+

WO2010046867A1 - Protéine de fusion présentant une activité antitumorale - Google Patents

Protéine de fusion présentant une activité antitumorale Download PDF

Info

Publication number
WO2010046867A1
WO2010046867A1 PCT/IB2009/054660 IB2009054660W WO2010046867A1 WO 2010046867 A1 WO2010046867 A1 WO 2010046867A1 IB 2009054660 W IB2009054660 W IB 2009054660W WO 2010046867 A1 WO2010046867 A1 WO 2010046867A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
tat
nucleic acid
acid sequence
grk5
Prior art date
Application number
PCT/IB2009/054660
Other languages
English (en)
Inventor
Guido Iaccarino
Bruno Trimarco
Daniela Sorriento
Original Assignee
Guido Iaccarino
Bruno Trimarco
Daniela Sorriento
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guido Iaccarino, Bruno Trimarco, Daniela Sorriento filed Critical Guido Iaccarino
Publication of WO2010046867A1 publication Critical patent/WO2010046867A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • a fusion protein having antitumor activity having antitumor activity
  • the present invention relates to a fusion protein designated as TAT-RH and its medical indications, particularly for treating rumor pathologies.
  • G protein-coupled receptor kinases are serine/threonine protein kinases that are structurally similar, characterized by a highly conserved central catalytic domain, an N- terminal 183-188 amino acid region that includes a domain homologous to the RGS (Regulator of G protein signaling) proteins and a C-terminal region variable in length. These kinases are well known to act as regulators of the signal from G protein-coupled receptors by events of receptor phosphorylation.
  • the present inventors have recently demonstrated that the GRK5 kinase regulates the gene transcription of the NFKB transcription factor [Sorriento D et al., Kinase independent inhibition of NFKB transcriptional activity by GRK5 through I ⁇ B ⁇ stabilization. PNAS 2008 in press].
  • the GRK5 kinase is capable of binding the NFKB inhibitory protein, I ⁇ B ⁇ , through its N-terminal RH domain (RGS homology domain) and stabilizing the NF ⁇ B/I ⁇ B ⁇ complex in the nucleus, thereby inhibiting NFKB transcriptional activity.
  • GRK5 RH GRK5 RH domain
  • NFKB is a dimeric transcription factor usually confined in the cytoplasm of non-stimulated cells, which has to translocate to the nucleus in order to carry out its own function.
  • the subcellular localization of NFKB is controlled by a family of inhibitory proteins called IKBS (among which the inhibitor I ⁇ B ⁇ is particularly mentioned) that bind NFKB masking its nuclear localization domain, thus preventing its translocation to the nucleus.
  • Extracellular stimulation results in the rapid phosphorylation, ubiquitination and proteolytic degradation of I ⁇ B ⁇ , thereby releasing NFKB that translocates to the nucleus activating gene transcription.
  • NFKB regulates the expression of different genes, among which there are genes encoding cytokines (IL-I, IL-2, IL-6, IL- 12, TNF ⁇ , IFN- ⁇ ), chemokines (IL-8, MIP- l ⁇ , MCPl), adhesion molecules (ICAM, VCAM, E-selectin), apoptosis and cell proliferation regulators (c-myc, Dl-cyclin, BCL-X). cytokines and chemokines generated in response to NFKB activation can induce lymphocyte migration and proliferation.
  • cytokines IL-I, IL-2, IL-6, IL- 12, TNF ⁇ , IFN- ⁇
  • chemokines IL-8, MIP- l ⁇ , MCPl
  • IAM adhesion molecules
  • VCAM VCAM
  • E-selectin apoptosis and cell proliferation regulators
  • c-myc Dl-cyclin, BCL-X
  • NFKB plays an important role in different biological phenomena, some of them pathological in nature, such as type II diabetes mellitus and insulin-resistance, cardiac hypertrophy, hypercholesterolemia, atherosclerosis, chronic heart failure, cancer and angiogenesis.
  • NFKB NFKB
  • the activity of this factor is constitu- tively high in many types of human tumors, such as melanomas, thyroid and colon carcinomas, and the persistent activation during chronic inflammation predisposes to transformation of normal cells into neoplastic cells.
