[go: up one dir, main page]

WO1998040093A1 - Procede permettant d'antagoniser le domaine humain src sh2 - Google Patents

Procede permettant d'antagoniser le domaine humain src sh2 Download PDF

Info

Publication number
WO1998040093A1
WO1998040093A1 PCT/US1998/004699 US9804699W WO9840093A1 WO 1998040093 A1 WO1998040093 A1 WO 1998040093A1 US 9804699 W US9804699 W US 9804699W WO 9840093 A1 WO9840093 A1 WO 9840093A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
glu
src
peptidomimetic
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1998/004699
Other languages
English (en)
Inventor
Dennis A. Holt
Daniel F. Veber
Dennis S. Yamashita
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Priority to CA002283472A priority Critical patent/CA2283472A1/fr
Priority to EP98910294A priority patent/EP1007076A4/fr
Priority to JP53972698A priority patent/JP2001514662A/ja
Publication of WO1998040093A1 publication Critical patent/WO1998040093A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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

Definitions

  • Mammalian bone is constantly undergoing bone remodeling, which is a dynamic process of bone resorption and bone formation. These processes are mediated by specialized cell types: bone formation is the result of the deposition of mineralized bone by osteoblast cells, and bone resorption is the result of the dissolution of bone matrix by osteoclast cells.
  • Many bone diseases are brought about by an imbalance of bone formation relative to bone resorption. For instance, diseases such as osteoporosis and Paget 's disease are characterized by a net loss of bone matrix. Thus, agents which inhibit bone resorption are useful for the treatment of such diseases.
  • An activated osteoclast resorbs bone by attaching to the bone matrix, and secreting proteolytic enzymes, organic acids and protons into the sealed compartment formed between its cell membrane and the bone matrix.
  • the acidic environment and proteolytic enzymes effect the dissolution of bone in the sealed compartment to crest pits, or lacuna, in the bone surface, which are apparent when the osteoclast detaches from the bone.
  • Tyrosine phosphorylation may be the primary, or possibly even the sole, indicator of signal transduction in multicellular organisms.
  • tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design.
  • tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design.
  • tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design.
  • many of the proteins comprising signal transduction pathways are present at low levels and often have opposing activities. The properties of these signaling molecules allow the cell to control transduction by means of the subcellular location and juxtaposition of effectors as well as by balancing activation with repression such that a small change in one pathway can achieve a switching effect.
  • SH2 domains which are conserved non-catalytic sequences of approximately 100 amino acids found in a variety of signaling 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.
  • antagonism of the src SH2 domain is indicated herein as effecting bone resorption while antagonism of the lck SH2 domain or the fyn SH2 domain induces immunosuppression.
  • the induction of immunosuppression would be undesirable in long term therapy for bone resorption disease.
  • the present invention provides a method of treating a bone resorption disease in a subject which comprises administering to the subject a therapeutically effective amount of a compound which forms a covalent bond or link to cysl85 of the src SH2 domain.
  • the present invention also provides a method of treating osteoporosis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which forms a covalent bond or link to cysl85 of the src SH2 domain.
  • the present invention also provides a method of impairing the function of osteoclasts in a subject which comprises administering to the subject an osteoclast function-inhibiting amount of a compound which forms a covalent bond or link to cys 185 of the src SH2 domain.
