Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • 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
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • 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
• • 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
• • 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
  • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• Integrins are involved in a number of pathological and physiological processes, including thrombosis, inflammation, and cancer. Other physiological and pathological conditions involving changes in cell adhesiveness are also mediated through integrins.
• transmembrane proteins are oligomeric, being noncovalent associations of two or more different types of polypeptide subunits.
• integrins are heterodimers of two different protein subunits, designated ⁇ and ⁇ .
• the subunits vary in size between 120 and 180 kDa and are each noncovalently associated with a ⁇ subunit.
• the extracellular domain of the integrin molecule forms a ligand binding site; both the ⁇ and ⁇ subunits are involved in forming the ligand binding site.
• integrins A number of different ligands for integrins are known, including collagens, laminin, fibronectin, vitronectin, complement components, thrombospondin, and integral membrane proteins of the immunoglobulin superfamily such as ICAM-1, ICAM-2, and VCAM-1.
• the integrins recognize various short peptide sequences in their ligands.
• integrin function is often caused by changes in the ligand binding affinity of the extracellular domain of the integrins (J.S. Bennett & G. Vilaire J. Clin . Invest . 64:1393-1401 (1979); Altieri et al. J " . Cell Biol .
• Integrin IIb ⁇ 3 (platelet GPIIb-IIIa) , a heterodimer of two type I transmembrane protein subunits, manifests highly regulated changes in ligand binding affinity.
• Affinity state-specific antibodies e.g., PAC1 (Shattil et al . J. Biol . Chem . 260:1107-1114 (1985)
• PAC1 Shattil et al . J. Biol . Chem . 260:1107-1114 (1985)
• Platelet agonists increase the affinity of ⁇ IIb ⁇ 3 (activation) probably by causing changes in the conformation of the extracellular domain (O'Toole et al .
• a method for preparation of proteins for use in structural models or mimics of the cytoplasmic face of multimeric transmembrane proteins such as integrins Proteins of the present invention may be prepared recombinantly or synthetically. However, by using recombinant proteins, limitations of polypeptide length and modest yield encountered in the initial synthetic approaches of the prior art are avoided. Accordingly, it is preferred that at least a portion of the structural model of the present invention be prepared recombinantly. In the model of the present invention, the heterodimeric nature of the ⁇ cytoplasmic domain is mimicked by use of covalent heterodimers of these domains. Helical coiled-coil architecture provides the desired parallel topology and vertical stagger of the tails.
• the model is useful in studying protein interactions with transmembrane proteins such as integrin and screening agents for integrin inhibitory activity and in obtaining structures of integrin cytoplasmic domains.
• transmembrane proteins such as integrin and screening agents for integrin inhibitory activity
• paxillin and paxillin related molecules such as leupaxin and Hie- 5 have high affinity interactions with 4 integrin.
• agents which inhibit the interaction of paxillin and paxillin related molecules with ⁇ 4 integrin are believed to be useful in inhibiting biological responses associated with ⁇ 4 integrins.
• these agents may be useful in inhibiting normal ⁇ 4 integrin activity such as that occurring in wound healing which can lead to scarring.
• These agents can also be used in inhibiting pathological responses of ⁇ 4 integrin such as in atherosclerosis and immune responses associated with conditions including, but not limited to inflammatory bowel disease, arthritis, multiple sclerosis and asthma.
• Figure 1 exemplifies amino acid sequences of recombinant model proteins of integrin cytoplasmic domains.
• Figure 1A shows the N-terminal (SEQ ID NO : 5) and heptad- repeat (SEQ ID NO: 6) structures common to all constructs. In the example shown, these are connected to the Gl- ⁇ lA cytoplasmic domain (SEQ ID NO: 7) .
• Arrows indicate the positions of hydrophobic residues corresponding to positions a and d of the heptad repeats. Positions of the additional Gly insertions in the
• Figure IB shows the integrin-specific sequences of the constructs used in experiments described herein including B1A (SEQ ID NO: 8), B1A (U788A) (SEQ ID NO: 9), BIB (SEQ ID NO: 10), BIC (SEQ ID NO: 11), BID (SEQ ID NO: 12) and B7 (SEQ ID NO: 13) . All integrin peptides correspond to the reported human integrin sequences . Detailed Description of the Invention
• the present invention relates to the production of mimics of the cytoplasmic face of occupied and clustered transmembrane proteins such as integrins consisting of polypeptides comprising a series of ⁇ -helical heptad repeats, preferably 2 to 20, more preferably 3 to 6 , most preferably 4, that mimic a transmembrane domain connected to a cytoplasmic domain of a selected multisubunit transmembrane receptors such as integrins.
