WO2004106370A1 - Peptides derives de m11l et leur utilisation therapeutique - Google Patents

Peptides derives de m11l et leur utilisation therapeutique Download PDF

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Publication number
WO2004106370A1
WO2004106370A1 PCT/EP2004/006581 EP2004006581W WO2004106370A1 WO 2004106370 A1 WO2004106370 A1 WO 2004106370A1 EP 2004006581 W EP2004006581 W EP 2004006581W WO 2004106370 A1 WO2004106370 A1 WO 2004106370A1
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Prior art keywords
apoptosis
amino acid
bak
peptide
bax
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PCT/EP2004/006581
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English (en)
Inventor
Etienne Jacotot
Grant Mcfadden
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Theraptosis Sa
Robarts Research Institute
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Publication of WO2004106370A1 publication Critical patent/WO2004106370A1/fr

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    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to a novel chimeric peptide derived from Ml IL protein of myxoma poxvirus capable of inhibiting apoptosis and its use as a cytoprotective molecule, as well as to pharmaceutical compositions containing the Ml lL derived chimeric peptides. Also provided are nucleic acid molecules that encode the chimeric anti-apoptotic peptides, and methods for using the proteins and nucleic acid molecules as cytoprotective molecules. The present invention further relates to a Ml lL fusion peptide possessing an uptake action and/or a targeted moiety into and to a cell and its use as apoptosis inhibitor.
  • the Ml lL derived peptides and fusions peptides are useful for treating AIDS, neurodegenerative diseases, osteomyelodysplasia, ischemic diseases, infectious multiple organ failure, hepatitis, ischemic reperfusion disorders, diabetes and the like.
  • Apoptotic cell death plays a pre-eminent role in host defense against viral infection as cellular initiation of a suicide program following infection serves as an effective means of curtailing viral propagation.
  • Apoptosis is a coordinated process of cellular destruction involving two groups of proteins, the caspase apoptotic proteins and the Bcl-2 family of regulatory proteins, both of which have also been targeted by viruses (2). Numerous viruses have adopted diverse strategies to circumvent the protective host response and extend the life of the infected cell to permit completion of the viral replication cycle (1-3).
  • Caspases are proteases that, once cleaved and activated, can function in the initiation phase of apoptosis by amplifying apoptotic cascades (as do caspases 8 and 9) whereas other caspases (such as caspase-3) take part in the effector phase of apoptosis by degrading cellular substrates (4). Viral inhibitors of both initiator and effector caspases are well documented (2).
  • Bcl-2 protein family such as Bak can modulate apoptosis.
  • Bcl-2 itself protects cells from apoptosis, whereas other members of this family, for example Bax and Bid, are activated by cell death stimuli and promote apoptosis.
  • Viral members of the Bcl-2 family all function to promote cell survival (5).
  • the precise functional role of the Bcl-2 proteins is presently unclear. However, the mitochondrial localization of many of the Bcl-2 proteins combined with recent experimental findings indicate that mitochondria play a key role in relaying many apoptotic signals. Bcl-2 proteins may modulate cell death signals that converge on the mitochondrial control point (6,7).
  • Apoptotic cell death is frequently accompanied by two distinct changes in mitochondrial properties: first, proteins that are normally retained in the intermembrane space between the inner and outer mitochondrial membranes may be released into the cytosol; and second, the electrochemical potential normally maintained across the inner membrane ( ⁇ m) is abruptly lost (6).
  • apoptotic effectors such as cytochrome c and Smac/Diablo from the inter membrane space into the cytosol and opening of the permeability transition PT pore with the resultant disruption of ATP generation and calcium ion, redox and pH homeostasis could result in irrevocable commitment to apoptosis (8).
  • the loss of membrane potential also known as the permeability transition occurs as a result of the sudden opening of a mitochondrial megachannel, the permeability transition (PT) pore.
  • the PT pore is reported to have three core components: the Voltage Dependent Anion Carrier (VDAC) that resides in the outer mitochondrial membrane; the Adenine nucleotide transporter (ANT) in the inner membrane and cyclophilin D that is associated with the matrix surface of the ANT (7).
  • VDAC Voltage Dependent Anion Carrier
  • ANT Adenine nucleotide transporter
  • cyclophilin D that is associated with the matrix surface of the ANT (7).
  • viral proteins are known to play a role in apoptosis are capable of modulating the channel activity of the PT pore and associating with its components. These viral proteins include vMIA of cytomegalovirus (9) and Vpr encoded by HIV (10), which associate with the ANT, and the VDAC-interacting protein, HBx, encoded by hepatitis B virus (11,12).
  • the pore also incorporates several accessory proteins including the Peripheral Benzodiazephine Receptor (PBR) and Hexokinase II associated with the ANT- VDAC complex in the intermembrane space (7).
  • PBR Peripheral Benzodiazephine Receptor
  • Hexokinase II associated with the ANT- VDAC complex in the intermembrane space (7).
  • the PBR is a highly hydrophobic 18 kDa protein which resides in the outer mitochondrial membrane where it associates with the VDAC component of the PT pore.
  • the PBR was first identified as a receptor present in the peripheral nervous system that binds strongly to benzodiazepines such as 4'- chlorodiazepam (Ro 5-4864) and the isoquinoline carboxamide analog PK11195 (13), a therapeutic agent used for its anxiolytic and anticonvulsant properties.
  • An important endogenous ligand of the PBR is thought to be protoporphyrin IX (PPIX).
  • the drug specificity of the PBR distinguishes this receptor from the benzodiazepine receptor present in the central nervous system, the GABAA receptor, which is a cell surface protein and has a different spectrum of benzodiazepine binding.
  • the PBR is present in somatic tissues such as adrenals, kidney and heart. Within leukocyte subsets, PBR is most abundant in monocytes and polymorphonuclear cells (14). Numerous functions have been ascribed to the PBR, including roles in steroidogenesis, cholesterol transport, heme transport (15) and the prevention of inflammation (16, 17). More recently, several lines of evidence have suggested a role for the PBR in apoptosis modulation.
