genes designated H-ras, K-ras, and N-ras, which encode highly similar proteins. See Barbacid (1987) Ann. Rev. Biochem. 56:779-827. Ras genes from different human tumors were characterized and found to have undergone point mutations leading to constitutive activation, especially codons 12, 13, and 61. These mutant versions are especially potent inducers of tumorigenic or oncogenic transformation. Mutations in the Ras genes may be responsible for as many as 90% of pancreatic adenocarcinomas.
  • the Ras proteins are guanosine triphosphate (GTP)- binding proteins, and serve as a molecular switch in signal transduction controlling the proliferation and differentiation of cells.
  • GTP guanosine triphosphate
  • the linkage of Ras with the nucleoside is non-covalent and designated Ras»GXP to distinguish from a "-" which would indicate a covalent bond.
  • Ras «GDP form is an inactive form which does not stimulate the downstream effector, e.g., target protein, to result in functional signal transduction.
  • Ras GTP conformation
  • the Ras GTP conformation is active, e.g., stimulates the effector to transmit an activation signal.
  • - Interconversion between the two conformations is enzymatically effected. Conversion from the protein «GDP conformation to the protein «GTP conformation causes activation, and is described as an activation step.
  • Somatic mutations which constitutively activate Ras e.g., oncogenic Ras, may contribute to tumorigenesis in up to 30% of human tumors. See, e.g., Bos (1989) Cancer Res. 49:4682- 4689; and Rodenhuis (1992) Seminars in Cancer Biol. 3:241-247. Most anti-cancer drugs currently available are not directed toward specific oncogenes, but rather inhibit even normal cellular processes.
  • the present invention provides methods for blocking Ras-induced effects on eukaryotic cells.
  • Different Ras mutations have been demonstrated to cause oncogenic transformation in eukaryotic cells by providing constitutive activation signaling to the cells.
  • Various fragments of GTPase Activating (GAP) proteins have been identified which specifically interact with defined Ras mutants to block signal transduction. These fragments likely function through a mechanism of interacting with the Ras»GTP activated conformation to block the natural interaction of the effector protein. These fragments thus block the constitutive signal transduction which results in Ras induced constitutive effects.
  • GAP GTPase Activating
  • the present invention provides methods of blocking a Ras-induced effect on a cell, comprising a step of introducing a GTPase Activating (GAP) protein to the cell.
  • GAP GTPase Activating
  • the Ras will be an oncogenic Ras or one which substantially lacks GTPase activity.
  • the Ras-induced effect will typically be induction of cell proliferation or transformation.
  • the cell will often be eukaryotic cell, e.g., a mammalian cell, including a human cell.
  • the step of introducing is by expression of a nucleic acid encoding the GAP protein.
  • the GAP protein will bind to the Ras protein with a Kd of less than 200 nM.
  • the GAP protein is selected from: (a) a fragment of a mammalian GAP protein; (b) a fragment of a mammalian NF1-GRD protein; and (c) a homologue or mimetic of (a) or (b) .
  • the GAP protein is selected from: (a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and (b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position 1063 through 1651 of NF1 or the corresponding region of other GAP proteins. Many of these substitutions will be a conservative substitution.
  • the GAP protein will interact with Ras and block interaction of an effector molecule which binds to Ras at a position corresponding to a position from 32 to 40 or from 59 to 65.
  • the GAP protein does not block signal transduction of non-oncogenic Ras. Greater specificity of action can be achieved by identifying the responsible oncogenic Ras and selecting a GAP protein which specifically blocks the identified oncogenic Ras.
  • the invention also provides methods of treating an oncogenic Ras transformed cell comprising the step of introducing to said cell a GAP protein capable of suppressing the transformation of said cell.
  • the oncogenic Ras transformed cell will be a mammalian cell, including a human cell.
  • the GAP protein does not block signal transduction of non-oncogenic Ras.
  • the method can be improved by adding steps of identifying the responsible oncogenic Ras and selecting a GAP protein which blocks transformation by the identified Ras.
  • the GAP protein does not block signal transduction of non-oncogenic Ras, e.g., exhibiting specificity.
  • the invention provides methods of identifying appropriate GAP proteins useful for treating a mutated Ras-induced condition of a eukaryote cell comprising: (a) identifying the mutated Ras which induces the condition; and (b) screening various GAP variants for specific variants which are capable of blocking the condition.
  • the eukaryote cell is a mammalian cell, including a human cell.
  • additional screening is performed to determine which GAP variants have minimal effect on non-mutated Ras effects.
  • the invention further provides GAP proteins capable of blocking transformation of a cell, where said transformation is due to an oncogenic Ras.
  • the GAP protein is selected from: (a) a fragment of a mammalian GAP protein; (b) a fragment of a mammalian NFl- GRD protein; and (c) a homologue or mimetic of (a) or (b) .
  • the GAP protein is selected from: (a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and (b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position from 1063 through 1651 of NFl or the corresponding region of other GAP proteins. Often the substitution will be a conservative substitution.
  • the protein interacts with Ras and blocks interaction of an effector molecule which binds to Ras at a position from 32 to 40 or from 59 to 65. Often the cell i ⁇ a eukaryotic cell, e.g., a mammalian cell, including a human cell.
  • the oncogenic Ras substantially lacks GTPase activity.
  • the protein binds to oncogenic Ras with a Kd of less than 200 nM.
  • the protein may interfere with interaction of Ras»GTP with an effector compound.
  • the invention provides an isolated nucleic acid encoding a protein normally expressed as a protein as described.
  • Figure 1 shows stimulation of GTPase activity of c-Ha- Ras Gl 12 and c-Ha-Ras Va112 proteins by yeast cell extracts containing wild-type and mutant NFl-GRDs.
