WO1999037669A1 - PEPTIDES POSSEDANT UNE ACTIVITE DE MODULATION DE L'ADHERENCE DES CELLULES DEPENDANT DE LA SOUS-UNITE D'INTEGRINE $g(b) - Google Patents

PEPTIDES POSSEDANT UNE ACTIVITE DE MODULATION DE L'ADHERENCE DES CELLULES DEPENDANT DE LA SOUS-UNITE D'INTEGRINE $g(b) Download PDF

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WO1999037669A1
WO1999037669A1 PCT/US1999/001236 US9901236W WO9937669A1 WO 1999037669 A1 WO1999037669 A1 WO 1999037669A1 US 9901236 W US9901236 W US 9901236W WO 9937669 A1 WO9937669 A1 WO 9937669A1
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seq
peptide
adhesion
terminal
tyr
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PCT/US1999/001236
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English (en)
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James B. Mccarthy
Leo T. Furcht
Angela Brienzo
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Regents Of The University Of Minnesota
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Priority to US09/600,432 priority Critical patent/US6849712B1/en
Priority to EP99902403A priority patent/EP1049710A1/fr
Priority to CA002319207A priority patent/CA2319207A1/fr
Priority to JP2000528590A priority patent/JP2002501082A/ja
Publication of WO1999037669A1 publication Critical patent/WO1999037669A1/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ECM extracellular matrix
  • Integrins are a family of receptors that are fundamentally important for mediating cell adhesion to ECM proteins. Tumor cells adhere to variety of ECM proteins and molecules on other cells as they invade and metastasize. These interactions of tumor cells have a profound effect on their phenotype. .although its exact role is complex and not completely understood, ⁇ 4 ⁇ l integrin has been implicated in tumor cell arrest and/or extravasation and is involved in tumor cell invasion and metastasis. This integrin is expressed on many hematopoietic malignancies and also on tumors such as melanomas.
  • ⁇ 4 ⁇ l integrin is unique among integrins in that it binds to both ECM components (e.g. fibronectin) and Ig superfamily adhesion receptors (e.g., VCAM-1) which are expressed on activated endothelial cells and other cell types.
  • ECM components e.g. fibronectin
  • Ig superfamily adhesion receptors e.g., VCAM-1
  • ⁇ 4 ⁇ l integrin also binds to itself and promotes homotypic cell adhesion.
  • 4 ⁇ l integrin Although a role for 4 ⁇ l integrin has been established in modulating various aspects of tumor cell biology, the mechanisms by which the function of the ⁇ 4 ⁇ l integrin is modulated are complex and not well understood. Understanding the nature of such interactions may help to explain cell- type specific behavior on ECM proteins that are often observed with integrins. There is, accordingly, a continuing need to identify peptides capable of modulating 4 ⁇ l dependent cell adhesion as a means of further
  • the present invention relates to peptides capable of modulating ⁇ l integrin subunit dependent cell adhesion.
  • the peptides include a C-terminal amino acid residue having a side chain which includes an aromatic group ("-Ar-”) and an amino acid residue with a lipophilic alkyl side chain group (“-Lip-”) as the penultimate C- terminal residue.
  • suitable peptides of the invention may include a C- terminal tyrosine residue .and .an isoleucine residue as the penultimate C-terminal residue, i.e., a C-terminal "IY motif (Ile-Tyr) .
  • the present peptides may include a relatively large number of .amino acid residues, e.g., up to about 100 amino acid residues or more, as disclosed herein even very small peptides which include the LipAr motif, such as the dipeptide Ile-Tyr and the tripeptide Arg-Ile-Tyr, are capable of modulating ⁇ l dependent adhesion.
  • the present peptides typically have no more than about 50 and, preferably, no more than about 25 amino acid residues.
  • the LipAr C-terminated peptides are preferably capable of inhibiting the ⁇ l integrin subunit dependent adhesion of cells, such as the ⁇ 4 ⁇ l integrin dependent adhesion of Ramos cells and the ⁇ 5 ⁇ l integrin dependent adhesion of erythroleukemic cells (e.g., the erythroleukemic cell line K562).
  • Figure 1 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of a number of alanine knockout analogs of FN-C/H V+Y.
  • FN C/H V+Y and a scrambled variant lacking a C- terminal IY motif (“sV"; RPQIPWARY (SEQ ID NO:2)) were included as controls.
  • Figure 2 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of a number of alanine knockout analogs of FN-C/H V+Y.
  • FN C/H V+Y and its scrambled analog sV were included as controls.
  • Figure 3 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of a number of fibronectin fragments tagged with a C-terminal tyrosine residue. FN C/H V+Y and its scrambled analog sV were included as controls. 3