  • a tumor cell is characterized by six alterations of the cell physiology: autonomous proliferation, non-response to anti-proliferation signals, resistance to apoptosis, tissue invasion and metastasis, sustained angiogenesis, immortality. Many of the genes that regulate these phenomena are under the transcriptional control of NFKB. This suggests that the modulation of NFKB activity may represent an effective therapeutic strategy for different pathologies, such as cancer.
  • the present invention provides a fusion protein consisting of a fusion between the GRK5 RH domain and the HIV TAT sequence.
  • the fusion protein of the invention hereinafter in short referred to as "TAT-RH”
  • TAT-RH is capable of penetrating into eukaryotic cells by crossing the barrier represented by the cell membrane, even without the aid of carrier o expression vectors, which represents a considerable advantage in that it allows for the direct administration of the purified protein, without having to use exogenous DNA constructs or potentially cytotoxic vectors.
  • the TAT-RH fusion protein of the invention proved to be as effective as the AdGRK5-RH adenovirus in inhibiting the growth of tumor cells, but extremely less toxic and immunogenic for the animals tested.
  • a first object of the invention is a fusion protein capable of penetrating into an eukaryotic cell without the help provided by vectors (e.g. carrier or expression vectors), and capable of increasing cell apoptosis, characterized in that it comprises a first segment consisting of the minimal sequence of the HIV-TAT transduction domain and a second segment consisting of the RH domain of the GRK5 kinase.
  • vectors e.g. carrier or expression vectors
  • the first segment of the fusion protein is N-terminal to the second segment, the first segment is preceded at the N-terminal end by a first linker sequence, and the first and second segments are linked to each other through a second linker sequence.
  • a linker sequence is a sequence linking two regions or segments of a fusion protein. It can perform different functions even simultaneously, as it may be composed of several segments designed to carry out different functions.
  • a linker sequence may serve as a cleavage site for a restriction enzyme, have chemo-physical features that are useful for the isolation or purification of the expressed protein, serve as a spacing element and/or contribute in conferring the desired conformation to the fusion protein.
  • the first segment of the fusion protein (HIV-TAT) consists of the amino acid sequence SEQ ID NO: 1 and the second segment of the fusion protein (GRK5-RH) comprises the amino acid sequence SEQ ID NO: 7.
  • SEQ ID NO: 7 is the minimal RH inhibitory sequence required to induce the anti-tumor effect.
  • the second segment of the fusion protein consists of the amino acid sequence SEQ ID NO: 2.
  • the fusion protein of the invention consists of the amino acid sequence SEQ ID NO: 3 or of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, capable of penetrating into a cell without the help provided by (carrier or expression) vectors, and capable of increasing cell apoptosis.
  • the first linker sequence of the fusion protein of the invention is the stretch comprised between amino acid positions 1 and 39 of SEQ ID NO: 3.
  • the first linker sequence comprises, among others, a labeling sequence consisting of 6 histidine residues useful for the purification of the fusion protein.
  • the second linker sequence is comprised between amino acid positions 51 and 65 of SEQ ED NO: 3 and comprises an HA-Tag.
  • a second object of the invention is a nucleic acid sequence encoding for the TAT-RH fusion protein, as defined above.
  • the nucleic acid sequence encodes for the amino acid sequence SEQ ID NO: 3 or for an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3. Further preferred identity percentages are those as indicated above.
  • the most preferred nucleic acid sequence is SEQ ID NO: 4.
  • a third object of the invention is a prokaryotic or eukaryotic host cell, which is transformed or transfected with the nucleic acid sequence encoding for the TAT-RH fusion protein, as defined above, so as to allow the host cell to express the fusion protein.
