EP0942931A2 - Modulation d'interactions entre myosine et integrines - Google Patents

Modulation d'interactions entre myosine et integrines

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Publication number
EP0942931A2
EP0942931A2 EP97952309A EP97952309A EP0942931A2 EP 0942931 A2 EP0942931 A2 EP 0942931A2 EP 97952309 A EP97952309 A EP 97952309A EP 97952309 A EP97952309 A EP 97952309A EP 0942931 A2 EP0942931 A2 EP 0942931A2
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EP
European Patent Office
Prior art keywords
integrin
myosin
peptide
agent
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97952309A
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German (de)
English (en)
Inventor
Alison L. Jenkins
David R. Phillips
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Millennium Pharmaceuticals Inc
Original Assignee
COR Therapeutics Inc
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Publication date
Application filed by COR Therapeutics Inc filed Critical COR Therapeutics Inc
Publication of EP0942931A2 publication Critical patent/EP0942931A2/fr
Withdrawn legal-status Critical Current

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70557—Integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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 present invention relates to the interaction of integrins with components of the cellular cytoskeleton.
  • integrins with components of the cellular cytoskeleton.
  • myosin and the cytoplasmic domain of ⁇ 3 integrin has been discovered and a method of identifying direct interactions between integrin cytoplasmic tails and cellular proteins is described.
  • Integrins are a family of ⁇ heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al, Science (1995) 268:233-239). Integrins also bind to the actin cytoskeleton through a series of intermediate proteins, and thus provide a link between the extracellular matrix and the intracellular cytoskeleton and its associated motile machinery. Such transmembrane linkages are required for cell migration.
  • integrin family also participate in signal transduction. This is evidenced by an alteration in the adhesive affinity of cell surface integrins in response to cellular activation, termed inside-out signal transduction. Additionally, effects on intracellular signaling pathways following integrin-mediated adhesion have been observed, termed outside-in signal transduction.
  • the integrin family consists of 15 related known ⁇ subunits ( ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ E, ⁇ V, ⁇ ll , ⁇ L, ⁇ M, and ⁇ X) and 8 related known ⁇ subunits ( ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, and ⁇ 8). Luscinskas et al, FASEB J, 8: 929-938 (1994). Integrin ⁇ and ⁇ subunits are known to exist in a variety of pairings as indicated in Figure 1. Integrin ligand specificity is determined by the specific pairing of the ⁇ and ⁇ subunits, although some redundancy exists as several of the integrins are known to bind the same ligand.
  • talin a 235 kD vinculin and actin binding protein, binds to the cytoplasmic domains of ⁇ llb and ⁇ 3 in a solid phase binding assay.
  • Paxillin a vinculin binding signaling protein also binds to the cytoplasmic domain of the ⁇ l integrin.
  • paxillin was postulated as being the substrate for and tyrosine phosphorylated by tyrosine kinase ppl25 FAK .
  • the actin binding protein filamin has been shown to bind to the cytoplasmic tail of the ⁇ 2 integrin subunit in vitro and co-immunoprecipitated and co- localized with ⁇ 2 integrins in vivo.
  • Platelet aggregation induced by a number of agonists results in the phosphorylation of tyrosine residues in the ⁇ 3 cytoplasmic tail. Law et al, J. Biol. Chem 271 : 10811-10815 (1996). In some respects, the phosphorylation of both tyrosine residues was necessary for binding to certain signaling proteins, whereas other signaling proteins bound following monophosphorylation. Furthermore, adhesion to vitronectin by cells transfected with ⁇ v ⁇ 3 induces a robust tyrosine phosphorylation of the ⁇ 3 subunit. Blystone et al, J. Biol. Chem 271:31458-31462 (1996).
  • Point mutations in homologous domains in ⁇ l- and ⁇ 2 -containing integrins also modulate function, as these mutations affect integrin-cytoskeletal interactions by reducting focal adhesions, A. A. Reszka et. al, J. Cell Biol. 117:1321-1330 (1992), and integrin activation, M. L. Hibbs et. al., J. Exp. Med. 174:1227-1238 (1991), respectively.
  • Tyrosine kinases similarly were found to be essential in regulating the cytoskeletal attachment of ⁇ llb ⁇ 3. Schoenwaelder et al, J. Biol. Chem. 269(51):32479-32487 (1994).
