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  • 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
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  • A61K31/12—Ketones
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
  • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
  • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• PTK regulatory protein tyrosine kinases
• tyrosine kinases Aberrant protein tyrosine kinase activity has been implicated or is suspected in a number of pathologies such as diabetes, atherosclerosis, psoriases, septic shock, bone loss, anemia, many cancers and other proliferative diseases. Accordingly, tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design. For a review, see Levitzki et al. in Science 267. 1782-1788 (1995).
• SH2 domains which are conserved non ⁇ catalytic sequences of approximately 100 amino acids found in a variety of signalling molecules such as non-receptor PTKs and kinase target effector molecules and in oncogenic proteins, play a critical role.
• the SH2 domains are highly specific for short phosphotyrosine-containing peptide sequences found in autophosphorylated PTK receptors or intracellular tyrosine kinases.
• the Stat (signal transduction and activation of transcription) proteins are SH2 containing intracellular proteins that transmit a signal from cytokine receptors to the nucleus and activate transcription of specific target genes (Darnell J. et al., Science 264. 1415-1421 (1994)). These proteins are recruited to phosphorylation sites on the receptor via their SH2 domains and are themselves phosphorylated on tyrosine residues by receptor-associated Janus (JAK) tyrosine kinases. Phosphorylation of Stat tyrosine residues provides additional binding ligands for the Stat SH2 domains which are displaced from the receptor and bind to the Stat phosphotyrosine motifs forming dimers.
• the dimeric Stats translocate to the nucleus where they bind to DNA and to other accessory proteins and activate transcription of target genes.
• Individual Stats or Stat heterodimers transduce signals from different cytokine receptors, for example, Stat 6 mediates interleukin 4 signalling while Stat 5 transduces signals from the erythropoietin (EPO) receptor (Hou J. et al.. Science 265. 1701-1706 (1994); Penta K. et al.. J. Biol. Chem. 270. 31282-31287 (1995)).
• EPO erythropoietin
• Stat proteins In disease states involving dysregulation of cytokine activity, compounds that activate or inhibit Stat proteins would be useful therapeutic agents. For example, activation of Stat 5 would mimic the effects of EPO and enhance erythropoiesis. Conversely, inhibition of Stat 6 would block IL-4 and D -13- mediated up-regulation of the IgE receptor (Izuhara K.et al., J. Biol. Chem. 271. 619-22 ( 1996)) and be useful in the treatment of allergic reactions.
• the SH2 domain of Stat proteins provides a means to activate or inhibit their function. Activation can be achieved by inducing dimer formation by means of a dyad-symmetric ligand for the relevant SH2 domain, while a monomeric ligand would inhibit Stat function. Discovery of such ligands requires targeting of the SH2 domains of specific Stat proteins to induce or inhibit the desired homo- or heterodimer formation.
• the Stat 5 SH2 domain and the Stat 6 SH2 domain are structurally similar, possessing a high degree of conservation between the domains.
• Activation of the Stat 5 SH2 domain is indicated as increasing red blood cell production while antagonism of the Stat 6 SH2 domain is indicated as treating allergic reactions. Therefore, an activator of Stat 5 with Stat 6 cross- reactivity would exacerbate allergic reactions, whereas a Stat 6 inhibitor cross- reacting with Stat 5 would inhibit erythrocyte production.
• the present invention provides a method of enhancing erythropoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity, which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• the present invention also provides a method of treating anemia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• the present invention also provides a method of enhancing hematopoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• enhancing erythropoiesis means increacing the production of erythrocytes.
• treating means prophylactic or therapeutic therapy.
• compound means a nonpeptide chemical compound.
• Stat 5 SH2 domain activation is meant.
• SH2 domains other than Stat 5 is meant.
• the term "Stat 5 SH2 domain activator" and derivatives thereof means a bidentate SH2 ligand which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred-fold higher, than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred ⁇ fold lower, than the binding
• the term "bidentate SH2 ligand” means a compound having two binding regions in the same molecule.
