US20110245167A1 - NaK-ATPase-Derived Peptide SRC Inhibitors and Ouabain Antagonists and Uses Thereof - Google Patents

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

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US20110245167A1
US20110245167A1 US13/133,252 US200913133252A US2011245167A1 US 20110245167 A1 US20110245167 A1 US 20110245167A1 US 200913133252 A US200913133252 A US 200913133252A US 2011245167 A1 US2011245167 A1 US 2011245167A1
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src
atpase
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Zi-Jian Xie
Zhichuan Li
Joseph I. Shapiro
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University of Toledo
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Assigned to THE UNIVERSITY OF TOLEDO reassignment THE UNIVERSITY OF TOLEDO ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LI, ZHICHUAN, XIE, ZI-JIAN, SHAPIRO, JOSEPH I.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is based, in part, on the discovery of a Na/K-ATPase/Src receptor complex, and its uses to develop novel agonists or antagonists of the receptor.
  • the receptor function of Na/K-ATPase/Src complex is altered in many diseases including cancer, tissue fibrosis, congestive heart failure and ischemia/reperfusion injury.
  • the Na/K-ATPase enzyme is ubiquitously expressed in most eukaryotic cells and is essential for maintaining the trans-membrane ion gradient by pumping Na + out and K + into cells (1).
  • the enzyme consists of two non-covalently linked ⁇ and ⁇ subunits. Similar to other P-ATPases, including the gastric H/K-ATPase and sarcoplasmic reticulum Ca-ATPase (SERCA), the Na/K-ATPase ⁇ subunit has 10 transmembrane domains with both N- and C-termini located in the cytoplasm (2,3).
  • the ⁇ subunit consists of several well-characterized domains: actuator (A) domain consists of the N-terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3; highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane; and a relatively isolated nucleotide-binding (N) domain (4).
  • actuator (A) domain consists of the N-terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3
  • P discontinuous phosphorylation
  • N nucleotide-binding
  • IP3Rs inositol 1,4,5-trisphosphate receptors
  • PI3K phosphoinositide 3′ kinase
  • PLC- ⁇ phospholipase C- ⁇
  • cofilin 7-12.
  • CTS cardiotonic steroids
  • CSK c-terminal Src kinase
  • CHK CSK-homologous kinase
  • WASP Wiscott-Aldrich syndrome protein
  • RACK1 caveolin (20-23).
  • the Na/K-ATPase interacts directly with Src via at least two binding motifs: one being between the CD2 of the ⁇ 1 subunit and Src SH2; and, other involving the third cytosolic domain (CD3) and Src kinase domain.
  • the formation of this Na/K-ATPase and Src complex serves as a receptor for ouabain to provoke protein kinase cascades. Specifically, binding of ouabain to Na/K-ATPase will disrupt the latter interaction, and then result in assembly and activation of different pathways including ERK cascades, PLC/PKC pathway and mitochondrial ROS production (24). Moreover, this interaction keeps Src in an inactive state.
  • the Na/K-ATPase functions as an endogenous negative Src regulator.
  • the basal Src activity is inversely correlated to the amount of Na/K-ATPase ⁇ 1 subunit in both cultured cells and in ⁇ 1 heterozygous mouse tissues (25,26).
  • PCT/US07/023,011 filed Oct. 17, 2007 (Pub. No. WO 2808/054792 on May 8, 2008), claiming priority from U.S. Ser. No. 60/855,482 filed Oct. 16, 2006, which are expressly incorporated herein by reference.
  • Such a method would also contribute to the development of increasingly more effective therapeutic, diagnostic, or prophylactic agents having fewer side effects.
  • a novel Src inhibitor comprising a composition that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades in one or more cells in need thereof.
  • a novel Src inhibitor comprising a composition that mimics the Na/K-ATPase-mediated regulation of Src and Src family kinases in one or more cells in need thereof.
  • a novel Src inhibitor comprising a composition that inhibits Src in vesicular or cytosolic compartments in one or more cells in need thereof.
  • FIGS. 1A-1E Identification of the N-terminus of N domain as a Src-interacting motif from the Na/K-ATPase ⁇ 1 subunit.
  • FIG. 1A Schematic presentations of different GST-fusion proteins.
  • FIGS. 1B , 1 D The Coomassie blue staining of purified GST-ND, GST-CD3 and GST-ND1, GST-ND1R, GST-ND2, GST-ND2R.
  • FIGS. 1C , 1 E Binding of GST-ND and GST-ND1 to Src. Purified His-Src (200 ng) was incubated with 5 ⁇ g GST fusion proteins in 0.5% Triton X-100 PBS for 30 min and followed by three washes with the same buffer. A representative Western blot from three independent experiments showed the pulldown products probed with anti-His antibody.
  • FIGS. 2A-2D Regulation of Src by ND1.
  • FIG. 2A Soluble GST fusion proteins (100 ng) was incubated with recombinant Src (4.5 U) for 15 min in PBS. Src activity was indicated by the phosphorylation of Y418 in the presence of ATP/Mg 2+ .
  • FIG. 2B Dose-dependent inhibition of Src by GST-ND1.
  • FIG. 2C-2D Effects of YFP-ND1 and other fusion proteins on Src activity in LLC-PK1 cells.
  • Cells were transiently transfected with pEYFP or pEYFP-ND1, pEYFP-ND, pEYFP-CD3 plasmids.
  • 24 h after transfection phosphorylation of Src in the cell lysates was measured by Western blot with anti-pY418 antibody. Representative Western blots and combined data from 3 to 5 independent experiments were shown. **p ⁇ 0.01 compared with the control.
  • FIGS. 3A-3B Targeting of YFP-ND1 to the Na/K-ATPase/Src complex in live cells.
  • FIG. 3A Localization of YFP-ND1 in LLC-PK1 cells. LLC-PK1 cells were transfected with pEYFP-ND1 and localization of YFP-ND1 was detected by confocal microscope. Arrow indicated the plasma membrane localization of YFP-ND1.
  • FIG. 3B Lysates from transfected cells were immunoprecipitated with anti-Na/K-ATPase ⁇ 1 antibody and then analyzed by Western blot using anti-GFP antibody and anti-Na/K-ATPase ⁇ 1 antibody. A representative Western blot of three separate experiments was shown.
  • FIGS. 4A-4F Effect of ND1-derived peptides on Src activity.
  • FIG. 4A Sequences of different ND1-derived peptides:
  • FIG. 4B Each peptide (1 ⁇ M) was incubated with recombinant Src (4.5 U) for 15 min, and then assayed for pY418 as in FIG. 1 .
  • FIG. 4C Dose-dependent inhibition of Src by the “NaKtide” (peptide 3). Curve fit analysis was performed with GraphPad software.