  • Ras gene family members are ubiquitous among eukaryotic cells. See, e.g., Barbacid (1987) Ann. Rev. Biochem. 56:779-827.
  • the genes were initially identified and studied as the viral oncogenes of several acute transforming retroviruses.
  • the relationship to human cancer was quickly established upon recognition that the retroviral oncogenes were derived from a group of mammalian cellular proto-oncogenes, e.g., endogenous genes which become oncogenic upon mutation.
  • Ras proteins are GTP-binding proteins involved in transduction of signals in response to extracellular stimuli.
  • the family of Ras proteins can be defined by a combination of functional and structural criteria. See, e.g., Bollag et al. (1991) Ann. Rev. Cell Biol. 7:601-632.
  • Ras-induced effects are the functional consequences of Ras activation.
  • the Ras-induced effects will be cell transformation, but may also include differentiation or proliferation effects which fail to satisfy the full criteria for transformation.
  • the yeast Saccharomvces cerevisiae possesses two members of the Ras family (Rasl and Ras2) which play an important role in cell growth through the regulation of adenylate cyclase. See, e.g., Broach et al. (1990) Adv.
  • Ras Ras family have also been studied in Xenopus laevis; Drosophila melanogaster, Caenorhabditis elegans; and Dictyostelium discoideum. See Bollag et al.
  • Ras proteins have been shown to be GTP-binding proteins. They can be either in GDP-bound conformation or a GTP-bound conformation. The GTP-bound conformation is the active and interacts with an as yet unidentified effector molecule.
  • Ras proteins become activated upon stimulation, transduce the signal to an as yet unidentified effector molecule, and subsequently become inactivated. Mutated, e.g., oncogenic, Ras proteins have lost their ability to become inactivated and thus constitutively send a stimulation signal.
  • Ras is active in its GTP-bound form. -The active Ras»GTP complex, which is a non-covalent association, is converted to an inactive Ras-guanosine diphosphate
  • Ras GTPase Activating protein
  • GAP GTPase Activating protein
  • oncogenic Ras lacks the intrinsic GTPase activity and GAP proteins have little, if any, effect on inactivating oncogenic Ras.
  • This substantial lack of GTPase activity in oncogenic Ras will typically be at least 20% less than the normal, more typically at least 35% less, usually at least 50%, more usually at least 60% less, preferably at least 70% less, and more preferably at least 80% or more less than normal Ras.
  • GAP proteins family, mammalian, NFl
  • GTPase activities are required to inactivate the Ras»GTP form of the protein in the cycling reaction.
  • a family of proteins stimulating endogenous GTPase activities of Ras proteins have been described which share structural and functional similarities. See Bollag et al. (1991) Ann. Rev. Cell Biol. 7:601-632.
  • Particularly relevant members of the GAP family include yeast and mammalian proteins, including the human neurofibromatosis type 1 (NFl) protein.
  • NFl human neurofibromatosis type 1
  • GAP protein refers to a protein which shares structural or functional properties with this family of proteins.
  • the protein will be a fragment shorter than the natural mammalian proteins so far described, normally less than about 600 amino acids, more normally less than about 550 amino acids, ordinarily less than about 500 amino acids, more ordinarily less than about 460 amino acids, usually less than about 420 amino acids, more usually less than about 380 amino acids, typically less than about 350 amino acids, more typically less than about 325 amino acids, preferably less than about 310 amino acids, more preferably less than about 300 amino acids, and in other embodiments, even fewer amino acids, down to 200 or fewer amino acids.
  • NFl was first identified as the gene responsible for the pathogenesis of the human genetic disorder, neurofibromatosis type 1.
  • cDNA cloning revealed that the NFl gene encodes a protein of 2818 amino acids.
  • This putative protein product has a domain showing a significant sequence homology with members of the Ras GTPase-activating protein (GAP) family.
  • GAP Ras GTPase-activating protein
  • NFl GAP Related Domain NFl GAP Related Domain
  • Iral and Ira2 Two yeast Saccharomvces cerevisiae proteins, Iral and Ira2, show particularly high sequence homology to the NFl.
  • members of the GAP family including the GAP-related domain of the NFl gene product (NFl-GRD; sometimes referred to as NFl fragment) , can stimulate guanosine triphosphatase (GTPase) activity of Ras proteins, i.e., converting Ras «GTP to Ras»GDP, and thereby negatively regulate the activity of Ras.
  • GTPase guanosine triphosphatase
  • Ras proteins Two proteins which regulate the activity of Ras proteins are the GTPase activating protein (GAP) and the protein encoded by NFl, the gene responsible for neurofibomatosis. type I disease. See Gutmann et al. (1992) Ann. Neurol. 31:555-561. III. Interaction of Ras and GAP proteins
  • the GAP proteins have been identified as one of the means by which activated Ras proteins are converted into the inactive form. Thus, the physical interaction of the GAP and Ras proteins are important in the understanding of the functional relationship between the entities.
  • the GAP protein effect on endogenous GTPase activity of RAS has been localized to a fragment of the natural GAP protein, e.g., wild-type sequences.
  • the catalytic domain has been localized to the carboxy terminal segment of the mammalian GAP proteins.
  • the active portion has been localized to a fragments of less than about 600 amino acids, corresponding to the NFl amino acids 1063- 1651.
  • the sites of GAP interaction with Ras have been proposed to be positions 32-40 and 59-63 of mammalian Ras.
  • the yeast S. cerevisiae possesses two NFl homologues, Iral and Ira2.
  • the human NFl is structurally closer to yeast Ira than human GAP and thus would be expected to interact well with the yeast Ras counterpart proteins. This structural similarity is reflected in a functional relationship, as NFl-GRD expressed in yeast cells can complement ira-deficient yeast. In ira ⁇ cells, the conversion of Ras*GTP to Ras*GDP is defective, and the cells show a phenotype which is very similar to that of activated Ras mutants, i.e., heat shock-sensitivity.
