Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 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
  • A61P35/02—Antineoplastic agents specific for leukemia
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

• This invention relates to a laminin receptor binding molecule. More particularly, the present invention relates to a laminin receptor binding molecule derived from PSP94 and its use for promoting signal transduction, such as laminin receptor mediated signal transduction, in a mammalian cell and its use to inhibit laminin and its receptor mediated signaling.
• Laminins are a family of heterotrimeric protein that resides primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and are important signaling molecules that can strongly influence cellular function.
• Laminins are composed of one each of the five known alpha, three known beta, and three known gamma chains. Laminins may be assembled into at least 15 isoforms made from combinations of the various alpha, beta and gamma chains. Laminin-1 , for example, is composed of alpha-1 , beta-1 and gamma-1 isoforms.
• the first group of five identified laminin molecules all share the beta-1 and gamma-1 chains, and vary by their alpha-chain composition (alpha-1 to alpha-5 chain).
• the second group of five identified laminin molecules all share the beta-1 and gamma-1 chain, and again vary by their alpha-chain composition.
• the third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of alpha-3, beta-3 and gamma-2.
• the fourth group of identified laminin molecules has one identified member, laminin 12, with the gamma-3 chain (alpha-2, beta-1 and gamma-3).
• Laminins mediate the attachment of both epithelial and neoplastic cells to the basement membrane, a ubiquitous, specialized type of extracellular matrix. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes. Normal cells appear to require an extracellular matrix for survival, proliferation, and differentiation, while migratory cells, both normal and neoplastic, must traverse the basement membrane in moving from one tissue to another. Laminins are therefore important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development. The laminin molecule integrates various matrix and cell interactive functions into one molecule.
• Laminin alpha chain may have specific as well as overlapping functions. Unique phenotypes have linked alpha-2 to a muscular dystrophy subset (Tome F.M., et al., C R Acad Sci III 317:351 , 1994). In fact, the absence of this laminin chain, in 13 patients seems to be associated with Fukuyama congenital muscular dystrophy. Impaired laminin alpha-3 chain expression seems to be involved in epidermolysis bullosa (Baudoin C, et al., J Invest Dermatol 104:597, 1995). The alpha-5 chain seems to contribute to vascular occlusion in sickle cell disease (Lee, SP. et al. Blood, Vol. 92 No. 8, 1998: pp. 2951-2958).
• integrins are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins may bind different laminin isoforms. (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam, pp. 127-158 (1996))
• laminin receptors include dystroglycan, heparan sulfate proteoglycans, sulfatides, HNK-1 (alpha-1 chain) and lutheran (alpha-5 chain).
• Laminin receptor by mediating the attachment of both epithelial and neoplastic cells to the basement membrane, plays a critical role in controlling, for example, the metastatic process.
• the relationship between laminin and MMP-9 regulation has been the subject of several papers (JBC, 1995, vol 270, p 10365-68; J. Immunology, 2003, vol 171, p 398-406; Cancer Research, 2004, vol 64, p 4810-4816)
• PSP94 inhibits the growth of tumor cells (see U.S. Pat. No.: 5,428,011 to Seth et al., the entire content of which is incorporated herein by reference).
• Tumor growth inhibition by PSP94 fragment such as PCK3145, has also been observed in animal models (see International application No. PCT/CA01/01463 to Garde, S. et al., published under No.: WO02/33090, the entire content of which is incorporated herein by reference).
• PSP94 also reduces the development of skeletal metastasis (see International application No.: PCT/CA02/01737 to Rabbani. S. et al., published under No.: WO03/039576, the entire content of which is incorporated herein by reference).
• This invention relates to molecule derived from PSP94 (PSP94 fragment) and its use to promote signal transduction in a mammalian cell, such as laminin receptor signal transduction.
• This invention also relates to the use of a molecule derived from PSP94 (PSP94 fragment) to inhibit laminin and its receptor mediated signaling.
• This invention also relates to targeting of cell surface laminin binding activities by a PSP94 fragment (PCK3145 (SEQ ID NO.:5, SEQ ID NO.:7 and derivatives).
• This invention also relates to regulation of HuR expression by a PSP94 fragment (PCK3145 (SEQ ID NO.:5, SEQ ID NO.:7 and derivatives) and the use of PSP94 fragments in the treatment of diseases affected by (associated with, linked with) HuR expression.
• PCK3145 SEQ ID NO.:5, SEQ ID NO.:7 and derivatives
• HuR regulation by a PSP94 fragment may be, for example, at the transcriptional level and/or at the level of protein expression.
• the present invention also relates to a laminin receptor binding molecule derived from PSP94 (PSP94 fragment) and its use to trigger (e.g., partially) laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event.
• PSP94 laminin receptor binding molecule
• the present invention further relates to the use of a PSP94 fragment in the treatment of a condition or disease associated with an inefficient activation of a laminin receptor or an inefficient (insufficient) activation of a laminin receptor downstream effector (e.g., ERK, etc.).
• a laminin receptor downstream effector e.g., ERK, etc.
• the present invention also relates to a soluble laminin-like peptidic molecule which may be useful to promote activation of a laminin receptor.
• the present invention also relates to the use of a laminin receptor binding molecule in the detection of a laminin receptor or a laminin receptor carrying cell.
• the present invention further relates to the use of a PSP94 fragment in the treatment of leukemia and for treating diseases mediated by laminin binding to a laminin receptor (e.g., other cancers such as prostate cancer, breast cancer, etc.).
• a laminin receptor e.g., other cancers such as prostate cancer, breast cancer, etc.
• the present invention provides, in one aspect thereof, a method of promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event in a mammal which may comprise the step of administering to the mammal, a compound (or pharmaceutical composition comprising a compound) selected from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other biologically active PSP94 derived molecule.
• the present invention further provides, in another aspect, a method of promoting laminin signal transduction-mediated event in a mammal which may comprise the step of administering to the mammal, a compound (or pharmaceutical composition comprising a compound) selected from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule.
• the present invention relates to the use of a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule for promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event in a mammal in need thereof.
• the present invention relates to the use of a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule in the manufacture of a pharmaceutical composition (or drug) for promoting laminin receptor signal transduction and laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event, for inhibiting laminin and laminin-induced laminin receptor mediated events (e.g., signal transduction) in a mammal.
• a pharmaceutical composition or drug
• the present invention provides the use of a compound which may be selected from the group consisting of; a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative, c) a SEQ ID NO.:5 biologically active fragment, d) a SEQ ID NO.:5 biologically active analog, and e) combination of any one of a) through d), in the preparation of a pharmaceutical composition (drug) for treating leukemia or other cancers (prostate cancer, breast cancer, etc.).
• a suitable biologically active fragment, derivative or analog is one that may be able to inhibit cell growth in an assay as described herein.
