WO2017149012A1 - Peptides et utilisations de ces derniers pour diminuer la motilité cellulaire induite par le cd95 - Google Patents

Peptides et utilisations de ces derniers pour diminuer la motilité cellulaire induite par le cd95 Download PDF

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WO2017149012A1
WO2017149012A1 PCT/EP2017/054771 EP2017054771W WO2017149012A1 WO 2017149012 A1 WO2017149012 A1 WO 2017149012A1 EP 2017054771 W EP2017054771 W EP 2017054771W WO 2017149012 A1 WO2017149012 A1 WO 2017149012A1
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syndrome
disease
autoimmune
peptide
chronic
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Patrick Legembre
Pierre Vacher
Amanda POISSONNIER
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Bergonie
Universite de Bordeaux
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Bergonie
Universite de Bordeaux
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to peptides and uses thereof for reducing cd95 -mediated cell motility.
  • CD95L is a transmembrane glycoprotein that acts locally through cell-to-cell contact (Suda et al, 1993).
  • the extracellular domain of CD95L comprises a juxtamembrane stalk region (amino acid residues (aa) 103-136)(Orlinick et al, 1997) cleavable by metalloproteases (Fouque et al, 2014), thereby releasing CD95L into the bloodstream.
  • CD95 (Fas/APO-l/TNFRSF6) belongs to the tumor necrosis factor receptor (TNF-R) family and is ubiquitously expressed in the body (Peter et al, 2015).
  • CD95L When membrane-bound CD95L binds to CD95, the intracellular region of CD95 (designated the death domain - DD) orchestrates the formation of a death-inducing signaling complex (DISC) by recruitment of the adaptor molecule, Fas-associated protein with death domain (FADD), which in turn induces caspase-8 aggregation and subsequent apoptosis (Kischkel et al, 1995).
  • DISC death-inducing signaling complex
  • FADD Fas-associated protein with death domain
  • the present invention relates to peptides and uses thereof for reducing cd95 -mediated cell motility.
  • the present invention is defined by the claims.
  • the present invention relates to a peptide comprising an amino sequence of formula of
  • Xi82-Xi83-RK-Xi86-Xi87-K (SEQ ID NO: l) wherein Xi82 represents an amino acid selected from the group consisting of A, T, C, K, Xi83 represents an amino acid selected from the group consisting of A and C, Xi86 represents an amino acid selected from the group consisting of A, H, N, and R, and Xi87 represents an amino acid selected from the group consisting of A, K, and L.
  • the term "A” or “Ala” has its general meaning in the art and refers to Alanine.
  • the term “R” or “Arg” has its general meaning in the art and refers to Arginine.
  • the term “N” or “Asn” has its general meaning in the art and refers to Asparagine.
  • the term “D” or “Asp” has its general meaning in the art and refers to Aspartic acid.
  • the term “C” or “Cys” has its general meaning in the art and refers to Cysteine.
  • E or “Glu” has its general meaning in the art and refers to Glutamic acid.
  • the term "Q" or “Gin” has its general meaning in the art and refers to Glutamine.
  • G or “Gly” has its general meaning in the art and refers to Glycine.
  • H or “His” has its general meaning in the art and refers to Histidine.
  • I or “He” has its general meaning in the art and refers to Isoleucine.
  • L or “Leu” has its general meaning in the art and refers to Leucine.
  • the term “K” or “Lys” has its general meaning in the art and refers to Lysine.
  • the term "M” or “Met” has its general meaning in the art and refers to Methionine.
  • the term “F” or “Phe” has its general meaning in the art and refers to Phenylalanine.
  • the term "P” or “Pro” has its general meaning in the art and refers to Proline.
  • the term “S” or “Ser” has its general meaning in the art and refers to Serine.
  • the term “T” or “Thr” has its general meaning in the art and refers to Threonine.
  • the term “Y” or “Tyr” has its general meaning in the art and refers to Tyrosine.
  • the term “V” or “Val” has its general meaning in the art and refers to Valine.
  • the peptide of the present invention comprises less than 15 amino acids, preferably less than 10 amino acids. In some embodiments, the peptide of the present invention comprises or consists of 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the peptide of the present invention comprise an amino acid sequence of Xi82-Xi83-RK-Xi 86 -Xi87-KE (SEQ ID NO:2).
  • the peptide of the present invention comprises or consists of the amino acid sequence TCRKHRK (SEQ ID NO:3) or TCRKHRKE (SEQ ID NO:4).
  • the peptide of the present invention is stapled.
  • a "stapled" peptide is a peptide comprising a selected number of standard or nonstandard amino acids, further comprising at least two moieties capable of undergoing reaction to promote carbon- carbon bond formation, that has been contacted with a reagent to generate at least one cross- linker between the at least two moieties, which modulates, for example, peptide stability.
  • peptide stapling is a term coined for a synthetic methodology used to covalently join two olefin-containing side chains present in a peptide chain using an olefin metathesis reaction (J. Org. Chem.