  • the myoxoma virus Ml lL protein was previously found to be antiapoptotic when expressed independently of other viral proteins. It was shown that the Ml IL viral protein is targeted specifically to mitochondria via its C-terminal region, which comprises a hydrophobic domain flanked by basic amino acid residues, adjacent to positively charged tail, and then prevents loss of mitochondrial membrane potential that accompanies cell death (21). Loss of mitochondrial membrane potential generally occurs as a result of an event known as permeability transition, which is associated with transmission of cell death signals through the mitochondrial cell death control point and arises from the opening of the permeability transition (PT) pore.
  • permeability transition which is associated with transmission of cell death signals through the mitochondrial cell death control point and arises from the opening of the permeability transition (PT) pore.
  • Ml lL viral protein physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR), a component of the permeability transition (PT) pore, and is capable of inhibiting apoptosis by modulating the mitochondrial permeability transition pore complex (28).
  • PBR mitochondrial peripheral benzodiazepine receptor
  • PT permeability transition
  • Ml lL was capable of preventing the release of mitochondrial cytochrome c induced by staurosporine or by PBR ligands, such as PK11195 or protoporphyrin IX (28).
  • the Applicants have characterized for the first time an amino acid sequence within the Ml lL viral protein which is capable of directly interacting with the BH3 domains of the pro-apoptotic members of the Bcl-2 family, Bax and Bak proteins.
  • the Bax and Bak proteins are well known to trigger apoptosis by opening the PT pore.
  • This novel amino acid sequence is located in the N-terminal of the Ml lL viral protein and is thus distinct from the PBR interacting sequence of Ml lL, which is located in C-terminus of the Ml lL protein.
  • Ml 1L/PBR interaction requires the C-terminal part of the Ml lL viral protein
  • Ml 1L/BH3 interaction requires the N-terminal part of Ml IL ( Figure 1).
  • the present application thus provides novel Ml lL derived peptides capable of recognizing and specifically protecting from apoptosis specific cell populations, such as for example neurons and cardiomyocytes, by blocking the (mitochondrial) effector phase of apoptosis.
  • An object of the present invention is to provide a Ml IL derived peptide capable of inhibiting apoptosis by blocking Bax and/or Bak and/or PBR dependant pathways. It also relates to the use of said Ml lL derived peptides as apoptosis inhibitors, methods and pharmaceutical compositions for treating AIDS, neurodegenerative diseases, osteomyelodysplasia, ischemic diseases, cardiac ischemia, cancer, infectious multiple organ failure, hepatitis, ischemic reperfusion disorders, diabetes and the like.
  • the present invention relates to a Ml lL derived peptide comprising at least the N-terminal portion of the Ml IL viral protein as set forth in SEQ ID NO: 1, capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking Bax and/or Bak apoptosis dependent pathway.
  • the present invention relates to a Ml lL derived peptide comprising at least the
  • the Ml lL derived peptide according to the present invention comprises at least 50-60 amino acid residues within the N-terminal of the Ml IL viral protein, and further comprises at least 50 to 60 amino acid residues within the C-terminal part of the Ml IL viral protein.
  • the Ml IL derived peptide according to the present invention comprises at least
  • the present invention also relates to a Ml IL derived peptide comprising at least the C-terminal and/or the N-terminal portion of the Ml lL viral protein as set forth in
  • SEQ ID NO: 1 capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking Bax and/or Bak and/or PBR dependent pathways, with the proviso that the
  • Ml IL derived peptide does not comprise the full length Ml lL viral protein.
  • the present invention also provides a Ml lL derived peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, wherein the Ml lL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking Bax and/or Bak apoptosis dependent pathway.
  • the present invention further provides a Ml lL derived peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, and an amino acid sequence as set forth in any one of SEQ ID NOs: 6 and 7, wherein the Ml IL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking Bax and/or Bak and/or PBR apoptosis dependent pathways.
  • the present invention further provides a Ml lL derived peptide comprising at least an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, wherein the Ml lL derived peptide is capable of inhibiting apoptosis.
  • the present invention further provides a Ml lL derived peptide comprising at least an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, and an amino acid sequence as set forth in any one of SEQ ID NOs: 6 and 7, wherein the Ml IL derived peptide is capable of inhibiting apoptosis.
  • the present invention also provides a Ml lL derived peptide comprising an amino acid sequence selected from the group consisting of: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3,
  • the present invention further relates to a Ml lL derived peptide comprising at least 10 contiguous amino acids of the N-terminal part of Ml IL viral protein as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 97%, 95%, or 90% identity to one of the above, and/or wherein the Ml IL derived peptide is capable of binding the
  • the present invention further relates to a Ml lL derived peptide comprising at least 10 contiguous amino acids of the N-terminal part of Ml IL viral protein as set forth in SEQ ID NO: 2 and at least 10 contiguous amino acids of the C-terminal part of Ml IL viral protein as set forth in SEQ ID NO: 6, or an amino acid sequence having at least
  • Ml lL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking a Bax and/or Bak and PBR dependent pathways and/or is capable of inhibiting apoptosis.
  • the present invention also encompasses Ml lL derived peptide conjugated or combined to a targeting moiety or cell uptake moiety or cell incorporation moiety to form a fusion, chimeric, or conjugate molecule, peptide, polypeptide, or protein.
  • Another object of the present invention is a pharmaceutical composition comprising said Ml lL derived peptides.
  • Still another object of the present invention relates to a method of treating AIDS, neurodegenerative diseases, osteomyelodysplasia, ischemic diseases, cardiac ischemia, infectious multiple organ failure, hepatitis, ischemic reperfusion disorders, stroke, diabetes and apoptosis-associated defects, comprising administering the Ml lL derived apoptosis inhibitor or conjugate thereof as described above to a patient to inhibit apoptosis in a effective amount to treat said apoptosis related diseases.
  • a further object of the present invention is the use of the Ml IL derived peptide for manufacturing a prophylactic or therapeutic cytoprotective drug.
  • FIG. 1A Truncated MllL was fused to pcDNA3.6C-TAP vector and transfected into 293T cells then the presence in elution products of Bak after immunoprecipitation of TAP -tagged protein is monitored by western Blot (presence of Bak: + on right panel).