  • Figure 4 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of a number of fibronectin fragments tagged with a C-terminal tyrosine residue.
  • FN C/H V+Y and its scrambled analog sV were included as controls.
  • Figure 5 shows a graph of % adhesion of 8 A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of two "IY" C-terminated peptides and their corresponding "des-Y” analogs.
  • FN C/H V+Y .and its scrambled analog sV were included as controls.
  • Figure 6 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of another "I Y" C-terminated peptide and its corresponding "des-Y” analogs.
  • FN C/H V+Y .and its scrambled analog sV were included as controls.
  • Figure 7 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of a number of truncated analogs of FN C/H V+Y. Controls included FN C/H V+Y and its scrambled analog sV.
  • Figure 8 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of a number of truncated analogs of
  • Figure 9 shows a graph of % adhesion of 8 A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of "IY” and its component single amino acid residues.
  • FN C/H V+Y and its scrambled analog sV were employed as controls.
  • Figure 10 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of a several C-terminal penultimate substitution variants of FN C/H V+Y. FN C/H V+Y and its scrambled analog sV were employed as controls.
  • Figure 11 shows a graph of % adhesion of 8A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of a several C-terminal penultimate substitution variants of FN C/H V+Y. FN C/H V+Y and its scrambled analog sV were employed as controls.
  • Figure 12 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of a several C-terminal substitution variants of FN C/H V+Y. FN C/H V+Y and its scrambled analog sV were employed as controls.
  • Figure 13 shows a graph of % adhesion of 8 A2 stimulated Ramos cells to
  • IIICS-GST as a function of the concentration of "IY" positional variants of FN C/H V+Y.
  • FN C/H V+Y, its scrambled analog sV, and untagged FN C/H V (WQPPRARI (SEQ ID NO: 37)) were employed as controls.
  • Figure 14 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of a negatively charged LipAr terminated peptide.
  • FN C/H V+Y and its scrambled analog sV were employed as controls.
  • Figure 15 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of PRARIY (SEQ ID NO: 24) and PRARI (SEQ ID NO: 39).
  • PRARIY SEQ ID NO: 24
  • PRARI SEQ ID NO: 39
  • Figure 16 shows a graph of % adhesion of the ⁇ 5 ⁇ l integrin dependent Mn +2 stimulated adhesion of erythroleukemic K562 cells to fibronectin ("FN") as a function of the concentration of PRARIY (SEQ ID NO: 24) and PRARI (SEQ ID NO: 39).
  • FN C/H V+Y, its scrambled analog sV, RGD and BSA (bovine serum albumin) were employed as controls.
  • Figure 17 shows a graph of % adhesion of the ⁇ 5 ⁇ l integrin dependent Mn +2 stimulated adhesion of erythroleukemic K562 cells to fibronectin ("FN") as a function of the concentration of RIY. FN C/H V+Y, its scr.ambled an.alog sV, RGD, CS1 .and BSA (bovine serum albumin) were employed as controls.
  • FN fibronectin
  • Figure 18 shows a graph of % adhesion of the ⁇ 2 ⁇ l, ⁇ 3 ⁇ l integrin dependent human melanoma M14#5 cell adhesion to laminin (“LM”) and type IV collagen (“TIV”) and bovine serum albumin (“BSA”).
  • LM laminin
  • TIV type IV collagen
  • BSA bovine serum albumin
  • Figure 19 shows a graph of % adhesion of 8A2 stimulated Ramos cells to IIICS-GST as a function of the concentration of all D-FN C/H V+Y (SEQ ID NO:l), and a retro inverso form of FN C/H V+Y (SEQ ID NO:40) versus various controls. 5 Detailed Description of the Invention
  • the present invention relates to peptides capable of modulating ⁇ l integrin subunit dependent cell adhesion.
  • These peptides include a C-terminal LipAr motif and are typically capable of inhibiting ⁇ l integrin subunit dependent cell adhesion and, in particular, of inhibiting ⁇ 4 ⁇ l integrin dependent cell adhesion.
  • the present peptides typically are also capable of inhibiting ⁇ 2 ⁇ l, ⁇ 3 ⁇ l and/or ⁇ 5 ⁇ l integrin dependent cell adhesion.
  • the term "LipAr motif refers to a dipeptide sequence in which C-terminal "Ar" residue has a side chain which includes an aromatic group.
  • Suitable amino acid residues having an aromatic group include tyrosine ("Tyr”), phenylalanine (“Phe”), histidine (“His”), and tryptophan (“Tip”).
  • the penultimate C-terminal "Lip” residue is an amino acid residue which includes a lipophilic alkyl side chain group.
  • the ⁇ -carboxyl group of the C-terminal amino acid residue of the present peptides is typically in the form of a carboxylic acid (-C0 2 H).
  • the "Lip” and “Ar” residues are L-amino acid residues.
  • amino acid residues which have a lipophilic alkyl side chain group include leucine ("Leu”), isoleucine ("He”), and valine (“Val”).