• the present invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d) thereof or any other PSP94 derived molecule for promoting laminin receptor signal transduction and/or laminin receptor signal transduction-mediated event, or for promoting laminin signal transduction-mediated event in a mammal, for inhibiting laminin and laminin- induced laminin receptor mediated signal transduction in a mammal and/or for treating cancer (leukemia) in
• the present invention further provides a method for detecting a laminin receptor (in a cell (e.g., on a cell surface), in a tissue, in a mammal, etc.) or a laminin receptor- expressing cell or tissue, the method may comprise contacting a cell or tissue with a laminin receptor binding molecule of the present invention.
• biologically active compounds may comprise, for example, at least one amino acid substitution in the SEQ ID NO.:5 sequence.
• the compound may be viewed, for example, as a laminin receptor agonist. More particularly, the compound may be viewed as a laminin receptor partial agonist. Also more particularly and further in accordance with the present invention, the compound may be viewed as a laminin receptor partial competitive agonist, laminin (and/or SIKVAV, and analogues thereof) being natural ligands of the laminin receptor.
• the laminin receptor binding molecule may be peptidic. Also in accordance with the present invention, the laminin receptor binding molecule may be a PSP94 derived molecule. In addition, further in accordance with the present invention, the laminin receptor binding molecule may be selected, for example, from the group consisting of a) a compound comprising SEQ ID NO.:5, b) a SEQ ID NO.:5 biologically active derivative which may be able to bind a laminin receptor, c) a SEQ ID NO.:5 biologically active fragment which may be able to bind a laminin receptor, d) a SEQ ID NO.:5 biologically active analog which may be able to bind a laminin receptor, and e) combination of any one of a) through d).
• the laminin-induced laminin receptor mediated events which may be inhibited by the compounds of the present invention, may comprise, for example, MMP (and/or pro-MMP) secretion, expression and/or activity, and metastatis as well as events other than MMP (and/or pro-MMP) secretion, expression and/or activity or metastasis.
• the compounds of the present invention may thus be used, for example, in the inhibition of differentiation of endothelial cells or the inhibition of leukemia cells (acute myeloid leukemia).
• a "molecule derived from PSP94" is to be understood herein as a polypeptide originating from PSP94.
• a "molecule derived from PSP94" may be selected, for example, from the group consisting of a) a SEQ ID NO.:1 biologically active derivative, b) a SEQ ID NO.:1 biologically active fragment, c) SEQ ID NO.:1 biologically active analogue, d) SEQ ID NO.:5, e) a SEQ ID NO.:5 biologically active derivative, f) a SEQ ID NO.:5 biologically active fragment, g) a SEQ ID NO.:5 biologically active analogue, h) SEQ ID NO.:7, and i) combination of any one of a) through h) thereof.
• a "molecule derived from PSP94" may also include, for example, SEQ ID NO.: 3, SEQ ID NO.:4, SEQ ID NO.:6, as well as SEQ ID NO.: 9 to 98.
• the SEQ ID NO.:1 fragment may be selected, for example, from the group consisting of SEQ ID NO.:4 and SEQ ID NO.:6.
• SEQ ID NO.:1 derivative may be selected, for example, from the group consisting of SEQ ID NO.:2 and SEQ ID NO.:3.
• a “fragment” is to be understood herein as a polypeptide originating from a portion of an original or parent sequence. Fragments encompass polypeptides having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus (C-terminus), or from the interior of the protein. A fragment may comprise the same sequence as the corresponding portion of the original sequence. For example, SEQ ID NO.: 4, SEQ ID NO.: 5 and SEQ ID NO.: 6 fall into the definition of "a PSP94 fragment”; when considering PSP94 (SEQ ID NO.:1) as an original sequence.
• a “derivative” is to be understood herein as a polypeptide originating from an original sequence or from a portion of an original sequence and which may comprise one or more modification; for example, one or more modification in the amino acid sequence (e.g., an amino acid addition, deletion, insertion, substitution etc.), one or more modification in the backbone or side-chain of one or more amino acid, or an addition of a group or another molecule to one or more amino acids (side-chains or backbone).
• SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 7 fall into the definition of "a PSP94 derivative"; when considering PSP94 (SEQ ID NO.:1) as an original sequence.
• SEQ ID NO.: 7 may fall into the definition of "a PCK3145 derivative” or “SEQ ID NO.:5 derivative when considering PCK3145 (SEQ ID NO.:5) as an original sequence.
• the addition of polyethylene glycol group (i.e., pegylation) to PCK3145 (SEQ ID NO.:5 or SEQ ID NO.: 7) also falls within the definition of "a PCK3145 derivative”.
• SEQ ID NO.: 5 and SEQ ID NO.: 7 are biologically equivalent as demonstrated herein and may be interchanged.
• an “analogue” is to be understood herein as a molecule having a biological activity and chemical structure similar to that of a polypeptide described herein.
• An “analogue” may have sequence similarity with that of an original sequence or a portion of an original sequence and may also have a modification of its structure as discussed herein.
• an “analogue” may have at least 90 % sequence similarity with an original sequence or a portion of an original sequence.
• An “analogue” may also have, for example; at least 70 % or even 50 % sequence similarity (or less, i.e., at least 40%) with an original sequence or a portion of an original sequence.
• an “analogue” may have, for example, 50 % sequence similarity to an original sequence with a combination of one or more modification in a backbone or side-chain of an amino acid, or an addition of a group or another molecule, etc.
• biologically active polypeptides in the form of the original polypeptides, fragments (modified or not), analogues (modified or not), derivatives (modified or not), homologues, (modified or not) of PSP94 and PCK3145 are encompassed by the present invention.
• any polypeptide having a modification compared to an original polypeptide e.g., PSP94, PCK3145
• an original polypeptide e.g., PSP94, PCK3145
• modifications may be made to the polypeptides of the present invention without deleteriously affecting their biological activity. These modifications may, on the other hand, keep or increase the biological activity of the original polypeptide or may optimize one or more of the particularity (e.g. stability, bioavailability, etc.) of the polypeptides of the present invention which, in some instance might be desirable.
• Polypeptides of the present invention comprises for example, those containing amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side-chains and the amino- or carboxy-terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching.
• Cyclic, branched and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Modifications comprise for example, without limitation, pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent, radioactive, etc.), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formy
• polypeptide modification may comprise, for example, amino acid insertion (i.e., addition), deletion and substitution (i.e., replacement), either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence where such changes do not substantially alter the overall biological activity of the polypeptide which is, for example, to reduce the level of expression of matrix metalloproteinases or pro-matrix metalloproteinases and/or to reduce their enzymatic activity.
• amino acid insertion i.e., addition
• deletion and substitution i.e., replacement
• conservative or non-conservative e.g., D-amino acids, desamino acids
• Example of substitutions may be those, which are conservative (i.e., wherein a residue is replaced by another of the same general type or group) or when wanted, non- conservative (i.e., wherein a residue is replaced by an amino acid of another type).
• a non-naturally occurring amino acid may substitute for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non- naturally occurring non-conservative amino acid substitution).