  • the present invention also provides compounds and pharmaceutical compositions of these compounds which are useful in antagonizing the human SRC SH2 domain.
  • a bone resorption disease means any disorder characterized by abnormal bone loss due to osteoclastic activity, preferably osteoporosis.
  • treating and derivatives thereof means prophylactic or therapeutic therapy.
  • compound means a peptide or chemical compound.
  • peptidomimetic is as defined in J. Med. Chem. 1993, 36, 3039-3049.
  • src SH2 domain antagonists means a compound which is capable of forming a covalent bond or link to cys 185 of the src SH2 domain.
  • cys 185 of the src SH2 domain refers to the Cysteine at the 185 position of the src gene following conventional numbering as described in Nature 1997, 385, 595-602. All of the src gene numbering references used herein follow conventional numbering as described in Nature 1997, 385, 595- 602.
  • the present invention provides a method of treating a bone resorption disease in a subject which comprises administering to the subject a therapeutically effective amount of a compound which forms a covalent bond or link to cys 185 of the src SH2 domain.
  • the invention also provides a method of treating osteoporosis in a subject which comprises administering to the subject a therapeutically effective amount of a compound which forms a covalent bond or link to cys 185 of the src SH2 domain.
  • the invention also provides a method of impairing the function of osteoclasts in a subject which comprises administering to the subject an osteoclast function-inhibiting amount of a compound which forms a covalent bond or link to cys 185 of the src SH2 domain.
  • the nonreceptor tyrosine kinase src is essential for resorption of bone by osteoclasts.
  • the src homology-2 (SH2) domain of src controls its association with other signaling molecules through binding to short peptide motifs containing phosphotyrosine. Inhibition of these interactions blocks src-mediated signal transduction by preventing recruitment of src into receptor-effector complexes.
  • cysteine 185 (cys 185) is located in the phosphotyrosine binding pocket, close to histidine 201, argl55, argl75 and lys203.
  • Compounds which form a covalent bond or link to cys 185 block the phosphotyrosine binding pocket of human src SH2 thereby irreversibly inhibiting human src SH2.
  • the compound which forms a covalent bond or link to cys 185 will also form a hydrogen bond with arg 175 and have a hydrophobic interaction with the sidechain portion of lys203.
  • Seq. ID No. 5 uses a portion of src gene.
  • cys 185 corresponds to cys67 in Seq. ID No. 5
  • his201 corresponds to his83 in Seq. ID No. 5
  • argl55 corresponds to arg37 in Seq. ID No. 5
  • argl75 corresponds to arg 57 in Seq. ID No. 5
  • lys203 corresponds to lys85 in Seq. ID. No. 5.
  • Presently preferred compounds of this invention which form a covalent bond or link to cys 185 of the src SH2 domain have the following Formula (I):
  • Scheme 1 depicts formation of (S)-alpha-(acetylamino)-l,3-dihydro-3- hydroxy-l-oxo-5-isobenzofuranpropanamido-glutamate-glutamate-isoleucine- glutamate-amine (Compound 1).
  • N-acetyl tyrosine ethyl ester was formylated with hexamethylene tetramine (methenamine) in TFA, AcOH (J. Ind. Chem. 1987, 26B, 7071). Then the aldehyde was protected as its 1,3-dithiane (Tet. Lett.
  • the inhibitory activity of compounds at the different human SH2 domains was determined in vitro using SH2 domains expressed as fusion proteins in E. coli as further described in detail in Example 2 below.
  • the present invention therefore provides a method of treating a bone resorption disease, which comprises administering a quantity of a src SH2 domain antagonists defined as herein in a quantity effective to inhibit bone resorption.
  • the drug may be administered to a patient afflicted with a bone resorption disease or in danger of contracting a bone resorption disease by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the quantity effective to inhibit bone resorption 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 a bone resorption disease disclosed in the present invention may also be carried out using a pharmaceutical composition comprising an src SH2 domain antagonists defined herein and a pharmaceutically acceptable carrier.