  • the GAP-Related Domain of the NF-1 gene product is a fragment from the NF-1 which can suppress the heat- sensitive phenotype of ira ⁇ , but not of RAS2 Va H9 or RAS 2Leu68. This is consistent with the fact that NFl-GRD stimulates GTPase activity of normal but not mutant Ras proteins. Thus, the natural GAP will have blocking effects of Ras functions of normal cells. IV. Downstream signal transduction
  • variant GAP segments may show great specificity in blocking Ras- induced effects.
  • the binding affinity of the GAP analogues which block Ras-induced effects are higher than normal GAP binding.
  • the GAP protein which is intended here to also encompass the concept of protein analogues and mimetics, will preferably be a relatively small polypeptide or analogue, including modified proteins and mimetics.
  • Mimetics include compounds possessing similar molecular shapes sufficient to confer the desired biological property.
  • Various amino acid substitutions may be designed, tested, or screened for activity in blocking Ras-induced functions. These may be effective in blocking effects of many different Ras mutants, or specific Ras variants.
  • the methodology described herein may be useful to define GAP proteins which exhibit high specificity for only interacting with oncogenic, e.g., mutant Ras, and having virtually no effect on natural Ras function.
  • the GAP proteins provided herein will be highly specific in affecting only oncogenic functions and will be innocuous in cells possessing normal Ras. Although the positions of GAP believed to be most important in the interaction with Ras are in the regions of 701-1047 of GAP, the NFl regions considered most likely to be useful herein will be within the region of 1063-1651 or the corresponding region of other GAP proteins, including 1175-1534, and more specifically in the regions of 1400- 1500. Mutations within this region are likely to interact with the Ras in the desired way, particularly in the region of 1421-1461 of NFl or the corresponding region of other GAP proteins.
  • the useful GAP proteins have high binding affinity for Ras or Ras-like proteins or GAP binding segments thereof.
  • the GAP protein will exhibit a Kd for Ras, or its oncogenic variant, of less than about 300 nM, more typically less than about 250 nM, usually less than about 200 nM, more usually less than about 150 nM, preferably less than about 100 nM, and more preferably even higher binding affinity.
  • a higher binding affinity will allow effective competitive effect on the effector binding at low concentrations of GAP protein.
  • GAP protein e.g., fragments, analogues, and mimetics
  • the GAP protein will be produced, e.g., by recombinant means, as are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, CSH, N.Y., and Ausubel (1987 and periodic supplements) Current Protocols in Molecular Biolo ⁇ v
  • the GAP protein can be purified and then administered to a patient.
  • These reagents can be combined for therapeutic use with additional active ingredient ⁇ , e.g., in conventional pharmaceutically acceptable carrier ⁇ or diluent ⁇ , along with phy ⁇ iologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • Drug screening using Ras or fragments thereof can be performed to identify compounds having binding affinity. Subsequent biological assays can then be utilized to determine if the compound has intrinsic activity and is therefore a blocker or antagonist in that it blocks the effects of oncogenic Ras.
  • Additional compounds may be screened or designed using the reagents described, or by molecular modeling and structural studies including, e.g., X-ray crystallography, multidimensional NMR, and other techniques. See, e.g., Blundell et al. (1976) Protein Crystallography Academic Press, New York.
  • reagents neces ⁇ ary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosage ⁇ used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective dose ⁇ for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed.
  • compositions for admini ⁇ tration are discus ⁇ ed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and other ⁇ .
  • Pharmaceutically acceptable carrier ⁇ will include water, saline, buffers, and other compound ⁇ de ⁇ cribed, e.g., in the Merck Index. Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 100 mM concentrations, typically less than about 10 mM concentrations, usually less than about 100 ⁇ M, preferably less than about 10 ⁇ M, and most preferably less than about
  • the GAP protein may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier protein ⁇ such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneou ⁇ , intramuscular, intravenous, and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed. , Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Perm.; each of which i ⁇ hereby incorporated herein by reference.
  • the therapy of this invention may be combined with or used in as ⁇ ociation with other chemotherapeutic or chemopreventive agent ⁇ .
  • the present invention allows for simple matching of a therapeutic agent to various oncogenic Ras variants.
  • This can provide highly ⁇ elective treatment of defined oncogenic condition ⁇ with a GAP having highly ⁇ elected ⁇ afety and efficacy combination ⁇ , virtually tailored to the relatively small number of oncogenic Ras mutations which cause defined proliferative conditions.
  • common variants of oncogenic Ras can be used to screen for GAP fragments which are effective in blocking the oncogenic effects. See, e.g. Kumar et al. (1990) Cancer Res. 52:6877-6884. Either the variants or equivalents thereof can be transformed into a cell, e.g., a yeast cell, and GAP mutants te ⁇ ted for their specific effect on the Ras variants. Once appropriate GAP proteins are identified for each of the common oncogenic Ras mutants, therapeutic reagents can be selected based upon the diagnosed mutant oncogenic Ras re ⁇ pon ⁇ ible for a given abnormality. Diagnosis of the responsible Ras mutation can be performed as described above.
  • compositions, analogues, mimetics Isolated GAP encoding DNA ⁇ can be readily modified by nucleotide substitution ⁇ , nucleotide deletion ⁇ , nucleotide in ⁇ ertion ⁇ , and inver ⁇ ion ⁇ of nucleotide stretches. These modifications result in novel DNA sequences which encode these modified GAP proteins, their derivative ⁇ , or protein ⁇ having the de ⁇ ired anti- oncogenic activity.
  • the ⁇ e modified sequences can be used to produce mutant GAP proteins or to enhance the expression of GAP. Enhanced expression may involve gene amplification, increased transcription, increased.... translation, and other mechanisms.
