Patent Grant
9234005
This application is the national phase application of International Patent Application Number PCT/EP2012/070200, filed Oct. 11, 2012, which claims the benefit of European Patent Application Number EP 11306318.4, filed Oct. 11, 2011, the contents of each of which are hereby incorporated by reference in their entirety.
The application relates to products, which can be used as anti-proliferative or tumoricid agents in the treatment and/or palliation and/or prevention of neoplasms of the human nervous system.
The application also relates to means deriving from said products, such as pharmaceutical compositions, as well as to the applications of said products and means, more particularly in the medical and/or biotechnological fields.
Tumors develop when, in a healthy organ, cells display uncontrolled growth and division beyond the normal limits and escape cell death mechanisms.
Viruses are masters for manipulating the proliferation or death of the host cell they infect, mainly by interfering with crucial endogenous interactions. Rabies virus (RABV) is a neurotropic virus, which causes fatal encephalitis in mammals. RABV has developed a particular strategy to ensure its propagation in the nervous system. Its virulence correlates with the ability of infected neurons to survive.
Virulent RABV strains have been described to preserve the integrity of the neuronal network, thereby favoring the spreading of the virulent strain within the Central Nervous System (CNS), although they have also been described to have no positive or a negative impact on neurite outgrowth, for example no positive or a negative impact on axon outgrowth of rat motoneurons in culture (Guigoni and Coulon, 2002).
Attenuation of laboratory strains in the search for candidate live anti-RABV vaccines led to the observation that RABV attenuation is linked to their ability to trigger cell death. WO 03/048198 relates to the G protein of RABV and to fragments thereof of at least 100 amino acids, which induce the disruption of the neuronal cell integrity and the formation of apoptotic bodies. WO 03/048198 describes that the effect of these apoptotic bodies is to stimulate a humoral immune response, preferably a B-dependent humoral immune response.
Préhaud et al., 2010 have described that the various partners of attenuated rabies virus strains include PTPN4, MAST2, MAST1, DLG2, MDPZ. A cell—penetrating peptide (Cyto13-att; SEQ ID NO: 11) encoding the PDZ binding site (PDZ-BS) of the attenuated ERA rabies virus strain induces cell death in the neuroblastoma cell line SK-N-SH, whereas peptides from the virulent VIR strain PDZ-BS (Cyto13-vir; SEQ ID NO: 14) or peptides lacking the last four amino acids (Cyto9-Δ; SEQ ID NO: 17) do not (Préhaud et al., 2010).
The application provides products, which may notably trigger pro-apoptotic or pro-death effects on neoplastic cells and/or tissues of the human nervous system, more particularly on neoplastic cells and/or tissues of the human CNS. The products of the application can be used as anti-proliferative or tumoricid agents in the treatment and/or palliation and/or prevention of neoplasms of the human nervous system, more particularly of glioblastoma or brain stem glioma. Therefore, the application also relates to compositions, such as pharmaceutical compositions or drugs, which comprise at least one of said products, as well as to the biotechnological and medical applications of said products and compositions.
The present application relates to the subject-matter as defined in the claims as filed and as herein described.
The application notably relates to products, which may trigger pro-apoptotic or pro-death effects on neoplastic cells and/or tissues of the human nervous system, and/or which may inhibit the effects of a neurotropic virus. The products of the application can notably be used as anti-proliferative or tumoricid agents in the treatment and/or palliation and/or prevention of neoplasms of the human nervous system, more particularly of human glioblastoma or human brain stem glioma.
The application relates to a peptide, the amino acid sequence of which comprises at least one of said SEQ ID NO: 1-13, more particularly at least one of SEQ ID NO: 1-10, 12-13, still more particularly at least one of SEQ ID NO: 1-10, even still more particularly at least one the peptides of SEQ ID NO: 1-9, even still more particularly at least one the peptides of SEQ ID NO: 1-8, even still more particularly at least one the peptides of SEQ ID NO: 1-7, even still more particularly at least one the peptides of SEQ ID NO: 1-6, even still more particularly at least one the peptides of SEQ ID NO: 1-5, even still more particularly at least one the peptides of SEQ ID NO: 1-4, even still more particularly at least one the peptides of SEQ ID NO: 1-3, even still more particularly at least one the peptides of SEQ ID NO: 1-2, even still more particularly the peptide of SEQ ID NO: 1.
As illustrated in the examples and in the figures below, said SEQ ID sequences are as follows:
The amino acid sequence of said peptide consists of less than 34 amino acids, more particularly of less than 30 amino acids, still more particularly of less than 29 amino acids, still more particularly of less than 28 amino acids, still more particularly of less than 27 amino acids, still more particularly of less than 26 amino acids, still more particularly of less than 25 amino acids, still more particularly of less than 24 amino acids, still more particularly of less than 23 amino acids, still more particularly of less than 22 amino acids, still more particularly of less than 21 amino acids, still more particularly of less than 20 amino acids, still more particularly of less than 19 amino acids, still more particularly of less than 18 amino acids, still more particularly of less than 17 amino acids.
According to an embodiment of the application, the amino acid sequence of said peptide consists of less than 16 amino acids, more particularly of less than 15 amino acids, more particularly of less than 14 amino acids.
The amino acid sequence of a peptide of the application may e.g., comprise one, two or three of said SEQ ID sequences.
When several of said SEQ ID sequences are contained in the amino acid sequence of a peptide of the application, said SEQ ID sequences can be the same or can different from each other. They can be directly linked to each or other, or can be indirectly linked to each via a molecular linker, such as a stretch or stretches comprises a few amino acids, e.g., 3 or 4 amino acids.