• the present invention provides a method of enhancing erythropoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which
• a preferred aspect of the invention provides a method of enhancing erythropoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing erythropoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing erythropoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• a preferred aspect of the invention provides a method of treating anemia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• a preferred aspect of the invention provides a method of treating anemia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• a preferred aspect of the invention provides a method of treating anemia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than one hundred ⁇ fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• a preferred aspect of the invention provides a method of treating anemia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing hamatopoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds.to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing hamatopoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing hamatopoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain, (b) binds to a human hep SH2 domain, a human Grb2 SH2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain, and (c) binds to a human src SH2 domain, a human lck SH2 domain and a human fyn SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such Stat 5 SH2 domain.
• a preferred aspect of the invention provides a method of enhancing hamatopoiesis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human Stat 5 SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human Stat 6 SH2 domain.
• SH2 domains The binding activity of compounds at the different human SH2 domains is determined in vitro using SH2 domains expressed as fusion proteins either in E. coli or in baculovirus as further described in detail in Example 11 below.
• Activity in these assays is recognized in the art as correlating with efficacy in enhancing erythropoiesis in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in treating anemia in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in enhancing hematopoiesis in vivo.
• the present invention therefore provides a method of enhancing erythropoiesis, which comprises administering a quantity of a Stat 5 SH2 domain activator defined as herein in a quantity effective to enhance erythropoiesis.
• the drug may be administered to a patient in need of enhanced erythropoiesis by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
• the quantity effective to enhance erythropoiesis is from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be administered from about 1-6 times daily.
• compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
• forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• the present invention further provides a method of treating anemia, which comprises administering a quantity of a Stat 5 SH2 domain activator defined as herein in a quantity effective against anemia.
• the drug may be administered to a patient in need of treatment for anemia by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
• the quantity effective to treat anemia is from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be administered from about 1-6 times daily.
• the method of treating anemia disclosed in the present invention may also be carried out using a pharmaceutical composition comprising a Stat 5 SH2 domain activator defined herein and a pharmaceutically acceptable carrier.
• the composition may contain between 0.05 mg and 500 mg of a Stat 5 SH2 domain activator, and may be constituted into any form suitable for the mode of administration selected.
• Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
• Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• the present invention further provides a method of enhancing hematopoiesis, which comprises administering a quantity of a Stat 5 SH2 domain activator defined as herein in a quantity effective enhance hematopoiesis.
• the drug may be administered to a patient in need of enhanced hematopoiesis by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
• the quantity effective to enhance hematopoiesis from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
• the selected dose will be administered from about 1-6 times daily.
• compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
• forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• the drug may otherwise be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings.
• Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular Stat 5 SH2 domain activator in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular patient being treated will result in a need to adjust dosages, including patient age, weight, diet, and time of administration.
• the invention also provides for the use of a Stat 5 SH2 domain activator in the manufacture of a medicament for use in the treatment of anemia.
• the invention also provides for the use of a Stat 5 SH2 domain activator in the manufacture of a medicament for use in enhancing hematopoiesis.
• the invention also provides for the use of a Stat SH2 domain activator in the manufacture of a medicament for use in enhancing erythropoiesis.
• the invention also provides for a pharmaceutical composition for use in the treatment anemia which comprises an Stat SH2 domain activator.
• the invention also provides for a pharmaceutical composition for use in enhancing hematopoiesis which comprises an Stat SH2 domain activator.
• the invention also provides for a pharmaceutical composition for use in enhancing erythropoiesis which comprises an Stat SH2 domain activator. No unacceptable toxicological effects are expected when the methods of the invention are utilized in accordance with the present invention.
• L-3,5-Dibromotyrosine can be prepared by methods known in the art, for example as described in "Thyoid Hormones and Analogues. I. Synthesis, Physical Properties and Theoretical Calculations” E. C. Jorgensen, Hormonal Proteins and Peptides, Vol. VI, 1978, Academic Press, N.Y. and references cited therein. L-3,5-dibromo-N-trifluoroacetyl-tyrosine methyl ester (for use in Example 2
• Example 2B (b)) can be prepared according to the following procedure.