  • Such mutant Ras or GAP derivatives include predetermined or site-specific mutations of the respective protein or its fragments.
  • a mutant GAP is a polypeptide otherwise falling within the homology defined by structure and function, but having an amino acid sequence which differ ⁇ from the corresponding segment of GAP as found in nature, whether by way of an amino acid deletion, sub ⁇ titution, or in ⁇ ertion. Similar protein ⁇ and nucleic acid ⁇ should be available from other warm blooded animals, e.g., mammals and birds. These description ⁇ are generally meant to encompa ⁇ ⁇ pecies and allelic variants of the GAP proteins, not limited to the specific embodiments discu ⁇ sed.
  • mutants need not be site specific.
  • GAP protein or Ras protein mutagenesis can be conducted by- making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include but are not limited to amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expres ⁇ ed GAP mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesi ⁇ . See also Sambrook et al. (1989) and Ausubel et al. (1987 and Supplements) .
  • the mutations in the DNA normally should not place coding sequence ⁇ out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such a ⁇ loop ⁇ or hairpin ⁇ .
  • the pre ⁇ ent invention al ⁇ o provide ⁇ recombinant protein ⁇ , e.g., heterologou ⁇ fu ⁇ ion protein ⁇ u ⁇ ing ⁇ egment ⁇ from these proteins.
  • a heterologous fusion protein is a fusion of protein ⁇ or segments which are naturally not normally fused in the ⁇ ame manner. Thu ⁇ , the fu ⁇ ion product of an immunoglobulin with a GAP polypeptide i ⁇ a continuous protein molecule having sequence ⁇ fused in a typical peptide linkage, e.g., typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar concept applies to heterologou ⁇ nucleic acid ⁇ equence ⁇ .
  • new con ⁇ truct ⁇ may be made from combining ⁇ imilar functional domain ⁇ from other proteins.
  • Ras-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham et al. (1989) Science 243:1330-1336; and O'Dowd et al. (1988) J. Biol. Chem. 263:15985-15992, each of which is incorporated herein by reference.
  • new chimeric polypeptide ⁇ exhibiting new combination ⁇ of specificities will result from the functional linkage of Ras-binding specificities.
  • the Ras-binding segment ⁇ from other related protein ⁇ may be added or combined with other binding segments from other proteins.
  • the resulting protein will often have hybrid function and properties.
  • a double ⁇ tranded fragment will often be obtained either by ⁇ ynthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand u ⁇ ing DNA polymera ⁇ e with an appropriate primer sequence.
  • Variou ⁇ GAP variant ⁇ may have ⁇ lightly different function ⁇ or biological activitie ⁇ , even though they share significant structural similaritie ⁇ . Dissection of structural elements which effect the various physiological functions or biological activities provided by the GAP proteins is possible using standard techniques of modern molecular biology, particularly in comparing variants within the related family of GAP proteins. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham et al. (1989) Science 243:1339-1336; and approaches used in O'Dowd et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter et al. (1990) EMBO J.
  • Ras binding segments can be sub ⁇ tituted between protein ⁇ to determine what ⁇ tructural feature ⁇ are important in both Ra ⁇ binding affinity and ⁇ pecificity for the natural or oncogenic Ras.
  • An array of different Ras variants e.g., allelic, will be used to screen for GAP proteins exhibiting desired properties of interaction with them, e.g., high binding affinity, blocking of effector function by conformational or competitive inhibition, or even forms which can induce GTPase action of the oncogenic Ras.
  • the specific segments of interaction of GAP with Ras may be identified by mutagenesi ⁇ or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will al ⁇ o be applicable. Identification of the ⁇ imilarities and differences between Ras oncogenic variants will lead to new diagnostic and therapeutic reagents or treatment ⁇ .
  • Structural ⁇ tudie ⁇ of the Ra ⁇ variant ⁇ will lead to design of new GAP proteins, particularly analogues exhibiting desired effect blocking properties. This can be combined with ⁇ creening methods to isolate new GAP proteins exhibiting desired spectra of activitie ⁇ .
  • Both the naturally occurring and the recombinant form ⁇ of Ra ⁇ are particularly u ⁇ eful in kit ⁇ and assay methods which are capable of ⁇ creening compound ⁇ for binding activity to them.
  • Several method ⁇ of automating a ⁇ ays have been developed in recent years so as to permit screening of tens of thousand ⁇ of compound ⁇ per year. See, e.g., Fodor et al.
  • a nucleic acid which encodes a Ras and GAP are readily available, or can be obtained by chemical ⁇ ynthe ⁇ i ⁇ , ⁇ creening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g., Marchuk et al. (1991) Genomics 11:931-940; and nucleic acid and protein data bases, e.g., Protein Identification Resource (PIR) , Georgetown University, Washington, D.C., SwissProt and other ⁇ , see intelliGenetics, Menlo Park, CA, or the Univ. Wi ⁇ con ⁇ in Biotechnology Center, Madison, Wiscon ⁇ in.
  • PIR Protein Identification Resource
  • Thi ⁇ DNA can be expre ⁇ ed in a wide variety of ho ⁇ t cells for the synthe ⁇ i ⁇ of a Ra ⁇ , GAP. or fragment ⁇ thereof which can in turn, for example, be u ⁇ ed to generate polyclonal or monoclonal antibodies; for construction and expression of modified Ra ⁇ or GAP molecule ⁇ ; and for structure/function ⁇ tudie ⁇ .
  • Each GAP can be expre ⁇ ed in host cells that are transformed or transfected with appropriate expre ⁇ ion vector ⁇ . These molecules can be sub ⁇ tantially free of protein or cellular contaminant ⁇ , other than tho ⁇ e derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent.