  • the composition may contain between 0.05 mg and 500 mg of a src SH2 domain antagonist, 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 impairing the function of osteoclasts, which comprises administering a quantity of a src SH2 domain antagonists defined as herein in a quantity effective to inhibit bone resorption.
  • the drug may be administered to a patient afflicted with a bone resorption disease or in danger of contracting a bone resorption disease by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the quantity effective to impair osteoclasts function 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 impairing the function of osteoclasts disclosed in the present invention may also be carried out using a pharmaceutical composition comprising an src SH2 domain antagonists defined herein and a pharmaceutically acceptable carrier.
  • the composition may contain between 0.05 mg and 500 mg of a src SH2 domain antagonist, 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 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 src SH2 domain antagonist 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 src SH2 domain antagonists in the manufacture of a medicament for use in the treatment of a bone resorption disease.
  • the invention also provides for the use of a src SH2 domain antagonists in the manufacture of a medicament for use in the treating osteoporosis.
  • the invention also provides for the use of a src SH2 domain antagonists in the manufacture of a medicament for use in inhibiting osteoclast function.
  • the invention also provides for a pharmaceutical composition for use in the treatment of a bone resorption disease which comprises a src SH2 domain antagonists.
  • the invention also provides for a pharmaceutical composition for use in the treatment of osteoporosis which comprises a src SH2 domain antagonists.
  • the invention also provides for a pharmaceutical composition for use in inhibiting osteoclast function which comprises a src SH2 domain antagonists.
  • Hexamethylene tetraamine (Aldrich, 25 g, 178 mmol) was added to a solution of N-acetyl tyrosine ethyl ester mono hydrate (Aldrich, 10 g, 37.1 mmol) in TFA (30 ml) and AcOH (30 ml) and the reaction was heated to 80 degrees C for 4.5 h. The reaction was cooled to RT, then H 2 O (200 ml) was added and the reaction mixture was extracted with EtOAc (3 x 200 ml).
  • N-phenyl trifluromethanesulfonimide (Aldrich, 1.0 g, 2.8 mmol) was added to a solution of N-acetyl-3-(l,3-dithiane)-tyrosine ethyl ester (1.0 g, 2.7 mmol) and triethyl amine (0.41 ml, 3.0 mmol) in CH 2 C1 2 (9.0 ml) at RT, and the reaction was stirred overnight. The reaction was diluted with H 2 O (20 ml), then the reaction mixture was extracted with EtOAc (3 x 20 ml). The combined organics were dried with magnesium sulfate, filtered, concentrated in vacuo, and chromatographed
  • the title peptide was prepared by standard solid-phase chemistry on a Symphony Multiple Peptide Synthesizer (Rainin) using standard FMOC protected amino acids (2 x 1.5 h, 6 equivalents using HBTU (2-lH-benzotriazole-l-yl)- 1,1 ,3 ,3-tetramethyluronium hexafluorophate)/ N-methyl morpholine in DMF coupling conditions) and 20% piperidine/ DMF deprotection conditions (10 min) starting with Rink Amide resin (Nova, 0.3 mmol/ g, PS/ 1% DVB, 100-200 mesh, H. Rink 7et. Lett. 1987, 28, 3787).
  • HBTU (2- 1 H-benzotriazole- 1 -yl)- 1 , 1 ,3 ,3-tetramethyluronium hexafluorophate) (61 mg, 0.16 mmol) was added to a slurry of N-Acetyl-3-(l,3- dithiane)-4-(t-butyl-carboxylate)-phenylalanine (68 mg, 0.16 mmol), ⁇ -t-butyl- glutamate- ⁇ -t-butyl-glutamate-isoleucine- ⁇ -t-butyl-glutamate-Rink resin (200 mg, 0.08 mmol), N-methyl morpholine (0.023 ml, 0.24 mmol) in DMF (5.0 ml) and was shaken at RT for 48 h. The reaction mixture was filtered, washed with DMF (300 ml), then CH 2 C1 2 (300 ml), and was dried under vacuum overnight.