  • the lipophilic alkyl side chain group has a SCDC (cyclohexane-water side chain distribution coefficient calculated as -RT In K D and expressed in kcal/mol) of at least about 3.0 and, preferably, at least about 4.0.
  • SCDC is defined according to Radzicka et al., Biochemistry, 27, 1664 (1988).
  • the SCDC value may be determined by measurement of the distribution coefficient between wet cyclohexane and water or by a comparison of a compound containing the same alkyl side chain group with other similar compounds using a hydrophobicity scale derived from HPLC retention according to the method of Parker et al., Biochemistry, 25, 5425 (1986).
  • a hydrophobicity scale derived from HPLC retention according to the method of Parker et al., Biochemistry, 25, 5425 (1986).
  • lipophilic alkyl side chain groups such as leucine, isoleucine, and valine
  • FN-C/H I+Y contains a C-terminal LipAr motif.
  • the .amino acid sequence of FN-C/H I+Y is YEKPGSPPREV-VPRPRPGVY (SEQ ID NO:38).
  • FN-C/H V+Y is WQPPRARIY (SEQ ID NO:l).
  • the other two Tyr-tagged fibronectin C-terminal heparin binding domain related peptides do not contain a C- terminal LipAr motif (both peptides end in "TY" (Thr-Tyr)).
  • the amino acid sequences of the these other two fibronectin C-terminal heparin binding domain fragments are KNNQKSEPLIGR-KKTY (FN-C/H II+Y; (SEQ ID NO:39)), and SPPRRARVTY (FN-C/H IV+Y; (SEQ ID NO:40)).
  • alanine knockout analogs of FN-C/H V+Y which preserve the C-terminal LipAr motif (i.e., retain the C-terminal Ile-Tyr dipeptide sequence) are capable of inhibiting ⁇ l integrin dependent cell adhesion.
  • alanine knockout analog refers to an analog of a peptide in which a single residue has been substituted by .an alanine residue.
  • Two of the alanine knockout analogs of FN-C/H V+Y have an alanine 7 residue substituted for one of the .arginine residues in the "PRARI" motif (Pro-Arg- Ala-Arg-Ile (SEQ ID NO:41)) within FN-C/H V+Y which has previously demonstrated to be the implicated in stimulated focal contact formation (see, e.g., Woods et al., Molec. Biol. Cell, 4, 605-613 (1993)).
  • These alanine knockout analogs have the amino acid sequences WQPPRAAIY (SEQ ID NO: 8) and WQPPAARIY (SEQ ID NO: 17).
  • AQPPRARIY SEQ ID NO: 3
  • WAPPRARIY SEQ ID NO: 4
  • FN-C/H V+Y by a non-conservative amino acid substitution (Ala for Tip .and Ala for Gin respectively).
  • FN-C/H V+Y by a non-conservative amino acid substitution but retain the C- terminal LipAr motif can be capable of modulating ⁇ l integrin subunit dependent cell adhesion even if the overall physical properties of the peptide differ substantially from FN-C/H V+Y.
  • an FN-C/ ⁇ V+Y analog in which the two arginine residues have been replaced by aspartic acid residues inhibits the 8A2 stimulated adhesion of Ramos cells at least as strongly as FN-C/H V+Y.
  • the analog, WQPPDADIY (SEQ ID NO: 38), exhibits this activity even though it has an overall net charge of -2 (in contrast to the +2 net charge of FN-C/H V+Y).
  • peptides examples include ARITGYIIY (SEQ ID NO: 14), RARITGYIY (SEQ ID NO: 13), PRQAWRPIY (SEQ ID NO: 18), and RPAPQRWIY (SEQ ID NO:20).
  • % homology refers to the percentage of amino acid residues of a peptide which are either identical to that of an original peptide sequence or differ from the original peptide sequence solely as a result of a conservative amino acid substitution.
  • the peptide PAIFDRSCGS has 8 40%) identity and 80% homology with respect to the peptide sequence
  • conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Ala, Gly, Ser, Thr, .and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr (representing side chains including an -OH or -SH group); Class III: Glu,
  • Asp, Asn and Gin (carboxyl group containing side chains): Class IV: His, Arg and
  • Lys (representing basic side chains); Class V: He, Val, Leu, Phe .and Met (representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr and His
  • the classes also include related amino acids such as 3 Hyp and 4Hyp in Class I; homocysteine in Class II; 2-aminoadipic acid, 2- aminopimelic acid, ⁇ -carboxyglutamic acid, ⁇ -carboxyaspartic acid, and the corresponding amino acid amides in Class III; ornithine, homoarginine, N-methyl lysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4- diaminobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IV; substituted phenylalanines, norleucine, norvaline, 2-aminooctanoic acid, 2- aminoheptanoic acid, statine and ⁇ -valine in Class V; and naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3-carboxylic acid,
  • the peptides contain no more than 10 amino acid residues and have a sequence which does not correspond substantially to the amino acid sequence of FN-C/H V+Y.
  • sequence of a particlar peptide does not correspond substantially to a reference amino acid sequence, if the particular peptide sequence has less than about 80% identity and preferably less than about 50%) homology with the reference sequence.