  • a fourth object of the invention is a method for recombinantly preparing the TAT-RH fusion protein of the invention, comprising culturing, under suitable nutrient conditions, the eukaryotic or prokaryotic host cell, which is transfected or transformed with a nucleic acid comprising the nucleic acid sequence encoding for the fusion protein, preferably the sequence SEQ ID NO: 4, so as to allow the host cell to express the fusion protein; and optionally isolating the fusion protein expressed by the host cell.
  • a fifth object of the invention is the TAT-RH fusion protein of the invention, or the nucleic acid sequence encoding the same, for use as a medicament, preferably an anti-tumor me- dicament, as well as the use of the TAT-RH fusion protein of the invention, or the nucleic acid sequence encoding the same, for the manufacture of an anti-tumor medicament, preferably for treating melanoma, pancreas tumor, colon tumor, thyroid carcinoma, osteosarcoma, muscle sarcoma, fibrosarcoma.
  • the modulation of NFKB activity by the TAT-RH fusion protein of the invention may represent an effective therapeutic strategy against cancer, since the inhibition of NFKB enhances the apoptotic processes induced by chemotherapy, decreases the high proliferation rate that characterizes tumor cells and inhibits metastasization.
  • the treatment with GRK5 RH was initially carried out by adenovirus-mediated gene therapy, as described previously in PNAS 2008 in press, cit. Such a treatment proved to be able to block tumor growth in a dose-dependent fashion, by induction of apoptosis. The obtained results thus confirmed that the RH domain can represent a therapeutic target for cancer.
  • the sequence encoding the RH domain was cloned into a vector comprising the HFV TAT sequence, in order to create the TAT-RH fusion protein of the invention that, thanks to the TAT sequence, is able to cross the plasma membrane and penetrate into the eukaryotic cell without having to be carried by a virus or other types of vectors.
  • TAT is a small 1 1 -amino acid sequence derived from the HIV TAT protein, which binds to the cell surface and enters the cytoplasm of the target cell.
  • a system is described in the prior art for transferring recombinant proteins and treating mouse models of cancer, inflammation and other pathologies.
  • the prior art does not include any indication that would have directed the person skilled in the art, with a desire to solve the cytotoxicity and immunogenicity problems of the AdGRK5-RH adenovirus, to apply such a system to the GRK5 RH domain with a reasonable expectation of success.
  • the TAT-RH fusion protein of the invention is capable of penetrating into tumor cells, inducing apoptosis. In such a way, the RH region blocks cell proliferation and consequently tumor growth.
  • One further therapeutic application for the fusion protein of the invention, or for a nucleic acid sequence encoding the same, relates to the therapeutic treatment of left ventricular hypertrophy.
  • Cell cultures Cells derived from anaplastic thyroid carcinoma (KAT4) were cultured in DMEM culture medium, integrated with 10% bovine serum (FBS), at 37 0 C in 5% CO 2 .
  • FBS bovine serum
  • a bicistronic adenovirus encoding the amino-terminal region of GRK5 was used, the which region includes the RH domain (AdGRK5-RH) and the Green Fluorescent Protein (GFP) under the control of different CMV promoters (Addgene).
  • Ad- Lac-Z (Biocompare), used as a control, is an adenovirus that encodes for the beta galacto- sidase under the control of a CMV promoter.
  • the expression of GRK5 after infection was assessed by fluorescence analysis of GFP and the expression of beta galactosidase by X- GAL staining.
  • the cells were lysed in lysis RIPA/SDS buffer [5OmM Tris-HCl (pH 7.5), 15OmM NaCL, 1% NP-40, 0.25% deoxycholate, 9.4mg/50ml sodium orthovanadate, 20% sodium dodecyl sulfate].
  • the protein concentration was determined by the BCA method (Pierce).
  • the endogenous I ⁇ B ⁇ and GRK5 proteins were immunoprecipitated from total cell extracts with the specific antibody (Santacruz) and protein A/G (Santacruz).
