NaK-ATPase-Derived Peptide SRC Inhibitors and Ouabain Antagonists and Uses Thereof

Abstract

A novel Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades and uses thereof are disclosed.

1 . A composition of matter comprising an amino acid peptide comprising at least ten consecutive amino acid residues of the sequence SATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3], or conservative substitutions of the amino acid residues, or substitutions with unnatural amino acids to improve pharmacodynamic or/and pharmacokinetic properties, wherein the peptide is capable of binding the kinase domain of Src or Src family kinases. 
     
     
         2 . A composition of  claim 1 , which further comprises a therapeutically acceptable excipient. 
     
     
         3 . A composition of matter of  claim 1 , wherein the amino acid peptide comprises the sequence SATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3]. 
     
     
         4 . A composition of  claim 1 , wherein the amino acid peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO: 5. 
     
     
         5 . A composition of  claim 1 , wherein the amino acid peptide comprises SEQ ID NO: 7 as pNaKtide. 
     
     
         6 . A composition of  claim 1 , wherein the amino acid peptide comprises SEQ ID NO: 8 as AP-NaKtide. 
     
     
         7 . A composition of  claim 1 , wherein the amino acid peptide comprises SEQ ID NO: 9 as A1N-NaKtide. 
     
     
         8 . A nucleic acid sequence encoding a composition of  claim 1 . 
     
     
         9 . A vector expressing a nucleic acid sequence encoding the peptide of  claim 5 . 
     
     
         10 . A vector expressing a nucleic acid sequence encoding the peptide of  claim 6 . 
     
     
         11 . A vector expressing a nucleic acid sequence encoding the peptide of  claim 7 . 
     
     
         12 . A cell comprising a vector of  claim 9  or  claim 10  or  claim 11 . 
     
     
         13 . A cell of  claim 12 , which is  E. coli.    
     
     
         14 . A cell of  claim 12 , which is mammalian. 
     
     
         15 . A cell of  claim 12 , which is a tumor cell. 
     
     
         16 . A monoclonal antibody selective for a composition of  claim 1 . 
     
     
         17 . A composition of  claim 1 , wherein the composition is capable of affecting a cellular process selected from the group consisting of: antagonizing a CTS-induced protein kinase cascades; Src inhibition; Na/K-ATPase mimic; Lyn inhibition; ouabain antagonism; FAK/ERK1/2 inhibition; anti-angiogenesis and inhibition of cell growth. 
     
     
         18 . A composition of  claim 1 , which is not an ATP analog. 
     
     
         19 . A composition of  claim 2 , which further comprises means to therapeutically permeate plasma membrane. 
     
     
         20 . A composition of  claim 2 , which further comprises at least one additional therapeutic composition useful to a treat a disease selected from the group consisting of: cancer; vascular disease; cardiovascular disease; heart disease; bone disease; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; ischemia/reperfusion injury and osteoporosis. 
     
     
         21 . A composition of  claim 3 , which further comprises a second compound bound with the amino acid in a location other than SEQ ID NO: 3, wherein the second compound is selected from the group consisting of: chemotherapeutic drug; toxin; immunological response modifier; enzyme; and radioisotope. 
     
     
         22 . A composition of  claim 3 , which further comprises a second compound bound with the amino acid peptide in a location other than SEQ ID NO: 3, wherein the second compound is selected from the group consisting of: HIV-Tat; Penetratin; and HIV-Tat-S-S; N-terminal sequence of Na/K-ATPase α1; GST; EYFP. 
     
     
         23 . A composition of  claim 3 , which comprises HIV-Tat-SEQ ID NO: 3. 
     
     
         24 . A composition of  claim 3 , which comprises a fusion protein, provided that the fusion does not disrupt the at least ten consecutive residues of SEQ ID NO: 3. 
     
     
         25 . A composition of  claim 3 , wherein the fusion is with GST. 
     
     
         26 . A method to bind a peptide to the kinase domain of Src in a Src-expressing cell, comprising contacting a peptide of  claim 1  to at least one Src-expressing cell. 
     
     
         27 . A method of  claim 26 , wherein the Src-expressing cell is a mammalian cell. 
     
     
         28 . A method of  claim 26 , wherein the at least one mammalian cell is a cell selected from the group consisting of: heart cell, liver cell, vascular cell; breast cell; prostate cell; kidney cell; muscle cell; bone cell; and brain cell. 
     
     
         29 . A method of  claim 26 , wherein the at least one mammalian cell is cultured in vitro. 
     
     
         30 . A method of treating a Src-associated disease in a mammal in need of such treatment, comprising administering a therapeutic composition of  claim 2 . 
     