• integrins consisting of polypeptides comprising a series of ⁇ -helical heptad repeats, preferably 2 to 20, more preferably 3 to 6 , most preferably 4, that mimic a transmembrane domain connected to a cytoplasmic domain of a selected multisubunit transmembrane receptors such as integrins.
• mimic it is meant that the series of heptad repeats, imitates or replaces the structural features of the transmembrane domain.
• an immobilizing epitope such as a His-Tag sequence or glutathione-S-transferase, is linked to the N-terminus for immobilization of the polypeptide in affinity chromatography .
• the immobilizing epitope be linked to the polypeptide via a Cys-Gly linker.
• a prokaryotic or chemical cleavage site such as a thrombin cleavage site can also be incorporated into the polypeptide at this linkage site.
• ⁇ - helical heptad-repeat it is meant a sequence consisting of substantially helical amphiphilic amino acids having hydrophobic residues at selected positions in the repeat, preferably positions a and d as depicted in Figure 1.
• each repeat is seven a ino acids with hydrophobic residues at the first and fourth positions.
• the heptad repeat comprises the amino acid sequence G-X 1 -L-X 2 -X 3 - -X 4 -G, (SEQ ID NO: 14) wherein X l is a lysine, arginine or ornithine, X 2 and X 4 are glutamic acid or aspartic acid, and X 3 is alanine, serine or threonine.
• the heptad repeats of the polypeptide are preferably identical. However, in some embodiments, each heptad repeat may differ in amino acid sequence .
• the cytoplasmic tail of a transmembrane receptor such as an integrin is linked to the heptad repeat via a glycine residue at the C-terminus of the heptad repeat.
• the polypeptide is predicted to form parallel coiled-coil dimers under physiological conditions.
• trimers and tetramers can also be designed based upon current methods for coiled coil protein design. These coiled-coil structures are likely to better mimic the proximity of transmembrane helices in the natural system and also ensure that a defined topology is maintained between the and ⁇ cytoplasmic tails.
• the coiled-coil of the ⁇ -helical heptad repeat can act as a structural template onto which the cytoplasmic domain of the integrin or other transmembrane protein is attached. This ensures that the two cytoplasmic tails are staggered with respect to one another in a manner that approximates the intact protein.
• a cystine bridge ensures a parallel orientation and a correct stagger of the coiled-coil sequences within this dimer configuration.
• cytoplasmic tails of integrins which can be used include, but are not limited to which, integrin ⁇ subunits such as ⁇ lA (SEQ ID NO: 8), ⁇ lA(Y788A) (SEQ ID NO: 9), ⁇ lB (SEQ ID NO: 10), ⁇ lC (SEQ ID NO: 11), ⁇ lB (SEQ ID NO: 12), ⁇ 7 (SEQ ID NO: 13), and ⁇ 3 and integrin subunits such as ⁇ llb, ⁇ 4 , ⁇ 3A, ⁇ 5 or ⁇ 6A.
• integrin ⁇ subunits such as ⁇ lA (SEQ ID NO: 8), ⁇ lA(Y788A) (SEQ ID NO: 9), ⁇ lB (SEQ ID NO: 10), ⁇ lC (SEQ ID NO: 11), ⁇ lB (SEQ ID NO: 12), ⁇ 7 (SEQ ID NO: 13), and ⁇ 3 and integrin subunits such as ⁇ llb
• polypeptides used in the mimics of the present invention be prepared recombinantly.
• Recombinant preparation of polypeptides overcomes limitations of polypeptide length and modest yield encountered in the initial synthetic approaches of the prior art .
• Methods for recombinant preparation of at least a portion of a polypeptide are well known in the art.
• Polypeptides of the mimics or portions thereof may also be prepared synthetically. Methods for synthetic preparation of polypeptides are well known in the art. Further, methods for combining portions of synthetically and recombinantly prepared peptides into a single polypeptide are known.