  • N-Acetyl-3-(l,3-dithiane)-4-(t-butyl-carboxylate)-phenylalanine- ⁇ -t-butyl- glutamate- ⁇ -t-butyl-glutamate-isoleucine- ⁇ -t-butyl-glutamate-Rink resin 100 mg, 0.04 mmol
  • N-Acetyl-3-(l,3-dithiane)-4-(t-butyl-carboxylate)-phenylalanine-glutamate- glutamate-isoleucine-glutamate-amine 5mg, 0.006 mmol was dissolved in 90 % acetone/ H 2 O (0.3 ml), then N-chlorosuccinimide (5 mg, 0.037 mmol) and silver perchlorate (10 mg, 0.048 mmol) were added and the reaction was stirred 10 minutes at RT.
  • the reaction mixture was chromatographed (C 18 reverse phase silica, MeCN, H 2 O), and the UV active fractions were combined, concentrated in vacuo, and dissolved in MeOH (0.2 ml).
  • the inhibitory activity of compounds at the human src SH2 domain was determined in vitro using the human src SH2 domain expressed as fusion proteins in E. coli.
  • the fusion protein containing the human SH2 domain was expressed as the general sequence: DETl-DET2-spacer-ek-SH2, where DETl, DET2, spacer, ek and SH2 are as described below.
  • DETl (“defined epitope tag 1 ") (SEQ ID NO: 1) is an 11 amino acid sequence found in the Human Immunodeficiency Virus Type 1 (HIV- 1) envelope protein gpl20 (or gpl60).
  • Monoclonal antibodies to various epitopes of HIV-1 gpl20 (or g ⁇ l60) are known in the art, see, for example U.S. Patent
  • 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-1 gpl60 molecule from strain BH10 (Ratner et al., Nature, 313:277-284 (1985)). This tag was used for detection of expression (by Western blot), for purification of the protein (by affinity chromatography), and for configuring assays in which the fusion protein was 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 BamHl 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 human src SH2 domain or, as described below, a construct used in the preparation of the human src SH2 domain.
  • each DETl-DET2-spacer-ek-SH2 was designed such that the indicated restriction sites (BamHl 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 or 3 below to create a DETl-DET2-spacer-ek-SH2 tagged protein.
  • the DNA sequence encoding each DETl-DET2-spacer-ek-SH2 constructs 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.
  • coli transcription and translation regulatory sequences and a downstream cloning site compatible with Xbal Although any suitable vector would yield similar results(e.g., pET-1 la; Novagen, Inc.), the vector used in the instant experiments was EL 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, Greene Publishing and Wiley-Interscience, N.Y. (hereinafter F.A. Ausubel et al.).
  • the specific vector pEAlKnRBS3 is described in Bergsma et al, 1991, J. Biol. Chem. 266:23204-23214.
  • the chicken src SH2 domain was expressed as DET1-DET2- spacer-SH2. Then, the other was inserted into this vector in place of chicken src to express protein in the form DETl-DET2-spacer-ek-spacer-SH2.
  • Procedure 1 Cloning and Expression of chicken src SH2 domain containing tags DETl 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. Proc. Natl. Acad. Sci. USA 83:4228) 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. coli strain AR120. In this host strain, 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 AR120 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,T. and Hanafusa, 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 MM294cI + .
  • the construct DETl-DET2-spacer-ek-src SH2 was confirmed by DNA sequencing (SEQ ID NO: 5) and induced in E. coli strain AR120 as described in Procedure 1 above.
  • a Coomassie-Blue-stained, Western- blot-positive induced protein band with an apparent molecular weight of 16,000 was observed after nalidixic acid induction.
  • Binding Assays The potency of compounds at the human SH2 domain was determined based on the ability of such compound to selectively inhibit the SH2 domain from binding to its respective specific pY peptide.
  • the binding assay for the human SH2 domain and pY peptide was 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. 17-0618-01
  • 2 ul of 2 mg/ml of MAB178.1 10 pmol of the subject SH2 domain fusion protein was added to one or more wells.
  • TBS-T tris buffered saline plus 0.05% tween-20
  • TBS-T tris buffered saline plus 0.05% tween-20