  • the platelet plasma membrane is coated by a lattice-like structure, known as the membrane skeleton, that is composed of short actin filaments, actin-binding protein, spectrin, vinculin and various other proteins, not all yet identified. Fox et al, J. Biol.
  • Myosin is a contractile protein that interacts with actin to produce contraction or movement.
  • the term "myosin” broadly refers to a diverse superfamily, comprised of at least 11 classes, of molecular motors capable of translocating actin filaments or of translocating vesicles or other cargo on fixed actin filaments by.
  • One characteristic of all myosins is their ability to reversibly bind to actin and to hydrolyze MgATP. See Figure 5 and J. R. Sellers and H.V. Goodson, Protein Profile 2:1323-1339 (1995).
  • the head or motor domain contains nucleotide and actin binding sites and is the most conserved region of the myosin superfamily.
  • the neck domain consists of a long single alpha helical strand from the heavy chain which is stabilized by the binding of light chain subunits.
  • the tail region which serves to anchor myosin so that it can translocate actin, is the most diverse primary sequence of all the regions and may serve to anchor certain myosin isoforms to cell or organelle membranes. It has been suggested that myosin clustering within a cell may occur on membranes or on actin filaments themselves. Titus, Trends in Cell Biology 7:119 (1997). However, the precise biochemical mechanism of interaction between the myosin tail and cytocellular structures has not heretofore been described.
  • the present invention is based in part on the discovery that the contractile protein myosin binds to the cytoplasmic domain of the ⁇ 3 integrin subunit. Discovery of this association was based on experiments involving the phosphorylation of tyrosine residues in the integrin cytoplasmic domain. Specifically, the integrin-myosin interaction typically is associated with the phosphorylation of one or more tyrosine residues on the integrin cytoplasmic domain. However, interactions that do not include phosphotyrosine residues are not excluded from the scope of the present invention.
  • the present invention provides peptide fragments of the cytoplasmic domains of integrin ⁇ subunits, which can be used to identify signaling and cytoskeletal proteins which bind directly to integrin cytoplasmic domains.
  • the binding of myosin proteins to integrins is detected; however, the binding of integrins to other cytoskeletal proteins or to other signaling partners also are contemplated.
  • These peptide fragments can also be tyrosine phosphorylated (either mono- or di-phosphorylated) to identify proteins that bind to integrin cytoplasmic domains in a phosphorylation-dependent manner.
  • such peptides do not necessarily require phosphorylation to be useful in the methods disclosed herein.
  • the present invention includes methods for identifying an agent which blocks or modulates the interaction of an integrin with myosin comprising the steps of: a) incubating a peptide comprising the phosphorylated cytoplasmic domain of the ⁇ subunit of the integrin with myosin and with an agent, and b) determining whether said agent blocks or modulates the binding of myosin to said peptide.
  • the present invention also provides methods for modulating, reducing, blocking and stimulating the association of an integrin with a cytoskeletal protein. Agents that block integrin/cytoskeletal associations can be used to modulate biological and pathological processes which require an integrin-mediated cytoskeletal attachment.
  • such methods and agents can be used to modulate cellular attachment or adhesion, migration, proliferation and differentiation, and clot retraction.
  • Pathological processes involving such cellular actions include thrombosis, inflammation, tumor metastasis, wound healing and others noted above.
  • the present invention further provides methods of reducing the severity of pathological processes which require integrin-mediated cytoskeletal association. Since phosphorylation is required for the association of integrins with certain cytoskeletal proteins, agents which block integrin/cytoskeletal association, such as agents which block tyrosine phosphorylation and agents which dephosphorylate phosphorylated tyrosines, and agents that otherwise interfere with integrin-myosin binding can be used in therapeutic methods.
  • Figure 1 shows the pairing of ⁇ and ⁇ integrin subunits.
  • Figure 2 shows the cytoplasmic domain of various integrin subunits.
  • Figure 3 shows the binding of ⁇ 3 peptides to platelet proteins.
  • FIG. 4 shows the binding of ⁇ 3 peptides to purified platelet myosin.
  • Figure 5A-5B demonstrates that the 200 kD ⁇ 3 -binding protein activity sedimented with the platelet cytoskeletal fraction.