  • the immunocomplexes were subjected to electrophoretic run (SDS/PAGE) and transferred onto nitrocellulose.
  • the total of the forms of GRK5 and I ⁇ B ⁇ were visualized by using selective antibodies, the chemiluminescence method and autoradiography on films.
  • the cells were lysed in a hypotonic buffer [10 mM Hepes pH 7.9; 10 mJVI KCl; 0.1 mM EDTA; 0.1 mM EGTA; 0.1 mM NaVO 3 ; 1 mM DTT; 0.5 mM PMSF; 10% NP40] and centrifuged to separate the cytosolic and nuclear extracts.
  • the nuclei were lysed in a hypertonic buffer [20 mM Hepes pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 20% gly- cerol; 0.1 mM NaVO 3 ; 1 mM DTT; 0.5 mM PMSF] and centrifuged.
  • the transcriptional activity of NFKB was assessed in control and transfected cells by lucife- rase assay, using luciferase as a gene reporter under the control of a NF ⁇ B-dependent promoter.
  • the Luciferase Assay System kit with Reporter Lysis Buffer Prome- ga
  • the DNA binding activity was tested by the Electrophoretic Mobility Shift Assay (EMSA) method.
  • ESA Electrophoretic Mobility Shift Assay
  • the sequence encoding for the GRK5 RH domain was amplified by PCR using the plasmid pCMV-GRK5-WT (ORIGENE) as a template. The correct sequence was confirmed by sequencing (3100-Avant Genetic Analyzer; Applied Biosystem).
  • the amplification primers were designed by adding the sequences corresponding to the restriction enzyme sites used for cloning, NCO I and KPN I: TAT-RH FOR: 5 ' CCCCC ATGGCCCG AG ATT ACTGC AGTTT
  • TAT-RH REV 5 'GGGGGTACCCTAG AG AAAGCGATC AAA
  • the sequence encoding for the RH domain was then cloned into the vector pT AT-HA (a kind gift by Dr. SF Dowdy and extensively described in the scientific literature), which, starting from the 5' end, encodes the following elements: a 4-amino acid linker sequence; a polyhistidine sequence (6 amino acids) useful as a labeling sequence for protein purification; a 29-amino acid linkage; the minimal sequence of the transduction HIV-TAT domain (11 amino acids, SEQ ID NO:1), which allows the fusion protein to enter the cell without the need of carrier vectors; a 20-amino acid linkage, including the sequence of the HA Tag (HA-Tag) that is useful for visualizing the protein in vitro; the GRK5 RH sequence, including 121 amino acids and ending with a stop sequence (SEQ ID NO:2).
  • the pT AT-HA plasmid which encodes for the RH sequence under the control of the T7 promoter, was transformed into BL21(DE3) cells (Invitrogen), a genetically engineered E. coli bacterial strain that contains in its own chromosome the gene encoding for the T7 RNA polymerase placed under the control of the inducible lac promoter.
  • the colonies were amplified in 2 liters of culture broth, LB, overnight.
  • isopropyl ⁇ -D-1-thiogalactopyranoside IPTG, 500 ⁇ M, 3 hours was added to the culture in order to induce expression of the recombinant protein.
  • the cells were lysed under denaturing conditions (Buffer A: 8 M UREA, 100 mM NaCl, 20 mM HEPES, pH 8).
  • the protein was then purified. The purification is carried out on a Nickel resin column, which has a high binding affinity to histidine. After the passage of the cell lysate through the column, the same was washed with Buffer A added with low concentrations of imidazole (20 and 50 ⁇ M).
  • the protein was eluted by incubating the resin with increasing concentrations of imidazole (100, 200 and 500 ⁇ M), which competes with the His-Tag for chelating the metal.
  • the analysis of the protein purification in the various steps was performed by a run on a poly- acrylamide gel and visualized by Coomassie blue staining.
  • the protein was stocked at - 80°C in 2M TRIS buffer, pH 6.8, added with 5% glycerol.
  • the change of the buffer was carried out with Amicon Ultra-4 Centrifugal Filters (Millipore) by centrifugation.
  • the purified TAT-RH protein is made of 191 amino acids (SEQ ID NO:3) and has a molecular weight of approximately 24 kD.
  • any type of host cells per se known and suitable for the purpose may be used.
  • mice About 6-week old nude mice (BALB/c) (Charles River) were used for the in vivo experiments. This mouse strain was chosen because of its immunodeficiency that allows injected tumors to take root under the cutis.