  • Figure IB The eluted products from TAP binding protein purification method were detected with anti-Bak using a Western blot approach.
  • Figure 1C The expression of the truncated Ml lL constructs was detected with anti- PAP which recognizes the C-TAP tag (20kDa) after immunoblotting.
  • Figure 2 displays various HIS TAG fusion in N-term of the Ml lL full length viral protein (SEQ ID NO: 1), HIS TAG fusions in C-terminal and N-terminal of HIV TAT(49-47) Ml lL conjugate, His Tag fusion in C-term of the N-term Ml lL protein, and HIS TAG fusion in N term of the C-terminal Ml IL protein.
  • Figure 3 represents amino acid sequences corresponding to 40 residues (SEQ ID NO: 2), 30 residues (SEQ ID NO: 3), 20 residues (SEQ ID NO: 4), and 10 residues (SEQ ID NO: 5) in N- terminal of the Ml IL protein.
  • Figure 4 represents amino acid sequences corresponding to 36 residues (SEQ ID NO: 6) and 25 residues (SEQ ID NO: 7) in the C-terminal part of the Ml lL protein.
  • Figure 5 is a table showing settings for the evaluation of the Ml lL derived peptides mitochondrio-protective effects on purified mitochondria by measuring inhibition of mitochondria swelling, inhibition of loss of mitochondrial membrane potential ( ⁇ ) and cytochrome c release, in presence of various apoptosis inducers, in comparison with references inhibitors.
  • Figure 6 is a table showing settings for the evaluation of the Ml lL derived peptides cytoprotective effects in HeLa cells, Jurkat cells, Neurons, and cardiomyocytes in presence of apoptosis inducers and in comparison with reference inhibitors as positive controls.
  • Parameters that are used to demonstrate Ml lL derived peptide cytoprotective activity are nuclear apoptosis, caspase activation, inhibition of the loss of mitochondrial membrane potential ( ⁇ ), and of the cytochrome c release.
  • An object of the present invention is to provide a Ml IL derived peptide capable of inhibiting apoptosis by blocking a Bax and/or Bak and/or PBR apoptosis dependant pathway, and its use as an apoptosis inhibitor, methods and pharmaceutical compositions for treating AIDS, neurodegenerative diseases, osteomyelodysplasia, ischemic diseases, cardiac ischemia, stroke, infectious multiple organ failure, hepatitis, ischemic reperfusion disorders, diabetes and the like.
  • the present invention relates to a Ml IL derived peptide comprising at least the
  • the present invention also relates to a Ml IL derived peptide comprising at least 50 to 60 amino acid residues within the N-terminal portion of the Ml IL viral protein as set forth in SEQ ID NO: 1, capable of binding the BH3 domains of Bax and/or Bak and/or PBR mitochondrial component.
  • the present invention also relates to a Ml IL derived peptide comprising at least the N-terminal and the C-terminal portion of the Ml IL viral protein as set forth in SEQ ID NO: 1, capable of binding the BH3 domains of Bax and/or Bak and/or PBR mitochondrial component.
  • the present invention relates to a Ml lL derived peptide comprising at least 50 to 60 residues within the C-terminal and/or the N-terminal portion of the Ml lL viral protein as set forth in SEQ ID NO: 1, capable of binding the BH3 domains of Bax and/or Bak and/or PBR mitochondrial component, and/or capable of efficiently blocking Bax and/or Bak and/or PBR apoptosis dependent pathways.
  • the present invention also relates to a Ml IL derived peptide comprising at least the C-terminal and/or the N-terminal portion of the Ml lL viral protein as set forth in SEQ ID NO: 1, capable of binding the BH3 domains of Bax and Bak, and efficiently blocking a Bax and/or Bak and/or PBR apoptosis dependent pathways, with the proviso that the Ml lL derived peptide does not comprise the native, full length Ml lL viral protein.
  • the present invention further provides a Ml lL derived peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, and an amino acid sequence as set forth in any one of SEQ ID NOs: 6 and 7, wherein the Ml IL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking a Bax and/or Bak and/or PBR apoptosis dependent pathway.
  • the present invention further provides a Ml lL derived peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, wherein the Ml lL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking a Bax and/or Bak apoptosis dependent pathway.
  • the present invention further provides a Ml lL derived peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5, and an amino acid sequence as set forth in any one of SEQ ID NOs: 6 and 7, wherein the Ml IL derived peptide is capable of binding the BH3 domains of Bax and/or Bak, and efficiently blocking a Bax and/or Bak and PBR apoptosis dependent pathways.
  • the Ml lL derived peptides according to the present invention are thus particularly efficient in inhibiting apoptosis.
  • the peptides disclosed are designed to block BH3 (Bax or bak) dependant pathways ( Figure 3) or block PBR dependant pathways ( Figure 4) and provide mitochondria-protective ( Figures 5) cytoprotective ( Figure 6) effects.
  • Peptides for example those described in Figures 3 and 4, can be combined at their N-ter or C-ter with peptidic or non-peptidic molecules to produce chimeric or conjugate molecules capable of entering cells (optionally followed by a specific recognition sequence or molecule) in order to prevent or treat apoptosis and provide mitochondria-protective (Figure 5) and cytoprotective effects (Figure 6).
  • the present invention also provides an amino acid sequence comprising at least an N-terminal portion of the Ml lL viral protein as set forth in SEQ ID NO: 1 and having anti-apoptotic effect through a Bax and/or Bak protein.
  • Preferred anti-apoptotic Bax and/or Bak peptides comprise at least 10 contiguous amino acid from the N-term part of the Ml IL protein, and most preferably comprise at least an amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
  • the present invention also provides an amino acid sequence comprising at least an N-terminal portion of the Ml lL viral protein as set forth in SEQ ID NO: 1 and having anti-apoptotic effect through a Bax and/or Bak protein and further comprising the C-terminal portion of the Ml IL viral protein as in SEQ ID NO: 1 for blocking PBR apoptosis dependent pathway.