According to an embodiment of the application, a peptide of the application has an affinity for the PDZ of PTPN4 that is lower than 200 microM, more particularly lower than 160 microM, more particularly lower than 130 microM, more particularly lower than 90 microM, more particularly lower than 50 microM, more particularly lower than 45 microM, more particularly lower than 40 microM, more particularly lower than 30 microM, more particularly lower than 20 microM, more particularly lower than 10 microM, more particularly lower than 5 microM, more particularly lower than 4.9 microM, more particularly lower than 4 microM, more particularly lower than 3 microM, more particularly lower than 2.6 microM, more particularly lower than 2.1 microM, more particularly lower than 2 microM, more particularly lower than 1.7 microM, more particularly lower than 1.6 microM, more particularly lower than 1.5 microM, more particularly lower than 1.4 microM, more particularly lower than 1.3 microM, more particularly lower than 1.2 microM, more particularly lower than 1.1 microM.
PTPN4 (also known as PTPMEG1) is a human non-receptor Protein Tyrosine Phosphatase (PTP) with functions in T Cell Receptor (TCR) cell signaling, learning, maintaining spatial memory and cerebellar synaptic plasticity.
PDZ (PSD-95, Discs Large, ZO-1) domains form globular structures of 80-100 amino acids organized into six beta-strands and two alpha helices creating a socket where the C terminal sequence of a partner protein can be inserted.
In the application, any means that the person of ordinary skill in the art finds appropriate to determine or measure the affinity of said peptide can be used, for example by Nuclear Magnetic Resonance (NMR) and/or Isothermal Titration calorimetry (ITC) and/or Surface Plasmonic Resonance (SPR). Preferred means comprise those which determine or measure the KD, for example by NMR, e.g., as illustrated in the examples below. The higher the KD value, the lower the affinity.
Affinity measured by NMR titration (following the method described in Préhaud et al., 2010), except for peptides of SEQ ID NO: 2-3 and 5-7 (affinity measured by ITC titration).
The symbol “-” means not measured.
“1285” means one thousand two hundred and eighty-five.
“1167” means one thousand one hundred and sixty-seven.
The amino acid sequence of a peptide of the invention may consist one of said SEQ ID sequences.
A peptide of the application can be glycosylated and/or biotynylated.
A peptide of the application can be linked to a His tag (e.g., a His tag intended for purification).
A peptide of the invention can be produced by synthesis. Any method of peptide synthesis that is known to the skilled person can be used. Examples of synthesis methods, such as the Merrifield solid phase synthesis, can e.g., be found in <<Solid Phase Peptide Synthesis>> (J. M. Steward & J. D. Young, 1969, Ed. W.H. Freeman Co., San Francisco), or in <<Peptide synthesis>> (M. Bodansky et al. 1976, John Wiley & Sons, 2nd Edition). A peptide of the invention can alternatively produced by recombinant expression of the nucleic acid encoding it, e.g., from a vector of the application (cf. below).
The application also relates to an antibody, which specifically binds to a peptide of the application, and to a fragment of such an antibody, which specifically binds to a peptide of the application.
Said antibody may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
Said antibody can be a polyclonal antibody or a monoclonal antibody, more particularly a monoclonal antibody. A monoclonal antibody of the application can e.g., be produced by the hybridoma technique of Kohler and Milstein (1975) Nature 256:495-497; and U.S. Pat. No. 4,376,110, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such hybridomas also are within the scope of the application. The hybridoma may be cultivated in vitro or in vivo.
Said antibody can be a recombinant antibody, more particularly a chimeric antibody, such as:
Said fragment can e.g., be a Fv, a Fab, a F(ab′)2, or a Fab/c having one Fab and a complete Fc. An antibody or antibody fragment of the application may e.g., be useful as a means to isolate and/or purify a peptide of the invention, or as a means to link a peptide of the application to another entity, moiety or biological target or to a solid support.
The application also relates to a nucleic acid coding for a peptide of the application (in accordance with the universal genetic code, taking due account of its degeneracy).
The application also relates to a nucleic acid construct, which is an expression cassette, wherein said expression cassette codes for the expression of a peptide of the invention.
The application also relates to a vector carrying a coding nucleic acid, wherein said coding nucleic acid is a nucleic acid of the application, or carrying an expression cassette, wherein said expression cassette is an expression cassette of the application
Said vector is a recombinant nucleic acid vector, more particularly an expression vector.
Such an expression vector comprises an expression cassette, in which the nucleic acid to be expressed is inserted. In such a vector, the nucleic acid encoding said at least one peptide of the application can be operably linked to at least one promoter and/or at least one regulatory sequence and/or to at least one secretion or excretion signal sequence and/or at least one polyadenylation signal sequence
Such a vector is notably useful for recombinantly producing said peptide (and optionally recovering it by purification and/or isolation), and may e.g., be a plasmid, a bacteriophage, a baculovirus.
Such a vector is notably useful for delivering the encoded peptide into a human organism in need thereof, more particularly to a human nervous system in need thereof, still more particularly to a human Sympathetic Nervous System (SNS) and/or to a human Central Nervous System (CNS) in need thereof, still more particularly to a human CNS in need thereof (cf. below).
Such a vector may e.g., be useful for penetrating and/or crossing the plasma membrane of a cell of said human organism (more particularly a cell of said human SNS and/or CNS, more particularly a cell of said human CNS, more particularly a cell of said human brain, more particularly a human neuron and/or a human glial cell, more particularly a human glial cell) and/or for crossing the blood-brain barrier of said human organism.