• amino acids may be sub-classified as acidic, basic, neutral and polar, or neutral and non-polar. Furthermore, three of the encoded amino acids are aromatic. It may be of use that encoded polypeptides differing from the determined polypeptide of the present invention contain substituted codons for amino acids, which are from the same type or group as that of the amino acid to be replaced.
• the basic amino acids Lys, Arg and His may be interchangeable; the acidic amino acids Asp and GIu may be interchangeable; the neutral polar amino acids Ser, Thr, Cys, GIn, and Asn may be interchangeable; the non-polar aliphatic amino acids GIy, Ala, VaI, lie, and Leu are interchangeable but because of size GIy and Ala are more closely related and VaI, lie and Leu are more closely related to each other, and the aromatic amino acids Phe, Trp and Tyr may be interchangeable.
• polypeptides are made synthetically, substitutions by amino acids, which are not naturally encoded by DNA (non-naturally occurring or unnatural amino acid) may also be made.
• a non-naturally occurring amino acid is to be understood herein as an amino acid which is not naturally produced or found in a mammal.
• a non-naturally occurring amino acid comprises a D-amino acid, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, etc.
• the inclusion of a non-naturally occurring amino acid in a defined polypeptide sequence will therefore generate a derivative of the original polypeptide.
• Non-naturally occurring amino acids include also the omega amino acids of the formula
• n 2-6, neutral nonpolar amino acids, such as sarcosine, t- butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, etc.
• Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
• Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
• analogues may be generated by substitutional mutagenesis and retain the biological activity of the polypeptides of the present invention. These analogues have at least one amino acid residue in the protein molecule removed and a different residue inserted in its place.
• one site of interest for substitutional mutagenesis may include but are not restricted to sites identified as the active site(s), or immunological site(s). Other sites of interest may be those, for example, in which particular residues obtained from various species are identical. These positions may be important for biological activity. Examples of substitutions identified as "conservative substitutions" are shown in table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in table 1 , or as further described herein in reference to amino acid classes, are introduced and the products screened.
• modification of a polypeptide may result in an increase in the polypeptide's biological activity, may modulate its toxicity, may result in changes in bioavailability or in stability, or may modulate its immunological activity or immunological identity.
• Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• Naturally occurring residues are divided into groups based on common side chain properties:
• Non-conservative substitutions will entail exchanging a member of one of these classes for another.
• Xi may be glutamic acid (i.e., glutamate) (GIu), aspartic acid (aspartate) (Asp), or asparagine (Asn), X 2 may be threonine (Thr) or serine (Ser) and X 3 may be tyrosine (Tyr) or phenylalanine (Phe). Any replacement of an original residue in SEQ ID NO.:5 with a conserved amino acid (i.e. conservative substitution) is encompassed by the present invention.
• PCK3145 SEQ ID NO: 5
• analogue may include, for example, a polypeptide as exemplified in SEQ ID NO.:88 or any other polypeptide having at least one conservative amino acid substitution (illustrated in bold below) as defined in Table 1 , such as, for example;
• Examples of a PCK3145 (SEQ ID NO: 5) derivative may include, for example, a polypeptide having an addition in one or both of the terminal region (amino-terminal or carboxy-terminal) as illustrated in SEQ IDs No.: 9 to 87, a peptide having a stabilizing group such as exemplified in SEQ ID NO.:7, or a peptide having one or more repeats of SEQ ID No.:5 such as exemplified in SEQ ID NOs.: 89 to 91, a polypeptide having at least one D-amino acid as exemplified in SEQ ID No. 98 and combination thereof.
• PCK3145 (SEQ ID NO: 5) fragment may include, for example, a polypeptide having a truncation in one or both of the terminal regions (amino-terminal or carboxy-terminal) as illustrated below.
• Polypeptides may be either naturally occurring (that is to say, substantially purified or isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the encoding DNA or made by other synthetic methods such as chemical synthesis). It is thus apparent that the polypeptides of the invention can be either naturally occurring or recombinant (that is to say prepared from the recombinant DNA techniques) or made by chemical synthesis (e.g., organic synthesis).
• compositions means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers.
• a “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
• Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts).
• Solubilizing agents e.g., glycerol, polyethylene glycerol
• anti-oxidants e.g., ascorbic acid, sodium metabisulfite
• preservatives e.g., thimerosal, benzyl alcohol, parabens
• bulking substances or tonicity modifiers e.g., lactose, mannitol
• covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
• Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
• particulate compositions coated with polymers e.g., poloxamers or poloxamines.
• Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes.
• the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermal ⁇ , subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.
• the formulations include those suitable for oral, rectal, ophthalmic, (including intravitreal or intracameral) nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
• Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
• Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.
• Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.
• Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier.
• An example of a topical delivery system is a transdermal patch containing the ingredient to be administered.
• Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
• Formulations suitable for nasal administration include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
• Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
• Formulations suitable for vaginal administration may be presented as pessaries, tamports, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• pharmaceutically acceptable carrier or “pharmaceutical carrier” are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8 % saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.
• any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
• any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
• polypeptides of the present invention each include each and every individual polypeptide described thereby as well as each and every possible mutant, variant, homolog, analogue or else whether such mutant, variant, homolog, analogue or else is defined as positively including particular polypeptides, as excluding particular polypeptides or a combination thereof; for example an exclusionary definition for a polypeptide analogue (e.g. X I WQX 2 DX I CX 1 X 2 CX 2 CX 3 XIX 2 (SEQ ID NO.88)) may read as follows: "provided that when one of Xi is glutamic acid and X 2 is threonine X 3 may not be phenylalanine" or else.
• Fig. 1A is a picture of a zymography gel showing the effect of the PCK3145 derivative (SEQ ID NO.:7) on MMP-9 levels and activity on collagen type 1- treated MatLyLu cells (first lane:marker; second lane:cells; third lane:cells and collagen; fourth lane:cells, collagen and 500 ⁇ g/ml of SEQ ID NO.:7; fifth laneicells, collagen and 1mg/ml of SEQ ID NO.:7), Fig.