  • the GAP, or portions thereof, may be expres ⁇ ed a ⁇ fu ⁇ ion ⁇ with other protein ⁇ .
  • Expre ⁇ ion vector ⁇ are typically ⁇ elf-replicating DNA or RNA con ⁇ truct ⁇ containing the de ⁇ ired Ra ⁇ or GAP gene or it ⁇ fragment ⁇ , u ⁇ ually operably linked to ⁇ uitable genetic control element ⁇ that are recognized in a ⁇ uitable ho ⁇ t cell.
  • the ⁇ e control elements are capable of effecting expres ⁇ ion within a suitable host. The specific type of control elements necessary to effect expres ⁇ ion will depend upon the eventual host cell used.
  • the genetic control elements can include a prokaryotic promoter sy ⁇ tem or a eukaryotic promoter expre ⁇ ion control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encode ⁇ a suitable ribosome binding site, and sequence ⁇ that terminate transcription and translation.
  • Expres ⁇ ion vector ⁇ also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • the vectors of this invention contain DNA which encodes a u ⁇ eful GAP-like peptide, or a fragment thereof encoding, e.g., an active polypeptide.
  • the DNA can be under the control of a viral promoter and can encode a ⁇ election marker.
  • This invention further contemplates u ⁇ e of such expres ⁇ ion vector ⁇ which are capable of expre ⁇ ing eukaryotic cDNA coding for a GAP in a prokaryotic or eukaryotic ho ⁇ t, where the vector i ⁇ compatible with the ho ⁇ t and where the eukaryotic cDNA coding for the GAP i ⁇ in ⁇ erted into the vector such that growth of the host containing the vector expres ⁇ e ⁇ the cDNA in que ⁇ tion.
  • expre ⁇ sion vectors are designed for stable replication in their ho ⁇ t cell ⁇ or for amplification to greatly increase the total number of copies of the desirable gene per cell.
  • an expression vector replicate in a host cell e.g., it is po ⁇ ible to effect tran ⁇ ient expres ⁇ ion of the GAP in variou ⁇ ho ⁇ t ⁇ u ⁇ ing vectors that do not contain a replication origin that is recognized by the host cell. It is al ⁇ o po ⁇ ible to u ⁇ e vector ⁇ that cau ⁇ e integration of GAP into the ho ⁇ t DNA by recombination.
  • Vectors as used herein, comprise pla ⁇ mids, viruse ⁇ , bacteriophage, integratable DNA fragment ⁇ , and other vehicle ⁇ which enable the integration of DNA fragment ⁇ into the genome of the ho ⁇ t.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual. Elsevier, N.Y. , and Rodriguez et al. (ed ⁇ ) Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Buttersworth, Boston, 1988, which are incorporated herein by reference.
  • DNA ⁇ equence ⁇ are operably linked when they are functionally related to each other.
  • DNA for a pre ⁇ equence or ⁇ ecretory leader is operably linked to a polypeptide if it is expres ⁇ ed a ⁇ a preprotein or participate ⁇ in directing the polypeptide to the cell membrane or in ⁇ ecretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it control ⁇ the tran ⁇ cription of the polypeptide;
  • a ribo ⁇ ome binding site is operably linked to a coding ⁇ equence if it i ⁇ po ⁇ itioned to permit tran ⁇ lation.
  • operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but ⁇ till bind to operator sequences that in turn control expression.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis.
  • Lower eukaryotes include yea ⁇ ts, e.g., S. cerevisiae and Pichia. and specie ⁇ of the genus pictyostelium.
  • Higher eukaryotes include establi ⁇ hed tis ⁇ ue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector system ⁇ include a wide variety of vectors for many different specie ⁇ . As used herein, E. coli and it ⁇ vectors will be used generically to include equivalent vector ⁇ used in other prokaryotes.
  • a representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the GAP protein include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN- ⁇ eries); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540) .
  • lac promoter pUC-series
  • trp promoter pBR322-trp
  • Ipp promoter the pIN- ⁇ eries
  • lambda-pP or pR promoters pOTS
  • hybrid promoters such as ptac (pDR540) .
  • Lower eukaryotes e.g., yeasts and Dictvostelium. may be transformed with GAP sequence containing vector ⁇ .
  • the most common lower eukaryotic host i ⁇ the baker' ⁇ yea ⁇ t, Saccharomvce ⁇ cerevi ⁇ iae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unles ⁇ of the integrating type) , a selection gene, a promoter, DNA encoding the Ra ⁇ or GAP protein or it ⁇ fragment ⁇ , and ⁇ equences for translation termination, polyadenylation, and transcription termination.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or ⁇ uch inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter.
  • Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp- ⁇ erie ⁇ ) , ⁇ elf-replicating high copy number (such a ⁇ the YEp-series); integrating type ⁇ (such as the Yip-series) , or mini-chromosome ⁇ ( ⁇ uch a ⁇ the YCp- ⁇ erie ⁇ ) .
  • eukaryotic cells grown in tissue culture are often the preferred host cells for expres ⁇ ion of the GAP protein.
  • any higher eukaryotic tissue culture cell line is workable, e.g., insect baculovirus expre ⁇ sion ⁇ ystems, whether from an invertebrate or vertebrate ⁇ ource.
  • mammalian cells are often preferred. Transformation or transfection and propagation of such cells ha ⁇ become a routine procedure.
  • Example ⁇ of u ⁇ eful cell line ⁇ include HeLa cell ⁇ , Chinese ham ⁇ ter ovary (CHO) cell line ⁇ , baby rat kidney (BRK) cell lines, insect cell line ⁇ , bird cell line ⁇ , and monkey (COS) cell lines.
  • Expres ⁇ ion vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used) , a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expres ⁇ ion vector ⁇ may be plasmids, viruses, or retroviruse ⁇ carrying promoter ⁇ derived, e.g., from ⁇ uch sources as from adenovirus, SV40, parvoviru ⁇ es, vaccinia virus, or cytomegaloviru ⁇ .