  • FIG. 4D The effect of ATP concentration on the “NaKtide” (peptide 3)-induced Src inhibition.
  • the “NaKtide” (peptide 3) (0.1 ⁇ M) was incubated with Src, and then assayed for pY418 in the presence of different concentrations of ATP/Mg 2+ .
  • FIG. 4E Effects of the “NaKtide” (peptide 3) on Lyn kinase activity. Recombinant Lyn (100 ng) was incubated with indicated amount of peptide 3 for 15 min, and then assayed for pY396 by Western blot. Curve fit analysis was done with GraphPad software.
  • FIG. 4F Effects of the “NaKtide” (peptide 3) on kinase activity of PKC mixture. Quantitative data were presented as mean ⁇ SE of at least three independent experiments. **p ⁇ 0.01 compared with control.
  • FIGS. 5A-5E Properties of cell permeable peptides.
  • FIG. 5A Sequence information of different peptides:
  • FIG. 5B Dose-dependent inhibition of Src by the “pNaKtide” and control “pC1”. Curve fit analysis was performed by GraphPad software.
  • FIG. 5C , 5 D Cell loading analyses of the “pNaKtide” and “AP-NaKtide” in LLC-PK1 cells.
  • Cells were serum-starved for 12 h and then exposed to 1 ⁇ M of FITC-pNaKtide or FITC-AP-NaKtide at 37° C. for 60 min. Cells were washed twice with PBS, and analyzed by confocal imaging. The scale bar represents 20 ⁇ m.
  • FIG. 5E Inhibition of Src by “A1N-NaKtide”.
  • FIGS. 6A-6C Effects of the “pNaKtide” on the formation of Na/K-ATPase/Src complex.
  • FIG. 6A LLC-PK1 cells were transfected with EYFP-rat ⁇ 1 (yellow) and Src-ECFP (cyan). FRET analysis was performed as described herein. Region of Interest 1 (Boxed area marked by ROI 1) was photobleached and analyzed for FRET. The same measurement was done in ROI 2 that was not photobleached.
  • FIGS. 6B , 6 C The same FRET analyses were conducted in transfected cells pretreated with 1 ⁇ M pC1 or different concentrations of “pNaKtide” for 1 h. Average FRET efficiency ( FIG. 6B ) and percentage of cells ( FIG. 6C ) showing FRET efficiency (cut-off value is 4.0%) were calculated. At least 20 cells from three experiments were measured for each condition.
  • FIGS. 7A-7B Effect of the “pNaKtide” on Src and Src-mediated signaling pathway.
  • FIGS. 8A-8C Effect of the “pNaKtide” on ouabain-induced signal transduction.
  • FIG. 9 Table 1 showing the effects of ND1, NaKtide, pNaKtide and PP2 on Src.
  • FIG. 10A-10D Correlation between Na/K-ATPase ⁇ 1 amount and Src activity.
  • FIG. 10A Expression level of Na/K-ATPase ⁇ 1 in different cell lines. Various cells were harvested at 95% density and lysates were analyzed by Western blot with anti-Na/K-ATPase ⁇ 1 antibody.
  • FIG. 10B Effects of Na/K-ATPase knockdown on Src activity in mouse liver.
  • Liver samples from wild type (WT) and Na/K-ATPase ⁇ 1 knockdown (+/ ⁇ ) mice were assessed by Western blot. N 6. **p ⁇ 0.01.
  • FIG. 10C , 10 D Changes of Na/K-ATPase ⁇ 1 amount in cells with different densities. Lysates of LLC-PK1 cells ( FIG. 10C ) and DU145 cells ( FIG. 10D ) under indicated cell densities were analyzed with anti-Na/K-ATPase ⁇ 1, pY418 and Src antibodies.
  • FIG. 11A-11C Regulation of Src and Src-mediated signaling by “pNaKtide”.
  • FIG. 11A Regulation of basal FAK and ERK phosphorylation by “pNaKtide”. TCN23-19 cells were serum-starved for 12 h and then exposed to 1 ⁇ M “pNaKtide” for indicated times. FAK and ERK phosphorylation was assessed by Western blot with anti-pFAK576/7 and anti-phospho-ERK antibodies, respectively.
  • FIG. 11B Effect of “pNaKtide” on Src in various cells.
  • Cultured LLC-PK1, LNCaP, and DU145 cells were exposed to 1 ⁇ M pNaKtide for 1 h. Then, cells were fixed with cold methanol and immunostaining was performed with anti-Na/K-ATPase ⁇ 1 and anti-pY418 antibodies.
  • FIG. 11C Regulation of Src and Src-mediated signaling by “pNaKtide” in DU145 cells.
  • Cell lysates were analyzed by Western blot with antibodies against pY418, phosphor-ERK, pFAK576/7 and c-Myc.
  • Membranes were striped and reprobed with antibodies against Src, ERK, and FAK.
  • FIG. 12 Effect of “pNaKtide” on prostate cancer cell migration. Scraped wound was introduced on confluent monolayer DU145 cells and the status of wound closure in the absence or presence of “pNaKtide” was monitored after 24 h.
  • FIG. 13A , 13 B Effect of ND1 overexpression on cell viability in cancer cells.
  • DU145 cells FIG. 13A
  • MCF-7 cells FIG. 13B
  • Fluorescence and phase-contrast images were collected at indicated time after transfection and then merged with SPOT Version 4.6 software (Diagnostic Instruments). Small boxed regions were enlarged and shown in the bottom right boxes. Original magnifications, ⁇ 400. The same experiments were repeated at least three times.
  • FIG. 14A “pNaKtide” caused dose-dependent inhibition of cell viability in LNCaP, DU145, and PC-3 prostate cancer cells.
  • FIG. 14B Time-dependent effect of “pNaKtide” on DU145 cell viability.
  • FIG. 14C , 14 D Dose-dependent inhibition of cell viability in neuroblastoma cells ( FIG. 14C ) and breast cancer cells ( FIG. 14D ).
  • FIG. 15A Effect of “pNaKtide” on tumorigenicity of DU145 cells in NOD/SCID mice. Tumors were removed and weighted after mice were sacrificed at 44 days. *, P ⁇ 0.05. **, P ⁇ 0.01.
  • FIG. 15B Mice bearing xenograft tumors. Arrowheads identify the location of the tumors.
  • FIG. 15C Growth of DU145 xenograft tumors in NOD/SCID mice treated with saline or “pNaKtide”.
  • FIG. 15D Inhibition of Src in “pNaKtide” treated xenograft tumors. After Xenograft tumors were removed and weighted, tumor homogenates were assessed by Western blot. **, P ⁇ 0.01.
  • FIG. 16A-16D Effect of “pNaKtide” on angiogenesis. *, P ⁇ 0.05. **, P ⁇ 0.01.