• At least one heptad repeat in the series of heptad repeats forming the coiled-coil sequences must differ in amino acid sequence from the other heptad repeats in the series.
• Polypeptides of the model of the present invention are preferably >90% homogenous as determined by reverse phase C18 high pressure liquid chromatography and have a monomer mass that varies by less than 0.1% from that of the desired monomer sequence as determined by electrospray mass spectrometry .
• formation of covalent dimers in aqueous solution can be observed by mass spectrometry and by SDS-PAGE, thus confirming the parallel orientation of the helices.
• the beginning of the integrin cytoplasmic domain sequence provides the hydrophobic residues of a fifth heptad repeat ( Figure 1) . Consequently, direct linkage of the coiled-coil sequence of the ⁇ -helical heptad repeat could induce helical structure in the tail.
• the four heptad repeats constitute 27% of the mass of the construct; therefore, 36% helical content is consistent with the helical structure being limited to these repeats.
• the Gly insertion appears to eliminate ⁇ -helical structure induced in the cytoplasmic domain coiled-coil sequence.
• the enriched 56, 58 and 140 kDa polypeptides have not been identified but have failed to react with antibodies specific for pp60 ⁇ rc , paxillin, ppl25 fak , ⁇ -actinin, vinculin and pp72 ⁇ yk in Western blotting experiments.
• Talin bound to the Gl- and G4- ⁇ lA construct but not to the Y788A-G4 ⁇ lA construct.
• Models of the present invention were also constructed with Gl- and G4- polypeptides of the muscle-specific splice variant ⁇ lD and the ⁇ 7 integrin subunits ( Figure 1) to study binding interactions of various integrin binding proteins.
• the ⁇ lD constructs bound more talin and ⁇ 7 constructs bound more filamin, compared to ⁇ lA.
• G4- constructs of ⁇ lA, ⁇ lD and ⁇ 7 integrin cytoplasmic domains bound more purified filamin than the corresponding Gl- constructs.
• the Gl- ⁇ 7 model protein still bound more filamin than G4- ⁇ lA or G4- ⁇ lD.
• a densitometric evaluation of the Coomassie blue-stained gels indicated that the ⁇ lD construct bound about nine times more talin, and the ⁇ 7 construct bound 8.4 times more filamin than the ⁇ lA model protein.
• the affinity of ⁇ lA for filamin is at least eight fold less than that of ⁇ 7, and its affinity for talin is at least nine fold less than that of ⁇ lD.
• Cytoplasmic domain mimics of the ⁇ 4 integrin have also been prepared in accordance with the present invention.
• the ⁇ 4 integrin subunit is indispensible for embryogenesis , hematopoiesis and the immune response (Stewart et al . Curr. Opin. Cell Biol. 7, 690-696 (1995); Shimizu et al . Adv. Immunol. 72, 325-380 (1999)). Because of their central role in the immune response ⁇ 4 integrins are strongly implicated as potential therapeutic targets for inflammatory bowel disease, arthritis, multiple sclerosis and asthma.
• ⁇ 4 may regulate cell migration, cytoskeletal organization and gene expression differently from other integrin ⁇ subunits (Hemler et al . Cold Spring Harbor Symposia on Quantitative Biology: The Cell Surface 57, 213-220 (1992)). These biological properties are dependent on the ⁇ 4 cytoplasmic domain (Stewart et al . Curr. Opin. Cell Biol. 7, 690-696 (1995); Hemler et al . Cold Spring Harbor Symposia on Quantitative Biology: The Cell Surface 57, 213-220 (1992) ; Newton et al . J. Leukocyte Biol. 61, 422-426 (1997)).
• Structural mimics of the present invention comprising the ⁇ 4 cytoplasmic tails were prepared and used to identify molecules involved in ⁇ 4 integrin-specific signaling.
• Lysates of Jurkat T lymphoblasts were then incubated with immobilized ⁇ 4 cytoplasmic domain mimics. Bound proteins were detected by immunoblotting for previously identified integrin cytoplasmic domain binding proteins. Within the bound fraction, it was found that paxillin was enriched greater than 57 fold as compared to the cell lysate. In contrast, while the ⁇ lA cytoplasmic domain bound paxillin, there was no enrichment relative to the cell lysate. The interactions with both the ⁇ 4 and ⁇ lA tails were specific in that binding was not seen to resin bearing no protein nor to the ⁇ llb cytoplasmic domain. Heterodimers of the ⁇ 4 ⁇ lA tails were also produced.