• L-3,5-Dibromotyrosine 500 g was suspended in methanol (5 liters) and dry hydrogen chloride passed through the stirred suspension for 5 hours. The reaction mixture was evaporated to dryness, the residue suspended in water (4 liters), and the pH adjusted to 6 with 40% sodium hydroxide. The precipitate was collected and washed with water to give L-3,5-dibromotyrosine methyl ester (467 g, 90%), m.p. 201°-203°. The ester (768 g) was suspended in chloroform (2.7 liters) and ethyl acetate (2.7 liters), then trifluoroacetic anhydride (565 g) was added over 0.5 hour, keeping the temperature below 35°.
• the amino group of 4-trans-aminomethyl-cyclohexyl-carboxylic acid 1 is protected with a standard protective group such as with a Boc group (Boc anhydride, NaOH, H j O, dioxane) to form 2, then is coupled to Kaiser oxime resin (Kaiser, E.T.; et al ] Am Chem Soc 1985, 107, 7087-7092) using a coupling reagent such as DCC to form 3.
• the amine is then deprotected under standard conditions (25% TFA, methylene chloride) to form 4, then is acylated with standard conditions (such as with HBTU, NMM in DMF or DCC or DIC in DMF or NMP) to form 5.
• the compound is then cleaved from the resin with various amines to form the final desired product 6.
• the tide compound was prepared by reaction of neat N,N'- diisopropylcarbodiimide (1.0 equiv) with 2-methyl-2-propanol (1.15 equiv) in the presence of CuCl (0.01 equiv) for 1 day at room temperature, according to the procedure of Santini et al. ( J. Org. Chem. 1994, 59, 2261).
• the iodonium trifluoroacetate salt can be used which is prepared as follows: Iodine (159 g) was suspended in trifluoroacetic anhydride (1 liter) and stirred under nitrogen whilst fuming nitric acid (350 ml) was added over 1.5 hours, keeping the temperature between 36° and 40°. Trifluoroacetic anhydride (300 ml) was then added and the mixture maintained at 40° under a stream of nitrogen until all nitrogen oxides were removed, then allowed to stand at room temperature overnight. The solvent was then removed under reduced pressure and the residual solvent removed by azeotroping with trifluoroacetic anhydride (2 X 300 ml).
• the organic layer was dried over magnesium sulfate and concentrated to a residue consisting of the desired product, 3-[4-[4-(4- methylbenzoyl)benzoyl]phenyl]-l -propene, and tin-containing by-products.
• This material was subjected to flash chromatography (silica gel, elution with 95:5 hexane : ethyl acetate) which removed most, but not all, of the tin impurities.
• Aqueous sodium hydroxide (IN, 100 ml, 100 mmol) was added to a solution of 4-trans-aminomethyl-cyclohexyl-carboxylic acid (9.0g, 60 mmol), in dioxane (100 ml), water (100 ml) at 0 degrees C.
• Boc anhydride (15.9 g, 66 mmol) was added and the reaction was warmed to rt and stirred ovemight.
• the solution was concentrated to 50 ml, then was diluted with EtOAc (100 ml) and acidified to pH 2 with adqueous KHSO 4 (IN). The organic layer was then extracted with water (100 ml) two times, and the organics were concentrated in vacuo.
• Kaiser oxime resin (20 g, 0.7 mmol/g loading, Advanced Chem Tech) was added to a solution of N-t-butyloxy carbonyl-trans-4-aminomethyl cyclohexyl carboxylic acid (5.0 g, 20 mmol) and DCC (4.4 g, 20 mmol) in methylene chloride (200 ml) and was gentle mixed at rt ovemight. The solid was filtered and collected, then washed with methylene chloride (5 x 100 ml).
• Trans-4-aminomethyl cyclohexyl (Kaiser oxime resin) carboxylate 200 mg was suspended in DMF (3.0 ml) and N-methyl morpholine (0.2 ml) and 4, 4'- benzophenone dicarboxylic acid (190 mg, 0.7 mmol) and HBTU (265 mg, 0.7 mmol) was added and the reaction was gently mixed for 3 h.
• the solid was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then was resuspended in DMF (3.0 ml) and N-methyl morpholine (0.1 ml) and 4,4'- benzophenone dicarboxylic acid (0.35 mmol) and HBTU (0.35 mmol) was added and the reaction was gently mixed for 3 h.
• the sohd was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then methylene chloride (5 x 20 ml), then was dried under vacuum.