  • FIG. 16A Inhibition of endothelial cell proliferation by “pNaKtide”.
  • Human Umbilical Vein Endothelial cells (HUVEC) and Human Aortic Endothelial cells (HAEC) cultured on 12-well plates were exposed to “pNaKtide” with indicated concentrations for 72 h. Cell numbers were counted with hemocytometer.
  • HUVEC Human Umbilical Vein Endothelial cells
  • HAEC Human Aortic Endothelial cells
  • FIG. 16B Immunohistostaining of CD31 in the formalin-fixed, paraffin-embedded xenograft tumors.
  • FIG. 16C Effect on the tumor vessel density by “pNaKtide”. The vessel density was calculated as the percent of tumor area occupied by vessels.
  • FIG. 16D Expression of VEGF in saline/pNaKtide-treated xenografted tumor homogenates. Tumor homogenates were analyzed by Western blot with anti-VEGF antibody.
  • This invention is based, at least in part, on the inventors' discovery that the Na/K-ATPase binds and inhibits Src.
  • the inventors now reveal the molecular mechanism of Na/K-ATPase-mediated Src regulation.
  • the invention relates to the generation of a novel peptide Src inhibitor from the Na/K-ATPase ⁇ 1 subunit that targets to the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades in cultured cells.
  • the invention is also based, at least in part, on the inventors' discovery that the Na/K-ATPase inhibits Src kinase by binding of the N-terminus of nucleotide binding domain to the Src kinase domain.
  • NaKtide 20 amino acid peptide
  • the invention is also based, at least in part, on the inventors' discovery that highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide), Penetratin-NaKtide (AP-NaKtide), ⁇ 1 N-terminus-NaKtide (A1N-NaKtide) readily enter cultured cells.
  • pNaKtide HIV-Tat-NaKtide
  • AP-NaKtide Penetratin-NaKtide
  • A1N-NaKtide ⁇ 1 N-terminus-NaKtide
  • the invention is also based, at least in part, on the inventors' discovery, using the functional studies of pNaKtide, that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and acts as a potent ouabain antagonist in cultured cells.
  • the invention is also based, at least in part, on the inventors' discovery, using the functional studies of AP-NaKtide, that this conjugate can specifically target the intracellular vesicles and can act as a potent Src inhibitor and ouabain antagonist in cultured cells.
  • the invention is also based, at least in part, on the inventors' discovery, using the functional studies of NaKtide, that loading the cells with soluble NaKtide by either detergent or HIV-Tat-SS-NaKtide (ssNaKtide) conjugate can produce potent inhibition of cellular Src activity and acts as a potent ouabain antagonist in cultured cells.
  • the invention is also based, at least in part, on the inventors' discovery, using the functional studies of A1N-NaKtide, that this conjugate can specifically target the intracellular compartments and can act as a potent Src inhibitor and ouabain antagonist in cultured cells.
  • pNaKtide unlike PP2, resides mainly in the plasma membrane. Consistently, it affects much less the basal Src activity than that of PP2.
  • AP-NaKtide and A1N-NaKtide reside mainly in vesicles and also have less effect on basal Src activity.
  • ssNaKtide would have significant effect on basal Src as PP2.
  • pNaKtide is effective in disrupting the formation of Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src and a down-stream signaling pathway such as ERK1/2 in cultured cells.
  • pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes.
  • the present invention relates to the use of pNaKtide as a ouabain antagonist.
  • the invention is also based, at least in part, on the inventors' discovery that some cancer cells express less Na/K-ATPase and have higher Src activity.
  • the invention is also based, at least in part, on the inventors' discovery that pNaKtide and AP-NaKtide can mimic the Na/K-ATPase and inhibit Src and then FAK in cancer cells.
  • the invention is also based, at least in part, on the inventors' discovery that pNaKtide and AP-NaKtide are effective in inhibiting cancer cell migration.
  • the invention is also based, at least in part, on the inventors' discovery that pNaKtide can inhibit proliferation of endothelial cells and prevent angiogenesis.
  • the invention is also based, at least in part, on the inventors' discovery that expression of Src inhibiting YFP-ND1 or addition of either pNaKtide or AP-NaKtide inhibits some cancer cell growth or causes cell death.
  • the invention is also based, at least in part, on the inventors' discovery that pNaKtide is effective in blocking the growth of xenografted prostate cancer in NOD/SCID mice.
  • a method for inhibiting Src activity or antagonizing CTS-induced signal transduction in a subject in need thereof comprising administering an effective amount of a peptide derived from Na/K-ATPase or biologically active fragments thereof.
  • compositions that functions as an effective Src inhibitor are 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.
  • a method to target NaKtide to different cellular compartments to achieve cellular compartment-specific inhibition of Src as exemplified by the following conjugates: HIV-Tat-NaKtide (pNaKtide) to the plasma membrane; Penetratin-NaKtide (AP-NaKtide) and Na/K-ATPase ⁇ 1-N-terminal-NaKtide (A1N-NaKtide) to vesicles; and HIV-Tat-S-S-NaKtide (ssNaKtide) to the cytosol.
  • HIV-Tat-NaKtide pNaKtide
  • AP-NaKtide Penetratin-NaKtide
  • A1N-NaKtide Na/K-ATPase ⁇ 1-N-terminal-NaKtide
  • ssNaKtide HIV-Tat-S-S-NaKtide
  • 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.
  • a method for targeting the Src in vesicles and acting as a potent Src inhibitor or ouabain antagonist in one or more cells in need thereof comprising administering an effective amount of AP-NaKtide or A1N-NaKtide.
  • a method for targeting Src in whole cell and acting as a potent Src inhibitor or ouabain antagonist in one or more cells in need thereof comprising administering an effective amount of ssNaKtide.
  • 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.
  • 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.
  • a method for determining the physiological and pathological significance of the inventors' discovery 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.
  • 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.
  • 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.
  • a Src inhibitor that is specific to Src shows no direct effect on PKC family of kinases, comprising one or more of ND1, NaKtide, pNaKtide, AP-NaKtide, A1N-NaKtide, ssNaKtide and biologically active fragments thereof.
  • 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.
  • the tagged peptide comprises HIV-Tat or Penetratin or Na/K-ATPase ⁇ 1 N-terminal peptide or other positively charged leader peptide tagged to NaKtide.
  • 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.
  • a new 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.
  • a method of inducing cell growth inhibition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of Src and a CTS antagonist.
  • compositions for preventing CTS-provoked signaling pathway comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof.
  • 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.
  • composition comprising one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, and a physiologically acceptable carrier.
  • the composition is adapted for use as a treatment for cardiac hypertrophy, tissue fibrosis and/or congestive heart failure.
  • the composition is adapted for use as a chemotherapeutic agent.
  • the composition is adapted for use as a treatment for a cancer related disorder.