  • Preferred anti-apoptotic Bax and/or Bak peptides comprise at least 10 contiguous amino acid from the N-term and C-term parts of the Ml lL protein, and most preferably comprise at least an amino acid sequence of SEQ ID NO: 2, 3, 4, or 5 and at least an amino acid sequence of SEQ ID NO: 6 or 7.
  • the Ml lL derived peptides of the invention exhibit an excellent biological characteristics and, in particular, can be capable of suppressing the mitochondrial ⁇ membrane potential ⁇ loss and/or cytochrome c release, so that apoptosis can be inhibited.
  • apoptosis refers to a manner of cell death characterized by changes represented by condensation or fragmentation of the nucleus of a cell, condensation or fragmentation of the cell itself, and fragmentation of chromosomal DNA of a cell into nucleosome units (about 180 bp), and includes those events caused by pathological factors and physiological factors (for instance, expression of physiological events such as immunization, hormone or development).
  • anti-apoptotic Bax and/or Bak peptide or protein means antagonists of apoptosis-inducing Bax or Bak protein and those possessing an action of inhibiting apoptosis induction pathway among proteins.
  • An activity of inhibiting apoptosis will herein be referred to as "anti-apoptotic activity.”
  • the inhibition of apoptosis can be evaluated by, for instance, the following method, without being limited thereto. The method comprises treating cells with VP16 for 24 hours, thereafter staining the cells with 1 ⁇ M Hoechst 33342, and then calculating an extent of apoptosis as the ratio of cells exhibiting nuclear fragmentation to the total cells.
  • the anti-apoptotic activity can be evaluated by the reduction of the mitochondrial membrane potential ⁇ and the inhibition of cytochrome c release.
  • the cytochrome c release can be evaluated by preparing mitochondria, centrifuging the mitochondria to obtain mitochondria pellets and supernatant, resuspending the resulting pellets, and subjecting the resulting suspension of mitochondria and the supernatant to Western blotting with an anti-cytochrome c antibody.
  • the present invention also provides a Ml lL derived peptide comprising an amino acid sequence selected from the group consisting of:
  • the present invention also relates to a Ml IL derived peptide comprising at least 10 contiguous amino acids of the N-terminal part of Ml IL viral protein as set for the in SEQ ID NO: 2, or an amino acid sequence having at least 97%, 95%, or 90% identity thereto, wherein the Ml IL derived peptide is capable binding the Bh3 domains of Bax and Bak, and efficiently blocking a Bax and/or Bak and/or PBR apoptosis dependent pathway and/or is capable of inhibiting apoptosis.
  • the present invention further relates to a Ml lL derived peptide comprising at least 10 contiguous amino acids of the N-terminal part of Ml IL viral protein as set for the in SEQ ID NO: 2 and at least 10 contiguous amino acids of the C-terminal part of Ml lL viral protein as set forth in SEQ ID NO: 6, or an amino acid sequence having at least 97%, 95%, or 90% identity thereto, wherein the Ml lL derived peptide is capable binding the BH3 domains of Bax and Bak, and efficiently blocking a Bax and/or Bak and/or PBR dependent pathway and/or is capable of inhibiting apoptosis.
  • the Ml lL derived peptides according to the present invention may have an amino acid sequence having substitution, deletion or insertion of 1, 2, 3, 4, 5 or more one amino acid residues in the amino acid sequence of SEQ ID NO: 1 to 7, as long as it conserves the ability to bind the BH3 domains of Bak and Bak, and to efficiently block a Bak and /or Bax and PBR apoptosis dependent pathway.
  • a polypeptide having the amino acid sequence comprising the mutation is also a polypeptide capable of exhibiting an anti-apoptotic activity.
  • the above-mentioned anti-apoptotic activity can be examined by evaluating the reduction of the mitochondrial membrane potential ⁇ and the inhibition of cytochrome c release according to the methods described above.
  • amino acid sequence having substitution, deletion or insertion of at least one amino acid residue can be arbitrarily prepared in accordance with the desired sequence when a peptide is synthesized by a known method.
  • the anti-apoptotic Ml IL derived peptides of the invention may have an amino acid sequence having at least about 50%, preferably about 70%, 80%, 85%, 90%, 95% or higher, and more preferably about 90% or higher sequence identity to the amino acid sequence of SEQ ID NOs: 1 to 7, as long as it conserves its ability to bind the BH3 domains of Bax and Bak, and to efficiently block Bak and /or Bax and PBR pathway.
  • the above Ml IL derived polypeptide having a particular sequence identity is a polypeptide capable of exhibiting anti-apoptotic activity.
  • the anti-apoptotic activity can be examined by evaluating the reduction of the mitochondrial membrane potential ⁇ and the inhibition of cytochrome c release according to the methods described above.
  • sequence identity refers to the identity of amino acid residues between two amino acid sequences, namely between the amino acid sequence of SEQ ID NOs: 1-7 and an amino acid sequence to be compared thereto.
  • sequence identity can be determined by comparing two amino acid sequences which are optimally aligned with each other over the region of the amino acid sequence to be compared.
  • the amino acid sequence to be compared may have an addition or deletion (for instance, gap, overhang or the like), as compared to the amino acid sequence of SEQ ID NOs: 1-7 given herein. If an identity is over the full length of a given SEQ ID NO: 1-7, for example, such a gap can be used to optimally align two sequences being compared.
  • the numerical value (percentage) of the sequence identity can be calculated by determining identical amino acid residues which are present in both of the sequences to determine the numbers of matched sites, thereafter dividing the above-mentioned number of the matched site by a total number of the amino acid residues present within the region of the sequence to be compared, and multiplying the resulting numerical value by 100.
  • An algorithm for obtaining optimal alignment and homology includes, for instance, BLAST and the like. Concretely, the optimal alignment and homology can be obtained by gene analysis software GENETYX-SV/RG Ver. 4.01 (manufactured by Software Development), gene analysis program BLAST [internet address: http://www.ncbi.nlm.nih.gov/BLAST; reference: Altschul, S. F. et al., J Mol. Biol, 215, 403-410, (1990), Altschul, S. F. et al., Nucl. Acid Res., 25, 3389-3402, (1997)], or the like.