• the ⁇ 4 tail also bound small amounts of the actin-binding proteins filamin and tails. However, these proteins were not enriched relative to the cell lysate. Further, the ⁇ 4 tail did not bind to vinculin or ⁇ -actinin.
• actin-binding proteins filamin and tails.
• the ⁇ 4 tail did not bind to vinculin or ⁇ -actinin.
• Immunoprecipitates of the surface biotin-labeled cells confirmed the immunoprecipitation of ⁇ 4 ⁇ l by the ⁇ 4 antibody, ⁇ 5 ⁇ l by the ⁇ 5 antibody and a mixture of these two plus a band with mobility of ⁇ l by the anti ⁇ l antibody.
• ⁇ 4 ⁇ l integrin did not co-precipitate with the irrelevant IgG.
• Paxillin' s tight association with the ⁇ 4 tails and its ready isolation with ⁇ 4 integrins is indicative of a significant fraction of ⁇ 4 ⁇ l being associated with paxillin in the cells.
• surface biotin labeled Jurkat cell lysate was sequentially immunoprecipitated with anti -paxillin antibody or irrelevant IgG.
• Western blotting with anti -paxillin antibody confirmed depletion of virtually all paxillin.
• Paxillin depletion resulted in almost complete loss of ⁇ 4 in the lysate.
• Immunoprecipitation with an irrelevant IgG did not result in significant loss of either ⁇ 4 ⁇ l or ⁇ 5 ⁇ l. Accordingly, a majority of or all of the ⁇ 4 appears to physically associate with paxillin.
• a chimera consisting of the ⁇ llb extracellular and transmembrane domain and the ⁇ 4 cytoplasmic domain was then constructed to determine whether the ⁇ 4 cytoplasmic tail alone is sufficient to connect paxillin to an integrin.
• the extracellular and transmembrane domains of ⁇ 3 were joined to the ⁇ lA or ⁇ 7 cytoplasmic domain.
• the ⁇ IIb ⁇ 4 ⁇ 3 ⁇ lA and ⁇ llb ⁇ 4 ⁇ 3 ⁇ 7 chimeric integrins were expressed in CHO cells.
• the functional effect of the ⁇ 4 tails was then examined by assaying cells adhesion and spreading on the ⁇ llb ⁇ 3 ligand, fibrinogen.
• the ⁇ 4 tail did not alter ⁇ llb ⁇ 3 -dependent cell adhesion.
• the ⁇ 4 tail opposed ⁇ llb ⁇ 3 -dependent cell spreading.
• These two cell lines adhered and spread equally well on a ligand for endogenous ⁇ 5 ⁇ l, fibronectin, confirming that the effect was specific to the recombinant integrin.
• an amino acid residue was identified, Y991A, that disrupted binding of paxillin.
• This mutation was introduced into ⁇ 4 chimera and ⁇ llb ⁇ 4 (Y991A) ⁇ 3 ⁇ lA was expressed in CHO cells. This mutation restored ⁇ llb ⁇ 3 -dependent cell spreading, but did not alter either ⁇ llb ⁇ 3 -dependent cell adhesions or cell spreading on fibronectin. Thus, interaction of ⁇ 4 tail with paxillin results in diminished cell spreading.
• the ⁇ 4 subunit was expressed in primary fibroblasts derived from wild-type or paxillin-deficient mice and cell spreading on VCAM-1 , an ⁇ 4 integrin- specific ligand, was assayed.
• a recombinant human paxillin-GST fusion protein was prepared.
• Paxillin binding to the ⁇ 4 tails was saturable and of high affinity.
• agents which inhibit activation will also be useful in inhibiting atherosclerosis.
• Further activation of ⁇ 4 integrin occurs during wound healing. More specifically, ⁇ 4 integrin activation signals monocytes to aggregate at the wound site. However, this aggregation can lead to scarring.
• inhibition of ⁇ 4 integrin activation is also useful in inhibiting scarring during wound healing.