  • 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.
  • 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 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.
  • 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 comprising: providing a model of the disorder; contacting the model with a test compound; 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 active Src to a reference, wherein a test compound that causes a significant difference in a level of the peptides as compared to the reference is a candidate compound for the treatment of the disorder.
  • a method of diagnosing a subject with a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer 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 these parameters 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.
  • a method of evaluating a treatment for a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer 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 and active Src to a reference, wherein a significant difference in a level of the peptides, as compared to an unaffected individual, as compared to the reference indicates the efficacy of the treatment.
  • the reference represents a level of the peptides prior to administration of the treatment.
  • the sample is from cardiac tissue or a cancer cell of the subject.
  • 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 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 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.
  • 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 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 as compared to the reference indicates whether the treatment should be initiated.
  • the reference represents a level of Na/K-ATPase peptides or Na/K-ATPase or CTS binding in an unaffected subject.
  • a method for preventing or treating a condition mediated by a ouabain steroid receptor 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.
  • the condition is one or more of cancer, cardiac hypertrophy, tissue fibrosis or congestive heart failure.
  • 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.
  • a method of conducting a drug discovery business comprising: a) providing a method for identifying a substance identified using one or more methods described herein; 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.
  • the receptor is a ouabain receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex.
  • a part of the peptide consists of a binding domain of the peptide that interacts with a ouabain receptor, wherein the part is the N-terminus of nucleotide binding domain that binds the Src kinase domain.
  • a method for regulating the Na/K-ATPase/Src receptor complex in a subject comprising inhibiting or stimulating the expression of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, a complex thereof; or the interactions thereof with a receptor.
  • a method for identifying one or more conditions selected from: cardiac hypertrophy, tissue fibrosis, congestive heart failure or cancer, in a subject comprising detecting changes in one or more peptides derived from Na/K-ATPase or Na/K-ATPase, CTS binding or biologically active fragments thereof in a sample from the subject.
  • the method comprising: collecting a sample from the subject; measuring the levels of one or more peptides in the sample; and comparing the levels of peptides in the sample to the levels in subjects not having cancer or a cardiac condition.
  • significantly decreased levels in the sample compared to levels in samples from subjects who do not suffer from the condition is indicative of an increased risk of the condition in the subject.
  • agents, compounds, and substances identified using the methods described herein are provided herein.
  • the agents, compounds, and substances are useful in the treatment or prevention of a condition mediated by a steroid receptor, including a condition mediated by a ouabain receptor.
  • antibodies specific for peptides derived from Na/K-ATPase or biologically active fragments thereof are provided herein.
  • antibodies labeled with a detectable substance and used to detect proteins or complexes derived from Na/K-ATPase or biologically active fragments thereof in biological samples, tissues, and cells.
  • 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.
  • 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 described herein, and a pharmaceutically acceptable carrier, diluent or excipient.
  • compositions adapted for the treatment of a patient suffering from a cardiac or cancer disorder which comprises a therapeutically effective amount of one or more peptides derived from Na/K-ATPase or biologically active fragments thereof, or a pharmaceutically acceptable salt thereof.
  • 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.
  • compositions 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.
  • 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.
  • a substance for inhibiting ouabain-provoked signal transduction comprising one or more of the peptides, or a complex thereof.
  • the complex is substantially cell permeable.
  • the present invention relates to 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 one or more amino acid residues, or substitutions with unnatural amino acids to improve pharmacodynamics or/and pharmacokinetics, wherein the peptide is capable of binding the kinase domain of Src.
  • compositions which further comprise a therapeutically acceptable excipient.
  • the amino acid peptide comprises the sequence SATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3].
  • 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.
  • the amino acid peptide comprises SEQ ID NO 7. In other particular embodiments, the amino acid peptide comprises SEQ ID NO: 8 or SEQ ID NO: 9.
  • nucleic acid sequences encoding a composition as described herein.
  • vectors comprising a nucleic acid sequence as described herein.
  • the vector comprises pEYFP.
  • cells comprising a vector as described herein.
  • the cell is E. coli .
  • the cell is mammalian.
  • the cell is a tumor cell.
  • a monoclonal antibody selective for a composition as described herein.
  • the monoclonal antibody further comprises a detectable label selected from the group consisting of: radioactive label; chemical label; fluorescent label; an antibody; and a protein.
  • compositions where the composition is capable of affecting a cellular process selected from the group consisting of: antagonizing a CTS-induced protein kinase cascade; upregulating a CTS induced protein kinase cascade; Src inhibition; Src stimulation; Na/K-ATPase mimic; Na/K-ATPase competitive inhibitor; Lyn inhibition; Lyn stimulation; ouabain antagonism; ouabain stimulation; ERK1/2 activation; ERK1/2 inhibition; membrane permeability by sodium ions; membrane permeability by potassium ions.
  • the composition is not an ATP analog.
  • compositions which further comprise means to therapeutically permeate plasma membrane.
  • compositions further comprise 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; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; ischemia/reperfusion injury.
  • a disease selected from the group consisting of: cancer; vascular disease; cardiovascular disease; heart disease; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; ischemia/reperfusion injury.
  • the composition 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: chemotherapeutic drug; toxin; immunological response modifier; enzyme; and radioisotope.
  • the composition 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; GST.
  • the composition comprises HIV-Tat-SEQ ID NO: 3.
  • the composition comprises a fusion protein, provided that the fusion does not disrupt the at least ten consecutive residues of SEQ ID NO: 3.
  • the fusion is with GST.
  • a method to bind a compound to the kinase domain of Src in a Src-expressing cell comprising contacting a compound described herein to at least one Src-expressing cell.
  • the Src-expressing cell is a mammalian cell.
  • 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; blood cell; and brain cell.
  • the at least one mammalian cell is cultured in vitro.
  • the at least one mammalian cell is an animal model.
  • the at least one mammalian cell is a human.
  • a method of treating a Src-associated disease in a mammal in need of such treatment comprising administering a therapeutic composition described herein.
  • the Src-associated disease is selected from the group consisting of: cancer; vascular disease; cardiovascular disease; heart disease; prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heart failure; and ischemia/reperfusion injury.
  • the mammal is a human.
  • 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.
  • a method of treating cancer in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • vascular disease in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating cardiovascular disease in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating heart disease in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating prostate cancer in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating breast cancer in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating neuroblastoma in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating cardiac hypertrophy in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating tissue fibrosis in a mammal in need of such treatment comprising administering a Src-inhibiting therapeutic composition described herein.
  • a method of treating congestive heart failure in a mammal in need of such treatment comprising administering a Src-stimulating therapeutic composition described herein.
  • ischemia/reperfusion injury in a mammal in need of such treatment, comprising administering a Src-stimulating therapeutic composition described herein.