• 1B is a picture of a western blot membrane showing the effect of the PCK3145 derivative (SEQ ID NO.:7) on MMP-9 expression level (fist lane: MMP9 standard; second lane:cells; third lane:cells and collagen; fourth lanexells, collagen and 100 ⁇ g/ml of SEQ ID NO.:7; fifth lane: cells, collagen and 500 ⁇ g/ml of SEQ ID N0..7; sixth lane: cells, collagen and 1mg/ml of SEQ ID NO.:7),
• Fig. 1C is a picture of a time-course/dose-response experiment of a MMP-9 secretion (upper panel) and a graph (lower panel) expressing these results in a quantitative manner;
• Fig. 2 is a picture of a MMP-9 zymography assay performed on cells induced with cytokines in the presence or absence of PCK3145;
• Fig. 3A is the result of a sequence homology search performed on the PCK3145 amino acid sequence (SEQ ID NO.:5) with the Gene Bank internet database,
• Fig. 3B is a picture of a westernblot illustrating the inhibitory effect of laminin on PCK3145 (PCK) induced ERK phosphorylation
• Fig. 3C is a picture of a further westernblot illustrating the inhibitory effect of laminin on PCK3145 (PCK) induced ERK phosphorylation
• Fig. 4A is a picture of a further westernblot illustrating expression of the laminin receptor in several cell lines
• Fig. 4B represents pictures of western blots of PCK3145-induced ERK phosphorylation in laminin receptor transfected U-87 cells or non-transfected cells (mock) and a graph illustrating the results in a quantitative manner
• Fig. 5A is a picture of a westernblot performed with anti-laminin receptor or beta-actin antibody on various fractions collected from an affinity column coupled with PCK3145 (Affi-PCK) or not (AfR-CtI) 1
• Fig. 5B is a histogram illustrating the results of Fig. 5A in a quantitative manner
• Fig. 6A is a graph illustrating cell binding of Iabeled-PCK3145 at 4 0 C and intracellular uptake of labeled-PCK3145 at 37 0 C and the dilution effect of added unlabelled PCK3145 (cold)
• Fig. 7A is dose-response of FITC-labeled PCK3145 binding to the cell surface measured by FACS analysis;
• Fig. 7B is a graph obtained from FACS analysis of FITC-labeled PCK3145 binding to the cell surface in the absence (upper panel) and in the presence of laminin (lower panel);
• Fig. 8A is a picture of a zymography gel showing the effect of the PCK3145 on MMP-9 levels in the presence or absence of laminin or the laminin-derived SIKVAV peptide;
• Fig. 8B is a histogram quantifying the results obtained in Fig. 8A;
• Fig. 9A is a picture illustrating the results of a time-course ERK phosphorylation experiment determined by a Western blot performed on lyzates of cells incubated with various concentration of PCK3145 for;
• Fig. 9B is a picture illustrating ERK-phophorylation determined by a Western blot performed on lyzates of cells incubated for 1 minute with various concentration of PCK3145 with or without the SIKVAV peptide;
• Fig. 10A are pictures of cells transfected with a laminin receptor or not (mock) adhering to PCK3145-coated dishes;
• Fig. 10B is a graph of a time-course of cell (transfected with a laminin receptor or not) adhesion experiment performed on PCK3145-coated dish;
• Fig. 11 A are pictures of stained PVDF membranes obtained following electrophoresis and transfer of proteins eluted from a PCK3145 coupled affinity matrix;
• Fig. 11B represents the sequence of a first PCK3145 binding protein; serum albumin;
• Fig. 11C represents the sequence of a first PCK3145 binding protein; enolase;
• Fig. 12A is a graph showing inhibition of cell adhesion to PCK3145-coated dishes by enolase or EGCg;
• Fig. 12B is a histogram showing results of competition of FITC-labeled PCK3145 binding to cells by enolase by FACS analysis;
• Fig. 13A -panel A represents graphs obtained from FACS analysis showing binding of biotinilated-laminin on cell surface (control; upper left panel) and inhibition of binding by excess unlabelled laminin (upper right panel), by PCK3145 (lower left panel) and by EGCg (lower right panel);
• panel B is a histogram illustrating the results of panel A. in a quantitative manner;
• Fig. 13B are pictures of PVDF membranes obtained following electrophoresis of cell lyzates or enolase and transfer to membranes; detection was performed with biotinilated-laminin and streptavidin-HRP (horse radish peroxidase) or biotinilated- laminin and an anti-biotin antibody;
• Fig. 14 -panel A is a picture of a gel loaded with RT-PCR-amplified HuR RNA obtained from cells incubated with PCK3145 in the presence or absence of laminin (Lam) or the SIKVAV peptide
• panel B is a picture of a western blot performed on lyzates obtained from cells incubated with PCK3145 in the presence or absence of laminin (Lam) or the SIKVAV peptide where the level of HuR protein expression is detected with an anti-HuR antibody;
• Fig. 15 -panel A represents graphs obtained from FACS analysis of cells incubated or not with PCK3145 and detected with either a labeled anti-CD44 antibody or a labeled anti-MMP-9 antibody;
• panel B are histograms illustrating the results of panel A. in a quantitative manner (white bars; without PCK3145, grey bars; with PCK3145);
• Fig. 16 is a schematic showing the biological effect of PCK3145;
• Fig.17A are graphs illustrating binding (left) of [ 14 C]labelled-PCK3145 to HT-1080 fibrosarcoma cells at 4 0 C and uptake (right) of [ 14 C]labelled-PCK3145 by HT-1080 fibrosarcoma cells at 37 0 C;
• Fig. 17B is a graph illustrating binding and uptake of increasing concentrations of [ 14 C]labelled-PCK3145 to HT-1080 fibrosarcoma cells;
• Fig. 18A is a histogram illustrating cell surface binding (white bars) or uptake (grey bars) of PCK3145 in the presence or not of laminin, hyaluronic acid (HA), or type-l collage, probability values of less than 0.05 were considered significant, and an asterisk ( ** ) identifies such significance cell surface binding and uptake experiments;
• Fig. 18B are graphs illustrating the relative PCK3145 binding or uptake in the presence or not of type-l collagen, insert represents the representative dose response as monitored for PCK3145 uptake in the presence of low type-l collagen concentrations (data are expressed as the % of control basal PCK3145 uptake and cell surface binding);
• Fig. 19-Left panel represents a picture of a Far-western blot of a 10% SDS-PAGE loaded with lysate from confluent HT-1080 cells and transferred to PVDF membranes incubated (+) or not (-) with biotin-labeled PCK3145 and bands revealed using Streptavidin-HRP coupled to ECL detection; -Right panel, represents a picture of the same PVDF membrane blotted with an anti-67 kDa laminin receptor (67LR) antibody.
• 67LR laminin receptor
• Fig. 20-panel A are pictures showing several cell density (10 4 to 10 6 cells/well);
• panel B s Cell lysates were isolated in duplicate from each cell confluence culture condition. SDS-PAGE and immunoblotting with the anti-67LR was performed at 24 hrs.;
• panel C is a histogram of results of panel B. expressed in a quantitative manner;
• panel D shows a time-course experiment of a laminin receptor expression in cells at a density of 10 5 by Western blot;
• Fig. 21 -panel A is an histogram representing the effect of the 67-kDa laminin receptor expression (dependent of cell confluence) on the inhibition of cell surface PCK3145 binding by EGCg in HT-1080 cells (10 4 cells/well, white bars; 10 6 cells/well, grey bars), control is without EGCg;
• -panel B. is a picture showing proMMP-9 secretion from low and high confluent cells monitored by gelatin zymography in serum-starved cells that were incubated or not with 300 ⁇ g/ml PCK3145 for 48 hrs
• -panel C. is an histogram representing the results of panel B (white bars; without PCK3145, grey bars; with PCK3145);
• Fig. 22A is an immunoblot performed on lysates of HT-1080 cells which have been transfected or not with a cDNA encoding the 67LR (laminin receptor of 67 Kda), and;
• Fig. 22B is a histogram representing PCK3145 cell surface binding and uptake on HT- 1080 cells which have been transfected or not with a cDNA encoding the 67LR Mock (white bars) and 67LR-transfected (grey bars).