• This structural model was used to identify a 15 mer peptide, SILQEENRRDSWSYI (SEQ ID NO: 15) derived from the ⁇ 4 cytoplasmic domain as an inhibitor of the binding of paxillin and the ⁇ 4 tail.
• the IC50 of inhibition of the interaction of paxillin and the ⁇ 4 tail by this peptide was 150 ⁇ M.
• SEQ ID NO: 15 Further substitution of various single amino acids within SEQ ID NO: 15 with alanine also abolished inhibitory activity.
• inhibition by the 15 mer peptide SILQEENRRDSWSYI (SEQ ID NO: 15) is structurally specific.
• the core active sequence of this peptide has been determined to comprise the 9 amino acid sequence ENRRDSWSY (SEQ ID NO: 18) .
• Knowledge of this core sequence and its structure are useful in the rational design of therapeutic agents which inhibit ⁇ 4 integrin biological responses.
• the structural models of the present invention provide a novel experimental tool for the analysis of various proteins associations with integrin tails in vi tro and the structural aspect of the cytoplasmic face of integrins .
• the structural models of the present invention thus have a number of applications based upon their ability to maintain the cytoplasmic tails of the construct in a configuration that is equivalent or similar to the configuration predominating in vivo while maintaining solubility and stability in an aqueous system, namely in staggered, parallel, and proximal topology.
• these models can be used to detect intracellular molecules capable of binding to integrins and modulating signals by inside-out signaling.
• these molecules can be used in vivo to disrupt or modulate inside-out signaling by binding to the cells in a manner such that the cytoplasmic domains of these recombinant models compete for intracellular molecules with the natural integrins.
• structural models of the present invention can be used to detect molecules capable of binding to the intracellular or cytoplasmic domain of integrins and other transmembrane molecules in vivo, such as by affinity chromatography .
• these models are useful in identifying various therapeutic compounds for selected cytoplasmic domains.
• therapeutic compounds it is meant to include, but is not limited to, molecules which are found to bind to a selected cytoplasmic domain of the model, molecules which bind to proteins that bind to the cytoplasmic domain of the model, and the models themselves.
• a structural model or mimic comprising an ⁇ 4 integrin cytoplasmic tail can be used in a high throughput screening assay to identify agents which inhibit binding of paxillin to the ⁇ 4 cytoplasmic tails.
• the structural model comprising an ⁇ 4 integrin cytoplasmic tail is exposed to paxillin or a paxillin related molecule in the presence or absence of a test agent. Binding of paxillin or the paxillin related molecule to the structural model in the presence and absence of the test agent is then determined.
• a test agent which decreases binding of paxillin or the paxillin related molecule to the structural model as compared to binding of paxillin or paxillin related molecules to the structural model in the absence of the test agent can inhibit biological responses relating to ⁇ 4 integrins.
• these agents may be useful in inhibiting normal wound healing response of ⁇ 4 integrin which can lead to scarring.
• compositions comprising such agents and a known pharmaceutically acceptable vehicle are believed to be useful therapeutically to inhibit biological responses of ⁇ 4 integrins.
• the following examples are provided for illustrative purposes only and are not intended to limit the invention.
• Antibodies and cDNAs Antibodies for the analysis of proteins bound to cytoplasmic domain model proteins on Western blots included: goat serum against filamin (Sigma Chemical Co., St. Louis, MO), rabbit serum against ⁇ -actinin (Sigma Chemical Co.), mAbs against talin (clone 8d4) (Sigma Chemical Co.), vinculin (clone hVIN-1) (Sigma Chemical Co.), pacillin (clone Z035) (Zy ed Laboratories Inc., S. San Francisco, CA) , filamin (MAB1680) (Chemicon International Inc.
• Biotin labeled anti -paxillin antibody was prepared by labeling commercial anti-paxillin (clone 349) with NHS-Biotin (Pierce) according to the manufacturer's instructions. Rabbit polyclonal anti- leupaxin was raised against the N- terminal 14 amino acids of human leupaxin (Lipsky et al . J. Biol. Chem. 273 11709-11713 (1998)).