     
         31 . A method of  claim 30 , wherein the Src-associated disease is selected from the group consisting of: cancer; vascular disease; cardiovascular disease; heart disease; bone disease; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; ischemia/reperfusion injury; and osteoporosis. 
     
     
         32 . A method of  claim 30 , wherein the mammal is a human. 
     
     
         33 . A method of  claim 30 , wherein the therapeutic composition comprises an amino acid peptide comprising a sequence selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9. 
     
     
         34 . A method of treating cancer in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         35 . A method of treating vascular disease in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         36 . A method of treating heart disease in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         37 . A method of treating prostate cancer in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         38 . A method of treating breast cancer in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         39 . A method of treating neuroblastoma in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         40 . A method of treating cardiac hypertrophy in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         41 . A method of treating tissue fibrosis in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         42 . A method of treating congestive heart failure in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         43 . A method of treating ischemia/reperfusion injury in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         44 . A method of treating osteoporosis in a mammal in need of such treatment, comprising administering a Src-inhibiting therapeutic composition of  claim 2 . 
     
     
         45 . A method to reduce increased basal Src activity in a tumor cell when the expression of Na/K-ATPase is reduced, comprising administering a Src-inhibiting composition of  claim 1  to a Src-expressing tumor cell. 
     
     
         46 . A method to inhibit FAK in a tumor cell comprising administering a Src-inhibiting composition of  claim 1  to a Src-expressing tumor cell. 
     
     
         47 . A method for screening at least one test composition to determine whether the at least one composition affects Src, comprising:
 introducing a test composition comprising a modified amino acid peptide of SATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3] to Src, wherein the modification is at least one conservative amino acid substitution; and   determining whether the test composition affects Src.   
     
     
         48 . A method of  claim 47 , wherein the affect is selected from the group consisting of: Src binding; Src inhibition; Src stimulation; Src function; Lyn binding; Lyn function; Lyn inhibition; ouabain antagonism; Na/K-ATPase function; ERK1/2 function; FAK inhibition. 
     
     
         49 . A method of  claim 47 , wherein introducing a test composition is accomplished in vitro. 
     
     
         50 . A method of  claim 47 , wherein introducing a test composition is accomplished in at least one mammalian cell. 
     
     
         51 . A method of  claim 47 , wherein introducing a test composition is accomplished in at least one tumor cell line. 
     
     
         52 . A method of  claim 51 , wherein determining whether the composition affects Src is measured by cell growth compared to control. 
     
     
         53 . A method of  claim 51 , wherein determining whether the composition affects Src is measured by cell migration compared to control. 
     
     
         54 . A method of  claim 51 , wherein the at least one mammalian cell is an animal model. 
     
     
         55 . A method of  claim 51 , wherein the animal model is a NOD/SCID mouse. 
     
     
         56 . A novel Src inhibitor, comprising a composition that targets the Na/K-ATPase/Src receptor complex and antagonizes CTS-induced protein kinase cascades in one or more cells in need thereof. 
     
     
         57 . A method for inhibiting Src activity or antagonizing CTS-induced signal transduction in a subject in need thereof, comprising administering an effective amount of one or more peptides derived from Na/K-ATPase or a biologically active fragments thereof. 
     
     
         58 . A composition that functions as an effective Src inhibitor, is not an ATP analog, does not directly affect PKC and ERK families of serine/threonine kinases, and inhibits Lyn, a Src family tyrosine kinase, comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         59 . A highly positively charged leader peptide conjugate comprising HIV-Tat-NaKtide (pNaKtide) which readily enters cells and resides in the plasma membrane. 
     
     
         60 . A highly positively charged leader peptide conjugate comprising Penetratin-NaKtide (AP-NaKtide) which readily enters cells and resides in vesicles. 
     
     
         61 . A highly positively charged leader peptide conjugate comprising HIV-Tat-SS-NaKtide (ssNaKtide) which readily enters cells and resides in the cytosol. 
     
     
         62 . A highly positively charged leader peptide conjugate comprising Na/K-ATPase α1 N-terminus-NaKtide (A1N-NaKtide) which readily enters cells and resides in cytosol. 
     
     
         63 . A method for targeting the Na/K-ATPase-interacting pool of Src and acting as a potent ouabain antagonist in one or more cells in need thereof, comprising administering an effective amount of pNaKtide. 
     
     
         64 . A method for targeting vesicular Src and acting as a potent ouabain antagonist in one or more cells in need thereof, comprising administering an effective amount of AP-NaKtide. 
     
     
         65 . A method for targeting whole cell Src and acting as a potent ouabain antagonist in one or more cells in need thereof, comprising administering an effective amount of ssNaKtide. 
     