  • the Ml lL derived peptides may have an amino acid sequence having one or more conservative substitutions.
  • conservative substitution encompasses substitution with an amino acid possessing similar properties (i.e., hydrophobicity, electric charge, pK, features in three-dimensional structure); and substitution with an amino acid which would cause any changes in the three- dimensional structure and folding structure of the polypeptide only to an extent so that the physiological activity inherently owned by the polypeptide is maintained.
  • the conservative substitution includes, for instance, the following specific substitutions within the groups of: 1) glycine and alanine; 2) valine, isoleucine and leucine; 3) aspartic acid, glutamic acid, aspartic acid and glutamine; 4) serine and threonine; 5) lysine and arginine; or 6) phenylalanine and tyrosine.
  • Ml lL derived peptides include recombinant proteins or synthetic peptides include:
  • the Ml lL derived peptides may have an amino acid sequence having a substitution, deletion or insertion of at least one amino acid residue in any one of the above-mentioned amino acid sequences (a) to (o), and may be an amino acid sequence having at least about 50%, preferably about 70% or higher, and more preferably about 90% or higher sequence identity to any one of the above-mentioned amino acid sequences (a) to (o), as long as the peptide exhibits the reduction of the mitochondrial membrane potential ⁇ and the inhibition of cytochrome c release, as for example determined according to the methods described above.
  • the present invention also encompasses one or more Ml lL derived peptides conjugated or linked to a recognition system, targeting moiety or cell uptake or incorporation moiety.
  • virus-encoded peptides capable of inducing permeability transition pore (PTP) closing constitute prototypes for the design of active anti-apoptotic molecules.
  • Non-specific recognition system include any peptidic or non-peptidic system for cellular delivery such as Protein Transduction domains (PTDs) (22); Bogoyevitch M.A. et al., DNA and CELL BIOLOGY 2002, 21(12):879-894), arginine containing peptides (23)(Tung CH.
  • Specific recognition systems also include antibodies or recombined antibody fragments, and peptidic or non-peptidic ligands of cell-surface receptors.
  • Peptidic and antibody-based cell penetrating systems optionally require a short peptidic linker (or spacer) to correctly associate with Ml lL derived peptides.
  • Spacers inserted between cytoprotective and cell penetrating functional domain include any peptidic sequence of 2 to 8 amino acids in length that allow a correct association between Ml lL derived sequences and the cell penetrating system.
  • this linker has one of the following sequences: -gly-gly- (-GG-), -GGGS-, -CGG-.
  • the additional polypeptide used in the Ml lL derived fusion or chimeric polypeptide of the present invention can be capable of exhibiting uptake action into a cell and includes, for instance, TAT from human immunodeficiency virus (HIV), p27 from HTLV-I, art/trs from HIV, VP22 from HSV-1, Antp from Drosophila and the like.
  • TAT human immunodeficiency virus
  • p27 from HTLV-I
  • art/trs from HIV
  • VP22 from HSV-1
  • Antp from Drosophila Antp from Drosophila and the like.
  • an amino acid sequence comprising a region consisting of at least amino acid nos: 49 to 57 (SEQ ID NO: 8) of TAT sequence from human immunodeficiency virus is preferable (30).
  • SEQ ID NO: 8 amino acid sequence comprising a region consisting of at least amino acid nos: 49 to 57 (SEQ ID NO: 8) of TAT sequence from human immunodefici
  • the targeting or homing moiety may be an antibody or a recombinant antibody, a recombinant antibody fragment or a ScFv (single chain fragment variable).
  • the antibody or antibody fragment can be all or part of a polyclonal or monoclonal antibody.
  • the term "antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind
  • binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al, Ann. N. Y Acad Sci., 51 :660 (1949).
  • antibody fragment includes the following.:
  • F(ab)'2 which is a dimer of Fab' resulting from pepsin cleavage below the hinge disulfides: this is bivalent and can precipitate antigen; • Fab' which is a a monomer resulting from mild reduction of F(ab)'2: an
  • Fragments of monoclonal antibodies are of particular interest as small antigen targeting molecules.
  • Antibody fragments are also useful for the assembly of the chimeric polypeptides of the invention designed to carry the Ml lL derived peptides such as a therapeutic conjugate.
  • fragments of antibodies are of interest due to their altered pharmacokinetic behavior and for their rapid penetration into body tissues, which offer advantages for therapy techniques.
  • An antibody fragment of particular interest for use in the invention is a minimal Fv fragment with antigen-binding activity. The two chains of the Fv fragment are less stably associated than the Fd and light chain of the Fab fragment with no covalent bond and less non covalent interaction, but nevertheless functional Fv fragments have been expressed for a number of different antibodies.
  • Two strategies can be employed to stabilize the Fv fragments used in the invention: first, mutating a selected residue on each of the VH and VL chains to a cysteine to allow formation of a disulphide bond between the two domains; and second, the introduction of a peptide linker between the C-terminus of one domain and the N-terminus of the other, such that the Fv is produced as a single polypeptide chain known as a single-chain Fv.
  • single-chain Fvs (ScFvs), recombinant VL and VH fragments covalently tethered together by a polypeptide link and forming one polypeptide chain, are useful in this invention.
  • Fv genes For expression of Fv genes, several systems can be effectively used, including myeloma cells, insect cells, yeasts and Escherichia coli cells. Expression in E. coli has been a frequently used production method, with both intracellular expression and secretion enabling high yields of ScFv to be made.
  • VH- linker-VL or VL-linker-VH are useful in the invention; however, for some antibodies one particular orientation may be preferable as a free N-terminus of one domain, or C- terminus of the other, may be required to retain the native conformation and thus full antigen binding.
  • the ScFv may be susceptible to aggregation, with dimers, trimers, and multimers formed. Due to small size, rapid clearance in vivo, stability, and easy engineering, ScFvs employed in this invention have various applications in the treatment of diseases, particularly cancer. ScFvs can exhibit the same affinity and specificity for antigen as monoclonal antibodies.
  • Polyclonal antibodies employed as a component of the fusions, chimerics, or conjugates of the invention can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art.