  • Figure 6A-6C shows the biding of ⁇ 3 peptides to purified platelet myosin.
  • Figure 7A-7D demonstrates the binding of diphosphorylated ⁇ 3 to purified platelet myosin cleaved by controlled proteolysis .
  • Figure 8A-8B presents flow cytometric analysis and clot retracting ability of cells expressing mutant ⁇ 3.
  • the present invention provides isolated peptides corresponding to the cytoplasmic domain of the ⁇ subunit of an integrin, as well as allelic variants of the integrin cytoplasmic domain and conservative amino acid substitutions of the cytoplasmic domain.
  • the preferred peptide comprises a sequence derived from the cytoplasmic domain of an integrin wherein the tyrosine residues are phosphorylated, typically at domain comprising a NPXY motif (Filardo et ⁇ /. (1995) J. Cell Biol. 130:441-50).
  • Such peptides are about 5, 10, 13, 15, 17, 18, 19, 20, 23, 25, 30, 35, 40, 45, 50 or more amino acids in length.
  • the most preferred embodiment consists essentially of the diphosphorylated peptide corresponding to residues 740-762 of ⁇ 3 having the following sequence: D-T-A-N-N-P-L-Y(PO3)-K-E-A-T-S-T-F-T-N-I-T-Y(PO3)-R-G-T-COOH (SEQ LD No. ).
  • the peptides of the invention include peptides corresponding the cytoplasmic domain of naturally occurring allelic variants of integrins. This variation results in peptides that have a slightly different amino acid sequence than that specifically recited above.
  • a peptide is said to be isolated when physical, mechanical or chemical methods are employed to remove the peptide protein from peptides having different primary amino acid sequences or from cellular constituents if the peptides are derived from natural cellular sources or recombinantly expressed in a suitable host cell.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated peptide.
  • the peptides of the present invention further include peptides having conservative amino acid substitutions compared to a peptide corresponding to the cytoplasmic domain of a naturally occurring integrin.
  • a conservative amino acid substitution refers to alterations in the amino acid sequence that do not adversely affect the ability of the peptide to bind to myosin.
  • a substitution, insertion or deletion is said to adversely affect the peptide when the altered sequence prevents the peptide from associating with myosin.
  • the overall charge, structure or hydrophobic/hydrophilic properties of a peptide can be altered without adversely affecting activity of the peptide.
  • amino acid sequence of a peptide can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the ability of the peptide to become associated with myosin.
  • peptides corresponding to the allelic variants of a given integrin or peptides having conservative amino acid substitutions will have an amino acid sequence having at least 75% amino acid sequence identity with a naturally occurring human integrin cytoplasmic domain, more preferably at least 80%), even more preferably at least 90%), and most preferably at least 95%>.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity.
  • the peptides of the present invention include: molecules having the amino acid sequence disclosed as Peptide 1 or peptides corresponding to peptide 1 from ⁇ - 1 , ⁇ - 2, ⁇ -5, ⁇ -6, or ⁇ -7; fragments thereof having a consecutive sequence of at least about 13, 15, 20 or 23 amino acid residues of Peptide 1 or corresponding peptides from ⁇ -1 , ⁇ -2, ⁇ - 5, ⁇ -6, and ⁇ -7, including both tyrosine residues found at positions 747 and 759 of the naturally occurring ⁇ 3; amino acid sequence variants of such sequences wherein at least one amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue.
  • Contemplated variants further include those containing predetermined mutations by, e.g. , homologous recombination, site-directed or PCR mutagenesis, and the corresponding peptides of other animal species, including but not limited to rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primate species; and derivatives wherein the peptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • a detectable moiety such as an enzyme or radioisotope
  • the present invention further provides recombinant DNA molecules (rDNAs) that contain a peptide encoding sequence.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • a peptide encoding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • vector and/or expression control sequences to which one of the peptide encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the peptide encoding sequence included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked peptide encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a peptide encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the peptide encoding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N.J.
  • Expression vectors compatible with eukaryotic cells can also be used to form a rDNA molecules the contains a peptide encoding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (we ⁇ ) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.)
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a peptide of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a peptide are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the peptide product.