     
         66 . A method for targeting vesicular Src and acting as a potent ouabain antagonist in one or more cells in need thereof, comprising administering an effective amount of A1N-NaKtide. 
     
     
         67 . A method for disrupting the formation of Na/K-ATPase/Src receptor complex in a dose-dependent manner, comprising administering an effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         68 . A method for blocking ouabain-induced activation of Src and a down-stream signaling pathway, such as ERK1/2, in a subject in need thereof, comprising administering an effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         69 . Use of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof as a ouabain antagonist. 
     
     
         70 . A method for determining the physiological and pathological significance of the signaling function of Na/K-ATPase and CTS, comprising using one or more peptides derived from Na/K-ATPase or biologically active fragments thereof as a probe. 
     
     
         71 . A Src inhibitor comprising a composition capable of targeting the plasma membrane Na/K-ATPase/Src complex selected from one or more of SEQ ID NOs: 1, 2, 3, 4 and 5, or biologically active fragments thereof. 
     
     
         72 . A composition capable of selectively targeting the Na/K-ATPase-interacting pool of Src and of functioning as an effective ouabain antagonist in one or more cells in need thereof, comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         73 . As a Src inhibitor that is specific to Src and shows no direct effect on PKC family of kinases, comprising one or more of ND1, NaKtide, pNaKtide, AP-NaKtide, ssNaKtide, A1N-NaKtide, and biologically active fragments thereof. 
     
     
         74 . A specific ouabain antagonist, comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof tagged with a positively charged leader peptide. 
     
     
         75 . The antagonist of  claim 74 , wherein the tagged peptide comprises HIV-Tat or Penetratin, HIV-Tat-SS (linked to NaKtide via SS bond), or Na/K-ATPase N-terminus tagged to NaKtide. 
     
     
         76 . A method for determining the physiology and/or probing the pathological significance of Na/K-ATPase and endogenous CTS, comprising using one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         77 . A therapeutic composition for cardiovascular diseases where the Na/K-ATPase/Src receptor is over-stimulated, comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         78 . A method of inducing cell growth inhibition and/or cell death in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a non ATP-competitive inhibitor of Src and a CTS antagonist. 
     
     
         79 . A composition for preventing CTS-provoked signaling pathway in a subject in need thereof, the composition comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         80 . A method for substantially abolishing ouabain-provoked signaling transduction in the heart in a subject in need thereof, comprising administering an effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         81 . The use of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, in the preparation of a medicament for the treatment of a cancer related disorder or a cardiac disease related disorder. 
     
     
         82 . A pharmaceutical composition comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, and a physiologically acceptable carrier. 
     
     
         83 . The composition of  claim 82 , wherein the composition is adapted for use as a treatment for cardiac hypertrophy, tissue fibrosis and/or congestive heart failure. 
     
     
         84 . The composition of  claim 82 , wherein the composition is adapted for use as a chemotherapeutic agent. 
     
     
         85 . The composition of  claim 82 , wherein the composition is adapted for use as a treatment for a cancer related disorder. 
     
     
         86 . The composition of  claim 82 , wherein the composition is adapted for use as a treatment for a cancer related disorder selected from one or more of prostate cancer, breast cancer, and neuroblastoma. 
     
     
         87 . A method of identifying a candidate compound for the treatment of a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, the method comprising:
 providing an assay for detecting an interaction between Na/K-ATPase and Src which inhibits Src activity;   conducting the assay with a test compound; and   identifying a test compound that is a non ATP-competitive Src inhibitor and a CTS antagonist,   
       wherein a test compound that significantly inhibits the disorder is a candidate compound for the treatment of the disorder. 
     
     
         88 . A method of identifying a candidate compound for the treatment of a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, the method comprising:
 providing a model of the disorder;   contacting the model with a test compound;   detecting a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   comparing the level of the peptides and Src to a reference,   
       wherein a test compound that causes a significant difference in a level of the peptides and active Src as compared to the reference is a candidate compound for the treatment of the disorder. 
     
     
         89 . A method of diagnosing a subject with a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, the method comprising:
 providing a sample from the subject;   detecting in the sample a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   comparing the level of peptides and active Src to a reference,   
       wherein a significant difference in a level of the peptides as compared to the reference indicates that the subject has the disorder. 
     
     
         90 . A method of evaluating a treatment for a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, the method comprising:
 providing a sample from the subject;   detecting in the sample a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   administering one or more doses of a treatment, and comparing the level of the peptides to a reference,   
       wherein a significant difference in a level of the peptides and Src, as compared to an unaffected individual, as compared to the reference indicates the efficacy of the treatment. 
     