  • One group of particularly suitable peptides of the invention are those which include a C-terminal "IIY” motif, i.e., the sequence of the three C-terminal most amino acid residues is Ile-Ile-Tyr.
  • One such peptide contains 9 amino acid residues and has the sequence ARITGYIIY (SEQ ID NO: 14).
  • one group of particularly advantageous peptides of the invention include the C-terminal IY motif and contain no more than ten and, preferably, no more than six .amino acid residues.
  • suitable examples of this group include PRARIY (SEQ ID NO:24), RARIY (SEQ ID NO:25), ARIY (SEQ ID NO:26) and RIY.
  • the peptides of the invention may be synthesized by the solid phase method using standard methods based on either t-butyloxycarbonyl (BOC) or 9- fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodology is described by G.B. Fields et al. in Synthetic Peptides: A User's Guide, W.M.
  • the present peptides may also be synthesized via recombinant techniques well known to those skilled in the art.
  • U.S. Patent 5,595,887 the disclosure of which is herein incorporated by reference, describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein which includes a binding protein and one or more copies of the desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide.
  • the peptides of the present invention may be employed in a monovalent state
  • the peptides may also be employed as conjugates having more than one (same or different) peptide fragment bound to a single carrier molecule.
  • the carrier may be a biological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan, albumin or the like) or a synthetic polymer (e.g., a polyalkyleneglycol or a synthetic chromatography support). Typically, ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like are employed as the carrier. Such modifications may increase the apparent affinity and or change the stability of a peptide.
  • the number of peptide fragments associated with or bound to each carrier can vary, but from about 4 to 8 peptide fragments per carrier molecule are typically obtained under standard coupling conditions.
  • peptide/carrier molecule conjugates may be prepared by treating a mixture of peptides and carrier molecules with a coupling agent, such as a carbodiimide.
  • the coupling agent may activate a carboxyl group on either the peptide or the carrier molecule so that the carboxyl group can react with a nucleophile (e.g., an amino or hydroxyl group) on the other member of the peptide/carrier molecule, resulting in the covalent linkage of the peptide and the carrier molecule.
  • the conjugate includes at least one peptide fragment which is not linked to the carrier molecule through an amide bond with the ⁇ - carboxyl group of the C-terminal aromatic amino acid residue of the LipAr- terminated fragment.
  • conjugates of a peptide coupled to ovalbumin may be prepared by dissolving equal amounts of lyophilized peptide .and ov.albumin in a small volume of water.
  • l-ethyl-3-(3-dimethylamino-propyl)-carboiimide hydrochloride (EDC; ten times the amount of peptide) is dissolved in a small amount of water.
  • EDC l-ethyl-3-(3-dimethylamino-propyl)-carboiimide hydrochloride
  • the EDC solution was added to the peptide/ovalbumin mixture and allowed to react for a number of hours.
  • the mixture may then dialyzed (e.g., into phosphate buffered saline) to obtain a purified solution of peptide/ovalbumin conjugate.
  • Peptide/carrier molecule conjugates prepared by this method typically contain about 4 to 5 peptide fragments per ovalbumin molecule. 11
  • the invention will be further described by reference to the following detailed examples. The examples are meant to provide illustration and should not be construed as limiting the scope of the present invention.
  • IIICS-GST is recombinantly produced fusion protein which contains a fragment from the type III CS region ("IIICS") of plamsa fibronectin fused to glutathione-S-transferase ("GST").
  • IIICS type III CS region
  • GST glutathione-S-transferase
  • the fibronectin fragment corresponds to fibronectin amino acid residues 1961 to 2039 (sequence numbering for fibronectin as designated in U.S. Patent 4,839,464) and includes the DELPQLVTLPHPNLHGPEILDVPST (SEQ ID NO:29) amino acid sequence
  • CS1 fibronectin residues 1961-1985.
  • a synthetically prepared peptide having the CS1 sequence has been shown to interact with ⁇ 4 ⁇ l integrin on human lymphocytes and promote cell adhesion but does not bind to heparin.
  • a 96-well plate was coated with the substrate IIICS-GST.
  • Ramos cells stimulated with the ⁇ l activating monoclonal antibody 8A2 (“Ab 8A2”) were preincubated with one of the peptides to be evaluated for their ability to adhere to IIICS-GST.
  • the fusion protein can be constructed by first using PCR primers to amplify the coding sequence for residues 1961-2039 of plama fibronectin.