• SH2 Domains The activity of compounds at the different human SH2 domains is determined in vitro using SH2 domains expressed as fusion proteins either in E coli or baculovirus.
• the SH2 domains used herein were the human forms of the Stat 6 SH2 domain, src SH2 domain, Grb2 SH2 domain, lck SH2 domain, Stat 5 SH2 domain, fyn SH2 domain, SH-PTP2 SH2 domain, ⁇ 85 SH2 domain and hep SH2 domain.
• DET1 defined epitope tag 1
• HV-l Human Immunodeficiency Virus Type 1 envelope protein gpl 20 (or gpl60).
• Monoclonal antibodies to various epitopes of HIV-l gpl 20 (or gpl 60) are known in the art, see, for example U.S. Patent 5,166,050.
• monoclonal antibody 178.1 (see, e.g., Thiriart et al., J. Immunol.. 143: 1832-1836 (1989)), which was prepared by immunization of mice with a yeast-expressed HIV-l gpl60 molecule from strain BH10 (Ratner et al., Nature. 313:277-284 (1985)). This tag was used for detection of expression (by Westem blot), for purification of the protein (by affinity chromatography), and for configuring assays in which the fusion protein is captured or immobilized using the 178.1 antibody.
• DET2 is a hexa- histidine sequence tag (SEQ ID NO: 2) which binds to nickel-containing resins and was used for purification purposes. Spacer (SEQ ID NO: 3) was utilized to design a BamHI restriction site at the indicated position of the construct.
• the term -ek- refers to a recognition sequence (SEQ ID NO: 4) for the enterokinase protease which provides for the optional removal of the tags from the SH2 domain, thus producing an SH2 domain that contains no extraneous amino acids.
• SH2 domains which contain no extraneous amino acids are preferable to tagged protein for crystallography studies.
• SH2 refers to the SH2 domains of different proteins.
• each DETl-DET2-spacer-ek-SH2 was designed such that the indicated restriction sites (BamHI and Xbal) flank the spacer- ek-SH2 region, thereby allowing different spacer-ek-SH2 contructs to be readily substituted into any one of the vectors described in Procedures 2, 3, 5 or 6 below to create a DETl-DET2-spacer-ek-SH2 tagged protein.
• the DNA sequence encoding each DETl-DET2-spacer-ek-SH2 construct was also designed such that the entire tagged SH2 domain can be moved as an Ndel-Xbal fragment into any expression vector containing an Ndel site at an appropriate distance downstream of E.
• the vector used in the instant experiments are the E. coli expression vector pEAlKnRBS3.
• This vector is a derivative of the series of vectors described in Shatzman, A, Gross, M, and Rosenberg, M, 1990, "Expression using vectors with phage lambda regulatory sequences", In: Current Protocols in Molecular Biology (F.A. Ausubel et al , eds.), pp. 16.3.1-16.3.11,
• Procedure 1 Cloning and Expression of chicken src SH2 domain containing tags DET1 and DET2 (DETl-DET2-spacer-SH2).
• a DNA sequence encoding the tagged protein DETl-DET2-spacer-SH2 was PCR amplified from a cDNA clone containing the chicken src gene (p5H; Levy et al 1986. Pro Natl. Acad. Sci. USA 8 :4228) by methods well known to those skilled in the art by using the following primers:
• the underlined sites are an Ndel recognition site (5') and a BamHI recognition site (3')-
• the underlined region is an Xbal recognition site.
• the PCR product was digested with Ndel and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was ligated into Ndel-Xbal- digested pEAlKnRBS3 vector (Bergsma et al, supra) that had been agarose gel purified as a 6.5 kbp fragment.
• the ligation reaction was used to transform E. coli MM294cI * (F.A. Ausubel et al., supra .
• a plasmid containing an insertion of the correct fragment was identified and confirmed by DNA sequencing.
• the resultant plasmid encodes DETl-DET2-spacer-SH2 under the control of the phage lamda P L promoter and regulatory system.
• Plasmid DNA was purified from MM294cI * and used to transform E. coji strain ARI 20.
• expression of the phage promoter can be induced by addition of nalidixic acid to the growing culture as described in F.A. Ausubel et al, supra. Nalidixic acid induction of ARI 20 containing this plasmid, followed by analysis of the cellular proteins on an SDS- polyacrylamide gel stained with Coomassie Blue (F.A.