• Human cDNA used in these experiments included: ⁇ lC cDNA; ⁇ l cDNA with the point mutation, Y788A1; a cDNA for the cytoplasmic domain of human integrin ⁇ lD obtained by RT-PCT of heart muscle total RNA; cDNA of human integrin ⁇ 7; and a cDNA coding for the human ⁇ lB subunit cytoplasmic domain synthesized in PCR reactions using a human ⁇ lA vector with a partially overlapping reverse-oligonucleotide containing the human ⁇ lB sequence.
• Oligonucleotides were synthesized and used in PCR reactions to create a cDNA for the ⁇ -helical heptad repeat protein sequence KLEALEGRLDALEGKLEALEGKLDALEG (SEQ ID NO: 6) Gl- ( [heptad] 4 ) . Variants containing 1 to 3 additional Gly residues (G2-4- ( [heptad] 4 ) ) at the C-terminus were synthesized by modification of the antisense oligonucleotide . These cDNAs were ligated into a Ndel- Hindlll restricted modified pET15b vector (Novagen, Madison, WI) .
• Integrin cytoplasmic domains were joined to the helix as a Hindlll-BamHI fragments.
• the final constructs coded for the N-terminal sequence GSSHHHHHHSSGLVPRGSHMCG (SEQ ID NO: 5) [heptad] 4 linked to the cytoplasmic domains of integrins.
• Different cytoplasmic domain cDNAs were cloned via PCR from appropriate cDNAs using forward oligonucleotides introducing a 5 ' -Hindlll site and reverse oligonucleotide creating a 3 ' -BamHI site directly after the Stop-codon.
• PCR products were first ligated into the pCRTM vector using the TA cloning ® kit (Invitrogen Corp., San Diego, CA) . After sequencing, Hindlll/BamHI inserts were ligated into a modified pET15b vector. Recombinant expression in BL21 (DE3)pLysS cells (Novagen) and purification of the recombinant products were performed according to the pET System Manual (Novagen) with an additional final purification step on a reverse phase C18 HPLC column (Vydac, Hesperia, CA) . Products were analyzed by electrospray mass spectrometry on an API -III quadruple spectrometer (Sciex, Toronto, Ontario, Canada) .
• Example 3 Ultraviolet circular dichroism spectroscopy
• Human platelets were obtained by centrifugation of freshly drawn blood samples at 1000 rpm for 20 minutes and sedimentation of the resulting platelet-rich plasma at 2600 rpm for 15 minutes. They were washed twice with 0.12 M NaCl, 0.0129 M trisodium citrate, 0.03 M glucose, pH 6.5, and once in Hepes-Saline (3.8 mM Hepes, 137 mM NaCl, 2.7 mM KC1, 5.6 mM D-Glucose, 3.3 mM Na 2 HP0 4 , pH 7.3-7.4).
• Human Jurkat and HT1080 cells and mouse C2C12 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in RPMI1680 (Jurkat) or DMEM with 10% fetal calf serum. For differentiation to myotubes, C2C12 myoblasts were kept confluent in DMEM with 5% horse serum for 6 days. Cultured cells were washed twice in phosphate- buffered saline (PBS) and biotinylated with 1 mM NHS-biotin (Pierce) in PBS during 30 minutes at room temperature. Platelets were biotinylated in Hepes-Saline.
• PBS phosphate- buffered saline
• NHS-biotin PBS
• Lysates were sonicated 5 times on ice for 10 seconds at a setting of 3 using an Astrason Ultrasonic Processor (Heart Systems, Farmingdale, NY). After 30 minutes, lysates were clarified by centrifugation at 12,000 g for 30 minutes.
• Example 5 Affinity chromatography experiments with integrin cytoplasmic domain mimics
• cytoplasmic domain proteins (500 ⁇ g) were dissolved in a mixture of 5 ml 20 mM Pipes, 50 mM NaCl, pH 6.8 and 1 ml 0.1 M sodium acetate, pH 3.5 and bound overnight to 80 ⁇ l of Ni 2t saturated His-bind resin (Novagen) . In control experiments, it was found that this leads to approximate saturation of the resin with peptide. Resins were washed twice with 20 mM Pipes, 50 mM NaCl, pH 6.8, and stored at 4°C with 0.1% sodium azide as suspensions with one volume of this buffer.
• Human uterus filamin (ABP-280) was prepared as a 1.5 mg/ml solution in 0.6 M KC1, 0.5 mM ATP, 0.5 mM DTT, 10 mM imidazole, pH 7.5.