An appropriate vector can e.g., be a recombinant plasmid, recombinant Herpes Simplex Virus (HSV) vector, a recombinant adeno-associated viral (AAV) vector, a recombinant adenoviral (Ad) vector, a recombinant retroviral vector, a recombinant lentiviral (LV) vector, a recombinant pox virus vector, as well as hybrid vectors thereof (which combine components from at least two different viral vectors), and the pseudo-typed forms of said viral vectors.
The application also relates to a host cell comprising a nucleic acid of the application and/or a vector of the application. Such a host cell can be a recombinant host cell. Examples of host cells notably comprise a prokaryotic cell, such as E. coli or B. subtilis, a eukaryotic cell, such as a yeast cell (e.g., S. cerevisiae), a plant cell (e.g., a Tobacco cell) or an animal cell, e.g., an insect cell, a mammalian cell, a non-human mammalian cell, a human cell.
The application relates to said peptides, nucleic acids, vectors, host cells in isolated or substantially purified form.
The application also relates to said peptides, nucleic acids, vectors in a form, wherein they are linked to, or inserted into other component(s).
The resulting compound can be defined as a compound, which comprises at least one peptide of the application or at least one nucleic acid of the application or at least one vector of the application, directly or indirectly linked to, or inserted into:
The terms <<linked to>> and <<inserted into>> are intended according to their respective meanings in the field.
By <<linked to>>, it is generally meant that said peptide, nucleic acid or vector is linked to the structure of said at least one neurotropic component or said at least one anti-neoplastic agent, without disrupting the primary or linear composition of said at least one neurotropic component or said at least one anti-neoplastic agent. Said linkage can be achieved at any location of the structure of said at least one neurotropic component or said at least one anti-neoplastic agent that the person of ordinary skill in the art may find appropriate.
By <<inserted into>>, it is generally meant that the inserted peptide, nucleic acid or vector is introduced between structural components of said at least one neurotropic component or said at least one anti-neoplastic agent, and is herein linked to said structural components. Preferably, said insertion is such that said at least one neurotropic component or said at least one anti-neoplastic agent retains a neurotropism or an anti-neoplastic activity, respectively.
Said linkage can be a covalent linkage or a non-covalent linkage, more particularly a covalent linkage.
The application more particularly relates to a compound of the application, wherein said at least one peptide, nucleic acid or vector is directly or indirectly linked to said at least one neurotropic component or said at least one anti-neoplastic agent.
The application more particularly relates to a compound of the application, which comprises at least one peptide of the application, directly or indirectly linked to, or inserted into:
Such a compound is herein referred to as <<peptide-containing compound of the application>>.
The application more particularly relates to those peptide-containing compounds of the application, wherein said at least one peptide of the application is directly or indirectly linked to, or inserted into at least one anti-neoplastic agent.
The application more particularly relates to those peptide-containing compounds of the application, wherein said at least one peptide of the application is directly or indirectly linked to at least one anti-neoplastic agent.
According to an embodiment of the application, said at least one neurotropic component is not a rabies virus G protein nor a neurotropic fragment thereof (e.g., a fragment of at least 4 amino acids (more particularly of at least 5 amino acids, more particularly of at least 6 amino acids, more particularly of at least 7 amino acids) of a rabies virus G protein, wherein said fragment has retained a tropism for human neurons). According to an embodiment of the application, said at least one neurotropic component is not a ligand of the PDZ of PTPN4, such as GluD2 or GluN2A. According to either one of said two embodiments, the amino acid sequence of said peptide can comprise SEQ ID NO: 11, said nucleic acid can be a nucleic acid coding for such a peptide, and said vector can be a vector carrying such a coding nucleic acid.
According to an embodiment of the application, said at least one neurotropic component is a rabies virus G protein or a neurotropic fragment thereof (e.g., a fragment of at least 4 amino acids (more particularly of at least 5 amino acids, more particularly of at least 6 amino acids, more particularly of at least 7 amino acids) of a rabies virus G protein, wherein said fragment has retained a tropism for human neurons). According to an embodiment of the application, said at least one neurotropic component is a ligand of the PDZ of PTPN4, such as GluD2 or GluN2A. According to either one of said two embodiments, the amino acid sequence of said peptide can comprise at least one of SEQ ID NO: 1-10, 12-13, said nucleic acid can be a nucleic acid coding for such a peptide, and said vector can be a vector carrying such a coding nucleic acid.
According to an embodiment of the application, said at least one neurotropic component is:
Said at least one polycation can e.g., be a cationic polymer, such as a lipopolyamine polymer, a polyamidoamine polymer, a cationic polymer polyethylenimine (PEI)
The application also relates to a compound of the application that comprises at least one peptide, nucleic acid or vector of the application, for its use in the treatment of a neoplasm of the human nervous system, wherein said compound carries said peptide, nucleic acid or vector through the blood-brain barrier.
According to an embodiment of the application, said at least one anti-neoplastic agent is an alkylating agent, an antimetabolite, an anthracycline, a plant alkaloid or a topoisomerase inhibitor. According to an embodiment of the application, a compound of the application has an affinity for the PDZ of PTPN4 that is lower than 200 microM, more particularly lower than 160 microM, more particularly lower than 130 microM, more particularly lower than 90 microM, more particularly lower than 50 microM, more particularly lower than 45 microM, more particularly lower than 40 microM, more particularly lower than 30 microM, more particularly lower than 20 microM, more particularly lower than 10 microM, more particularly lower than 5 microM, more particularly lower than 4.9 microM, more particularly lower than 4 microM, more particularly lower than 3 microM, more particularly lower than 2.6 microM, more particularly lower than 2.1 microM, more particularly lower than 2 microM, more particularly lower than 1.7 microM, more particularly lower than 1.6 microM, more particularly lower than 1.5 microM, more particularly lower than 1.4 microM, more particularly lower than 1.3 microM, more particularly lower than 1.2 microM, more particularly lower than 1.1 microM.