• Procedure 2 Cloning, expression and purification of human src SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-src SH2).
• a DNA sequence encoding protein ek-src SH2 was PCR amplified from a cDNA clone containing the human src gene (c-src SH2 DNA sequence identical to that described in Takeya . and Hanaf sa, H, 1983 Cell 32:881-890) using the following primers:
• the underlined site is a BamHI recognition site
• the underlined region is an Xbal recognition site.
• the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1- DET2-spacer-SH2 described in Procedure 1 above.
• the BamHI site is located between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
• the ligation reaction was used to transform E. coli MM294cr.
• the construct DETl-DET2-spacer-ek-src SH2 was confirmed by DNA sequencing (SEQ ID NO: 5) and induced in E. coli strain ARI 20 as described in Procedure 1 above.
• a Coomassie-Blue-stained, Westem-blot- positive induced protein band with an apparent molecular weight of 16,000 was observed after nalidixic acid induction.
• Procedure 3 Cloning and expression of human lck SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer-ek-lck SH2).
• a DNA sequence encoding protein ek-lck SH2 was PCR amplified from a cDNA clone containing the human lck gene (Genbank accession number M36881) using the following primers:
• the underlined site is a BamHI recognition site.
• the underlined region is an Xbal recognition site.
• the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1- DET2-spacer-SH2 described in Procedure 1 above.
• the BamHI site is located in between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
• the ligation reaction was used to transform E. coli MM294cI * .
• the construct containing DETl-DET2-spacer-ek-lck SH2 was confirmed by DNA sequencing (SEQ ID NO: 6) and induced in E_. coli strain ARI 20 as described in Procedure 1 above.
• Procedure 4 Cloning and expression of human hep SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer-ek-hcp SH2).
• a DNA sequence encoding protein ek-hcp SH2 (hep SH2 DNA sequence identical to that described in Shen, S-H. Nature (1991) 352: 736-739) was reverse transcriptase-PCR amplified from human fetal liver RNA.
• RNA isolation used Tri- Reagent (Molecular Research Center Inc.) and the Reverse Transcriptase system (GIBCO-BRL) according to the manufacture's instructions. PCR was carried out using the following primers:
• the underlined site is a Bglll recognition site.
• the underlined region is an Xbal recognition site.
• the PCR product was digested with Bgi ⁇ and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was hgated into BamHI- Xbal-digested expression vector containing the tagged human src gene DET1- DET2-spacer-ek-src SH2 described in Procedure 2 above.
• the BamHI site is located in between the coding regions for DET2 and ek
• the Xbal site is located after the 3' end of the SH2 coding region.
• the ek-hcp SH2 sequence replaced the ek-src SH2 sequence in the above vector.
• the Ugation reaction was used to transform £. coli MM294cI * .
• the construct containing DET1- DET2-spacer-ek-hcp SH2 was confirmed by DNA sequencing (SEQ ID NO: 7) and used to transform I c ⁇ Ji GI698 (Invitrogen Co ⁇ oration, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions. A Coomassie-Blue- stained, Western-blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 30° C. Cells were lysed at neutral pH by sonication in the presance of lysozyme.
• the insoluble pellet was solubilized with 8 M urea in Tris buffer pH 8 and bound onto a Ni NTA column.
• the resin was washed with equilibration buffer (Tris buffer pH 8 containing 0.5 M NaCl, 8 M urea and 5 mM BME) and the same buffer containing 15 mM imidazole.
• the protein was refolded on the column during the removal of urea in the presence of 5 mM BME and the purified refolded protein eluted with 300 mM imidazole in Tris buffer pH 8.
• the SH2 domain purified in this fashion, was found to bind with high affinity in a specific, saturable fashion to the appropriate pY peptide in the "Binding Assays" described below, demonstrating that the tag did not interfere with function and that the protein was refolded successfully.
• This expressed fusion protein, DETl-DET2-spacer-ek-hcp SH2 was utilized in the "Binding Assays” described below in order to determine the specificity of compounds to selectively inhibit the human hep SH2 domain.