  • a method to reduce increased basal Src activity in a tumor cell comprising administering a Src-inhibiting composition described herein to a Src-expressing tumor cell.
  • a method to inhibit FAK in a tumor cell comprising administering a Src-inhibiting composition described herein to a Src-expressing tumor cell.
  • the Src-expressing cell is a TCN23-19 cell.
  • a method to reduce tumor cell migration in a tumor cell test model comprising administering a Src-inhibiting composition described herein to a Src-expressing tumor cell.
  • a method to kill cancer cells when the expression of Na/K-ATPase is reduced comprising administering a Src-inhibiting composition described herein to a Src-expressing tumor cell having reduced Na/K-ATPase expression.
  • a method of inhibiting cell growth in a tumor cell line comprising administering a Src-inhibiting composition described herein to a Src-expressing tumor cell line.
  • a method which further comprises comparison of the ability of a composition described herein to inhibit cell growth in a tumor cell line to a test compound's ability to inhibit cell growth in the same tumor cell line.
  • a method of inhibiting prostate tumor cell growth in a prostate tumor cell line comprising administering a Src-inhibiting composition described herein to a Src-expressing prostate tumor cell line.
  • a method which further comprises comparison of the ability of a composition described herein inhibiting prostate tumor cell growth in a prostate tumor cell line to a test compound's ability to inhibit cell growth in the same prostate tumor cell line.
  • a method of inhibiting breast tumor cell growth in a breast tumor cell line comprising administering a Src-inhibiting composition described herein to a Src-expressing breast tumor cell line.
  • the method further comprises comparison of the ability of a composition described herein inhibiting prostate tumor cell growth in a breast tumor cell line to a test compound's ability to inhibit cell growth in the same breast tumor cell line.
  • a method of inhibiting neuroblastoma cell growth in a neuroblastoma tumor cell line comprising administering a Src-inhibiting composition described herein to a Src-expressing neuroblastoma tumor cell line.
  • the method further comprises comparison of the ability of a composition described herein inhibiting neuroblastoma tumor cell growth in a prostate tumor cell line to a test compound's ability to inhibit cell growth in the same neuroblastoma tumor cell line.
  • 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.
  • 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.
  • the method includes introducing a test composition is accomplished in vitro.
  • the method includes introducing a test composition is accomplished in at least one mammalian cell.
  • the method includes introducing a test composition is accomplished in at least one tumor cell line.
  • the method includes determining whether the composition affects Src is measured by cell growth compared to control.
  • the method includes determining whether the composition affects Src is measured by cell migration compared to control.
  • the at least one mammalian cell is an animal model.
  • the animal model is a NOD/SCID mouse.
  • determining whether the composition affects Src is measured by tumor growth compared to control.
  • the at least one mammalian cell is a human.
  • the method includes determining whether the composition affects Src is measured by tumor growth compared to control.
  • Polyclonal antibodies against phosphor-Akt (Ser473), Phosphor-FAK (576/7), Akt and FAK were purchased from Cell Signaling Technology (Danvers, Mass.).
  • the polyclonal anti-VEGF antibody and monoclonal anti- ⁇ 1 antibody ( ⁇ 6F) was obtained from Abcam (Cambridge, Mass.) and the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, Iowa), respectively.
  • Glutathione beads from Amersham Bioscience (Uppsala, Sweden) and ProBond Purification System from Invitrogen (Carlsbad, Calif.) were used respectively.
  • Recombinant human Src and Lyn expressed in Sf9 insect cells for kinase activity assay and IGF-1 expressed in Escherichia coli were obtained from Upstate Biotechnology (Lake Placid, N.Y.). Plasmids pEYFP-C1 was purchased from Clontech (Palo Alto, Calif.), and pGEX-4T-1 and pTrc-His A vectors were from Invitrogen (Carlsbad, Calif.). The Optitran nitrocellulose membranes used for Western blotting were obtained from Schleicher and Schuell (Dassel, Germany). All the peptides were synthesized with the purity of 95%. Identity and purity were confirmed by high-performance liquid chromatography-mass spectroscopy.
  • Plasmid Constructs The preparation of plasmid constructs expressing GST fusion proteins were done as described (24). GST-CD3 (amino acid residue 350-785), GST-ND (amino acid residue 379-594), GST-ND2 (amino acid residue 379-475), GST-ND2R (amino acid residue 476-594), GST-ND1 (amino acid residue 379-435) and GST-ND1R (amino acid residue 436-594) expression vectors were constructed based on the sequence of pig kidney Na/K-ATPase ⁇ 1 subunit.
  • His-tagged Src constructs were generated by excising the corresponding Src cDNA from the GST-Src vector (27) and then inserting them into pTrc-His A vector.
  • pEYFP-ND1, pEYFP-ND and pEYFP-CD3 were made by directional subcloning the corresponding cDNAs from the GST-Src vector into pEYFP-C1 vector. All constructs were verified by DNA sequencing.
  • Pig kidney proximal LLC-PK1 and mouse fibroblast SYF and SYF+Src cells were obtained from American Type Culture Collection (Manassas, Va.) and cultured in DMEM medium containing 10% fetal bovine serum and penicillin (100 U/ml)/streptomycin (100 ⁇ g/ml).
  • Na/K-ATPase ⁇ 1 knockdown cells PY-17 and TCN23-19 were generated from LLC-PK1 cells as described (25).
  • Myocytes were plated at a density of 8 ⁇ 10 2 cells/mm 2 in 100-mm Corning cell culture dishes in Dulbecco's modified Eagle's medium-M199 (4:1) containing 10% (vol/vol) fetal bovine serum (24 h, 37° C.) and then incubated in serum-free medium for 48 h before experimentation. All research on rats was done according to procedures and guidelines approved by the Institutional Animal Care and Use Committee.
  • Src Preparation of Src, Na/K-ATPase, GST-fused Proteins, and His-tagged Proteins.
  • Src without the first 85 amino acid residues, was purified from sf-9 cells as described (27) and used in the initial binding assays to ensure that Src binds to the Na/K-ATPase.
  • purified recombinant full-length Src from Upstate Biotechnology was used.
  • Na/K-ATPase was purified from pig kidney outer medulla using the Jorgensen method as we previously described (27) and the preparations with specific activities between 1200 and 1400 ⁇ mmol Pi/mg/h were used.
  • GST-fused proteins or His-tagged proteins were expressed in Escherichia coli BL21 (Invitrogen) and purified using glutathione beads or ProBond Purification System (Invitrogen). Soluble GST-fused proteins were eluted from the glutathione beads with elution buffer [10 mM reduced glutathione, 0.1% Triton X-100, 50 mM Tris-HCl, (pH8.0)] and then dialyzed in the buffer containing 0.1% Triton X-100, 50 mM Tris-HCl (pH8.0) to remove remnant glutathione.