In the application, any means that the person of ordinary skill in the art finds appropriate to determine or measure the affinity of said peptide can be used, for example by Nuclear Magnetic Resonance (NMR) and/or Isothermal Titration calorimetry (ITC) and/or Surface Plasmonic Resonance (SPR). Preferred means comprise those which determine or measure the KD, for example by NMR, e.g., as illustrated in the examples below. The higher the KD value, the lower the affinity.
The application also relates to nanoparticles and liposomes.
Said peptide, nucleic acid, vector, host cell or compound of the application can be contained in and/or on nanoparticles.
Said peptide, nucleic acid, vector, host cell or compound of the application can be contained in and/or on a liposome, e.g., it can be encapsulated inside a liposome or associated to a liposome delivery system. Said liposome can e.g., be a cationic liposome, a pegylated liposome. Said liposome can be loaded with nanoparticles.
The nanoparticle and/or liposome formulation of the peptide, nucleic acid, vector, host cell or compound of the application is notably useful for improved crossing of the blood-brain barrier and/or for protection against serum degradation.
The application relates to said nanoparticles and to said liposomes.
The application also relates to a composition, comprising:
Said composition can e.g., be a pharmaceutical composition, a medicament or a drug.
Said at least one anti-neoplastic agent can e.g., be an alkylating agent, an antimetabolite, an anthracycline, a plant alkaloid or a topoisomerase inhibitor.
The composition of the invention may further comprise at least one pharmaceutically and/or physiologically acceptable vehicle (diluent, excipient, additive, pH adjuster, emulsifier or dispersing agent, preservative, surfactant, gelling agent, as well as buffering and other stabilizing and solubilizing agent, etc.). The composition of the invention can for example be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Preferably, it is formulated under a form suitable for parenteral administration. The composition of the invention can be a non-immunogenic pharmaceutical composition or drug, or an immunogenic pharmaceutical composition or drug, for example a vaccine. Any administration mode that the skilled person may find appropriate is encompassed by the present invention. Depending on how the product of the invention is formulated, it can administered by parenteral or enteral (e.g., oral) administration, preferably by parenteral administration, more preferably by parenteral injection.
The application also relates to a method of treatment of a subject, more particularly of a human being, in need thereof, which comprises administering to said subject or human being at least one peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application as above-described.
The application also relates to a method of producing a pharmaceutical composition, which comprises formulating a peptide, nucleic acid, vector, host cell, compound or liposome or nanoparticle of the application with at least one pharmaceutically physiologically acceptable vehicle, carrier or diluent.
A peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application is notably useful in the treatment and/or palliation and/or prevention (e.g., by administration to a human in need thereof) of a disease, disorder or condition involving an insufficient cell degeneration of the human nervous system, more particularly a disease, disorder or condition involving an insufficient cell degeneration of the Sympathetic Nervous System (SNS) and/or a disease, disorder or condition involving an insufficient cell degeneration of the Central Nervous System (CNS), more particularly a disease, disorder or condition involving an insufficient cell degeneration of the CNS, even still more particularly a disease, disorder or condition involving an insufficient cell degeneration of the brain, even still more particularly a disease, disorder or condition involving an insufficient cell degeneration of human neurons and/or of human glial cells. Such a disease, disorder or condition notably comprises a disease, disorder or condition, which involves the presence or spreading of undesired cells, such as:
A peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application is useful in stimulating the apoptosis and/or the membrane rupture of human nervous system cells, more particularly of cells of the human SNS and/or human CNS, more particularly of cells of the human CNS, more particularly of cells of the human brain, more particularly of human neurons and/or human glial cells, more particularly of human glial cells.
A peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application is notably useful in stimulating the apoptosis and/or the membrane rupture of such human cells, when said human cells are dividing cells.
Such dividing cells notably comprise tumor or neoplastic cells.
The tumor or neoplasm is a tumor or neoplasm, which affects the human nervous system either as a primary tumor or neoplasm or as a secondary tumor or neoplasm, more particularly as a primary tumor or neoplasm. The tumor or neoplasm can e.g., be a solid tumor.
Hence, a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application is useful as an active principle in a composition intended for the treatment and/or palliation and/or prevention of tumor or neoplastic cells of the human nervous system, more particularly of the human SNS and/or CNS, more particularly of the human CNS, more particularly of the human brain, more particularly of human neurons and/or human glial cells, more particularly of human glial cells.
Said tumor or neoplastic cells can e.g., be malignant or pre-malignant or potentially malignant cells, more particularly malignant cells.
Illustrative tumor or neoplastic cells of the human SNS notably comprise neuroblastoma cells.
Illustrative tumor or neoplastic cells of the human CNS notably comprise glioblastoma cells (e.g., glioblastoma multiforme cells), oligodendroglioma cells, brain stem glioma ells, more particularly glioblastoma cells.
Said disease, disorder or condition involving an insufficient cell degeneration of the SNS can be an extracranial solid tumor, such as a neuroblastoma.
Said disease, disorder or condition involving an insufficient cell degeneration of the CNS can e.g., be an intracranial solid tumor, such as a glioblastoma.
A peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application can also be useful as an active principle in a composition intended for the treatment and/or palliation and/or prevention of an infection of a human being by a neurotropic virus, such as rabies virus, HSV-1 or an influenza virus, more particularly an infection of the human nervous system (more particularly the SNS and/or the CNS, more particularly the CNS, still more particularly the brain). Indeed, a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application may e.g., compete with a neurotropic virus and inhibit its effects.
A peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application can be immunogenic or non-immunogenic. The main mode of action of such a compound is a non-immunogenic one, according to which it competes with the virus. Hence, such a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application can be comprised as an active principle in a pharmaceutical composition which is a non-immunogenic pharmaceutical composition. In such a composition, said compound is non-immunogenic.
If desired, such a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application may nevertheless be chosen to be immunogenic or may be rendered immunogenic (e.g., by linking or otherwise associating it with an immunogenicity enhancer or adjuvant). Alternatively, such a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application may be non-immunogenic and be nevertheless used for simultaneous or differed administration with another active principle that is an immunogenic one.
Therefore, such a peptide, nucleic acid, vector, host cell, compound, liposome or nanoparticle of the application may be comprised as an active principle in a composition, more particularly a pharmaceutical composition, which is an immunogenic composition or a vaccine. In such a composition, said compound can be immunogenic or can be non-immunogenic.
The application also relates to a complex comprising:
In the application, said complex is being herein referred to as <<PTPN4 complex of the application>>.
The application also relates to a crystal comprising:
The atomic coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank under:
The person ordinary skill in the art can also refer to the guidelines and information available from the worldwide Protein Data Bank (wwPDB), more particularly the “Protein Data Bank Contents Guide: Atomic coordinate Entry Format Description” e.g., version 3.20, and the wwPDB Processing Procedures and Policies Document March 2009, e.g., version 2.3. (http://www.wwpdb.org/docs.html).
A crystal of the application may further comprise molecule(s) originating from the solution in which the complex has been crystallized, such as molecules of:
The application also relates to a computer device or computer device system comprising:
Said atomic coordinates advantageously are at least the atomic coordinates of a peptide or complex of the application.
Said computer system device may further comprise means displaying molecular representations (and/or 3D conformation) and/or means for calculating binding affinities. For example, said computer system device may further comprise:
and/or
The application also relates to a solid support, onto which at least one peptide, nucleic acid, vector, host cell, antibody, compound, liposome or nanoparticle, or complex of the application is bound, by covalent or non-covalent linkage.
Said solid support may e.g., be a solid support suitable for the screening of organic molecules, preferably a chip.
Said solid support can e.g., be an essentially polymeric material, or an essentially plastic material, notably a polystyrene material, or a glass material, or a silicon material, or a magnetic material, or a non-magnetic material.
Said solid support may more particularly be a plastic plate, notably a polystyrene plaque, comprising several wells suitable for molecular binding analysis, such as a protein titration or microtitration plate, e.g., an ELISA plate.
Said solid support may more particularly be microbeads, e.g., magnetic microbeads or non-magnetic microbeads, more particularly microbeads suitable for microtitration, e.g., according to a Luminex® technology (12212 Technology Blvd.; Austin, Tex. 78727; United States of America).
Said at least one peptide, nucleic acid, vector, host cell, antibody, compound, liposome or nanoparticle, or complex of the application can be directly bound to said solid support, or indirectly bound thereto via at least one capture or linking agent that is attached to said solid support. Said at least one capture or linking agent may comprise a portion that is attached to said solid support and a portion that comprises a ligand, which specifically binds to said at least one peptide, nucleic acid, vector, host cell, antibody, compound, liposome or nanoparticle, or complex of the application. Such a ligand can e.g., an antibody, a monoclonal antibody, or a fragment thereof which has retained said binding specificity (e.g., a Fv, Fab or F(ab′)2 fragment thereof, or a polypeptide containing one or several CDR thereof).
The application also relates to a kit, comprising at least one solid support or chip of the application.
In the application, unless specified otherwise or unless a context dictates otherwise, all the terms have their ordinary meaning in the relevant field(s).
The term “comprising”, which is synonymous with “including” or “containing”, is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term “consisting of” is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited.
The term “essentially consisting of” is a partially open term, which does not exclude additional, unrecited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention.
The term “comprising” (or “comprise(s)”) hence includes the term “consisting of” (“consist(s) of”), as well as the term “essentially consisting of” (“essentially consist(s) of”). Accordingly, the term “comprising” (or “comprise(s)”) is, in the present application, meant as more particularly encompassing the term “consisting of” (“consist(s) of”), and the term “essentially consisting of” (“essentially consist(s) of”).
In the application, the term “at least x” relating to a set or group of n elements (wherein x is different from zero, and n is a number that is higher than x), explicitly encompasses each value, which is comprises between x and n. For example, the term “at least one” relating to a group or set of six elements explicitly encompasses one, two, three, four, five and six of said elements, as well as at least two, at least three, at least four, at least five of said elements.
In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.
Each of the relevant disclosures of all references cited herein is specifically incorporated by reference. The following examples are offered by way of illustration, and not by way of limitation.
Experimental Procedures
Cells
Human grade III U373MG astrocytoma cells (ATCC, HTB 17) were propagated as previously described (Lafon et al, 2005).
ATCC is American Type Culture Collection [10801 University Blvd.; Manassas, Va. 20110-2209; United States of America].