  • a cell suspension comprising approximately 2.5 millions of human anaplastic carcinoma cells from the thyroid (KAT4) (a kind gift by Dott.ssa Maddalena Illario, Universita Federico II di Napoli, Dipartimento di Biologia e Patologia Cellulare e Molecolare) was inoculated subcutaneously into the dorsal side of the nude mice. 10 Days after injection of the tumor cells, small tumors having a diameter of approximately 0.5 cm are visible.
  • the mice were divided into groups: one control group and three groups of treated animals.
  • the three groups of treated animals received 10 8 pfu (low dose) and 10" pfu (high dose) injections of the AdGRK5-RH adenovirus and 200 ⁇ g of the purified TAT-RH protein of the invention, respectively, whereas the controls were injected with the same volume of physiological solution.
  • the animals received two treatments per week for about three weeks. At completion of the treatment period, the animals were sacrificed and the tumors were collected and frozen at -80°C.
  • the tumors were homogenized in RIPA/SDS buffer.
  • the levels of I ⁇ B ⁇ , p-JNK and cleaved caspase-3 were analyzed by the western blot method.
  • VEGF expression was as- sessed by immunoprecipitation and western blot.
  • NFKB activity was evaluated by EMSA on nuclear extracts.
  • the statistical analysis was performed by variance analysis with the software SPSS.
  • GRK5 RH expression in tumor cells is shown by fluorescence analysis of paraffin-embedded tissue sections. Expression of the control adenovirus is assessed by X-GAL staining. The total levels of I ⁇ B ⁇ were assessed in the homogenized samples. Consistent with the in vitro results, treatment with GRK5 RH causes build-up of IicB ⁇ .
  • Pathological angiogenesis is essential for the malignant progression of solid tumors and the central factor that regulates this process is the Vascular Endothelial Growth Factor (VEGF). The expression levels of VEGF were thus evaluated in the tumor samples.
  • VEGF Vascular Endothelial Growth Factor
  • a reduction in the VEGF expression levels is seen in treated versus control samples, which is consistent with the reduction of tumor growth.
  • Apoptosis is the main effect of inhibition of NFKB activity, as in tumor development the malignant cell escapes the mechanisms that induce cell death by activation of NF ⁇ B-dependent anti-apoptotic mechanisms.
  • An increase in p-JNK and cleaved caspase-3 is seen in treated tumors.
  • the fusion protein TAT-RH was generated as described in Materials and Methods.
  • the effectiveness of the TAT RH protein was thus assessed by analyzing in vitro, in KAT4 cells, the pro-apoptotic effects by fluorescent Annexin V staining.
  • Apoptosis is determined by measuring the binding of Annexin V to the phosphatidylserine residues, which, during the apoptotic process, translocate from the plasma membrane inner sheet to the outer one.
  • AV fluoresceinated Annexin V
  • PI Propidium Iodide
  • the KAT4 cells were plated onto a slide, treated with the TAT-RH protein and subsequently incubated with Annexin V and Propidium Iodide. After 15 minutes of incubation, the slides were mounted with aqueous mounting solution added with DAPI (for the staining of the nuclei) and observed under a fluorescence microscope. Table 7 shows that at increasing doses of fusion protein the apoptotic events increase in the tumor cells.
  • the effects of the protein were then tested in vivo.
  • the animals were intratumorally injected with 200 ⁇ g of purified protein (2 injections/week for 4 weeks).
  • the treatment can reproduce the effects previously observed with high dose AdGRK5-RH treatment.
  • Table 8 shows that treatment with the purified protein can slow down the tumor growth compared to the control. As in the treatment with the adenovirus, no significant changes in body weight of control versus treated animals were found by treatment with the purified protein.