• PCK3145 The wild type amino acid sequence of PCK3145 has been disclosed, for example, in international application No.: PCT/CA01/01463 and is defined herein in SEQ ID NO.: 5.
• a PCK3145 derivative has been generated by attaching an acetylaminomethyl group to the sulfur atom of each of the three cysteines of PCK3145. These groups stabilize the compound by preventing formation of peptide dinners or polymer by blocking the sulfhydryl group of cysteines.
• This PCK3145 derivative is defined in SEQ ID NO.: 7.
• the drug was manufactured by Multiple Peptide Systems (3550) (General Atomics Court, San Diego, Calif.) using standard solid-phase peptide chemistry and lyophilized into a powder.
• PCK3145 derivatives, analogs and fragments may be generated similarly.
• SDS sodium dodecylsulfate
• BSA bovine serum albumin
• Electrophoresis reagents were purchased from Bio-Propanediol.
• 67LR was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), while the mouse ⁇ -actin monoclonal antibody was from Sigma.
• ECL enhanced chemiluminescence
• micro bicinchoninic acid protein assay reagents were from Pierce
• Fluorescein isothiocyanate (FITC)-N-terminal-labeled PCK3145 was synthesized by EZBiolab (Westfield, IN). All other reagents were from Sigma-AIdrich Canada.
• HT- 1080 cell line was purchased from American Type Culture Collection and maintained in Dulbecco Minimum Essential Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and were cultured at 37 0 C under a humidified atmosphere containing 5% CO 2 .
• the human 67LR full-length cDNA was from OriGene Technologies (Rockville, MD).
• HT-1080 cells were transiently transfected with cDNA using the non-liposomal formulation FUGENE-6 transfection reagent. Transfection efficiency was confirmed by western blotting. All experiments involving these cells were performed 36 hrs following transfection. Mock transfections of HT-1080 cultures with pcDNA (3.1+) expression vector alone were used as controls.
• Membrane protein labeling Endothelial or cancer cell membrane proteins will be biotin labeled with EZ-Link Sulfo-NHS-Biotin (Pierce). Confluent dishes containing approximately 3*10 6 cells will be rinsed with cold PBS and incubated at 4 0 C with 4 mg of Sulfo-NHS-Biotin. The biotin reagent will be neutralized with Tris-buffer saline. Cells will then be scraped off the dish, pelleted by centrifugation, and lysed with 0.5% Nonidet P-40 in PBS (NP/PBS). Soluble-labeled membrane proteins will be collected after centrifugation at 11,000g at 4°C and used for either BIAcore studies, immunoprecipitations or affinity gel chromatography approaches.
• Affinity chromatography will be performed with PCK3145 or scrambled peptide linked to Affi-gel 10 or 15.
• Four mg of peptide will be coupled overnight to Affi-gel resin in 100 mmol/L sodium carbonate buffer, pH 8.5.
• Remaining active groups blocked with 100 mmol/L diethanolamine, and the resin equilibrated with NP/PBS.
• Beads blocked with diethanolamine or to which scrambled peptide or PCK3145 were coupled will be used as control.
• Biotin-labeled membrane proteins will be passed through the column and allowed to incubate for 30 min.
• Cell culture media were obtained from Life Technologies (Burlington, Ontario, Canada) and serum was purchased from Hyclone Laboratories (Logan, UT). Electrophoresis reagents were purchased from Bio-Rad (Mississauga, Ontario, Canada).
• the polyclonal (C-1158) and monoclonal (A3) antibodies, used for precipitation and detection, respectively, of VEGFR-2, and the anti-PDGFR pAb (958) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).
• Antiphosphotyrosine mAb PY99 was also purchased from Santa Cruz Biotechnologies.
• Anti-phospho-ERK polyclonal antibodies were from Cell Signaling Technology (Beverly, MA).
• Antimouse and anti-rabbit horseradish peroxidase-linked secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia Biotech (Baie d'Urfe, Quebec, Canada). Human recombinant PDGF was obtained from R&D Systems (Minneapolis, MN). Micro bicinchoninic acid protein assay reagents were from Pierce (Rockford, IL). All other reagents were from Sigma-Aldrich Canada.
• Protein concentrations were determined using the micro bicinchoninic acid method.
• lysates were clarified by a 1 h incubation at 4 0 C with a mixture of Protein A/Protein G Sepharose beads. After removal of the Sepharose beads by low-speed centrifugation, identical amounts of protein (200 ⁇ g) from each sample were transferred to fresh tubes and incubated in lysis buffer overnight at 4°C in the presence of 2 ⁇ g/ml of specific antibodies. Immunocomplexes were collected by incubating the mixture with 25 ⁇ l (50% suspension) of Protein A- (rabbit primary antibody) or Protein G- (mouse primary antibody) Sepharose beads, for 2 h.
• Nonspecifically-bound material was removed by washing the beads three times in 1 ml of lysis buffer containing 1 mM sodium orthovanadate, and bound material was solubilized in 25 ⁇ l of two-fold concentrated Laemmli sample buffer, boiled 5 min, and resolved by SDS-PAGE.
• the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, blocked 1 h at room temperature with Tris-buffered saline/Tween 20 (147 mM NaCI, 20 mM Tris/HCI, pH 7.5, and 0.1% Tween 20) containing 2% bovine serum albumin and incubated overnight at 4 0 C with primary antibody.
• PVDF polyvinylidene difluoride
• Immunoreactive bands were revealed after a 1 h incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies, and the signals were visualized by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfee, QC).
• Membranes were further washed in TBST and incubated with the primary antibodies (1/1 ,000 dilution) in TBST containing 3% bovine serum albumin, followed by a 1 hr incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1/10,000 dilution for 67LR) or anti- mouse IgG (1/5,000 dilution) in TBST containing 5% non-fat dry milk. Immunoreactive material was visualized by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfee, QC).
• Biologically active molecule; Fragments, derivatives and analogues may be prepared by techniques known in the art (recombinant technology, solid phase synthesis, etc.).
• the biological activity of derivatives, fragments and analogues may be determined by any of the techniques described herein (effect on ERK phosphorylation) or known in the field to be relevant for any of the biological activity described herein.
• Fig. 1A 2.5x10 5 MatLyLu tumor cells (American Type Culture Collection No.: JHU-5)) were seeded in T-25 flasks containing RPMI with 10% fetal bovine serum (FBS). After overnight incubation, the cells were washed once with serum free medium and treated with various concentrations of the PCK3145 derivative (500ug/ml and 1 mg/ml) in the presence of 50ug/ml collagen type-l in serum free RPMI for 72 hrs. Control cells received 50ug/ml collagen or only serum free medium.
• FBS fetal bovine serum
• the media were collected after 72 hours of exposure to the PCK3145 derivative and subjected to gelatin zymography.