Detection of Synthetic Peptide Entry and Cell Death
Peptides were conjugated to the HIV-1 Tat domain and labelled with a FITC molecule as previously described (Préhaud et al, 2010). U373MG cells (250,000 cells per well in 1 mL culture medium) were treated with FITC-conjugated peptides (25 microM). Three hours post-treatment, cells were detached by trypsin treatment, washed in phosphate buffer (SPB) Ca2+Mg2+, and assayed for peptide entry and cell death. Membrane permeation, a marker of cell death, was measured by detecting the passage of PI (R&D system Annexin V kit). Cells were analyzed by flow cytometry. 30,000 cells were sampled for each analysis. Results were given as percentages of dead cells among peptide loaded cells.
Sample Preparation
Peptides were synthesized in solid phase using an Fmoc strategy (Covalab).
Expression and purification of PTPN4-PDZ samples for NMR and crystallogenesis were performed as follows. PTPN4-PDZ (also know as PTPMEG1) is known to the person of average skill in the art and is for example available under UniProtKB: P29074. PTPN4-PDZ (residues G499-N604, G499 being a non-native residue due to the TEV cleavage site) was encoded as an N-terminal glutathione S-transferase (GST)-tagged protein in the pDEST15 expression plasmid (Gateway System, Invitrogen). The vector was used to transform Escherichia coli BL21 (DE3) star (Invitrogen). Cells were grown in a 1.6 L bioreactor of either stable isotopically labeled M9 minimal medium containing 1.0 g/L 15NH4Cl as the sole nitrogen source for NMR experiments or high-density medium. Protein expression was induced at OD600 nm 2.5 with 0.5 mM IPTG at 30° C. for 3 h. Harvested cells were resuspended in buffer A (50 mM Tris-HCl, 150 mM NaCl, pH7.5) with 2 mM β-mercaptoethanol and a protease inhibitor cocktail (Roche), and then disrupted in a French press. Clarified cell lysate was then loaded on a GST column (GSTrap HP, GE) equilibrated with buffer A containing 2 mM dithiothreitol (DTT). The GST tag was cleaved by the TEV protease (1% M/M), which was directly injected on the column, overnight at 4° C. The samples containing PTPN4-PDZ were pooled and concentrated to 1.0 ml, and then loaded onto a size exclusion column (Sephacryl S-100 HP 16/60, GE) equilibrated with buffer A containing 2 mM DTT. All purification steps were performed at 4° C. and in the presence of a protease inhibitor cocktail (Roche). The size, purity, and sequence of the PTPN4-PDZ samples were checked by SDS-PAGE, mass spectroscopy, and microsequencing using Edman degradation. Protein concentrations were estimated from the absorbance at 280 nm, assuming a calculated extinction coefficient of 1850 M−1 cm−1 as determined by amino acid analysis of aliquots from a purified PTPN4-PDZ solution of known absorbance, after 6 N HCl hydrolysis for 24 h at 110° C. D2O (12%) was added to NMR samples.
Crystallization, Data Collection, and Structure Determination
Initial crystallization screening was carried out by the vapor diffusion method using a Cartesian™ nanoliter dispensing system (Santarsiero et al, 2002). Sitting drops containing 200 nL of protein complex (6 mg/mL) and 200 nL of the crystallization solution were equilibrated against 150 mL of the buffer solution in Greiner plates. Screening trials (576 conditions) were performed using commercially available sparse-matrix kits. The crystallization conditions were reproduced and optimized using crystallization solutions generated by the Matrix Maker automated formulation system from Emerald BioSystems.
Crystals were grown at 18° C. using the sitting drop vapor diffusion method. The protein solution was mixed with crystallization buffer and equilibrated against a reservoir of crystallization buffer. The Cyto13-att and GluN2A-16 peptides (SEQ ID NO: 11 and 8, respectively) used for PDZ-peptide co-crystallization were added in excess to form more than 95% complex. The crystallization conditions and mixing ratios of protein to crystallization buffer are given in Table 3. Crystals were cryo-cooled by plunging them into liquid nitrogen, and X-ray data were collected at 100 K using a nitrogen stream. When appropriate, the crystals were transferred first into a cryoprotectant solution consisting of crystallization condition plus glycerol (Table 3) before being cryo-cooled. X-ray data were collected for the PTPN4-PDZ/Cyto13-att complex on beamline Proxima I at the Soleil synchrotron (St. Aubin, France); diffraction images were measured with a rotation of 1° per image at a wavelength of 0.918 Å (Table 3). X-ray data were collected for the PTPN4-PDZ/GluN2A-16 complex on beamline PX1 of the Swiss Light Source for the PTPN4-PDZ/GluN2A-16 complex using the high-resolution diffractometer set up; diffraction images were measured with a rotation of 0.25° per image at a wavelength of 0.9795 Å (Table 3). The data were processed with MOSFLM or XDS, SCALA, and other programs from the CCP4 suite (1994). Structures were solved by molecular replacement using PHASER (McCoy, 2007) and the atomic model of PTPN4-PDZ (PDB accession code 2VPH). The locations of the bound peptides were determined from a Fo-Fc difference electron density map. Models were rebuilt using COOT (Emsley & Cowtan, 2004), and refinement was performed using REFMAC5 (Murshudov et al, 1997) and BUSTER (Blanc et al, 2004). The overall assessment of model quality was performed using MolProbity (Lovell et al, 2003).
NMR Experiments
NMR data were recorded at 298 K on a Varian Inova 600-MHz spectrometer equipped with a cryogenically cooled triple-resonance pulsed field gradient probe. NMR titration experiments to measure PTPN4-PDZ/peptide affinities were performed as previously described (Préhaud et al, 2010) using 0.16 mM 15N-labeled PTPN4-PDZ with stepwise additions of unlabeled peptides.