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NTH Swiss mouse embryo cells NTH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines.
  • CCL61 Chinese hamster ovary (CHO) cells available from the ATCC as CCL61
  • NTH Swiss mouse embryo cells NTH/3T3 available from the ATCC as CRL 1658
  • BHK baby hamster kidney cells
  • Any prokaryotic host can be used to express a peptide-encoding rDNA molecule.
  • the preferred prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al. , Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al. , Molecular Cloning. A
  • Successfully transformed cells i.e., cells that contain a rD ⁇ A molecule of the present invention
  • cells resulting from the introduction of an rD ⁇ A of the present invention can be cloned to produce single colonies.
  • Cells from those colonies can be harvested, lysed and their D ⁇ A content examined for the presence of the rD ⁇ A using a method such as that described by Southern, J Mol. Biol. 98:503, 1975, or Berent et al, Biotech. 3:208, 1985 or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a peptide that uses one of the peptide encoding nucleic acid molecules herein described.
  • the production of a recombinant form of a peptide of the invention typically involves the following steps:
  • a nucleic acid molecule is obtained that encodes a peptide.
  • the peptide encoding nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the peptide encoding sequences.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the peptide.
  • the peptide is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with peptide encoding sequences to produce peptide according to the invention.
  • Recombinant peptide tyrosine residues may be phosphorylated using standard procedures.
  • Another embodiment of the present invention provides methods for identifying agents that reduce or block the association of an integrin with a cytoskeletal protein, such as myosin.
  • a cytoskeletal protein such as myosin.
  • an integrin or integrin peptide comprising the tyrosin phosphorylated ⁇ subunit cytoplasmic domain (such as Peptide 1 disclosed herein) is mixed with a cytoskeletal protein such as myosin in solution or attached to a solid support, in the presence and absence of an agent to be tested.
  • the two mixtures are analyzed and compared to determine if the agent reduced or blocked the association of the integrin with the cytoskeletal protein.
  • Agents that block or reduce the association of an integrin with the cytoskeletal protein will be identified as decreasing the amount of association present in the sample containing the tested agent.
  • Assays that result in the modulation of integrin association with a cytoskeletal protein such as myosin may utilize any available means to detect or monitor integrin- myosin interactions. Such methods include the direct monitoring of integrin-myosin binding or the monitoring of secondary endpoints such as those exhibited when using permeabilized platelets (Pumiglia et al, (1992) Biochem J. 286:441-9). Such methods of detection are widely available as disclosed by Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • solid phase assays are widely employed wherein either binding partner is attached to a solid support.
  • the integrin or peptide derived from the tyrosine phosphorylated ⁇ subunit cytoplasmic domain is typically labeled and incubated with myosin attached to the solid support.
  • Any label may be used, including, but not limited to radioactive, enzymatic, florescent or other dye labels.
  • the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to the integrin or integrin peptide or to interfere with the association of the integrin or integrin peptide and myosin.
  • the amount of bound label is used as a measure of the ability of the particular agent to bind to the integrin or integrin peptide or to interfere with the association of the integrin or integrin peptide and myosin.
  • Another technique useful for screening for agents which modulate the interaction between an integrin or integrin peptide comprising the tyrosine phosphorylated ⁇ subunit cytoplasmic domain and a cytoskeletal protein such as myosin is the use of high throughput screening for compounds having suitable binding affinity to the integrin or integrin peptide comprising the ⁇ subunit cytoplasmic domain.
  • Such high throughput screening systems are widely available such as those described in European Patent Application 84/03564. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with the integrin or integrin peptide comprising the tyrosine phosphorylated ⁇ subunit cytoplasmic domain and washed. Bound integrin or integrin peptide comprising the ⁇ subunit cytoplasmic domain is then detected by methods well known in the art. Purified integrin or integrin peptide comprising the ⁇ subunit cytoplasmic domain can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
  • Alternative assay formats include the use of competitive screening assays in which neutralizing antibodies capable of specifically binding integrin or an integrin peptide comprising the tyrosine phosphorylated ⁇ subunit cytoplasmic domain compete with a test compound or agent for binding to the integrin or integrin peptide.