  • purified cell surface proteins or glycoproteins or a peptide based on the amino acid sequence of cell surface proteins or glycoproteins that is appropriately conjugated is administered to the host animal typically through parenteral injection.
  • the immunogenicity of cell surface proteins or glycoproteins can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to cell surface proteins or glycoproteins.
  • Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno- electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Patent Nos. 4,376,110 and 4,486,530.
  • Monoclonal antibodies employed as a component of the fusions, chimerics, or conjugates of the invention can be readily prepared using well known procedures. See, for example, the procedures, described in U.S. Patent Nos US 4.902,614, US 4,543,439, and US 4.411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980.
  • the host animals such as mice
  • the host animals are injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified cell surface proteins or glycoproteins, conjugated cell surface proteins or glycoproteins, optionally in the presence of adjuvant.
  • Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse.
  • ABSC antibody capture
  • the mice are given an intravenous boost of cell surface proteins or glycoproteins or conjugated cell surface proteins or glycoproteins.
  • Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Agg.653 (ATCC), following established protocols.
  • Agg.653 Agg.653
  • the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out in plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti -mouse
  • a label such as 125 I-labeled cell surface proteins or glycoproteins, is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).
  • the monoclonal antibodies of the Ml lL derived conjugates can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).
  • the monoclonal antibodies and fragments thereof employed as a component of the Ml lL derived conjugate include chimeric antibodies, e ⁇ ., humanized versions of murine monoclonal antibodies.
  • Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
  • the humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (BioTechnology 7:934, 1989), and Winter and Harris (TIPS 14:139, May 1993). Procedures to generate antibodies transgenically can be found in GB 2 440, US Patent Nos. 5,569,825 and 5,545,806, all of which are incorporated by reference herein.
  • the Ml lL derived peptide according to the present invention may be advantageously fused or combined with a further anti-apoptotic peptide.
  • novel cytoprotective conjugates according to the present invention advantageously retain their efficient anti-apoptotic properties.
  • Ml lL derived conjugates of the invention can be generated by a variety of conventional techniques. Such techniques include those described in B. Merrifield, Methods Enzymol, 289:313, 1997; H. Ball and P. Mascagni, Int. J. Pept. Protein Res. 48:31-47, 1996; F. Molina et al., Pept. Res. 9:151-155, 1996; J. Fox, Mol. Biotechnol. 3:249-258, 1995; and P. Lepage et al., Anal. Biochem. 213: 40-48, 1993.
  • the Ml lL conjugates of the invention can be prepared by subcloning a DNA sequence encoding a desired peptide sequence into an expression vector for the production of the desired peptide.
  • the DNA sequence encoding the peptide is advantageously fused to a sequence encoding a suitable leader or signal peptide.
  • the DNA fragment may be chemically synthesized using conventional techniques.
  • the DNA fragment can also be produced by restriction endonuclease digestion of a clone of, for example HIV A, DNA using known restriction enzymes (New England Biolabs 1997 Catalog, Stratagene 1997 Catalog, Promega 1997 Catalog) and isolated by conventional means, such as by agarose gel electrophoresis.
  • PCR polymerase chain reaction
  • Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers.
  • the oligonucleotides can contain recognition sites for restriction endonucleases to facilitate insertion of the amplified DNA fragment into an expression vector.
  • Still another object of the present invention relates to a method of treating AIDS, neurodegenerative diseases, osteomyelodysplasia, ischemic diseases, infectious multiple organ failure, hepatitis, ischemic reperfusion disorders, diabetes and apoptosis- associated defects, comprising administering the Ml lL derived apoptosis inhibitor as described above to an animal to inhibit apoptosis in an effective amount to treat said apoptosis related diseases.
  • treatment or treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treating can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • an amount effective, at dosages and for periods of time necessary to achieve the desired result means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • the term "animal” as used herein includes all members of the animal kingdom, including humans. Preferably, the animal to be treated is a human.
  • a further object of the present invention is the use of the Ml IL derived peptide for manufacturing a prophylactic or therapeutic cytoprotective drug.
  • recombinant or synthetic molecules may be used for the prevention and treatment of a disease or condition that is characterized by excessive apoptosis.
  • a disease or condition that is characterized by excessive apoptosis.
  • the Ml lL derived peptides are used to prevent and/or treat mammalian disorders characterized by abnormal or excessive apoptosis specifically apoptosis in humans.
  • the recombinant proteins are used to prevent and/or treat apoptosis during chronic degenerative diseases e.g. neurodegenerative disease including Alzheimer's disease, Huntingtons' disease, Multiple sclerosis etc.
  • the Ml lL derived peptides are used to prevent and/or treat cytotoxic T cell and natural killer cell-mediated apoptosis associated with autoimmune disease and transplant rejection.
  • the Ml lL derived peptides are used to prevent and/or treat mitochondrial drug toxicity e.g. as a result of
  • Toxicity can arise, for example, during anti-HIV therapy and has also been associated with aspirin that can cause Reye's syndrome when taken during a prodromal viral illness.
  • the Ml lL derived peptides are used to prevent and/or treat inflammation or inflammatory diseases.
  • the present invention provides a use of Ml lL derived peptides incorporating the Bax, Bak or PBR binding domain of Ml IL for treating disorders in animals characterized by excessive apoptosis.
  • the present invention also provides a use of an effective amount of the Ml lL derived peptides to treat the disorders characterized by excessive apoptosis.
  • Ml lL derived peptides according to the present invention have therapeutic effects for AIDS, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa or cerebellar degeneration), osteomyelodysplasia (aplastic anemia and the like), ischemic diseases (myocardial infarction, brain infarction, apoplectic stroke, ischemic reperfusion disorder and the like), fulminant hepatitis, hepatic failures caused by addictions such as an alcoholism, infectious multiple organ failure, diabetes and the like.
  • neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa or cerebellar degeneration
  • the present invention also encompasses a method for treating the above-mentioned diseases, characterized by administering the above-mentioned apoptosis-inhibitor to a patient with the above- mentioned diseases to inhibit apoptosis, thereby treating those diseases.
  • the treatment method is, among other options, suitable for treatment of ischemic diseases, particularly myocardial infarction and brain infarction, and especially myocardial infarction.