Data Collection and Structure Determination
Further details on X-ray data collection, processing, molecular replacement using the atomic model of PTPN4 (PDB code 2VPH), and refinement are given in Table 3. The atomic coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank under accession codes 3NFK for PTPN4-PDZ/Cyto13-att and 3NFL for PTPN4-PDZ/GluN2A-16).
aValues in parentheses refer to statistics in the highest bin.
bRmerge = Σ|(I − <I>)/Σ (I).
cR factor = Σ|Fobs − Fcalc|/Σ Fobs
dRfree was calculated with 5% of the data excluded from the refinement.
eCategories were defined by MolProbity
Results
Peptide affinity for PTPN4-PDZ and killing efficiency
We synthesized nine peptides:
The affinity of these peptides for PTPN4-PDZ was measured by NMR titration (
After a 3 h exposure of U373MG to the peptides, a period of time that allows for maximal peptide entry (
Altogether, these data indicate that the affinity of the last 4 residues of a putative PDZ-BS for PTPN4-PDZ is a critical determinant for the induction of cell death. We established that the killing efficiency of the peptide is closely reflected by their respective affinity for the PTPN4-PDZ. According to the scenario depicted in
Crystal Structures of PTPN4-PDZ in Complex with the Peptides
PTPN4-PDZ/Cyto13-att Structure
Two molecules in the asymmetric unit are disulfide-bonded. Each molecule corresponds to the complex formed by PTPN4-PDZ (chains A and B) and the Cyto13-att peptide (chains C and D). In order to check the oligomeric state of the complex, NMR experiments (
PTPN4-PDZ/GluN2A-16 Structure
Four molecules were found in the asymmetric unit, constituting four monomers. Each molecule corresponds to the complex formed by PTPN4-PDZ (chains A to D) and the GluN2A-16 peptide (chains E to H). Chain A is disulfide-bonded to chain D and chain B is disulfide-bonded to chain C. NMR experiments and analytical ultracentrifugation (data not shown) also confirmed that the disulfide bonds are due to the crystalline conditions. In all peptide chains, the electron density of only five of the sixteen residues was well defined in the crystal. Superimposition of all PTPN4-PDZ chains displayed a Cα RMSD of less than 0.3 Å, indicating that no crystal packing influence exists in the overall structure of the PDZ domain. Because peptide chain E does not experience any crystal contacts, we described the PTPN4-PDZ/GluN2A-16 complex formed by the A and E chains.
Structure of the PTPN4-PDZ/Viral Peptide Complex
To investigate the specificity of PTPN4-PDZ for the attenuated RABV G protein, the crystal structure of the complex formed by PTPN4-PDZ and Cyto13-att was solved by molecular replacement at 1.4 Å resolution (Table 4).
PTPN4-PDZ adopts a typical PDZ fold comprising of five β-strands and two α-helices (
PTPN4-PDZ possesses the interaction network specific to class I PDZ domains and recognizes the consensus peptide sequence X-S/T-X-ΦCOOH, where X is any residue and Φ is a hydrophobic residue. At position −1, the electron density beyond the Cγ atom of the R residue is not clearly assigned. This cationic side chain is completely exposed to the solvent, surrounded by positively charged residues (R527, R546, and K587), and does not contact any PTPN4-PDZ residues. Position −3 is a critical determinant of the PTPN4-PDZ/Cyto13-att interaction. The side chain carboxyl of E-3 forms a bifurcated H-bond with the hydroxyl of S545 and N531 amine group. E-3 is also strongly stabilized by hydrophobic contacts involving its Cβ-Cγ carbon chain and the long aliphatic side chain of K533 in a manner quite reminiscent of the bonding patterns observed for complexes involving PDZ domains highly structurally related to PTPN4-PDZ, the α1- and β2-syntrophin-PDZ (PDB entry 2PDZ and 2VRF, respectively). Lastly, the carbonyl oxygen of G-4 forms an H-bond with the amide nitrogen of Q538.
Though PDZ domain classification is essentially based on positions 0 and −2, numerous studies have emphasized the importance of the other positions −1, −3, and −4, crucial for PDZ/PDZ-BS interactions. Indeed as described above, E-3 is involved in an extended interaction network with residues of the PTPN4-PDZ β-sheet. In contrast, residues occupying positions −1 and −4 of Cyto13-att do not appear to be optimal for the interaction with PTPN4-PDZ (Kd=160 μM), likely accounting for the fact that the overall affinity remains rather weak for a PDZ/PDZ-BS interaction, more generally in the 1-10 μM range.
Structural Consequences of the E/Q Mutation
When affinities of the Cyto13-vir and Cyto13-att peptides for PTPN4-PDZ were compared, a 4 fold difference in Kd was noted, Q-3 having a higher Kd than E-3 (
In conclusion, slight modifications in the interaction network at the PDZ binding site lead to the drastic functional effect observed for the E>Q change, which is sufficient to abrogate the ability of the PDZ-BS to recruit PTPN4 and to deny the virulent strain the capacity to trigger cell death.
The vampire bat strain of RABV is another RABV strain that does not trigger apoptosis (data not shown). We analyzed the complex formed by PTPN4-PDZ with the C-terminal sequence of G protein of this strain, which is terminated by a PDZ-BS, −DARLCOOH
Structure of the PTPN4-PDZ/Endogenous Peptide Complex
Two endogenous partners of PTPN4-PDZ are known: the orphan ionotropic glutamate receptor, GluD2 terminated by −RGTSICOOH and GluN2A terminated by −ESDVCOOH. Their terminal peptides (GluD2-13 and GluN2A-16, respectively) display affinities for PTPN4-PDZ, slightly better than the one observed for the viral peptide Cyto13-att (
The structure of the PTPN4-PDZ/GluN2A-16 complex solved at 1.9 Å resolution was found to be very similar to the PTPN4-PDZ/Cyto13-att complex, with a RMSD of 0.21 Å (FIG. 3A,C). The overall structure of PTPN4-PDZ was not affected by peptide binding. Only the last five C-terminal residues of GluN2A-16 were resolved.