  • 90 ul of TBS-T was then added to each well.
  • the specific pY biotinylated peptide was diluted to a concentration of 1.0 uM in TBS-T (this peptide can be obtained from Bachem Bioscience of Pennsylvania, Genosys Biotechnologies of Texas and California Peptide Research of California). 10 ul was aliquoted per well to yield a final concentration of 0.1 uM (approx.
  • 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 ligand used in the above Binding Assays is as follows.
  • Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for the human src SH2 domain.
  • Tables I and II illustrate the activity of SH2 antagonists at the human src SH2 domain.
  • Example 3 Activity of src SH2 Domain Antagonists The compounds of this invention which are antagonist of the human src SH2 domain are tested for their potency to inhibit osteoclast mediated bone reso ⁇ tion in the fetal rat long bone (FRLB) assay as described in Raisz LG (1965) J Clin Invest 44: 103-116, Stern PH et al., (1979) Skeletal Research: An experimental Approach. New York:Academic Press, 21-59 and Votta BJ et al., (1994) Bone 15:533-538).
  • FRLB fetal rat long bone
  • Sprague Dawley rats (Taconic Farms, Germantown, NY) are injected subcutaneously with 200 ⁇ Ci of 45caCl2 on day 18 of gestation, housed overnight, then anesthetized with Innovar- Vet (Pittman- Moore, Mundelein, IL) and sacrificed by cervical dislocation. Fetuses are removed aseptically and radii and ulnae were dissected free of surrounding soft tissue and cartilaginous ends.
  • the bones are cultured 18-24 hours in BGJt ⁇ medium (Sigma) containing 1 mg/ml bovine serum albumin, then transferred to fresh medium and cultured for an additional 48 hours in the absence or presence of a compound which is an antagonist of the human src SH2 domain.
  • BGJt ⁇ medium Sigma
  • bovine serum albumin 1 mg/ml bovine serum albumin
  • Data is expressed as the % calcium released from treated bones as compared to corresponding control bones.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 610-270-5023 (B) TELEFAX:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • Gly Lys lie Thr Arg Arg Glu Ser Glu Arg Leu Leu Leu Asn Ala Glu 35 40 45 Asn Pro Arg Gly Thr Phe Leu Val Arg Glu Ser Glu Thr Thr Lys Gly 50 55 60
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Physical Education & Sports Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une méthode de traitement d'une maladie de résorption osseuse chez un patient, cette méthode consistant à administrer au sujet une dose thérapeutique efficace d'un composé qui forme une fixation ou une liaison covalente avec cys 185 du domaine src SH2. Les composés préférés pour une utilisation dans ladite méthode sont les composés de l'invention qui sont représentés par la formule (I) ou bien leur sel, hydrate ou solvate pharmaceutiquement acceptables. Dans la formule, X représente OR'', SR'', NR'', R'''; R'' représente H, méthyle, alkyle; R''' représente CONH2, CONHMe, CO NHalkyle, SONH2, SONHMe, SONH alkyle, SO2NH2, SO2NHMe, SO2NH alkyle; n représente 0,1 ou 2; R représente H, CH2CH(NHCOR'''')CONHR'''''; une fraction organique; R'''' représente glu-glu-ileu-glu-NH2, peptide, peptidomimétique, alkyle, alkyle substitué, aryle, aryle substitué, hétéroaryle, hétéroaryle substitué; R' représente H, peptidomimétique; ou R, R' représentent un système d'anneau fusionné substitué par H ou peptidomimétique. Cette invention concerne également une composition pharmaceutique contenant un excipient pharmaceutique approprié et un composé de formule (I).
PCT/US1998/004699 1997-03-10 1998-03-10 Procede permettant d'antagoniser le domaine humain src sh2 Ceased WO1998040093A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002283472A CA2283472A1 (fr) 1997-03-10 1998-03-10 Procede permettant d'antagoniser le domaine humain src sh2
EP98910294A EP1007076A4 (fr) 1997-03-10 1998-03-10 Procede permettant d'antagoniser le domaine humain src sh2
JP53972698A JP2001514662A (ja) 1997-03-10 1998-03-10 ヒトｓｒｃ ＳＨ２ドメインの拮抗方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4065897P 1997-03-10 1997-03-10
US60/040,658 1997-03-10