  • the organ can be stored in a more preferable state until implantation.
  • the apoptosis-inhibitor of the present invention may comprise the Ml lL derived peptides in any modified form so that the uptake of the above-mentioned Ml lL derived polypeptide into a particular cell is facilitated.
  • the apoptosis- inhibitor of the present invention may contain various aids which are usually employed within a range so that anti-apoptotic activity can be exhibited.
  • the administration form, the administration method and the dose can be suitably set, depending upon age, weight and conditions of an individual to be administered.
  • Ml lL derived polypeptide for the manufacture of a prophylactic or therapeutic agent for the above-mentioned diseases, notably ischemic diseases, particularly myocardial infarction and brain infarction, and especially myocardial infarction.
  • compositions comprising said Ml lL derived peptides.
  • Compositions comprising an effective amount of a Ml lL polypeptide of the present invention, in combination with other components, such as a physiologically acceptable diluent, carrier, or excipient, are provided herein.
  • the chimeric polypeptide can be formulated according to known methods used to prepare pharmaceutically useful compositions.
  • Suitable formulations for pharmaceutical compositions include those described in Renfington's Pharmaceutical Sciences, 16 ed. 1980, Mack Publishing Company, Easton, PA.
  • compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic, acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamiellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • PEG polyethylene glycol
  • metal ions or incorporated into polymeric compounds such as polyacetic, acid, polyglycolic acid, hydrogels, dextran, etc.
  • liposomes such as polyacetic, acid, polyglycolic acid, hydrogels, dextran, etc.
  • Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance and are thus chosen according to the intended application.
  • compositions of the invention comprising the Ml IL derived peptide can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation.
  • parenteral includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g.,. at a site of disease or injury. Sustained release from implants is also contemplated.
  • suitable dosages will vary depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
  • Compositions comprising nucleic acids in physiologically acceptable formulations are also contemplated. DNA may be formulated for injection, for example.
  • the invention in one of its most general applications, relates to a recombinant vector incorporating a DNA segment having a sequence encoding the chimeric polypeptide of the invention.
  • the term "chimeric polypeptide" is defined as including any polypeptide where at least a portion of a viral anti-apoptotic peptide is coupled to a cell uptake or penetration system such as the HIV TAT PTD as described above or a portion of an antibody or antibody fragment. The coupling can be achieved in a manner that provides for a functional transcribing and translating of the DNA segment and message derived therefrom, respectively.
  • the vectors of the invention will generally be constructed such that the Ml lL derived peptide encoding sequence is positioned adjacent to and under the control of an effective promoter.
  • the promoter will comprise a prokaryotic promoter where the vector is being adapted for expression in a prokaryotic host.
  • the promoter will comprise a eukaryotic promoter where the vector is being adapted for expression in a eukaryotic host.
  • the vector will typically further include a polyadenylation signal position 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded chimeric polypeptide.
  • Promoters of particular utility in the vectors of the invention are cytomegalovirus promoters and baculovirus promoters, depending upon the cell used for expression. Regardless of the exact nature of the vector's promoters, the recombinant vectors of the invention will incorporate a DNA segment as defined below.
  • a recombinant host cell is also claimed herein, which incorporates a vector of the invention.
  • the recombinant host cell may be either a eukaryotic cell or a prokaryotic host cell.
  • a Chinese Hamster Ovary (CHO) cell has utility.
  • the insect cell lines SF9 or SF21 can be used.
  • Ml IL derived peptides of the invention were constructed to contain a TAG having 6 histidine: HIS TAG in order to allow appropriate purification of the peptide Also detection of the full length protein was performed using a polyclonal antibody as described by Graham et al. (Virology, 1992, 191 :112-124). Alternatively to the production of Ml lL derived peptides using the Baculovirus
  • Ml lL derived peptides and conjugates according to the present invention can be synthesized by Fmoc method using Fmoc-linked amide resin in ABI433A automated synthesizer.
  • the resulting protected peptide resin is treated with a reagent containing trifluoroacetic acid, and free peptide is then purified by reverse phase HPLC.
  • eosin-5-iodoacetamide dissolved in DMF was mixed with the above- mentioned purified peptide dissolved in 50 mm phosphate buffer (ph 7.7), and the mixture was allowed to react at room temperature for 1 hour. After the reaction, the peptide is purified again by reverse phase HPLC, and collected as a single peak. The molecular weight of the purified sample is determined by MALDI-TOF MS.
  • Example 1 Construction of TAP tag vectors and TAP-tagged protein purification and demonstration of a physical interaction between the N-terminal portion of MllL with Bak. As shown in Figure 1A, truncated Ml lL was fused to pcDNA3.6C-TAP vector and transfected into 293T cells then the presence in elution products of Bak after immunoprecipitation of TAP-tagged protein is monitored by western Blot (presence of Bak: + on right panel).
  • FIG. 1(B) shows the expression of the truncated Ml IL constructs was detected with anti-PAP which recognizes the C-TAP tag (20kDa) after immunoblotting.
  • the C-terminal C-TAP epitope is subcloned into pcDNA3, resulting in the pcDNA3.6 C-TAP fusion vector, which is used for further subcloning of Ml IL and deletion mutants of Ml lL (M11L-C-TAP) ( Figure 1A).
  • Logarithmic-phase 293T cells were seeded in 100-mm-diameter tissue culture dishes (5 x 10 6 cells/dish) in fresh medium the day before transfection. Cells were transiently transfected or cotransfected with 20 ⁇ g of each of the expression plasmids by using a calciumphosphate mammalian transfection kit (BD Clontech) according to the manufacturer's recommended procedure.
  • Cells expressing the TAP-tagged Ml lL protein were lysed 24 h after transfection in cell lysis buffer (50 mM Tris-HCI [pH 8.0], 137 mM NaCl, 2% CHAPS) supplemented with a protease inhibitor cocktail. Following centrifugation at 14,000 ⁇ g for 30 min at 4°C, the supernatant was recovered and added to rabbit immuno globulin G (IgG) agarose beads (Sigma), which were then rotated for 2 h at 4°C to allow the binding of the fusion protein.