  • Particularly useful antibodies are those raised against small peptides comprising an integrin ⁇ subunit cytoplasmic domain such as Peptide 1 wherein the tyrosine residues have been phosphorylated. In this manner, the antibodies can be used to detect the ability of the agent to competitively bind to the integrin or peptide.
  • an agent is said to reduce or block integrin or integrin peptide/myosin association when the presence of the agent decreases or prevents the integrin or peptide from becoming associated with myosin.
  • One class of agents will reduce or block the association by binding to the integrin or integrin peptide while another class of agents will reduce or block the association by binding to the integrin binding domain of myosin.
  • Other classes of agents include those that block the phosphorylation of the cytoplasmic domain or dephosphorylate the cytoplasmic domain.
  • Agents that are assayed in the above methods can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the integrin with myosin.
  • An example of randomly selected agents is the use a chemical library, a peptide combinatorial library or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the contact sites of the integrin/myosin complex pair.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the tyrosine phosphorylated cytoplasmic domain of the integrin or the integrin contact site on myosin.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or " peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem. 30:1229, which are incorporated herein by reference). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • agents of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of the tyrosine phosphorylated cytoplasmic domain of the integrin ⁇ subunit.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of the tyrosine phosphorylated cytoplasmic domain of an integrin or with the integrin binding domain of myosin.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the integrin ⁇ subunit cytoplasmic domain intended to be targeted by the antibodies.
  • a peptide is preferably Peptide 1.
  • Critical regions include the contact sites involved in the association of the integrin with myosin, including the antigenic sites which encompass or include the phosphorylated tyrosine residues of Peptide 1.
  • Antibody agents are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptide haptens alone, if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten or is the integrin or signaling complex itself.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab,
  • Fab' of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of receptor can also be produced in the context of chimeras with multiple species origin.
  • the antibodies thus produced are useful not only as modulators of the association of an integrin with myosin, but are also useful in immunoassays for detecting integrin mediated signaling and for the purification of integrin-associated signaling proteins.
  • integrins play important roles in intracellular signaling, cellular attachment, cellular aggregation and cellular migration.
  • Agents that modulate, reduce or block the interactions of an integrin with a cytoskeletal component such as myosin can be used to modulate biological and pathologic processes associated with integrin function and activity.
  • Eosinophils although they represent only a minor fraction of circulating leukocytes, are major inflammatory infiltrators in a number of pathological conditions, including asthma, atopic skin reactions, parasitic infestation and some delayed-type hypersensitivity reactions.
  • Kuijpers et al J. Exp. Med. 178:279-284 (1993).
  • ⁇ l integrin deficient mice possess impaired blood cell migration.
  • Hirsch et al Nature 380:171 (1996). Together these studies suggest that integrins are important players in cell migration.
  • ⁇ 3 integrins in particular are often expressed on highly motile cells, such as melanoma cells. Albeda et al, Cancer Res. 50:6757-6764 (1991).
  • myosin classes themselves play different roles in mediating cell motility, and no single myosin is believed to be fully responsible for cell movement. Forces must be generated to extend membrane processes at the front of the cell, and also at the rear to extend the cell body forward.
  • myosin I is thought to be involved in the extension of pseudopods in the front portion of migrating cells, while myosin Il-based contraction occurs in the rear of the cell (Lauffenburger et al 1996, 84; 359-369).
  • Integrins are also differentially expressed within the cell. It has been hypothesized that cell migration is regulated by transient activation of an integrin at the cell front, with subsequent deactivation at the cell rear.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by an integrin.
  • the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
  • a biological or pathological process mediated by an integrin or integrin signaling refers to the wide variety of cellular events in which an integrin binds a cytoskeletal component such as myosin.
  • biological processes include, but are not limited to, cellular attachment or adhesion to substrates and other cells, cellular aggregation, cellular migration, cell proliferation, and cell differentiation.