  • ⁇ uitable expre ⁇ ion vector ⁇ include pCDNAl (Invitrogen, San Diego, CA) ; pCD, see Okayama et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, ⁇ ee Thoma ⁇ et al.
  • GAP polypeptide in a system which provides a specific or defined glycosylation pattern.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the GAP gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells.
  • a yea ⁇ t Ra ⁇ system was used to isolate NFl-GRD mutants which can suppres ⁇ specifically the activity of oncogenic Ra ⁇ .
  • Yeast cells carrying activated mutations in Ras (such as RAS2 Va119 and ⁇ __2.
  • Le ⁇ 68 ) are defective in re ⁇ ponding to environmental condition ⁇ , and show a variety of phenotypes including a heat shock- sensitive phenotype.
  • mutant ⁇ of NFl-GRD mo ⁇ t likely bind tightly with the oncogenic, e.g., mutated, Ra ⁇ proteins to sequester the latter proteins from the signal transduction for normal cell growth.
  • oncogenic, e.g., mutated, Ra ⁇ proteins to sequester the latter proteins from the signal transduction for normal cell growth.
  • Detailed analysis of the ⁇ tructures involved in the interaction between mutant NFl- GRD ⁇ and Ras will enable testing of compounds, e.g., analogues and mimetics, which can mimic the action of NFl- GRDs, and inhibit specifically transforming Ras activity.
  • a plasmid pKPll which expres ⁇ es a domain of NFl (amino acid residues 1063-1651; the numbers of amino acid residues are referred to according to Marchuk et al. (1991) Genomics 11:931-940, and a yeast strain carrying RAS2 Va119 mutation were used to obtain mutant NFl-GAP Related Domains (GRDs) which can ⁇ uppre ⁇ the phenotype of activated Ra ⁇ . In a previou ⁇ study, this plasmid was shown to suppre ⁇ ira2 " but not RAS2 Va119 .
  • Plasmid DNAs were recovered from these cell ⁇ , re-tran ⁇ formed into TK161-R2V-D, and phenotypic rever ⁇ ion wa ⁇ examined. Twelve positive colonies were obtained in the initial screening. Subsequently, two clones, NF201 (SEQ ID NO: 1) and NF204 (SEQ ID NO: 2), which had a relatively strong suppression activity for R S2 Va ⁇ 19 , were selected, and subjected to further analysis.
  • NF201 SEQ ID NO: 1
  • NF204 SEQ ID NO: 2
  • Wild-type NFl-GRD could weakly revert the phenotype of RAS2 Leu ⁇ 8, fc> u t was totally ineffective on RAS2 Va119 and RAS2 Ser41 .
  • NF201 ⁇ uppressed the heat shock-sensitive phenotype of all three alleles of RAS2 examined, including RAS2 Va ⁇ - ⁇ 9 .
  • RA£2 Leu68 RA£2 Ser41 (Tanaka et al. (1992) Mol. Cell.
  • NF204 preferentially suppressed RAS2 Va l 19 but not the other two alleles .
  • these two mutant NFl-GRDs could suppres ⁇ ira2 ⁇ cells, in which normal Ras proteins are activated, to the same extent as wild-type NFl-GRD, sugge ⁇ ting that NF201 and NF204 retain the ability to ⁇ timulate GTPase activity of normal Ras.
  • mutant NFl-GRDs were sequenced to identify mutations in NF201 and NF204, and the ⁇ equence ⁇ compared the ⁇ equence ⁇ with that of wild-type NFl-GRD.
  • NF201 SEQ ID NO: 1
  • the codon TT£ for Phe at residue 1434 was changed to TTA coding for Leu
  • NF204 SEQ ID NO: 2
  • the codon Aj__G for Lys at residue 1436 was replaced by AG.A coding for Arg.
  • Ly ⁇ at po ⁇ ition 1423 in NFl- GRD which i ⁇ located ju ⁇ t 11 and 13 amino acids upstream of the mutation sites of NF201 and NF204, respectively, is important for the structure and function of NFl.
  • the substitution of Glu for Lys at position 1423 has been identified in some human tumors as well as in a family of neurofibromatosis patients (Li et al. (1992) Cell 69:275- 281) .
  • the GAP activity of this mutant NFl-GRD was 200- to 400-fold lower than that of the wild-type NFl-GRD. It was also reported that the substitution of Met for Lys at the same position resulted in a decrease in stability of the protein (Wiesmuller et al. (1992) J. Biol. Chem. 267:10207- 10219). Thus, the amino acid residues at 1423, 1434 and 1436, and their surrounding sequence, are likely to be important for the structure and/or function of NFl proteins.
  • EXAMPLE 3 Effect of mutant NFl-GRDs in mammalian cells
  • the effect of these mutant NFl-GRDs in mammalian cells was investigated.
  • the cDNA fragments of the wild-type and mutant NFl-GRDs were recloned into a mammalian expression vector, and transfected into cell line ⁇ .
  • the size of the NFl-GRD protein tran ⁇ iently expre ⁇ ed in Cos7 cells was checked.
  • Western blot analysi ⁇ with an anti-NFl-GRD anti- ⁇ erum ⁇ ee Hattori et al. (1992) Oncogene 7:481-485
  • transfection of the plasmid ⁇ expre ⁇ ing NF201 and NF204 could induce flat reversion at dramatically high frequencies (8-9% of total G418-re ⁇ i ⁇ tant colonie ⁇ ) .
  • the frequency was even higher than that obtained by transfection of a K ⁇ e ⁇ -1 plasmid which ha ⁇ been ⁇ hown to posse ⁇ s anti-oncogenic activity in DT cells (Kitamura et al. (1990) Proc. Natl Acad. Sci. USA 87:4284-4288).