• this solution was diluted 1/12 with buffer A, 0.05% TRITON X-100, 3 mM MgCl 2 , 2 mg/ml BSA, protease-inhibitors (see Example 5) , omitting the 50 mM NaCl (see Example 5) , and resins with bound model proteins were added. Washing was performed in this buffer without BSA and with additional 50 mM Kcl.
• Talin was purified from human platelets in accordance with well known procedures with an additional purification step using chromatography on phosphocellulose and stored at 1 mg/ml in 10 mM NaCl, 50% glycerol .
• This solution was diluted to either 87 or 17 ⁇ g/ml talin with buffer A, 0.05% TRITON X-100, 3 mM MgCl 2 , 2 mg/ml BSA and protease inhibitors (see Example 5, including 0.1 mM E-64) and processed as indicated in the binding assays with cell lysates.
• scans of Coomassie- stained gels were processed using the program NIH- Image
• the ⁇ llb ⁇ 4 and ⁇ IIb ⁇ 4*Y991A) chimeras were formed by connecting human ⁇ llb extracellular and transmembrane domains to human ⁇ 4 or ⁇ 4 (Y991A) cytoplasmic domain.
• ⁇ 3 ⁇ lA or ⁇ 3 ⁇ 7 chimeras were formed by connecting human ⁇ 3 extracellular and transmembrane domains to human ⁇ lA or ⁇ 7 cytoplasmic domains.
• CHO cells stably expressing ⁇ IIb ⁇ 4 ⁇ 3 ⁇ lA, ⁇ llb ⁇ 4 (Y991A) ⁇ 3 ⁇ lA, or cell lines expressing these chimeras were transfected and isolated as described by Hughes et al .
• Precipitated cell surface biotin-labeled polypeptides were separated under non- reducing conditions and detected with streptavidin- peroxidase followed by ECL (Amersham) .
• Immunoprecipitation for detection of co-precipitated paxillin was performed as above except cells were not surface-labeled with biotin; and immunoprecipitated proteins were separated under reducing conditions and paxillin co-precipitation was detected with biotin-labeled anti-paxillin .
• surface biotin-labeled Jurkat cell lysate was precipitated with antibodies reactive to paxillin, ⁇ 4 , ⁇ 5 or irrelevant IgG.
• Immunoprecipitates were separated on 6% SDS-PAGE under non- reducing conditions and surface polypeptides were detected with streptavidin-peroxidase and ECL.
• paxillin- depletion assay aliquots of cell surface biotinylated Jurkat T cell lysate were subjected to varying rounds of immunoprecipitation using anti-paxillin antibody or irrelevant IgG. The degree of paxillin-depletion in the cell lysate was assessed by Western blot analysis. Cell lysates with or without paxillin-depletion, as well as with the irrelevant IgG precipitation, were then immunoprecipitated with either anti- ⁇ 4 or ⁇ 5 antibody. Immunoprecipitates of surface proteins were separated on 6% SDS-PAGE under non-reducing conditions and polypeptides were detected with streptavidin-peroxidase and ECL.
• Example 9 Cell Adhesion and Spreading Assays

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biochemistry (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• Toxicology (AREA)
• Zoology (AREA)
• Gastroenterology & Hepatology (AREA)
• Pulmonology (AREA)
• Hematology (AREA)
• Food Science & Technology (AREA)
• Neurosurgery (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Heart & Thoracic Surgery (AREA)
• Microbiology (AREA)
• Cardiology (AREA)
• Physics & Mathematics (AREA)
• Vascular Medicine (AREA)
• Neurology (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=23259240

Family Applications (1)

Country Status (5)

Cited By (2)

• 2000
  • 2000-06-01 EP EP00936458A patent/EP1180122A4/en not_active Withdrawn
  • 2000-06-01 WO PCT/US2000/015153 patent/WO2000073342A1/en not_active Application Discontinuation
  • 2000-06-01 JP JP2001500666A patent/JP2003504308A/ja not_active Ceased
  • 2000-06-01 CA CA002374133A patent/CA2374133A1/en not_active Abandoned
  • 2000-06-01 AU AU51773/00A patent/AU765990B2/en not_active Ceased

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events