  • TAT-RH protein In order to assess the possible toxic effects caused by treatment with the TAT-RH protein, some animals were intraperitoneally injected with 200 ⁇ g of TAT RH protein. The amount of protein, number of injections and duration of treatment were the same as those that resulted to be effective in the inhibition of tumor growth. The parameters considered for the evaluation of the adverse effects from the purified protein were:
  • mice treated with the intra-peritoneal injection of the protein for the entire duration of treatment no changes were observed in the body weight of treated animals compared to the control animals, nor inappetence or dehydration signs, changes in trophism and in skin pigmentation.
  • the animals were sacrificed and the internal organs collected. No changes in weight or in the morphology of lung, kidney and liver were observed, which shows absence of organ-specific damages in the treated animals compared to the control animals.
  • EXAMPLE 5 inhibition of heart hypertrophic response.
  • GRK5-RH adenovirus-mediated gene transfer and the activity of NFKB and of the Atrial Natriuretic Factor (ANF) promoter was assessed by luciferase assays.
  • AdGRK5-RH adenovirus-mediated gene transfer
  • AdGRK5-RH adenovirus-mediated gene transfer
  • ANF Atrial Natriuretic Factor
  • GRK5-RH The effects of GRK5-RH on hypertrophy were thus assessed in vitro, by analyzing ANF promoter activity, which is a marker for hypertrophy. Stimulation with phenylephrine induces increased ANF activity and over-expression of GRK5-RH is capable of decreasing this activity. These in vitro data suggest that GRK5-RH is able to regulate hypertrophy by modulating NFKB activity.
  • SHR spontaneously hypertensive rat
  • Treatment was performed by intramural injection with an adenovirus encoding the GRK5 RH domain (AdGRK5-RH) or Lac-Z in the control animals (AdLac-Z): gene therapy.
  • AdGRK5-RH an adenovirus encoding the GRK5 RH domain
  • AdLac-Z Lac-Z in the control animals
  • Hypertrophy markers expression levels of the Atrial Natriuretic Factor, ANF; size of the cardiomyocytes; fibrosis
  • Paraffin-embedded heart sections were stained by Masson's trichrome staining, which shows a decrease in the cardiomyocyte size and a decrease in the collagen deposits, and consequently in fibrosis.
  • Gene expression of ANF was assessed in RNA extracts from these hearts by Real Time- PCR, which shows that it is considerably lower in GRK5-RH-treated hearts compared to control hearts.
  • NFKB transcriptional activity and the molecular signaling associated therewith were then analyzed.
  • NFKB activity increases in hypertrophic hearts compared to normal hearts and decreases significantly in GRK5-RH-treated animals. This is accompanied by an increase in the total levels of the IKB inhibitory protein and of the NFKB inactive form in the nuclear extracts.
  • Apoptosis under NFKB transcriptional control was then assessed and usually it increases in hypertrophic hearts.
  • increased expression levels of cleaved caspase-3, an apoptosis marker is observed in SHR hearts, the which levels, in contrast, decrease following GRK5-RH treatment.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une protéine de fusion, dénommée TAT-RH, comprenant un premier segment constitué de la séquence minimale du domaine de transduction HIV-TAT et d'un second segment constitué de la région RH de la kinase GRK5. La protéine de fusion TAT-RH est avantageusement capable de pénétrer dans une cellule eucaryote sans l'aide fournie par les vecteurs et est capable d'augmenter l'apoptose cellulaire. Ces propriétés font de la protéine de fusion TAT-RH selon l'invention une protéine particulièrement adaptée pour être utilisée comme médicament antitumoral. L'invention concerne en outre la séquence d'acide nucléique codant pour la protéine de fusion TAT-RH, une cellule hôte transformée par l'acide nucléique codant pour TAT-RH, ainsi qu'un procédé de production de TAT-RH par un moyen recombinant.
PCT/IB2009/054660 2008-10-22 2009-10-21 Protéine de fusion présentant une activité antitumorale WO2010046867A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000772A ITTO20080772A1 (it) 2008-10-22 2008-10-22 Proteina di fusione avente attivita' antitumorale
ITTO2008A000772 2008-10-22