     
         91 . The method of  claim 90 , wherein the reference represents a level of the peptides and Src prior to administration of the treatment. 
     
     
         92 . The method of  claim 90  wherein the sample is from cardiac tissue or a cancer cell of the subject. 
     
     
         93 . A method of determining a subject's risk for development of a complication of a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, the method comprising:
 providing a sample from the subject;   detecting in the sample a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   comparing the level of the peptides and Src to a reference,   
       wherein a significant difference in a level of the peptides as compared to the reference indicates the subject's risk of developing the complication. 
     
     
         94 . A method of determining when a treatment modality administered to a subject to treat a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis and/or congestive heart failure can be stopped, the method comprising:
 providing a sample from the subject;   detecting in the sample a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   comparing the level of the peptides and Src to a reference,   
       wherein a level of the peptides and Src that approaches the level of the peptides and Src in the reference indicates whether the treatment can be stopped. 
     
     
         95 . A method of determining when a treatment for a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, should be initiated in a subject, the method comprising:
 providing a sample from the subject;   detecting in the sample a level of: i) one or more peptides derived from Na/K-ATPase or biologically active fragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; and   comparing the level of to a reference,   
       wherein a significant difference in a level of the peptides and active Src as compared to the reference indicates whether the treatment should be initiated. 
     
     
         96 . The method of  claim 95 , wherein the reference represents a level of peptides and active Src in an unaffected subject. 
     
     
         97 . A method for preventing or treating a condition mediated by a ouabain steroid in a subject, comprising administering one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, and/or an agonist or antagonist thereof. 
     
     
         98 . The method of  claim 97 , wherein the condition is one or more of cancer, cardiac hypertrophy, tissue fibrosis or congestive heart failure. 
     
     
         99 . A method for identifying one or more of:
 i) a substance that modulates a ouabain steroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex,   ii) a process mediated by a ouabain steroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex,   iii) degradation of a ouabain steroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex,   iv) a ouabain steroid receptor and/or Na/K-ATPase receptor signaling transduction pathway,   v) a condition mediated by a ouabain steroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex,   vi) a steroid receptor transactivation, and/or inhibits or potentiates the interaction of a ouabain steroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex,   
       comprising
 assaying for a substance that inhibits or stimulates a ouabain steroid receptor, Na/K-ATPase receptor and/or a Na/K-ATPase receptor/Src complex. 
 
     
     
         100 . A method for evaluating a substance, comprising:
 reacting one or more peptides derived from Na/K-ATPase or biologically active fragments thereof and a receptor therefore with a test substance, wherein the peptides and receptor bind to form a complex; and   comparing to a control in the absence of the test substance to determine if the substance stimulates or inhibits the binding of the peptides to the receptor.   
     
     
         101 . A method of conducting a drug discovery business comprising:
 a) providing a method for identifying a substance as claimed in  claim 100 ;   b) conducting therapeutic profiling of substances identified in step a), or further analogs thereof, for efficacy and toxicity in animals; and   c) formulating a pharmaceutical preparation including one or more substances identified in step b) as having an acceptable therapeutic profile.   
     
     
         102 . A method for regulating the Na/K-ATPase/Src receptor complex in a subject comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, a complex thereof; or interaction thereof with a receptor. 
     
     
         103 . Antibodies specific for peptides derived from Na/K-ATPase or biologically active fragments thereof. 
     
     
         104 . Antibodies labeled with a detectable substance and used to detect proteins or complexes peptides derived from Na/K-ATPase or biologically active fragments thereof in biological samples, tissues, and cells. 
     
     
         105 . Antibodies having uses in therapeutic applications, and in conjugates and immunotoxins as target selective carriers of various agents which have therapeutic effects including chemotherapeutic drugs, toxins, immunological response modifiers, enzymes, and radioisotopes. 
     
     
         106 . A pharmaceutical composition adapted for administration to a subject for the prevention or treatment of a condition mediated by a steroid receptor comprising an effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, or agonists or antagonists thereof, or an agent, compound or substance identified using a method of  claim 99  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         107 . A pharmaceutical composition comprising an effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof or an agonist or antagonist thereof, and an appropriate carrier, diluent, or excipient. 
     
     
         108 . A pharmaceutical composition adapted for administration to a subject for the prevention or treatment of a condition mediated by a steroid receptor, in particular a condition mediated by a ouabain receptor, and an appropriate carrier, diluent, or excipient. 
     
     
         109 . Use of peptides derived from Na/K-ATPase, biologically active fragments thereof or agonists or antagonists thereof, for the manufacture of, or in the preparation of a medicament.