  • IgG immuno globulin G
  • the beads were washed three times with wash buffer (10 mM Tris-HCI [pH 8.0], 150 mM NaCl, 0.1% CHAPS) and resuspended in 1 ml of TEV cleavage buffer (10 mM Tris-HCI [pH 8.0], 0.5 mM EDTA, 1 mM dithiothreitol) containing 200 U of TEV protease (Invitrogen).
  • wash buffer (10 mM Tris-HCI [pH 8.0], 150 mM NaCl, 0.1% CHAPS)
  • TEV cleavage buffer 10 mM Tris-HCI [pH 8.0], 0.5 mM EDTA, 1 mM dithiothreitol
  • TEV protease Invitrogen
  • Calmodulin binding buffer (10 mM ⁇ -mercaptoethanol, lOmM Tris-HCI [pH 8.0], 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaC12, and 0.1% CHAPS) was added to the eluted product together with calmodulin affinity resin (Stratagene) and mixed for 1 h at 4°C to allow binding to the beads.
  • calmodulin binding buffer 10 mM ⁇ -mercaptoethanol, 10 mM Tris-HCI [pH 8.0], 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM EGTA, 0.1% CHAPS.
  • the eluted purified product was concentrated to an appropriate volume through a protein concentrator (NANOSEP 10K OMEGA; Pall Gelman Laboratory) and resolved by denaturing SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were immunoblotted with the proper antibodies (anti Bak and anti- PAP which recognize Bak (Figure IB) and the TAP epitope respectively(Figure 1C)).
  • Example 2 Evaluation of the MllL derived peptides mitochondrio-protective effects on purified mitochondria
  • the mitochondrio-protective potential of the Ml IL derived polypeptides as well as the full length protein is evaluated using mitochondria from mouse liver and mitochondria purified from HeLa cells.
  • inductors of mitochondrio-toxicity are used such as Ca2+, Atractyloside PK11195, Vpr52-96, Alamethicin in order to provide the mitochondrio-toxic stimulus whereas reference inhibitors such as CsA, Bongkrekate, Bcl-Xl(Rec) are used as positive controls of inhibition.
  • Example 3 In cellula validation of the MllL derived peptides cytoprotective effects The cellular anti-apoptotic potential Ml lL derived polypeptides as well as the full length protein is evaluated on human cell lines, HeLa cells and Jurkat, and on primary cells, such as cortical neurons from mouse and cardiomyocytes from rat.
  • Murhammer DW. "The use of insect cell cultures for recombinant protein synthesis: Engineering aspects", Appl Biochem Biotechnol. 1991 Dec;31(3):283-310.

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Abstract

L'invention se rapporte à un nouveau peptide chimère dérivé d'une protéine M11L d'un poxvirus de myxome apte à inhiber l'apoptose et à son utilisation comme molécule cytoprotectrice, ainsi qu'à des compositions pharmaceutiques contenant les peptides chimères dérivés de M11L. Font également l'objet de cette invention des molécules d'acide nucléique codant les peptides anti-apoptotiques chimères et des procédés d'utilisation des protéines et des molécules d'acides nucléiques tenant lieu de molécules cytoprotectrices. La présente invention se rapporte également à un peptide de fusion avec M11L possédant une action de reprise et/ou un fragment ciblé dirigé dans une cellule et à son utilisation comme inhibiteur d'apoptose. Les peptides de cette invention et les peptides de fusion sont utilisés pour traiter le sida, les maladies neurodégénératives, l'ostéomyelodysplasie, les maladies ischémiques, la défaillance multiviscérale infectieuse, l'hépatite, les manifestations de reperfusion ischémiques, le diabète et analogues.
PCT/EP2004/006581 2003-05-28 2004-05-28 Peptides derives de m11l et leur utilisation therapeutique WO2004106370A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2007101925A1 (fr) * 2006-03-09 2007-09-13 Trophos Utilisation de derives du 3,5-seco-4-nor-cholestane pour l'obtention d'un medicament cytoprotecteur
WO2007108749A1 (fr) * 2006-03-20 2007-09-27 Cepep Iii Ab Constructions chimériques entre peptides de localisation du cancer et peptides de pénétration cellulaire, couplées à des médicaments et/ou un ou des agents de diagnostic anticancer
WO2007128555A1 (fr) * 2006-05-08 2007-11-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Inhibition de la gliotoxine

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WO2002074908A2 (fr) * 2001-03-02 2002-09-26 Mds Proteomics, Inc. Methodes et reactifs de regulation de l'apoptose

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EVERETT HELEN ET AL: "M11L: A novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 191, no. 9, 1 May 2000 (2000-05-01), pages 1487 - 1498, XP002295776, ISSN: 0022-1007 *
GRAHAM KATHRYN A ET AL: "Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical in manifestation of viral virulence", VIROLOGY, vol. 191, no. 1, 1992, pages 112 - 124, XP001183493, ISSN: 0042-6822 *
WANG GEN ET AL: "Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak", JOURNAL OF VIROLOGY, vol. 78, no. 13, July 2004 (2004-07-01), pages 7097 - 7111, XP009036379, ISSN: 0022-538X *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007101925A1 (fr) * 2006-03-09 2007-09-13 Trophos Utilisation de derives du 3,5-seco-4-nor-cholestane pour l'obtention d'un medicament cytoprotecteur
FR2898272A1 (fr) * 2006-03-09 2007-09-14 Trophos Sa Utilisation de derives du 3,5-seco-4-nor-cholestrane pour l'obtention d'un medicament cytoprotecteur
US7985774B2 (en) 2006-03-09 2011-07-26 Trophos Use of 3,5-seco-4-nor-cholestane derivatives for obtaining a cytoprotective drug
WO2007108749A1 (fr) * 2006-03-20 2007-09-27 Cepep Iii Ab Constructions chimériques entre peptides de localisation du cancer et peptides de pénétration cellulaire, couplées à des médicaments et/ou un ou des agents de diagnostic anticancer
WO2007128555A1 (fr) * 2006-05-08 2007-11-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Inhibition de la gliotoxine

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