• Zymography for MMP-2 and MMP-9 was performed in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) containing 0.1% gelatin (Invitrogen). Twenty-four microliters of culture media was mixed with non- reducing sample buffer and subjected to electrophoresis without boiling. After electrophoresis, gels were soaked for 30 minutes in 2.5% Triton X-100 solution with 2- 3 washing steps.
• the gels were then incubated for 18 hours at 37 0 C in buffer containing 50 mM Tris/HCI, pH 7.6, 50 mM NaCI, 1OmM CaCI 2 and 0.05% Brij-35. After incubation, the gels were stained with 0.2% Coomassie blue and de-stained until clear proteolytic bands appeared. Gels were scanned with Microtek flatbed scanner (Scanmaker 5 software; Microtek lab, Redondo Beach, CA). The band intensities were determined using the Image Quant software (version 5.0) from molecular Dynamics.
• the MMP-9 and MMP-2 gelatinase zymography standard were purchased from Chemicon (catalogue no. CC073). One nanogram of purified human pro-MMP-2 and pro-MMP-9 standards were used in every gel run.
• the membranes were washed three times in PBS (5 minutes each wash) to remove non-specific binding and they were incubated with the secondary antibody (Rabbit anti- mouse IgG horseradish peroxidase-conjugated (Dako no. 0260)) at a dilution of 1 :5000 for one hour.
• the secondary antibody Rabbit anti- mouse IgG horseradish peroxidase-conjugated (Dako no. 0260)
• Detection of specific MMP-9 protein was made by incubating the membrane in ECLTM reagent (enhanced-chemoluminescence, Roche) and exposing to the X-ray film.
• ECLTM reagent enhanced-chemoluminescence, Roche
• PCK3145 In order to investigate the effect of PCK3145 on MMP-9 secretion in vitro, we treated serum-starved HT-1080 fibrosarcoma cells with increasing doses of PCK3145 for 24 and 48 hrs. Doses of PCK3145 were found not to be cytotoxic as assessed by the measurement of the pro-apoptotic caspase-3 activity (not shown). MMP-9 extracellular levels were then assessed by western blotting and immunodetection. While MMP-9 extracellular levels continued to increase from 24 to 48 hrs in untreated cells, those from PCK3145-treatments decreased in a dose-dependent manner.
• PCK3145 has been shown to inhibit basal MMP-9 secretion from cells including HT- 1080 cells, a human fibrosarcoma cell line provided by American Type Culture Collection (ATCC, Manassas, USA) (data not shown). The inhibitory effect was also observed on phorbol ester (PMA)- and tumor necrosis factor (TNF)-induced MMP-9 secretion (Fig. 2). These two agents trigger specific intracellular signaling that lead either to intracellular Ca ++ release and protein kinase C (PKC) activation, or to TNF- induced signaling that collectively result into strong MMP-9 production.
• PCK3145 can thus be considered as a potential inhibitor of intracellular signal transduction as it antagonizes both PMA- and TNF-induced MMP-9 secretion as observed by gelatin- zymography (Fig 2).
• PCK3145 shares, to some extent, homology with different structural chains of laminin suggests that it may indeed also share the same cell surface receptor.
• EXAMPLE 4 Competition assays Competitions experiments using several extra-cellular matrix (ECM) purified proteins (including laminin), on the potential of PCK3145 to induce ERK ⁇ Extracellular-signal- Regulated protein Kinases 1 or 2) phosphorylation in U-87 cells were then performed. Indeed, laminin was able to inhibit PCK3145-induced ERK phosphorylation but not Fibrinogen (FN), hyaluronic acid (HA), or Fibrin (Fib) (Fig. 3B). The specific antagonistic effect of laminin on PCK3145 was further confirmed in Fig. 3C.
• ECM extra-cellular matrix
• Affinity chromatography was performed to analyze PCK3145 binding to laminin receptor. Briefly, native PCK3145 was cross-linked to Sepharose-activated gel beads, followed by the loading of U-87 cell lysate and extensive washing of the gel. Immunodetection was then performed for laminin receptor in gels that have been either coupled or not to PCK3145. This experiment indicate that in fact the laminin receptor possesses affinity to PCK3145 (Fig. 5A) as shown by the increase in laminin receptor immunoreactivity.
• PCK3145 In order to shed some light into the kinetics of PCK3145 binding and uptake, PCK3145 was iodinated on its unique tyrosine and was used to perform binding assays at 4 0 C and uptake at 37 0 C. PCK3145 is seen to be rapidly taken up into the U-87 cells within the first 20 minutes of incubation, and this uptake kinetic reached a plateau after about
• HBSS buffer and stabilized at either 4 0 C (binding assay) or 37 0 C (uptake assay) for approximately 15 min.
• Reactions were initiated by adding 500 ⁇ l of Ringer/HEPES containing 0.5% ovalbumin and 0.5-1.0 ⁇ Ci of [ 14 C]PCK3145 (30 ⁇ g/ml [ 14 C]labelled- PCK3145).
• PCK3145 binding/uptake assays were performed for up to 5 minutes.
• Data illustrated in Fig. 17A are from one representative experiment. Reactions were stopped with rapid aspiration of the media, and cells washed rapidly with 1 ml Ringer/HEPES containing 0.5% ovalbumin. Radioactivity was then counted in the whole cells after the addition of 500 ⁇ l NaOH 1 N. Similarly, increasing concentrations of PCK3145 were used to monitor cell surface binding and uptake (Fig. 17B.)
• Dissociation constants were also computed and were not statistically different for both processes (9.0 ⁇ 1.1 ⁇ g/ml vs 7.7 ⁇ 0.5 ⁇ g/ml for binding and uptake respectively) and one can conclude that the relative affinity for PCK3145, whether it is in binding or internalization processes, is approximately equivalent.
• PCK3145 binds to the cell surface of HT-1080 cells
• Binding (and internalization) of iodinated-PCK3145 to the cell surface of cancer cells has been demonstrated herein.
• An assay has been designed to monitor such binding to the cell surface with the use of flow cytometry (FACS).
• Fluoresceinisothiocyanate (FITC) is currently the most commonly-used fluorescent dye for FACS analysis and was conjugated to the N-terminus of PCK3145. This assay enables to visualize cell binding of FITC-PCK3145 through the shift in fluorescence associated with cells that bind to it. 80-90% confluent HT-1080 cells were trypsinized and counted. Labeling was performed on 10 6 cells for 1 hr at 4 0 C.
• FITC-PCK3145 effectively bound to the cell surface in a dose-dependent manner with a plateau reached around 10 ⁇ g/ml (Fig 7A). More importantly, FITC-PCK3145 (10 ⁇ g/ml) cell surface binding was substantially inhibited by the incubation of excess 30 ⁇ g/ml laminin-1 (Fig 7B). This latter observation supports that a common cell surface receptor or that some laminin- binding activity is shared between PCK3145 and laminin and may trigger the subsequent signaling by PCK3145.
• PCK3145 e.g., biotin, etc.
• FACS analysis it is useful to use a fluorescent marker.