  • the wild-type NFl-GRD could also induce flat reversion of DT cells, although it wa ⁇ 5 to 6 time ⁇ le ⁇ potent than mutant clone ⁇ .
  • DT cell ⁇ were cotran ⁇ fected with 20 ⁇ g of NFl-GRD pla ⁇ mids and 2 ⁇ g of pSV2n_ej_. as described by Kitamura et al. (1990) proc. Natl Acad. Sci. USA 87:4284-4288, and transfectants were selected in a medium containing 0.5 mg/ml G418.
  • pKrev-1 plasmid it ⁇ elf contained the neo gene, 2 ⁇ g of the pla ⁇ mid wa ⁇ cotran ⁇ fected with 20 ⁇ g of pEF-BOS (the vector for NFl-GRD) .
  • the pEF-GAP contained rat full-length GAP cDNA in pEF-BOS. Frequency of rever ⁇ ion in DT cell ⁇ i ⁇ defined a ⁇ the ratio (%) of morphologically flat cell colonies to total G418-resistant colonies. N.D. : not determined.
  • EXAMPLE 4 Biochemical properties of the mutant. NFl-GRDs The biological properties of the mutant NFl-GRDs were studied to under ⁇ tand the molecular mechanism of anti- oncogenic activity. Extracts were prepared from yeast cells expres ⁇ ing wild-type and mutant NFl-GRD ⁇ , and GTPase- ⁇ timulating activity wa ⁇ mea ⁇ ured in vitro by u ⁇ ing recombinant c-Ha-Ra ⁇ protein ⁇ as sub ⁇ trate ⁇ .
  • Recombinant c-Ha-Ras G ly!2 (A) or c-Ha-Ras Va l 12 (B) protein ⁇ were loaded with [ ⁇ - 32 P]GTP (30 Ci/mmol) in buffer B (50 mM tri ⁇ -HCl [pH 7.4], 50 mM KCl, 1 mM MgCl2, 2.5 mM EDTA, and 0.2 mg/ml BSA) at 30 'C for 10 minute ⁇ . The reaction was stopped by the addition of MgCl2 to the final concentration of 7 mM.
  • Yeast cell extracts were prepared from wild-type yeast cells, RAY-3A-D, carrying various NFl-GRD plasmids.
  • yeast extract protein ⁇ and Ras* [ ⁇ -3 2 p]GTP were 1 mg/ml and 11.5 nM, respectively.
  • the cell extracts assayed were from the cell carrying the following plasmids: ., wild-type NFl-GRD; o, NF201; ⁇ , NF204; [solid square], vector alone; or [open square] , buffer A plus 1 mg/ml BSA.
  • Two mutant NFl-GRD ⁇ , NF201 and NF204, stimulated the GTPase activity of to the ⁇ ame extent a ⁇ wild-type NFl-GRD ( Figure 1) .
  • NFl can potentially act as a specific block of effector function by normal Ras.
  • oncogenic Ras lacks the intrinsic GTPase activity, and thus, natural GAP sequence ⁇ cannot stimulate the inactivation of the activated oncogenic Ras.
  • NFl-GRD suppre ⁇ e ⁇ the heat shock-sensitive phenotype of ira ⁇ cells, but not the same phenotype of activated mutants of Ras, e.g., E____2 Va119 and BA__2.
  • eu68 which correspond to mammalian oncogenic Ras, ras Va ⁇ - ⁇ - and £ja__ Leu 61, respectively.
  • Various mammalian oncogenic Ras mutants may be simulated by corresponding mutations in yeast Ra ⁇ protein ⁇ . The ⁇ e ob ⁇ ervation ⁇ lead to a model which i ⁇ useful for testing interaction of Ras variants with GAP variants, and which predict ⁇ u ⁇ eful blocking or reversal of mutant or oncogenic Ras-induced effects.
  • mutant NFl- GRD has higher affinity for oncogenic Ras*GTP as compared to the wild-type NFl-GRD.
  • the GAP binding region, and the effector binding regions on the Ras protein are in clo ⁇ e phy ⁇ ical proximity.
  • mutant NFl-GRD binding to oncogenic Ra ⁇ e.g., high affinity binding, could form an irreversible NFl*Ras*GTP complex. This could prevent interaction with putative downstream effector molecules, e.g., by conformational changes or competition.
  • the oncogenic Ras would be sequestered from signal transduction pathways. Two observations support this hypothe ⁇ is.
  • RAS2 Leu68 and wild-type NFl-GRD can explain the phenotypic ⁇ uppression.
  • Likewi ⁇ e, thi ⁇ model can also explain the differences in transformation- ⁇ uppressor activitie ⁇ among GAP, wild-type NFl-GRD, and mutant NFl-GRD ⁇ .
  • Table 2 shows that wild-type NFl-GRD, but not GAP, can suppre ⁇ tran ⁇ formation by v-Ra ⁇ ; two mutant NFl-GRD ⁇ are more potent suppres ⁇ or ⁇ than wild-type NFl-GRD.
  • Thi ⁇ order of potency a ⁇ tran ⁇ formation ⁇ uppressors may reflect the relative affinity for Ra ⁇ proteins; that is, wild-type NFl- GRD has 20 times higher affinity for Ras than GAP (see Martin et al. Cell 63:843-850); mutant NFl-GRDs may have even greater affinitie ⁇ .
  • thi ⁇ it ⁇ hould be noted that Ballester et al. (1990) Cell 63:851-859 previously observed the inhibitory effect of wild-type NF1- GRD but not of GAP on c-Ha-Ras Va H 2 expressed in yeast cells.