  • pathological state in reference to the direct interaction of an integrin to myosin includes, but is not limited to thrombosis, inflammation, angiogenesis, tumor metastasis, wound healing (including cutaneous wounds such as burn wounds, donor site wounds from skin transplants and cutaneous, decubitis, venous stasis and diabetic ulcers), acute coronary syndrome, myocardial infarction, unstable angina, refractory angina, occlusive coronary thrombus occurring post-thrombolytic therapy or post-coronary angioplasty, a thrombotically mediated cerebrovascular syndrome, embolic stroke, thrombotic stroke, transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus, coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic pu ⁇ ura, thromboangiitis obliterans, thro
  • the phrase "cell mobility impairment” is associated with conditions related to sperm motility, inflammation, resistance to infection, immune function, autoimmune disease, wound repair, cancer, immune diseases, and spastic diseases and disorders such as gastrointestinal cramps and contractions related to pregnancy.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, thrombosis is the deleterious attachment and aggregation of platelets while metastasis is the deleterious migration and proliferation of tumor cells.
  • These pathological processes can be modulated using agents which reduce or block integrin association to a cytoskeletal component such as myosin.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • an agent is said to modulate thrombosis when the agent reduces the attachment or aggregation of platelets.
  • agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention that reduces thrombosis by blocking integrin cytoskeletal association can be administered in combination with other anti-thrombotic agents.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which block integrin/cytoskeletal association. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt.
  • the preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt.
  • the most preferred dosages comprise 0.1 to 1 ⁇ g kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as anticoagulant agents, thrombolytic agents, or other antithrombotics, including platelet aggregation inhibitors, tissue plasminogen activators, urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • Lysates from either the supernatant fraction of the insoluble pellet were subjected to 2D-gel analysis and the ratio of ⁇ 3 phosphorylation (with basal phosphorylation subtracted)/total amount of ⁇ 3 present in the samples as determined by densitometry of five separate experiments.
  • Only about 5% of the ⁇ llb ⁇ 3 was found in the cytoskeleton of unstimulated platelets.
  • Platelet samples of 0.5 ml were then stirred at 37°C in a whole blood lumiaggregometer and various agonists and conditions were examined.
  • 4X non- reducing Laemmli sample buffer containing vanadate 37mM Tris pH 6.8, 11.8%> (v/v) glycerol, 2.36% (w/v) SDS, 2mM sodium orthovanadate and 0.002%> (w/v) bromophenol blue (final concentration) was added immediately after aggregation and samples were boiled for 5 minutes.
  • platelets were lysed immediately after aggregation by the addition of an equal volume of ice cold 2X Triton X-100 lysis buffer (1% (v/v)Triton X-100, lOOmM NaCl, 20mM Tris pH 7.0, 2 mM ethylenedinitrilo-tetraacetic acid, 2 mM [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid, 20 ⁇ g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 ⁇ M leupeptin, 4 mM sodium orthovanadate, 2 mM benzamidine, 50 ⁇ g/ml diisopropyl fluorophosphate, 5 mM sodium pyrophosphate (final concentrations)).
  • 2X Triton X-100 lysis buffer 1% (v/v)Triton X-100, lOOmM NaCl, 20mM Tris pH 7.0
  • the lysate was then centrifuged for 6 minutes at 15,000 x g to remove any Triton X-100 insoluble material formed during aggregation.
  • the supernatant was reserved and 100 ⁇ l of 2X RIPA was added to the pellet and sonicated for 20 minutes in a Branson 5120 Sonicator to resolubilize the pellet.
  • Non-reducing sample buffer (as described above) was added to each of the samples (supernatant and resolubilized pellet) and boiled for 5 minutes.
  • platelets are lysed in RIPA buffer (1% (w/v) Triton X- 100, 1% (w/v) deoxycholic acid, 0. 1% sodium dodecyl sulfate, 5 mM ethylenedinitrilo- tetraacetic acid, 20 mM Tris pH 7.5, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 ⁇ g/ml leupeptin, 20 ⁇ g/ml aprotinin (final concentrations)).
  • RIPA buffer 1% (w/v) Triton X- 100, 1% (w/v) deoxycholic acid, 0. 1% sodium dodecyl sulfate, 5 mM ethylenedinitrilo- tetraacetic acid, 20 mM Tris pH 7.5, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 ⁇ g
  • platelets were lysed in Triton X-100 buffer (1% (v/v) Triton X-100, 137 mM NaCl, 2 mM ethylenedinitrilo-tetraacetic acid, 20 mM Tris PH 8, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 ⁇ /ml leupeptin, 20 ⁇ /ml aprotinin (final concentrations)) and cytoplasmic actin filaments were sedimented by centrifugation at 15600 x g for l5 min at 4°C.