• Procedure 5 Cloning, expression and purification of human Stat 6 SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-Stat 6 SH2).
• a DNA sequence encoding protein ek-Stat 6-SH2 was PCR amphfied from a cDNA clone containing the human Stat 6 gene (Stat 6 SH2 DNA sequence identical to that described in Science 265. (1994) 1701) using the following primers:
• the underhned site is a BamHI recognition site.
• the underhned region is an Xbal recognition site.
• the PCR product was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was ligated with the vector fragment derived from BamHI- Xbal digestion of the expression vector containing the tagged human src gene DETl-DET2-spacer-ek-src SH2 described in Procedure 2 above.
• the BamHI site is located between the coding regions for DET2 and SH2
• the Xbal site is located after the 3' end of the SH2 coding region.
• the ligation reaction was used to transform E. coli MM294cI+.
• DETl-DET2-spacer-ek-Stat 6 SH2 was confirmed by DNA sequencing (SEQ ID NO: 29) and induced in E. co]i strain GI698 (Invitrogen Co ⁇ oration, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions, also as described in procedure 4. A Coomassie-Blue-stained, Western-blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 27° C.
• Procedure 6 Cloning, expression and purification of human Stat 5 SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-Stat 5 SH2).
• Restriction sites for BamHI and Xbal are underlined in the sequence above.
• the ek-Stat 5-SH2 gene sequence was digested with BamHI and Xbal, followed by isolation of the digested fragment on an agarose gel.
• the fragment was ligated with the vector fragment derived from BamHI-Xbal digestion of the expression vector containing the tagged human src gene DETl-DET2-spacer-ek-src SH2 described in Procedure 2 above.
• the BamHI site is located between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
• the ligation reaction was used to transform E. coli MM294cI+.
• the construct DETl-DET2-spacer-ek-Stat 5 SH2 was confirmed by DNA sequencing (SEQ ID NO: 32) and induced in E. coh strain GI698 (Invitrogen Co ⁇ oration, San Diego, CA). Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions, also as described in procedure 4. A Coomassie-Blue-stained, Western- blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 27° C.
• Fusion proteins having the structure GST-X-SH2 are prepared as described in the GST gene fusion kit system available from Pharmacia (New Jersey).
• GST is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 8) for fyn, Grb2 and SH-PTP2 and is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 9) for p85.
• SH2 refers to the SH2 domains of fyn, Grb2, p85 and SH-PTP2 which were expressed and purified using glutathione Sepharose 4B (Pharmacia) according to "Current Protocols in Molecular Biology", ed. FM Ausubel et al., pub.
• X is an appropriate linker, preferably of 6 to 21 base pairs, used to keep the SH2 construct in frame and complement cloning. As such, the sequence of X is not critical. One skilled in the art can readily construct the appropriate linker.
• the DNA sequence encoding each GST-X-SH2 fusion protein was designed such that the indicated restriction sites (BamHI and EcoRI) flank the SH2 region.
• the vector used in the instant experiments was the E. coh expression vector pGEX-2T (Pharmacia) for fyn, Grb2 and SH-PTP2, and pGEX-3X (Pharmacia) for p85. Each of these vectors result in SH2 constructs having additional C-terminal amino acids as described below.
• the sequence encoding the SH2 domain of human fyn (amino acids 143- 252) (Yamamoto, T. et al. Proc. Natl. Acad. Sci. USA 83_, 5459-5463 (1986)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
• the SH2 domain including the additional C-terminal amino acids leucine-threonine- asparagine-serine-serine (SEQ ID NO: 10) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-fyn. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human fyn SH2 domain.
• Human p85 SH2 domain The sequence encoding the SH2 domain of human p85 (amino acids 321-440) (Skolnik, E. et al., £e ⁇ . 83-90 (1991)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-3X.
• the SH2 domain including the additional C-terminal amino acids asparagine-serine-serine (SEQ ID NO: 1 l) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-p85. This expressed fusion protein was
• Human SH-PTP2 SH2 domain The sequence encoding the SH2 domain of human SH-PTP2 (amino acids l-106))(Bastien, L. et al., Biochem. Biophvs. Res. Commun. 196. 124-133 (1993)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
• Human Grb2 SH2 domain The sequence encoding the SH2 domain of human Grb2 (amino acids 58-159) (Lowenstein, E. et al., Qsii IQ, 431-442 (1992)) was cloned into the BamHI and EcoRI sites of the expression vector pGEX-2T.