  • elution buffer 10 mM reduced glutathione, 0.1% Triton X-100, 50 mM Tris-HCl, (pH8.0)
  • GST pulldown assay were performed as following: 5 ⁇ g GST-fused proteins were conjugated on glutathione beads and incubated with 100 ng purified his-Src in 500 ⁇ l PBS in the presence of 0.5% Triton X-100 at room temperature for 30 min. The beads were washed with the same buffer for four times. The bound his-Src was resolved on 10% SDS-PAGE and detected by Western blot with anti-His antibody.
  • Cell lysates were cleared by centrifugation at 16,000 ⁇ g for 15 min, and the supernatants were separated by SDS-PAGE (60 ⁇ g/lane) and transferred to an Optitran membrane and were analyzed with anti-pY418 antibody.
  • the pY418 signal was detected using the enhanced chemiluminescence kit (Pierce) and quantified using a Bio-Rad GS-670 imaging densitometer as we previously described (29).
  • PKC Kinase Activity Assay Activity of PKC was measured by PepTag phosphorylation assay for non-radioactive detection of PKC (Promega) as described in the product instruction. Briefly, 40 ng PKC were incubated for 30 min at 30° C. with the reaction mixture containing 5 ⁇ l reaction buffer, 5 ⁇ l PepTag C1 (0.4 ⁇ g/ ⁇ l), 5 ⁇ l PKC activator solution, 1 ⁇ l peptide protection solution and 10 ⁇ M staurosporine or peptide. Then the reaction mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for 20 min.
  • Image Analysis with Confocal Fluorescence Microscope Cells cultured on coverslips were subjected to the indicated treatment. Then cells were washed twice with PBS and fixed for 15 min with methanol prechilled at ⁇ 20° C. The fixed cells were then rinsed with PBS three times and blocked with 200 ⁇ l of Image-iT FX signal enhancer (Invitrogen) for 30 min at room temperature. The cells were washed again and incubated with anti-pY418 antibody in PBS containing 1% bovine serum albumin for 1 h at room temperature. After three washes with PBS, the cells were incubated with Alexa Fluor-conjugated anti-rabbit secondary antibody (Invitrogen). Image visualization was performed using a Leica DMIRE2 confocal microscope (Leica, Mannheim, Germany). Leica confocal software was used for data analysis.
  • DU145 cells were seeded in 6-well plates in growth medium containing 10% fetal bovine serum. The cells were allowed to grow to confluent monolayer. The wound-induced migration was triggered by scraping the cells with a plastic pipette tip. The cells were then were treated with or without pNaKtide at different concentrations. The wound was imaged immediately (0 h) and at 24 h with an inverted phase-contrast microscope with a ⁇ 10 objective.
  • NOD/SCID mice nude mice (NCI) were housed in laminar airflow cabinets under pathogen-free conditions with a 12 h light/12 h dark schedule and fed autoclaved standard chow and water.
  • mice were injected subcutaneously near the tumor site with pNaKtide in saline at the dose of 2 mg/kg and 10 mg/kg (body weight) every other day for one week. All research on NOD/SCID mice was done according to procedures and guidelines approved by the Institutional Animal Care and Use Committee of University of Toledo Health Science Campus.
  • the N domain contains over 200 amino acid residues.
  • the inventors constructed ND2 and ND2 remaining (ND2R) GST fusion proteins as illustrated in FIG. 1A .
  • GST pull-down assay showed that GST-ND2, but not GST-ND2R, bound Src ( FIG. 1E ).
  • ND2 N-terminus of ND2 is highly unstructural and may undergo induced-fit (30). Moreover, this domain is also highly exposed and is less important for ATP binding (31), and thus the catalytic function of the Na/K-ATPase.
  • the inventors constructed two more GST-fusion proteins (ND1 and ND1R), and assessed their binding to Src using GST pull-down assay. As depicted in FIG. 1E , the ND1, but not the ND1R, interacted with Src.
  • ND1 as a potent Src inhibitor can target to the Na/K-ATPase/Src complex in cultured cells: To test whether ND1 inhibits Src as did the CD3, the inventors incubated Src with 100 ng soluble GST fusion proteins in the test tube and measured Src pY418 level by Western blot. GST-CD3 was used in the experiment as a positive control and GST-ND was also tested.
  • both GST-ND1 and GST-ND were as effective as the positive control in inhibiting Src activity.
  • IC 50 50 nM
  • the inventors measured Src pY418 level changes in LLC-PK1 cells transfected with different YFP expression vectors. The expression of YFP-ND1, but not YFP, reduced Src activity in cell lysates as did by YFP-ND and YFP-CD3 ( FIG. 2C , 2 D).
  • the YFP-ND1 was expressed as a soluble protein. However, the inventors did detect a pool of YFP-ND1 resided near the plasma membrane. Second, to test whether this pool of YFP-ND1 interacts with Src, the inventors co-transfected LLC-PK1 cells with YFP-ND1 and Src-CFP, and then performed FRET analysis. A significant FRET was detected in the cotransfected cells (13.4 ⁇ 2.4%), which showed that YFP-ND1 and the plasma membrane Src-CFP were likely to associate.
  • peptide 1 and 2 are un-structural whereas peptides 3 and 4 may form ⁇ -helix.
  • 1 ⁇ M peptide 3 caused almost a 100% inhibition of Src while peptide 4 produced a partial inhibition.
  • both peptide 1 and peptide 2 showed no effect.
  • the inventors showed that peptide 3 was quite potent in inhibiting Src with an IC 50 of about 70 nM ( FIG. 4C ), comparable to that of GST-ND1.
  • the peptide 1 had no effect. Since peptide 3 is derived from the Na/K-ATPase, the inventors named it “NaKtide”. Because peptide 1 has no effect on Src, it was used as a control. Thus, the inventors named it “C1.”
  • NaKtide acts as an ATP analog as a generic Src inhibitor PP2
  • the inventors measured its effect on Src in the presence of different concentrations of ATP. As shown in FIG. 4D , changes in ATP concentration from 0.1 to 2 mM did not affect NaKtide-induced Src inhibition.
  • the inventors measured its dose-dependent effect on another Src family kinase Lyn. As shown in FIG. 4E , the NaKtide produced a dose-dependent inhibition of Lyn. However, the IC 50 is about 2.5 ⁇ M, 40 times higher than that of Src inhibition.
  • NaKtide affects serine/threonine kinases
  • the inventors incubated a PKC family kinase cocktail with NaKtide and C1, and then measured for the kinase activity.
  • FIG. 4F unlike staurosporine (a non-specific PKC family kinase inhibitor), NaKtide, as well as C1, showed no effect up to 10 ⁇ M.