  • the PCR product can be introduced into a suitable bacterial expression vector in frame with the gene for GST.
  • the resulting vector can be transformed .and expressed in a suitable host cell, such as E.Coli, to produce the fusion protein.
  • the fusion protein can be purified using a glutathione column. In control experiments in which GST alone was coated onto a 96-well plate, no adhesion of 8A2 activated Ramos cells was observed.
  • a 96-well plate was coated in triplicate with 50 ⁇ l/well of IIICS-GST diluted to 3-5 ⁇ g/ml in PBS containing lmM CaCl 2 , MgCl 2 ("PBS/cations") and incubated overnight at 37°C.
  • the IIICS-GST solution was removed and the wells were 12 blocked with 150 ⁇ l/well of PBS/cations containing 0.3% BSA for 1-2 hours at 37°C.
  • each well contained 100 ⁇ l of Ramos cells (10,000 cells/well) with or without peptide. Ramos cells were washed 3 times in adhesion media (DMEM without phenol red containing 20mM HEPES and 3 mg/ml BSA).
  • the dose-dependent dilutions of peptides were prepared using adhesion media to dilute the stock peptide. Labeled cells were mixed with peptide dilutions for 5 minutes at 37°C at a final concentration of 100,000 cells/ml and appropriate final peptide concentrations.
  • the blocking solution was removed from the 96-well plate and the cell/peptide mixture is added at lOO ⁇ l/well (10,000 cells/well) and incubated for 30 minutes at 37°C. An aliquot of standard cells/peptide (1000 ⁇ l) was placed at 37°C for quantitating adhesion. Using aspiration, non-adherent cells were removed from the plate. The standard cells were centrifuged .and resuspended in 1000 ⁇ l of adhesion media. The standard cells were added to empty wells at 100, 80, 60, 40, 20 and 0 ⁇ l/well representing 100%, 80%, 60%, 40%, 20% and 0% adhesion, respectively. The plate fluorescence was read at excitation 485 and emission 530. Cell adhesion was represented as percent input cells remaining adherent and was determined by a standard curve of the fluorescence obtained with the standard cells. 13 The experimental fluorescence readings were extrapolated from the standard curve to obtain percent adhesion.
  • Example 1 Alanine Knockout Analogs of FN-C/H V+Y To determine which amino acid residues were required for the ⁇ 4 ⁇ l dependent cell adhesion inhibiting activity of FN C/H V+Y, a series of analogs having a single individual residue substituted by alanine were examined. The results are shown in Figures 1 and 2. The only alanine substitution which resulted in loss of the ability to inhibit adhesion was substitution of alanine for the isoleucine residue at the penultimate C-terminal position. All of the other alanine knockout peptides showed cell adhesion inhibition comparable to that of FN C/H V+Y.
  • PRARIY 6 residue peptide
  • RARIY 5 residue peptide
  • R arginine residues
  • Other short IY-terminated peptides with the sequences QPPRARIY (SEQ ID NO:22), PPRARIY (SEQ ID NO:23), ARIY (SEQ ID NO:26) and RIY also exhibited ⁇ 4 ⁇ l integrin dependent cell adhesion inhibition activity.
  • SEQ ID NO:35 were inactive in the assay. Switching the order of the He and Tyr residues at the C-terminus of an FN C/H V+Y analog, WQPPRARYI (SEQ ID NO:35), also resulted in a peptide which was inactive in the ⁇ 4 ⁇ l dependent Ramos cell adhesion inhibition assay. Finally, control peptide having the tyrosine tag removed from the C-terminus of FN C/H V+Y, WQPPRARI (SEQ ID NO:35), was also inactive in the assay.
  • Figure 14 clearly demonstrates that substitution of the .arginines with aspartic acid residues does not alter the ability of the peptide to inhibit ⁇ l integrin subunit dependent adhesion, thereby further demonstrating the importance of the "LipAr" motif to this activity.
  • peptide RIY and peptides ending in isoleucine-tyrosine (PRARIY) and isoleucine (PRARI) were evaluated for the ability to inhibit ⁇ 5 ⁇ l integrin-mediated cell adhesion.
  • PRARIY isoleucine-tyrosine
  • PRARI isoleucine
  • Adhesion of the erythroleukemic cell line K562, which expresses ⁇ 5 ⁇ l but not 4 ⁇ l integrin, to FN is completely inhibited following preincubation with soluble RGD or FN-C/H V-Y at the maximal concentration tested, 0.84 mM
  • Laminin, type IV collagen and BSA were coated overnight in a 96 well microtiter plate at 10 ⁇ g/ml and blocked with 0.3%) BSA.
  • M14#5 cells were 18 preincubated with 0.5 mg/ml of peptide (equivalent to 0.42 mM FN-C/H V and scrambled FN-C/H V and 0.17 mM CSI) and allowed to adhere to substrates for 30 minutes.
  • Soluble peptide FN-C/H V inhibited human melanoma M14#5 cell adhesion to laminin and type IV collagen coated substrates, whereas scrambled FN-C/H V has no effect (see Figure 18). This adhesion is dependent on ⁇ 2 ⁇ l and ⁇ 3 ⁇ l integrin as determined using specific anti-integrin blocking mAbs (data not shown).