• HT-1080 cells were incubated with either the native laminin-1 protein (Fig 8A, 8B), or with a laminin-1 -derived peptide SIKVAV known for its capacity to induce MMP-9 secretion [Freitas et a/., (2004) Oral Oncology 40:483-489].
• Confluent serum-starved cells were treated with these agents in the presence or not of 300 ⁇ g/ml PCK3145 for 24 hours.
• Conditioned media was isolated and gelatin zymography performed to monitor the extent of MMP-9 extracellular levels.
• PCK3145 triggers rapid intracellular ERK phosphorylation
• PCK3145 has the capacity to trigger ERK (Extracellular-signal-Regulated protein Kinases) phosphorylation.
• ERK Extracellular-signal-Regulated protein Kinases
• a time-course/dose- response experiment was performed. Overnight serum-starved quiescent HT-1080 cells were incubated with vehicle (phosphate-buffered saline (PBS) pH 7.4) or PCK3145 (3-100 ⁇ g/ml) for 1 , 2 or 5 minutes at 37 0 C. The cells were scraped from the culture dishes in PBS containing NaF/Na 3 VO 4 and incubated in the same medium buffer for 1 h at 4°C. The resulting lysates were further clarified by centrifugation. Western blotting and immunodetection using anti-phosphoERK and anti-ERK antibodies was then performed.
• HT-1080 cells adhere to PCK3145-coated dishes through the 67-kDa laminin receptor
• the overexpression of the recombinant 67-kDa laminin receptor is able to sustain PCK-induced ERK phosphorylation.
• a cell adhesion assay was designed where increasing concentrations of PCK3145 were coated on dishes and cells subsequently incubated. This coating may be performed for either 1 hour at 37 0 C or overnight at 4 0 C. Blocking with albumin was avoided due to a potential interaction between PCK3145 and albumin (see below).
• Confluent HT-1080 cells were trypsinised, seeded onto the PCK3145-coated dishes, and adhesion left to proceed for 1 hour at 37 0 C. Results show that cells adhered with more avidity to increasing doses of PCK3145 (Fig 10A).
• HT-1080 cells were seeded in individual wells of a 6-well plate at increasing confluence of 10 4 to 10 6 cells/well and pictures taken 24 hrs later (Fig 20-panel A). Cell lysates were isolated in duplicate from each cell confluence culture condition. SDS-PAGE and immunoblotting with the anti-67LR was performed at 24 hrs (Fig 20-panels B and C). HT-1080 cells were seeded at 10 5 cells/well in a 6-well culture plate and lysates were isolated at 24, 48, and 72 hrs (Fig 20-panel D).
• proMMP-9 secretion from low and high confluent cells was also monitored by gelatin zymography in serum-starved cells that were incubated or not with 300 ⁇ g/ml PCK3145 for 48 hrs (Fig. 21-panel B and panel C).;
• PCK3145-binding proteins revealed by affinity-gel chromatography
• PCK3145-binding proteins In order to identify potential PCK3145-binding proteins, an affinity chromatography assay was designed in which PCK3145 was coupled to activated Sepharose-beads. Cell lysates were then overlaid onto the respective gels and columns washed extensively to minimize non-specific protein binding. Three independent PCK3145 coupling were performed. It was systematically observed that proteins at approximately MW of 58, 47, and 35 kDa were differentially bound to PCK3145- coupled gels (Fig 11A). The corresponding PVDF membranes were cut and sent for protein sequencing. The identity of two of these three PCK-binding candidates was found to be Albumin (Fig 11B) and enolase (Fig 11C).
• enolase as a potential PCK3145-binding protein is supported by its recently published ability to also bind to laminin [Cameiro et al. (2004) Microbes Infect. 6:604-608].
• a test of cell adhesion to 10 ug/ml PCK3145-coated dished was performed. Cell adhesion was performed for 2 hours at 37 0 C in the presence or not of 30 ⁇ g/ml enolase or with 30 ⁇ M EGCg, a green tea catechin that we have been shown herein to both inhibit MMP secretion and PCK3145 internalization.
• PCK3145 was biotin labeled according to Pierce and purified by FPLC- chromatography using Akta-explorer with a resource RPC-30mI column (Amersham Bioscience, QC). The biotin-labeled peptide was eluted with a gradient from 20% CH 3 CN to 80% CH 3 CN (+0.05%TFA) at a flow rate of 4 ml/min. Products were monitored at different wavelengths (205, 229 and 254 nm) with a UV-900 cell-10. Confluent HT-1080 cell lysates (-50-100 ⁇ g/well) were migrated using 10% SDS-gels and then electrotransferred onto PVDF membranes.
• Membranes were then incubated with 10 ⁇ g/ml of unlabeled PCK3145 or 10 ⁇ g/ml biotin-labeled PCK3145 for 1 hr, followed with incubation with HRP-Streptavidin for another hour and ECL detection.
• the results illustrated in the left panel of Fig 19 shows proteins from HT-1080 cell lysate that are recognized by biotin-labeled PCK3145 ((+) incubated or (-) not incubated with biotin-labeled PCK3145).
• the right panel of Fig. 19 represents the same PVDF membrane blotted with an anti-67 kDa laminin receptor (67LR) antibody.
• Numbers (1 to 11) next to the left panel represent the potential proteins that interact with PCK3145.
• Numbers (6, 8, and 9) represent the 67LR immunoreactive bands that are similar to those identified in the left panel.
• HT-1080 cells were transiently transfected with a cDNA encoding the 67LR. Cell lysates were isolated and immunoblot performed as described herein (mock; white bars and 67LR-transfected cells; grey bars). HT-1080 cells were trypsinized and PCK3145 cell surface binding and uptake monitored as described herein.
• Results of Figs. 22A and B indicate that the expression of the 67-kDa laminin receptor regulates PCK3145 cell surface binding but not internalization.
• PCK3145 inhibits HuR gene and protein expression
• MMP-9 expression to be induced through a stabilizing nuclear factor HuR [Huwiler et al. (2003) J Biol Chem. 278:51748-69]
• a3b1 integrin an integrin known to bind laminin regulates MMP-9 mRNA [Lyer et al., (2005) J Cell Science 118:1185-1195].
• AREs AU-rich elements
• RNAs encoding for instance, VEGF and MMP-9.
• PCK3145 may regulate HuR expression was therefore tested. Serum-starved cells were treated or not with 300 ⁇ g/ml PCK3145 for 24 hours in the presence or not of laminin or SIKVAV, and cells harvested for either RNA extraction or cell homogenates. RT-PCR with specific primers revealed that HuR gene expression was downregulated by PCK3145 and that both laminin and laminin-derived peptide SIKVAV antagonized PCK3145's inhibitory effect (Fig 14). This was further confirmed independently with specific anti-HuR antibody and immunodetection. This effect of PCK3145 on HuR may explain partly the inhibition of MMP-9 expression and subsequent diminished extracellular levels.