  • HIV-Tat-P1 (pC1) was also synthesized and used as a control. Meanwhile, after careful review on the Na/K-ATPase ⁇ 1 sequence, the inventors found an N-terminal polybasic fragment which may facilitate the uptake of NaKtide. The inventors synthesized the Na/K-ATPase ⁇ 1 N-terminal-NaKtide (A1N-NaKtide). To compare, the inventors also synthesized Penetratin-NaKtide (“AP-NaKtide”). In vitro kinase assay showed that NaKtide was a highly potent Src inhibitor while the control pC1 was inactive ( FIG. 5B ).
  • both pNaKtide and AP-NaKtide were labeled with FITC.
  • FITC confocal imaging analysis of live cells indicated that pNaKtide, but not NaKtide, was cell-permeable. Maximal loading was achieved after 30 to 60 min of incubation with 1 ⁇ M pNaKtide in almost every LLC-PK1 cell in culture.
  • pNaKtide resided mainly in the plasma membrane with some distribution to the intracellular membrane compartments.
  • pNaKtide As a potential ouabain antagonist, the inventors performed FRET analysis to determine the effect of NaKtide on the formation of Na/K-ATPase/Src receptor complex. LLC-PK1 cells were co-transfected with YFP- ⁇ 1 and Src-CFP, and then exposed to different concentrations of pNaKtide. The inventors focused on pNaKtide because this conjugate resided mainly in the plasma membrane where the receptor Na/K-ATPase/Src is located.
  • both YFP- ⁇ 1 and Src-CFP were targeted to the plasma membrane.
  • a significant FRET was detected in control LLC-PK1 cells.
  • Addition of pNaKtide produced a dose-dependent reduction in overall FRET efficiency ( FIG. 6B ) as well as the percentage of cells that exhibited detectable FRET ( FIG. 6C ), indicating that pNaKtide is effective as YFP-ND1 in interacting with the plasma membrane pool of Src, thus blocking the formation of a stable Na/K-ATPase/Src complex. Since significant effect was observed when the cells were exposed to 100 nM to 1 ⁇ M pNaKtide, 1 ⁇ M pNaKtide was used in the following three sets of experiments to test its effectiveness and specificity as a ouabain antagonist.
  • TCN23-19 cells were derived from LLC-PK1 cells (25). Knockdown of the Na/K-ATPase in these cells reduces the pool of Src-interacting Na/K-ATPase and thus increases the basal Src and ERK1/2 activity (25). As depicted in FIG.
  • the inventors measured the effect of pNaKtide in ouabain-induced activation of ERK1/2 in LLC-PK1 cells. This showed that 1 ⁇ M pNaKtide completely abolished ouabain-induced ERK1/2 activation in LLC-PK1 cells ( FIG. 8A ). To confirm that this is not a cell-specific effect, the inventors repeated the same experiments in primary cultures of cardiac myocytes. As shown in FIG. 8B , ouabain-induced activation of ERK1/2 was also blocked by pNaKtide.
  • the inventors also compared pNaKtide and PP2, a generic Src inhibitor. As shown in FIG. 9 —Table 1, both pNaKtide and PP2 have a similar IC 50 on Src kinase. However, PP2 produced more inhibition on basal Src activity than that of pNaKtide in both LLC-PK1 and cardiac myocytes. Moreover, when cardiac myocytes were stimulated by IGF, PP2, but not pNaKtide, caused a significant inhibition of ERK1/2 activation ( FIG. 8C ).
  • the inventors also found that the ⁇ 1 expression was more than doubled with an increase in cell density from 50% to 90% whereas active Src was reduced more than 80% in LLC-PK1 cells ( FIG. 10C ). However, this regulation was lost in DU145 cells, apparently because of defects in both ⁇ 1 and Src expression ( FIG. 10D ). When the same experiments were repeated in LNCaP and PC3 cells, we observed essentially the same defect (data not shown).
  • pNaKtide inhibits FAK and reduces cell migration: Because Src is the affector of FAK, a key kinase involved in control of cell migration and thus tumor metastasis, the inventors tested whether pNaKtide can inhibit FAK and tumor cell migration. As depicted in FIG. 11A , pNaKtide caused a time-dependent inhibition of FAK and ERK in TCN23-19 cells where FAK is activated because of Na/K-ATPase-knockdown-induced Src activation (25).
  • ND1 is sufficient in killing cancer cells where the expression of Na/K-ATPase is reduced. Some tumor cells express less Na/K-ATPase and exhibits higher Src activity. The inventors tested whether expression of a Src-inhibiting Na/K-ATPase fragment (ND1) inhibits the cell growth. As depicted in FIG. 13A and FIG. 13B , expression of YFP-ND1 caused cell growth inhibition or cell death in DU145 and MCF-7 cells.
  • pNaKtide is effective in inhibiting cell growth in some tumor cell lines: The inventors tested whether pNaKtide can be used to inhibit tumor cell growth. As shown in FIG. 14 , pNaKtide is effective in blocking the growth of several tumor cell lines including prostate cancer, breast cancer and neuroblastoma.
  • pNaKtide is effective in inhibiting tumor growth in vivo: The inventors further evaluated the effect of pNaKtide in inhibiting tumor growth in NOD/SCID mice. As depicted in FIG. 15A and FIG. 15B , injection of pNaKtide to NOD/SCID mice bearing xenografted prostate tumors produced a dose-dependent inhibition in both the incidence rate as well as the tumor weight. Quantitative measurement of the tumor volume confirmed the effect of pNaKtide in inhibiting the tumor development is rapid and in a dose-dependent manner ( FIG. 15C ). Administration of 10 mg/kg pNaKtide resulted in over 75% reduction in both tumor volume and actual tumor weight. And apparently, significant reduction of Src activity was found in the tumors after pNaKtide administration ( FIG. 15D ).
  • pNaKtide inhibits angiogenesis: Endothelial cell proliferation is the prerequisite of angiogenesis. As depicted in FIG. 16A , pNaKtide inhibited the proliferation of both HUVEC and HAEC cells in a dose-dependent manner. Moreover, the vessel density of tumors from control mice indicated by CD31 staining was about 10% and pNaKtide treatment reduced the density to about 2.5% ( FIG. 16B and FIG. 16C ). When the level of angiogenic factor VEGF was assessed, pNaKtide treatment produced significant reduction in the tumor homogenates ( FIG. 16D ).
  • the inventors herein now show a molecular structure of the Na/K-ATPase ⁇ 1 subunit that interacts and inhibits Src.
  • the inventors have also engineered a novel peptide Src inhibitor that can target the Na/K-ATPase/Src receptor complex and thus function as an effective ouabain antagonist in cultured cells.
  • the CD3 of Na/K-ATPase ⁇ 1 subunit consists of both N and P domains.