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des peptides capables de moduler l'adhérence des cellules dépendant de la sous-unité d'intégrine β qui comprennent un résidu d'acides aminés aromatiques à terminal C et un résidu d'acides aminés possédant une chaîne latérale d'alkyle lipophile se présentant comme un dernier résidu du terminal C. Ces peptides 'LipAr' à terminaison C sont, en règle générale, capables de moduler l'adhérence des cellules dépendant de la sous-unité d'intégrine β, par exemple des cellules de Ramos.
PCT/US1999/001236 1998-01-22 1999-01-21 PEPTIDES POSSEDANT UNE ACTIVITE DE MODULATION DE L'ADHERENCE DES CELLULES DEPENDANT DE LA SOUS-UNITE D'INTEGRINE $g(b) WO1999037669A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US09/600,432 US6849712B1 (en) 1998-01-22 1999-01-21 Peptides with β1 integrin subunit dependent cell adhesion modulating activity
EP99902403A EP1049710A1 (fr) 1998-01-22 1999-01-21 Peptides possedant une activite de modulation de l'adherence des cellules dependant de la sous-unite d'integrine beta-1
CA002319207A CA2319207A1 (fr) 1998-01-22 1999-01-21 Peptides possedant une activite de modulation de l'adherence des cellules dependant de la sous-unite d'integrine .beta.
JP2000528590A JP2002501082A (ja) 1998-01-22 1999-01-21 β１インテグリンサブユニット依存性細胞接着調節活性を有するペプチド