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14218498A A-371-Of-International 1997-03-10 1998-09-02
US10/119,235 Continuation US20030096760A1 (en) 1997-03-10 2002-04-08 Method of antagonizing the human SRC SH2 domain

Publications (1)

Publication Number Publication Date
WO1998040093A1 true WO1998040093A1 (fr) 1998-09-17

Family

ID=21912217

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/004699 Ceased WO1998040093A1 (fr) 1997-03-10 1998-03-10 Procede permettant d'antagoniser le domaine humain src sh2

Country Status (4)

Country Link
EP (1) EP1007076A4 (fr)
JP (1) JP2001514662A (fr)
CA (1) CA2283472A1 (fr)
WO (1) WO1998040093A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6576766B1 (en) 1997-11-12 2003-06-10 Ariad Pharmaceuticals, Inc. Signal transduction inhibitors, compositions containing them

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580979A (en) * 1994-03-15 1996-12-03 Trustees Of Tufts University Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions
AU4440496A (en) * 1995-02-10 1996-08-22 Smithkline Beecham Corporation Use of src SH2 specific compounds to treat a bone resorption disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP1007076A4 *
WANG Y-C J, HANSON M A: "PARENTERAL FORMULATIONS OF PROTEINS AND PEPTIDES: STABILITY AND STABILIZERS", JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY., PARENTAL DRUF ASSOCIATION, US, vol. 42, no. 25, 1 January 1988 (1988-01-01), US, pages S04 - S25, XP002911772, ISSN: 0279-7976 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6576766B1 (en) 1997-11-12 2003-06-10 Ariad Pharmaceuticals, Inc. Signal transduction inhibitors, compositions containing them

Also Published As

Publication number Publication date
CA2283472A1 (fr) 1998-09-17
EP1007076A4 (fr) 2004-10-27
JP2001514662A (ja) 2001-09-11
EP1007076A1 (fr) 2000-06-14

Similar Documents

Publication Publication Date Title
US6794144B1 (en) Methods and materials for generating SH3 domains with tailored binding properties
EP0846127A1 (fr) Derives oligopeptidiques acyles presentant une activite inhibant des signaux cellulaires
WO2019148194A2 (fr) Inhibiteurs peptidyliques de l'interaction calcineurine-nfat
EP0772773B1 (fr) PEPTIDES SE FIXANT AUX SH3 DE Src ET LEURS METHODES D'ISOLEMENT ET D'UTILISATION
WO1997007131A1 (fr) Oligopeptides acyles divers
JP4603240B2 (ja) ＮＦ−κＢ活性化受容体のインヒビターおよびその使用
JP2001103993A (ja) 環状ペプチド及びセリンプロテアーゼ阻害剤
EP0727211A1 (fr) Emploi de composés spécifiques src SH2 dans le traitement de maladies de résorption osseuse
WO1997002023A1 (fr) UTILISATION DE COMPOSES SPECIFIQUES DU DOMAINE Stat 6 SH2 POUR LE TRAITEMENT DES REACTIONS ALLERGIQUES
WO1998040093A1 (fr) Procede permettant d'antagoniser le domaine humain src sh2
US20030096760A1 (en) Method of antagonizing the human SRC SH2 domain
WO1997002024A1 (fr) Utilisation de composes se liant specifiquement au domaine stat 5 sh2 pour augmenter l'erythropoiese
US6303574B1 (en) Scr SH3 binding peptides and methods of isolating and using same
JPH092974A (ja) 血球増生を促進するためのｈｃｐ ｓｈ２特異的化合物の使用
US7045617B2 (en) Bisbubstrate inhibitors of kinases
JP3819712B2 (ja) 細胞内信号伝達を攪乱させる合成ペプチド
Marastoni et al. HIV protease inhibitors: synthesis and activity of N-aryl-N′-hydroxyalkyl hydrazide pseudopeptides
AU4923796A (en) Use of lck sh2 specific compounds to treat autoimmune diseases and allograft rejection
US20030148377A1 (en) Binding compounds and methods for identifying binding compounds
WO2005117932A1 (fr) Inhibiteurs du substrat de src tyrosine kinases tenant lieu d'agents therapeutiques agents
WO2008069290A1 (fr) Médicament anti-hiv comprenant une protéine de fusion contenant de l'actinohivine multimérique, polypeptide constituant le médicament, gène codant pour le polypeptide et procédé de fabrication du médicament anti-hiv
KR100236473B1 (ko) 항암 효과를 갖는 아자펩타이드 파네실 전이효소 저해제
MXPA97006130A (en) Use of specific compounds of lck sh2 to treat autoimmune diseases and the rejection of aloinjer
US20110294728A1 (en) Antiviral Polypeptides
JPWO2007043715A1 (ja) Ｒｎａ結合ペプチド

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 09142184

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1998910294

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2283472

Country of ref document: CA

Ref country code: JP

Ref document number: 1998 539726

Kind code of ref document: A

Format of ref document f/p: F

Ref country code: CA

Ref document number: 2283472

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1998910294

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998910294

Country of ref document: EP