  • the inventors have shown that CD3 binds the Src kinase domain and inhibits Src kinase activity in vitro (24). Based on the newly released crystal structure of Na/K-ATPase, the N domain is exposed, whereas the P domain is relatively close to the membrane (3,5,6).
  • the inventors now show that the N domain binds and inhibits Src. Interestingly, it is known that the less structural N-terminus of SERCA N domain interacts with phospholamban. The inventors now demonstrate that the ND1 ( FIG. 2 ), the first 50 amino acid residues of the ⁇ 1 N domain, inhibits Src. However, further mapping analyses reveal that the corresponding phospholamban-binding domain in ND1 (peptide 2, see FIG. 4A ) actually had no effect on Src kinase activity.
  • peptide 3 and peptide 4 showed strong inhibitory effect on Src. Based on the crystal structure as well as NMR data, both peptides possessed a helix structure, suggesting that helix/helix interaction between the ND1 and the Src kinase may be responsible for the Na/K-ATPase-induced Src inhibition.
  • Literature review reveals that several endogenous proteins interact and inhibit Src. Noteworthy are RACK1 and WASP. While RACK1 inhibits Src via its interaction with the SH2 domain (34), WASP does so by binding to the Src kinase domain (22). However, no detailed structural information is available for further comparison.
  • Src inhibitors Like inhibitors of other tyrosine kinases, most Src inhibitors are ATP mimetics (35). When the ATP dependence was assessed, the inventors found that changes in ATP concentration did not affect NaKtide-induced Src inhibition. Moreover, it is unlikely that NaKtide acts as a substrate Src inhibitor (36) since the peptide does not contain Tyr residue. Furthermore, limited structural analyses show that NaKtide interacts with N-lobe, but not the substrate pocket-containing C-lobe, of the kinase domain (Li and Xie, unpublished data). Thus, NaKtide represents a novel class of Src inhibitor.
  • both NaKtide and ND1 are potent.
  • the IC 50 is close to 50 nM, comparable to most of reported Src inhibitors.
  • the inventors found that NaKtide appears to be relatively specific to Src. NaKtide showed no effect on PKC family of kinases. Its effect on ERK1/2 depends on the expression of Src, indicating that it is not an ERK inhibitor but can affect ERK signaling by inhibiting Src. This is consistent with the fact that ERK1/2 are well-known effectors of Src kinase.
  • NaKtide inhibits Lyn, another Src family kinase, it exhibits much lower potency toward Lyn than Src ( FIG. 4E ).
  • the pNaKtide was very effective in blocking the formation of Na/K-ATPase/Src receptor complex ( FIG. 6B ).
  • ouabain-induced activation of ERK1/2 in LLC-PK1 cells was completely abolished by 1 ⁇ M pNaKtide ( FIG. 8A ).
  • pNaKtide also resided mainly in the plasma membrane in cardiac myocytes and was effective in inhibiting ouabain-induced activation of ERK1/2 ( FIG. 8B ).
  • ouabain and other CTS have been considered only as drugs since their discovery, recent studies have identified both ouabain and marinobufagenin (MBG) as endogenous steroids whose production and secretion are regulated by multiple physiological stimuli including ACTH and angiotensin II (37-41). Moreover, they are found to play an important role in the regulation of renal salt handling, vascular and cardiac contractions (42). Pathologically, they are likely to be involved in cardiovascular remodeling seen during chronic renal failure and the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) by stimulating the proliferation of renal epithelial cells (43).
  • ADPKD autosomal dominant polycystic kidney disease
  • the pNaKtide can be useful in determining the physiology, as well as to probe the pathological significance, of Na/K-ATPase and endogenous CTS. This is of particular importance since it is now believed that physiologically relevant doses of ouabain and MBG are sufficient to stimulate Src and its down-stream protein kinase cascades in the heart and kidney (44,45). Moreover, PST 2238, an ouabain antagonist, has been demonstrated as an effective anti-hypertensive drug (44). Therefore, pNaKtide can also be useful as a new therapeutics for cardiovascular diseases where the Na/K-ATPase/Src receptor is over-stimulated.
  • Src family kinases include at least nine members.
  • Src and Lyn are highly homologous, NaKtide appears to be more potent toward Src than Lyn. Thus, it is likely that this regulation could be isoform-specific. Also, determining whether NaKtide affects kinases other than PKC, ERK and Src can be further useful.
  • the AP-NaKtide and A1N-NaKtide resided mainly in intracellular vesicles. Like pNaKtide, it also had almost no effect on basal Src activity. As such, determining whether AP-NaKtide or A1N-NaKtide can block ouabain-induced ERK1/2 activation can also be useful. Moreover, AP-NaKtide and A1N-NaKtide may have a relative specific effect on endocytosis, exocytosis or vesicle recycling since Src is known to play a role in these events. Further, assessment of the ability of pNaKtide as a ouabain antagonist in intact animals or isolated organs can provide further proof of the usefulness of the novel NaKtide.
  • pNaKtide as a potential anti-cancer therapeutics: Many tumors have elevated Src activity. Both in vitro and in vivo studies have demonstrated that cellular Src activity is inversely correlated with the amount of Na/K-ATPase. Thus, supplement of Src-inhibiting Na/K-ATPase or its equivalent (ND1 or pNaKtide) may be useful for reducing the tumor growth. Moreover, because Src controls FAK activity that is required for tumor metastasis, the Na/K-ATPase and its equivalent may also be effective in preventing tumor metastasis. Consistently, the inventors demonstrate that many tumor cell lines express less Na/K-ATPase and have higher Src activity ( FIG.
  • FIG. 10 and FIG. 11 Rescuing these cells with YFP-ND1 or pNaKtide is effective in inhibiting the growth of these tumor cells ( FIG. 13 and FIG. 14 ). Furthermore, pNaKtide inhibits FAK and blocks tumor cell migration in vitro ( FIG. 11 and FIG. 12 ). Also, in vivo studies show that IP injection of pNaKtide can block the growth of xenografted prostate tumor in NOD/SCID mice ( FIG. 15 ). Inhibition of Src in tumors may be involved in the regulation of angiogenesis by pNaKtide ( FIG. 16 ), which will further limit the nutrients supply for the tumor growth. Taken together, these findings indicate that pNaKtide may be useful as an anti-cancer therapeutic agent.
  • GRKKRRQRRRPPQMTVAHMWFDNQIHEADTTEN [SEQ ID NO: 7] pNaKtide . . . GRKKRRQRRRPPQSATWLALSRIAGLCNRAVFQ [SEQ ID NO: 8] AP-NaKtide. . . RQIKIWFQNRRMKWKKSATWLALSRIAGLCNR AVFQ [SEQ ID NO: 9] A1N-NaKtide . . . KKGKKGKKSATWLALSRIAGLCNRAVFQ

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