At position −1, the D side chain was well defined, in contrast to R-1 of Cyto13-att. D-1 forms an H-bond with the Q531 side chain amine group of the PDZ domain. Therefore, an acidic residue is more favorable at the penultimate position of the peptide. The nature of the residue at position −1 probably makes a significant contribution to the four-fold increase in affinity between Cyto13-att and GluN2A-16 for PTPN4-PDZ. Furthermore, the interaction network of the conserved E-3 residue was similar in both cases. 1-4 was well defined in the crystal; its side chain is at a Van der Waals distance from the imidazole group of H579, and its carbonyl group is H-bonded to the amino group of the Q538 side chain. A cationic residue at position −4 is strongly preferred in highly structurally related PDZ domains. In the case of α1-syntrophin, an ionic bond occurs between K-4 of the ligand and D151 at the N-terminus of the α2 helix, an anionic position conserved in PTPN4-PDZ (D580). Lastly, in the chimeric peptide Cyto9-ESDV in which the PDZ-BS of GluN2A is inserted into the Cyto-att core, the affinity for PTPN4-PDZ dropped by a factor of 2 compared to the native GluN2A peptide. The flexibility of Cyto9-ESDV due to the presence of the glycine doublet at positions −4 and −5, and the substitution of 1-4 by a glycine residue, are likely responsible for this effect.
Tailoring Best-Suited Peptides by Structural Analysis
The identification of the most crucial determinants of ligand recognition by PTPN4-PDZ led us to conceive an optimized peptide leading to tighter interactions with the phosphatase and consequently to enhanced capacity of cell death. Considering that positions 0, −2 and −3 are involved in an extended interaction network with residues of the PTPN4-PDZ, we focused our sequence optimization on positions −1 and −4. We conceived that a long acidic residue at position −1 and cationic residue at position −4 could induce additional crucial contacts with surrounding residues of the PDZ domain, and that the PDZ-BS -RETEVCOOH could be an optimized binding partner for PTPN4-PDZ. The chimeric peptide encoding the PDZ-BS-RETEVCOOH with the core of Cyto13-att (Cyto8-RETEV) displays a Kd for PTPN4-PDZ that drastically decreases to 1 μM (
Discussion
We established a relationship between the affinity of peptides for PTPN4-PDZ and their ability to provoke glioblastoma cell death. The structures of PTPN4-PDZ complexed to viral and endogenous peptides allowed us to identify the structural determinants required for ligand recognition and to design a new sequence displaying a higher affinity for PTPN4-PDZ. Among the peptides tested in this example, this optimized peptide is indeed the best PTPN4-PDZ binding partner and the best inducer of cell death. These findings identify the key role of the PTPN4 PDZ domain in the mechanism controlling death in tumor cells. They strongly support the simple mechanism, depicted in
Two other members of the non-receptor phosphotyrosine phosphatase family contain either one (PTPN3) or five (PTPN13) PDZ domains. Intriguingly, they are both involved in the control of tumor cell death. PTPN13 (also termed PTP-BAS, PTPL1, PTP1E or FAP-1) is a negative regulator of CD95 (Fas)-mediated apoptosis involved in human pancreatic adenocarcinoma and colon cancer cells. The interaction between PTPN13-PDZ and the PDZ-BS of CD95 is crucial for PTPN13 activity, likely by allowing dephosphorylation of CD95 and by altering its membrane localization. In addition, the recruitment of the second PTPN13 PDZ domain by a peptide mimicking the PDZ-BS of CD95 is sufficient to promote Fas-mediated apoptosis in a colon cancer cell line. PTPN3 (also termed PTPH1) has been described as an activator of RAS oncogenesis in colorectal cancer cells. These results point out the critical role played by the interaction of phosphatase PDZ domains with the PDZ-BS of their respective ligands for controlling apoptosis or oncogenesis.
An enhanced specific abrogation of the anti-apoptotic function of PTPN4 may be obtained by targeting both the PDZ site of interaction and the catalytic center. This embodiment is under investigation. In any case, the present results show that an efficient disruption of specific PTP-PDZ/ligand interactions can be obtained via a simple optimization of candidate pro-death sequences with the help of structural design, already opening new avenues for anti-proliferative therapies.
Eight chimeric peptides were synthetized as described in Example 1. The values of the affinity of these peptides for the PTPN4-PDZ are given in Table 2 above. U373MG cells were treated with these peptides as described in Example 1 above. These eight chimeric peptides were SWYERETEV (SEQ ID NO: 2); SWEERETEV (SEQ ID NO: 3); SWARVSKETPL (SEQ ID NO: 4); SWERRETEV (SEQ ID NO: 5); SWEERETEF (SEQ ID NO: 6); SWEDRETEV (SEQ ID NO: 7); SWPDRDRESIV (SEQ ID NO: 12); SWRVDSKETEC (SEQ ID NO: 13).
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| Number | Date | Country | Kind |
|---|---|---|---|
| 11306318 | Oct 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2012/070200 | 10/11/2012 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/053848 | 4/18/2013 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050282743 | Lu et al. | Dec 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| 2239330 | Oct 2010 | EP |
| 2004045535 | Jun 2004 | WO |
| 2008109010 | Sep 2008 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20140302123 A1 | Oct 2014 | US |