• PCK3145 triggers CD44 cell surface shedding and inhibits MMP-9 docking at the cell surface
• Increased cell migration and adhesion on HA was observed when cells were pretreated with PCK3145 (data not shown). It was shown that this was the results of an increased CD44 cell surface shedding as demonstrated by a strong immunoreactive band observed in the cells which had been pre-treated with PCK3145. This effect was also shown to happen in parallel with MT1-MMP-transfected cells. Interestingly, an increase in MT1-MMP expression in the PCK3145-treated cells was shown which may partially explain how PCK may lead to CD44 shedding. The potential implication of these observations on MMP-9 cell surface binding was investigated, since CD44 is also reported to be the docking receptor for MMP-9.
• HT- 1080 cells were treated or not with 300 ⁇ g/ml PCK3145 for 24 hours, trypsinised and either labeled with anti-CD44 or anti-MMP-9 antibodies.
• Flow cytometry measurements clearly show a 25% downregulation in CD44 cell surface expression that is correlated to a 50% inhibition of cell surface-associated MMP-9 (Fig 15).
• PCK3145 inhibits extracellular matrix (ECM) degradation by MMP-9; a matrix metalloproteinase involved in prostate cancer progression and several other conditions such as, for example, angiogenesis, wound healing, etc. While PCK3145 did not affect MMP-9 enzymatic activity per se, it however significantly reduces its (type-l collagen-induced) gene expression which, consequently, led to decreased extracellular MMP-9 secreted levels.
• ECM extracellular matrix
• MMP-9 MMP-9
• its expression can also be regulated at the steps of mRNA stability, translation and protein secretion.
• the ability to modulate MMP-9 expression at multiple steps through distinct signaling pathways may be particularly important during malignant conversion and metastasis, when tumor cells need to induce or maintain MMP-9 levels in response to changing environmental cues.
• PCK3145 inhibits growth of leukemia cell
• the therapeutic effect of PCK3145 on the inhibition of leukemia cell growth was evaluated.
• Human leukemia cells RPMI-8226 and SR available at the National Cancer Institute (http://www.dtp.nci.nih.gov/branches/btb/services.html) were incubated with PCK3145 and cell growth was monitored. Results are expressed in Table 2A and 2B below.
• 67LR laminin receptor
• FITC-labeled PCK3145 effectively binds to the cell surface, and that this binding is inhibited by either laminin-1 or a laminin-1 -derived peptide (SIKVAV)
• EGCg a green tea catechin that inhibits MMP-9 secretion, is also a laminin receptor ligand that antagonized cell binding to PCK3145
• PCK3145-affinity chromatography as identified enolase, an enzyme recently thought to also possess laminin-binding properties, and which have been shown herein to inhibit cell binding to PCK3145.
• 67-kDa laminin receptor Increased expression of the 67-kDa laminin receptor has been reported in a variety of human carcinomas (colon, breast, stomach, liver, and ovary) and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize.
• the 67-kDa laminin receptor is detectable in anaplastic large cell lymphomas and in small subsets of high-grade B-cell non-Hodgkin's or Hodgkin's lymphomas. More recently, expression of the 67-kDa laminin receptor has been found to mediate acute myeloid leukemia cell adhesion to laminin and to be frequently associated with monocytic differentiation.
• cancer e.g., of lymphoid or monocytic origin, etc.
• specific stage of development such as in monocytic-oriented acute myeloid leukemia may be targeted by PCK3145.
• PCK3145 triggers intracellular signaling that regulates HuR expression.
• PCK3145 shares, to some extent, homology with different structural chains of laminin suggests that it may indeed also share the same cell surface receptor.
• laminin and PCK3145 competed for ERK phosphorylation as well as for the binding/uptake of PCK3145.
• intracellular signaling leads to MMP-9 decreased secretion and that HuR, a MMP-9 mRNA stabilizing factor, is targeted by PCK3145.
• HuR a MMP-9 mRNA stabilizing factor
• both of HuR gene and protein expression were downregulated by PCK3145, and this downregulation was reversed by laminin receptor ligands.
• PCK3145 is therefore a laminin receptor-mediated signal transduction inhibitor.
• HuR also binds to the AU-rich elements of RNAs encoding genes for cytokines, growth factors, tumor suppressor genes, proto-oncogene, and cell cycle regulators, one can envisioned that downregulation of HuR by PCK3145 inhibits several cellular processes and may therefore be used to treat several diseases associated with HuR expression.
• Fig. 16 is a schematic summarizing the effect of PCK on its target and the biological event and signaling event occurring thereafter.
• SEQ ID NO: 1 Ser Cys Tyr Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg 1 5 10 15
• GIn Arg lie Phe Lys Lys GIu Asp Cys Lys Tyr lie VaI VaI GIu Lys 65 70 75 80 Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie lie lie
• SEQ ID NO : 6 lie VaI VaI GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu 1 5 10 15
• SEQ ID NO: 7 an acetylaminomethyl group may be attached to the sulfur atom of cysteine 7, of cysteine 10 and/or of cysteine 12
• TCATGCTATT TCATACCTAA TGAGGGAGTT CCAGGAGATT CAACCAGGAA ATGCATGGAT 60 CTCAAAGGAA ACAAACACCC A ⁇ TAAACTCG GAGTGGCAGA CTGACAACTG TGAGACATGC 120
• SEQ ID NO: 22 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr 20 25
• SEQUENCE DESCRIPTION SEQ ID NO: 23 :
• SEQ ID NO : 24 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys
• SEQ ID NO: 32 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15
• SEQ ID NO: 38 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15
• SEQ ID NO: 42 GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15
• SEQ ID NO: 45 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15 lie Ser Cys Cys Thr Leu VaI Ser Thr Pro VaI GIy Tyr Asp Lys Asp
• SEQ ID NO: 50 GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15
• SEQ ID NO: 54 GIu Trp GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu 1 5 10 15
• GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie 50 55 60
• GIu Lys Lys Asp Pro Lys Lys Thr Cys Ser VaI Ser GIu Trp lie 50 55 60
• SEQ ID NO: 62 His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys Thr 1 5 10 15
• Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu Thr Cys 1 5 10 15
• GIy Asn Lys His Pro lie Asn Ser GIu Trp Gin Thr Asp Asn Cys GIu 1 5 10 15
• Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys 1 5 10 15
• SEQ ID NO: 71 Lys Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp 1 5 10 15
• GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys GIu 20 25 30
• Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn Thr Asp Asn Cys 20 25 30
• Cys Tyr Phe lie Pro Asn GIu GIy VaI Pro GIy Asp Ser Thr Arg Lys 1 5 10 15 Cys Met Asp Leu Lys GIy Asn Lys His Pro lie Asn Ser GIu Trp GIn 20 25 30
• Trp GIn Thr Asp Asn Cys Glu Thr Cys Thr Cys Tyr Glu Thr Glu Trp 20 25 30 GIn Thr Asp Asn Cys GIu Thr Cys Thr Cys Tyr GIu Thr GIu Trp GIn
• SEQ ID NO: 94 GIu Trp Gin Thr Asp GIn Ser GIu Thr Cys Thr Cys Tyr Asp Thr 1 5 10 15

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