Publications (1)

Publication Number Publication Date
WO2010046867A1 true WO2010046867A1 (fr) 2010-04-29

Family

ID=41009899

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2009/054660 WO2010046867A1 (fr) 2008-10-22 2009-10-21 Protéine de fusion présentant une activité antitumorale

Country Status (2)

Country Link
IT (1) ITTO20080772A1 (fr)
WO (1) WO2010046867A1 (fr)

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
EL-ANDALOUSSI S ET AL: "Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: A comparative study", BIOCHEMICAL JOURNAL 20071015 PORTLAND PRESS LTD GB, vol. 407, no. 2, 15 October 2007 (2007-10-15), pages 285 - 292, XP002566102 *
HARADA HIROSHI ET AL: "Antitumor protein therapy; application of the protein transduction domain to the development of a protein drug for cancer treatment.", BREAST CANCER (TOKYO, JAPAN) 2006, vol. 13, no. 1, 2006, pages 16 - 26, XP002544010, ISSN: 1340-6868 *
JONES SIMON W ET AL: "Characterisation of cell-penetrating peptide-mediated peptide delivery", BRITISH JOURNAL OF PHARMACOLOGY, vol. 145, no. 8, August 2005 (2005-08-01), pages 1093 - 1102, XP002566101, ISSN: 0007-1188 *
LI YUEHUA ET AL: "NF-kappaB activation is required for the development of cardiac hypertrophy in vivo", AMERICAN JOURNAL OF PHYSIOLOGY - HEART AND CIRCULATORY PHYSIOLOGY, vol. 287, no. 4, October 2004 (2004-10-01), pages H1712 - H1720, XP002566103, ISSN: 0363-6135 *
MÉTAYÉ THIERRY ET AL: "Expression and activity of g protein-coupled receptor kinases in differentiated thyroid carcinoma.", THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM JUL 2002, vol. 87, no. 7, July 2002 (2002-07-01), pages 3279 - 3286, XP002544009, ISSN: 0021-972X *
SAAR ET AL: "Cell-penetrating peptides: A comparative membrane toxicity study", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 345, no. 1, 1 October 2005 (2005-10-01), pages 55 - 65, XP005078986, ISSN: 0003-2697 *
SORRIENTO DANIELA ET AL: "Rgs homology domain of GRK5 regulates postischemic neoangiogenesis by inhibiting NFkB trascriptional activity", CIRCULATION, vol. 116, no. 16, Suppl. S, October 2007 (2007-10-01), & 80TH ANNUAL SCIENTIFIC SESSION OF THE AMERICAN-HEART-ASSOCIATION; ORLANDO, FL, USA; NOVEMBER 04 -07, 2007, pages 129, XP002544008, ISSN: 0009-7322 *
SORRIENTO DANIELA ET AL: "The G-protein-coupled receptor kinase 5 inhibits NFkappaB transcriptional activity by inducing nuclear accumulation of IkappaB alpha.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 18 NOV 2008, vol. 105, no. 46, 18 November 2008 (2008-11-18), pages 17818 - 17823, XP002544011, ISSN: 1091-6490 *
SORRIENTO ET AL: "Kinase independent inhibition of NFkB transcriptional activity by GRK5 through IkBa stabilization", NATURE PRECEEDINGS, 1 October 2007 (2007-10-01), XP002544007, Retrieved from the Internet <URL:http://precedings.nature.com/documents/1196/version/1/files/npre20071196-1.pdf> [retrieved on 20090901] *
TILSTRA J ET AL: "Protein transduction: identification, characterization and optimization.", BIOCHEMICAL SOCIETY TRANSACTIONS AUG 2007, vol. 35, no. Pt 4, August 2007 (2007-08-01), pages 811 - 815, XP008111293, ISSN: 0300-5127 *
WADIA J S ET AL: "MODULATION OF CELLULAR FUNCTION BY TAT MEDIATED TRANSDUCTION OF FULL LENGTH PROTEINS", CURRENT PROTEIN AND PEPTIDE SCIENCE, BENTHAM SCIENCE PULBISHERS, NL, vol. 4, no. 2, 1 January 2003 (2003-01-01), pages 97 - 104, XP009057910, ISSN: 1389-2037 *
WADIA J S ET AL: "Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER BV, AMSTERDAM, NL, vol. 57, no. 4, 28 February 2005 (2005-02-28), pages 579 - 596, XP025283894, ISSN: 0169-409X, [retrieved on 20050228] *