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US7211998P 1998-01-22 1998-01-22
US60/072,119 1998-01-22
US9621198P 1998-08-12 1998-08-12
US9621298P 1998-08-12 1998-08-12
US60/096,211 1998-08-12
US60/096,212 1998-08-12

Publications (1)

Publication Number Publication Date
WO1999037669A1 true WO1999037669A1 (fr) 1999-07-29

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PCT/US1999/001236 WO1999037669A1 (fr) 1998-01-22 1999-01-21 PEPTIDES POSSEDANT UNE ACTIVITE DE MODULATION DE L'ADHERENCE DES CELLULES DEPENDANT DE LA SOUS-UNITE D'INTEGRINE $g(b)

Country Status (4)

Country Link
EP (1) EP1049710A1 (fr)
JP (1) JP2002501082A (fr)
CA (1) CA2319207A1 (fr)
WO (1) WO1999037669A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056350A2 (fr) * 1999-03-22 2000-09-28 Regents Of The University Of Minnesota Procede d'utilisation des inhibiteurs de la beta1-integrine
US9382322B2 (en) 2003-10-17 2016-07-05 Rehab Al-Jamal Tissue repair by modulation of beta-1 integrin biological function

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4570402B2 (ja) * 2004-06-25 2010-10-27 日本サプリメント株式会社 中枢機能改善剤

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WO1989001942A1 (fr) * 1987-08-25 1989-03-09 Regents Of The University Of Minnesota Polypeptides ayant une activite de fibronectine
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WO1994017097A1 (fr) * 1993-01-19 1994-08-04 Regents Of The University Of Minnesota Fragments de fibronectine synthetique utilises comme inhibiteurs d'infections retrovirales
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WO1989001942A1 (fr) * 1987-08-25 1989-03-09 Regents Of The University Of Minnesota Polypeptides ayant une activite de fibronectine
EP0347890A1 (fr) * 1988-06-22 1989-12-27 Roussel Morishita Co., Ltd. Compositions nutritives contenant des acides aminés
US5382569A (en) * 1991-05-16 1995-01-17 Warner-Lambert Company Endotherlin antagonists
EP0567898A1 (fr) * 1992-05-01 1993-11-03 Jürgen Dr. Manthey Méthode de mesure de la posture et du mouvement du corps
WO1994017097A1 (fr) * 1993-01-19 1994-08-04 Regents Of The University Of Minnesota Fragments de fibronectine synthetique utilises comme inhibiteurs d'infections retrovirales

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J.L LAUSER ET AL.: "INHIBITION OF MELANOMA CELL BINDING TO TYPE IV COLLAGEN BY ANALOGS OF CELL ADHESION REGULATOR", J. MED. CHEM., vol. 40, 1997, pages 3077 - 3084, XP002107028 *
LASZ E C ET AL: "B3 INTEGRIN DERIVED PEPTIDE 217-230 INHIBITS FIBRINOGEN BINDING AND PLATELET AGGREGATION: SIGNIFICANCE OF RGD SEQUENCES AND FIBRINOGEN AA-CHAIN", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 190, no. 1, 15 January 1993 (1993-01-15), pages 118 - 124, XP000327808 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056350A2 (fr) * 1999-03-22 2000-09-28 Regents Of The University Of Minnesota Procede d'utilisation des inhibiteurs de la beta1-integrine
WO2000056350A3 (fr) * 1999-03-22 2001-05-31 Univ Minnesota Procede d'utilisation des inhibiteurs de la beta1-integrine
US9382322B2 (en) 2003-10-17 2016-07-05 Rehab Al-Jamal Tissue repair by modulation of beta-1 integrin biological function

Also Published As

Publication number Publication date
JP2002501082A (ja) 2002-01-15
EP1049710A1 (fr) 2000-11-08
CA2319207A1 (fr) 1999-07-29

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