  • NF201 can inhibit the activity of RAS2 Ser41 ⁇ trongly ⁇ ugge ⁇ t ⁇ that the mutation in NF201 re ⁇ tores the interaction between RAS2 Ser41 and NFl-GRD.
  • Comparison of the relative affinities of wild-type and mutant NFl-GRDs for oncogenic Ra ⁇ protein ⁇ ⁇ hould provide a te ⁇ t for thi ⁇ model. Thi ⁇ model predict ⁇ that highly specific reagents could be produced having specificity only for blocking oncogenic Ras effects while having virtually no effects on normal Ras.
  • mutant NFl-GRD ⁇ could inhibit specifically oncogenic but not normal Ra ⁇ .
  • bound GTP would be rapidly hydrolyzed to GDP upon interaction with NFl-GRD, and NFl- GRD would be released from Ras*GDP.
  • a mutant NFl-GRD was expre ⁇ sed as a protein of 578 amino acid ⁇ , which is still a substantially large protein.
  • the yeast screening sy ⁇ tem described will allow determination of the minimum fragment of NFl-GRD which retain ⁇ anti- oncogenic activity.
  • Thi ⁇ approach will allow development of Ra ⁇ -specific anti-oncogenic compounds.
  • Trp Glu Asp Asn Ser Val lie Phe Leu Leu Val Gin Ser Met Val 1 5 10 15
  • Cys Phe Arg lie Ser Pro His Asn Asn Gin His Phe Lys lie Cys Leu 50 55 60
  • His Arg lie lie Thr Asn Ser Ala Leu Asp Trp Trp Pro Lys lie Asp 85 90 95
  • ORGANISM Saccharomyces cerevisiae
  • ORGANISM Saccharomyces cerevisiae

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Abstract

Sont décrits des procédés pour bloquer des affections induites par les Ras, telles que des anomalies prolifératives au sein de cellules eucaryotes, par exemple mammifères. Sont également décrits des protéines et des imitateurs, ainsi que leurs utilisations, qui peuvent bloquer les signaux intracellulaires anormaux entraînant souvent une prolifération incontrôlée, par exemple des cancers.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2727116A1 (fr) * 1994-11-22 1996-05-24 Rhone Poulenc Rorer Sa Peptides capables de se lier au domaine sh3 de la proteine gap, sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
WO1996016169A1 (fr) * 1994-11-22 1996-05-30 Rhône-Poulenc Rorer S.A. Peptides capables de se lier au domaine sh3 de la proteine gap, sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
FR2734266A1 (fr) * 1995-05-16 1996-11-22 Rhone Poulenc Rorer Sa Peptides capables de se lier au domaine sh3 de la proteine gap sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
WO1998037196A1 (fr) * 1997-02-25 1998-08-27 Ludwig Institute For Cancer Research Parg, proteine d'activation de gtpase exerçant une interaction avec ptpl1
WO2000031263A2 (fr) * 1998-11-23 2000-06-02 Incyte Pharmaceuticals, Inc. Proteines associees a la gtpase
US7148002B2 (en) 1996-11-06 2006-12-12 Onyx Pharmaceuticals, Inc. Nucleic acids and polypeptides related to a guanine exchange factor of Rho GTPase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL. vol. 63 , 16 November 1990 , CAMBRIDGE, NA US pages 835 - 841 XU ET AL. 'The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae' *
NATURE. vol. 346 , 23 August 1990 , LONDON GB pages 754 - 756 ZHANG ET AL. 'Suppression of c-ras transformation by GTPase-activating protein' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87 , June 1990 , WASHINGTON US pages 4284 - 4288 KITAYAMA ET AL. 'Genetic analysis of the Kirsten-ras-revertant 1 gene:Potentiation of its tumor suppressor activity by specific point mutations' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 90, no. 14 , 15 July 1993 , WASHINGTON US pages 6706 - 6710 NAKAFUKU ET AL. 'Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions' *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2727116A1 (fr) * 1994-11-22 1996-05-24 Rhone Poulenc Rorer Sa Peptides capables de se lier au domaine sh3 de la proteine gap, sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
WO1996016169A1 (fr) * 1994-11-22 1996-05-30 Rhône-Poulenc Rorer S.A. Peptides capables de se lier au domaine sh3 de la proteine gap, sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
US5886150A (en) * 1994-11-22 1999-03-23 Rhone-Poulenc Rorer Sa Peptides capable of binding to the GAP protein SH3 domain, nucleotide sequences coding therefor, and preparation and use thereof
FR2734266A1 (fr) * 1995-05-16 1996-11-22 Rhone Poulenc Rorer Sa Peptides capables de se lier au domaine sh3 de la proteine gap sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation
US7148002B2 (en) 1996-11-06 2006-12-12 Onyx Pharmaceuticals, Inc. Nucleic acids and polypeptides related to a guanine exchange factor of Rho GTPase
US7994294B2 (en) 1996-11-06 2011-08-09 Onyx Pharmaceuticals, Inc. Nucleic acids and polypeptides related to a guanine exchange factor of Rho GTPase
WO1998037196A1 (fr) * 1997-02-25 1998-08-27 Ludwig Institute For Cancer Research Parg, proteine d'activation de gtpase exerçant une interaction avec ptpl1
US6083721A (en) * 1997-02-25 2000-07-04 Ludwig Institute For Cancer Research Isolated nucleic acid molecules encoding PARG, a GTPase activating protein which interacts with PTPL1
US6475775B1 (en) 1997-02-25 2002-11-05 Ludwig Institute For Cancer Research PARG, a GTPase activating protein which interacts with PTPL1
WO2000031263A2 (fr) * 1998-11-23 2000-06-02 Incyte Pharmaceuticals, Inc. Proteines associees a la gtpase
WO2000031263A3 (fr) * 1998-11-23 2000-09-14 Incyte Pharma Inc Proteines associees a la gtpase

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