  • Triton X-100 buffer 1% (v/v) Triton X-100, 137 mM NaCl, 2 mM ethylenedinitrilo-tetraacetic acid, 20 mM Tris PH 8, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 ⁇
  • Peptides consisting of cytoplasmic regions of ⁇ 3 were synthesized by SynPep corporation using solid phase Fmoc chemistry. Peptides were dissolved in water and diluted as needed.
  • HBB HEPES blot buffer
  • the blot is blocked in HBB containing 4% bovine serum albumin overnight at 4° C and probed with 1 ⁇ M biotinylated peptide in HBB containing 0.5% bovine serum albumin for 3 hours at room temperature. After washing in TBS/0.01% NP40 three times at 4° C, peptide- reactive bands are visualized by incubating the blots in horseradish peroxidase-conjugated streptavidin and employing ECL detection.
  • Myosin was purified from human platelets as described by J.L. Daniel and J.R. Sellers, Methods Enzymol. 215:78-88 (1992).
  • Solid Phase Binding Assay Immulon 4 plates were coated with platelet myosin overnight at 4°C. Plates were washed in TBS 0.01% Brij, blocked in 4%BSA/TBS 0.1%Tween (TBST) and incubated with 10 ⁇ M biotinylated peptides for 3 hours at room temperature. Plates were incubated with bound peptide was detected with peroxidase substrate (ABTS) at 650 nm using a plate reader (Molecular Devices).
  • ABTS peroxidase substrate
  • a ligand blot protocol was developed in order to detect direct interactions between phosphorylated integrin cytoplasmic tail peptides and proteins in cellular lysates. See, Crowley et «/., J. Biol. Chem. 271 :1145-1152 (1996).
  • the basic ligand blotting methodology has not previously been applied to the discovery of proteins that bind to integrin cytoplasmic domains, phosphorylated or not. Proteins separated on SDS-PAGE are transferred to nitrocellulose, denatured with Guanidine HCI under reducing conditions and renatured gradually by dilution of Guanidine HCI in reducing conditions as set forth above. The blot is then blocked overnight and probed with biotinylated phosphopeptides, detected with streptavidin HRP and visualized using chemiluminescence.
  • a phosphorylated peptide corresponding residues 740-762 of ⁇ 3 was synthesized and coupled to biotin at the amino terminus:
  • Clot retraction experiments were performed as described by Chen et al. ((1995) Blood. 86:2606-15) with minor modifications. In brief, cells were trypsinized, washed twice and resuspended in Dulbecco's Modified Eagle Medium + 25 mM HEPES. 0.5 ml of cell suspension containing 5 x 10 6 cells was mixed with 0. 1 ml fibronectin-depleted plasma in a 12 x 70 mm glass tube treated with Sigmacote. In some experiments, the tyrosine kinase inhibitor genistein was added to 250 ⁇ M. Fibrin clots were formed by adding I U/ml thrombin and allowed to retract at 37°C over a 2 to 3 hour period. Extent of clot retraction was measured by removing and weighing the clot.
  • myosin is the diphosphorylated ⁇ 3-peptide binding protein
  • myosin heavy chain was found to display the same peptide binding specificity as the 200 kD protein in platelet lysates (Fig 4B and 6B): it bound the di-phosphorylated ⁇ 3 peptide but not unphosphorylated ⁇ 3, doubly phosphorylated S752P ⁇ 3, or CD3 zeta IT AM (Peptides 2,3,4, respectively).
  • 10 ⁇ g of purified platelet myosin (lane 1) and platelet lysate (lane 2) were probed with the diphosphorylated ⁇ 3 peptide (1 ⁇ M).
  • phenylphosphate a compound known to compete for phosphotyrosine binding sites was used (see Glenney et al. (1988) J Immunol Methods. 109:277-85). 10 mM phenylphosphate inhibited completely the binding of the diphosphorylated ⁇ 3 peptide to purified myosin in renatured blots (Fig. 6C), indicating that the ⁇ 3-myosin interaction was indeed phosphotyrosine-dependent under these binding conditions.
  • myosin type II
  • myosin II found in most cell types including platelets, is a hexameric molecule consisting of 2 heavy chains and 2 pairs of light chains. The coiled- coil tail regions of myosin II self-associate to form myosin filaments.