Also Published As

Publication number Publication date
ITTO20080772A1 (it) 2010-04-23

Similar Documents

Publication Publication Date Title
Lee et al. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.
CA2432111A1 (fr) Proteine jfy induisant l&#39;apoptose rapide
CN102311493B (zh) 一种抑制端粒酶活性的肽及其制备方法和应用
CN101812127A (zh) 微管结合蛋白及其编码基因与应用
JP2023181361A (ja) 過増殖性疾患を治療するためのｐｃｂｐ１の使用
US5948643A (en) Modulators of BRCA1 activity
CN105999227B (zh) FOXM1蛋白氮端（1-234 aa）的表达及其用途
CA2517285A1 (fr) Facteur transcriptionnel induisant l&#39;apoptose dans une cellule cancereuse
CN112088163A (zh) 微肽及其用途
Liu et al. AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation
WO2010046867A1 (fr) Protéine de fusion présentant une activité antitumorale
US9585380B2 (en) SSAT mRNA translation repression and activation
KR100739118B1 (ko) Ｍｓｘ1 단백질 또는 이를 코딩하는 유전자의 신규한 용도
US7741464B2 (en) Compositions and methods of using CRMP-1 and its fragments for treating cancer
JP2004502416A (ja) 血管新生および腫瘍進行に関連する抑制性ｐａｓドメインタンパク質（ｉｐａｓ）およびスクリーニング法
KR101051135B1 (ko) 엘라핀을 포함하는 아팝토시스 유도제 조성물 및 이의 제조방법
Luhn et al. Identification of FAKTS as a novel 14‐3‐3‐associated nuclear protein
Dunkelberg et al. LZ-FYVE: a novel developmental stage-specific leucine zipper, FYVE-finger protein
KR101108476B1 (ko) 야생형 ＥＷＳ-Ｏｃｔ-4 단백질의 전사 촉진 활성을 억제하는 ＥＷＳ-Ｏｃｔ-4 단백질 변이체 및 이를 코딩하는 뉴클레오타이드 서열
RU2772733C2 (ru) Hmgb1 мутанты
WO2008051048A2 (fr) Méthode et matières pour l&#39;inhibition d&#39;un export nucléaire de gsk-3
KR20050098800A (ko) 임포틴 α유전자 및/또는 재조합 ｐ５３ 유전자를 포함하는항암제
Elliott Transduction of E2F1 Tat fusion proteins into primary invasive ductal breast carcinoma cell lines and subsequent effects on gene transcription
US20080044436A1 (en) Method for Screening and Compositions for Treating Cancers
WO2012010946A2 (fr) Isoformes de bnip3 et leurs procédés d&#39;utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09756814

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09756814

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载