• the SH2 domain including the additional C-terminal amino acids isoleucine-histidine- arginine-aspartate (SEQ ID NO: 25) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-Grb2.
• a six nucleotide linker was used and resulted in the amino acids glycine and serine between the GST and SH2 domain. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human Grb2 SH2 domain.
• Binding Assays The potency of compounds at the SH2 domains is determined based on the ability of such compounds to selectively inhibit such SH2 domain from binding to its respective specific pY peptide.
• the binding assays for the SH2 domains and pY peptides are performed in an ELISA-based 96 well plate assay.
• hydrophilic Durapore® pore size 0.65um Cat. No. MADVN6550
• 2 ul 50% suspension
• Protein-G Sepharose available from Pharmacia of N.J. Cat. No.
• Specific pY biotinylated peptides are diluted to a concentration of 1.0 uM in TBS-T (these peptides can be obtained from Bachem Bioscience of Pennsylvania, Genosys Biotechnologies of Texas and California Peptide Research of California). 10 ul is aliquoted per well to yield a final concentration of 0.1 uM (approx. the I for each SH2 domain/peptide pair) and a final volume of 100 ul. The assay plates are incubated until equilibrium binding was attained (3 hr at 4°C with shaking).
• the assay plates are washed 2 X per well TBS-T (4°C), then 100 ul of SABC (Strepavidin biotinylated horseradish peroxidase complex, available from the Zymed co ⁇ oration of California cat. no. 93-0043, 1 drop reagent A (streptavidin) and 1 drop of reagent B (AH-biotin conjugated-horseradish peroxidase) per 10 ml of TBS- T, incubated at 37°C for 30 minutes, then cooled to 4°C) is added per well, then incubated at 4°C for 30-60 minutes. The plates are then washed 4 X with TBS-T (4°C) (250 ul/well)/wash).
• SABC Stepavidin biotinylated horseradish peroxidase complex, available from the Zymed co ⁇ oration of California cat. no. 93-0043
• 1 drop reagent A streptavidin
• the Ki for respective compounds is calculated via the following equation (see below). This expanded equation must be used under the conditions of this assay, due to the fact that the pY biotinylated peptide is not in vast excess concentration (lOOX) over the SH2 domain fusion protein.
• the IC 50 is an extrapolated value from a nonlinear curve fit using Kaleidagraph.
• Rtot and *D are known values for reagents input into the assay.
• KD generally must be experimentally determined for each combination of SH2 domain fusion protein and pY biotinylated peptide.
• KD (uM)KD value for the specific pY and biotinylated peptide for each SH2 domain
• IC 50 is the concentration of inhibitor at which the response or signal is inhibited by
• KD is the dissociation constant for a ligand in a receptor/ligand interaction, normally equaling the concentration of ligand which is at 1/2 Vmax on a saturation binding curve>
• the pY peptide ligands used in the above Binding Assays are as follows:
• Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for SH-PTP2 SH2
• Tables I and II illustrate the cross reactivity compounds at the indicated SH2 domains. From the methods described herein, compounds which have binding affinities which are greater than fifty-fold higher at the Stat 5 SH2 domain than the binding affinities at other SH2 domains can be readily identified.
• a dimeric form is prepared by attaching two molecules of the ligand to one molecule of a bifunctional spacer by methods well known to those skilled in the art.
• the size of the spacer may be varied to optimize activation of Stat 5 as determined by incubation of unstimulated cell extracts with the compound and assaying for Stat 5 activation in a DNA gel shift assaying as described in Hou et al.. Immunity 2. (1995). 321 to 329. Contemplated herein is the method, of activating other Stat SH2 domains
• Lys Ser lie Arg Ile Gin Arg Gly Pro Gly Arg 1 5 10
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE (vi) ORIGINAL SOURCE:
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE (vi) ORIGINAL SOURCE:
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE (vi) ORIGINAL SOURCE:
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTISENSE NO
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• MOLECULE TYPE peptide
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE

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