  • the present inventors have discovered that the doubly phosphorylated ⁇ 3 integrin tail binds to myosin II purified from platelets. In subsequent experiments, the present inventors have found that the peptide also binds to skeletal muscle myosin.
  • Example 3 Diphospho- ⁇ 3 peptide binds the tail region of myosin.
  • Example 4 The tyrosine residues within the ⁇ 3 cytoplasmic domain are important for ⁇ 3- dependent clot retraction in transfected CHO cells.
  • the tyrosine kinase inhibitor herbimycin A was used as another means of assaying the role of ⁇ 3 phosporylation in myosin binding because of its ability to inhibit platelet- mediated clot retraction.
  • Example 5 Other Mvosin Binding Interactions
  • the presence of novel classes of myosin in platelets has not been formally investigated.
  • the invention includes all types and subtypes of myosin, not limited to those expressed in platelets.
  • Myosin-integrin binding itself could represent a general mechanism for connecting adhesive proteins to the contractile apparatus in platelets and other cells.
  • diphosphorylated ⁇ 3 integrin tail peptide binds to the coiled-coil tail region of myosin.
  • myosin II Members of the myosin II class are found in all animal cells and share many common structural properties. Based on sequence and functional differences myosins are divided into the following subclasses: 1) skeletal and cardiac muscle myosin II and 2) smooth muscle and nonmuscle myosin II. There are at least two genes for nonmuscle myosin II, namely IIA and IIB, that are differentially expressed in cells.
  • Myosin IIA is the subtype found predominantly in platelets, lymphocytes, neutrophils and brush border enterocytes, while neuronal tissue mainly expressly myosin IIB.
  • the diphosphorylated ⁇ 3 peptide binds to skeletal muscle myosin II.
  • the integrin tail binds to a common structural element in the myosin II family, and not just to platelet myosin.
  • NPXY motifs i.e., ⁇ l, ⁇ 5, ⁇ 6 and ⁇ 7 and therefore would be envisaged to bind to members of the myosin superfamily in a similar manner.
  • other proteins that have this motif would be expected to bind to members of the myosin superfamily and related proteins. This binding may or may not require the involvement of phosphorylated tyrosine residues.
  • Zambrano et al, J. Biol. Chem 272:6399-6405 discusses a phosphorylation independent interaction between the Fe65 protein and the ⁇ -amyloid precursor protein which has this motif.
  • Agents which modulate the interaction between myosin and integrins are isolated by incubating a peptide comprising the phosphorylated cytoplasmic domain of the ⁇ subunit of an integrin with myosin in the presence of the agent to be tested. Agents which inhibit or interfere with the interaction(s) between the peptide and myosin are identified by the modulation, decrease or inhibition in peptide-myosin interactions. Any means available to detect agent mediated quantitative or qualitative differences in the interaction between the peptide and myosin can be used. For instance, the ligand blot procedure set forth above is employed.
  • HBB HEPES blot buffer
  • Immulon 4 plates are coated with platelet myosin overnight at 4°C. Plates were washed in TBS 0.01% Brij, blocked in 4%BSA/TBS 0.1%Tween (TBST) and incubated with 10 ⁇ M biotinylated peptide for 3 hours at room temperature. Plates were incubated with bound peptide was detected with peroxidase substrate (ABTS) at 650 nm using a plate reader (Molecular Devices). Modulation of the interaction between an integrin and myosin is detected by a decrease or increase in the detected binding of the biotinylated peptide to the myosin or myosin fragment in the presence of the agent.
  • ABTS peroxidase substrate

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Abstract

La présente invention décrit une interaction directe entre la myosine et le domaine cytoplasmique de protéines membranaires, en particulier les domaines cytoplasmiques phosphorylés de la sous-unité β d'intégrines. L'invention concerne des méthodes d'identification d'agents bloquant la liaison d'intégrine à la myosine; des méthodes d'utilisation d'agents bloquant la liaison d'intégrine à la myosine afin de moduler des procédés biologiques et pathologiques; et des agents bloquant la liaison assistée d'intégrine à la myosine, modulant ainsi le déplacement associé d'une cellule ou d'un composant cellulaire.
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