  • the stapled peptide strategy in which an all- hydrocarbon cross-link is generated by olefin metathesis is an efficient approach to increase the helical character of peptides to target a- helical binding motifs. Unlike their unstapled analogues these hydrocarbon- stapled peptides have shown to be a-helical, protease-resistant, and cell permeable.
  • the peptide of the present invention is fused to at least one heterologous peptide (i.e. a peptide which is not derived to CD95).
  • the peptide according to the invention is fused directly or via a spacer to at least one heterologous peptide.
  • the peptide according to the invention is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous peptide, or at its N-terminal end to the C-terminal end of the heterologous peptide.
  • the term "directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the peptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous peptide.
  • the last amino acid of the C- terminal end of said peptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous peptide, or the first amino acid of the N-terminal end of said peptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous peptide.
  • the term “spacer” refers to a sequence of at least one amino acid that links the peptide of the present invention to the heterologous peptide. Such a spacer may be useful to prevent steric hindrances.
  • the heterologous peptide is a cell-penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence.
  • TAT Transactivator of Transcription
  • cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • the peptides of the present invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. For instance, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said peptides, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, typically using a commercially available peptide synthesis apparatus and following the manufacturer's instructions. Alternatively, the peptides of the present invention can be synthesized by recombinant DNA techniques as is now well- known in the art.
  • these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired peptide, from which they can be later isolated using well-known techniques.
  • Peptides of the present invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).
  • a further object of the present invention relates to a nucleic acid molecule encoding for a peptide according to the invention.
  • nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art.
  • said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
  • a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a peptide of the present invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted.
  • the peptides of the present invention are particularly suitable of reducing CD95- mediated cell motility and thus may find various therapeutic applications.
  • the peptide of the present invention is particularly suitable for reducing CD95 -mediated cancer cell motility. In some embodiments, the peptides of the present invention are particularly suitable for the treatment of cancer in a subject in need thereof.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the peptide of the present is particularly suitable for the treatment of triple negative breast cancer.
  • Triple negative breast cancer has its general meaning in the art and means that said breast cancer lacks receptors for the hormones estrogen (ER-negative) and progesterone (PR-negative), and for the protein HER2.
  • the peptide of the present invention is particularly suitable for the prevention of metastases (e.g. in a subject suffering from a triple negative breast cancer).
  • the present invention relates to the peptide of the present invention for use in enhancing therapeutic efficacy of cancer treatment in a subject in need thereof.
  • the peptide of the present invention may be administered sequentially or concomitantly with one or more therapeutic active agent such as chemotherapeutic or radiotherapeutic agents.
  • chemotherapeutics include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, epipodophyllotoxins such as etoposide and teniposide, camptothecins such as irinotecan and topotecan, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil and 5- fluorouracil combined with leucovorin, taxa
  • additional therapeutic active agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, anthracyclines, MDR inhibitors and Ca 2+ ATPase inhibitors.
  • radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • the peptide of the present invention is particularly suitable for reducing CD95-mediated lymphocyte (e.g. T cell) motility.
  • the peptide of the present invention is particularly suitable for the treatment of an auto-immune disease.
  • an "autoimmune disease” is a disease or disorder arising from and directed at an individual's own tissues.
  • the peptide of the present invention is particularly suitable for the treatment of an inflammatory condition.
  • inflammatory condition refers to acute or chronic localized or systemic responses to harmful stimuli, such as pathogens, damaged cells, physical injury or irritants, that are mediated in part by the activity of cytokines, chemokines, or inflammatory cells (e.g., neutrophils, monocytes, lymphocytes, macrophages) and is characterized in most instances by pain, redness, swelling, and impairment of tissue function.
  • harmful stimuli such as pathogens, damaged cells, physical injury or irritants
  • the autoimmune disease or inflammatory condition is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and
  • the peptide of the present invention is particularly suitable for the treatment of systemic lupus erythematosus.
  • the peptide of the present invention is particularly suitable for preventing Thl7 cell transmigration. Accordingly, the peptide of the present invention is particularly suitable for treating Thl7 mediated diseases.
  • Thl7-mediated disease is used herein in the broadest sense and includes all diseases and pathological conditions the pathogenesis of which involves abnormalities of Thl7 cells.
  • Thl7 cells has its general meaning in the art and refers to a subset of T helper cells producing interleukin 17 (IL-17). "A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage". Nat. Med. 13 (2): 139-145.).
  • IL-17 has its general meaning in the art and refers to the interleukin- 17A protein.
  • Thl7 cells are characterized by classical expression of Th cell markers at their cell surface such as CD4, and by the expression of IL17.
  • a Thl7 cell is a IL-17+ cell.
  • Thl7 mediated diseases include but are not limited to autoimmune diseases, inflammatory diseases, osteoclasia, and transplantation rejection of cells, tissue and organs.
  • Thl7-mediated diseases may be one or more selected from the group consisting of Beliefs disease, polymyositis/dermatomyositis, autoimmune cytopenias, autoimmune myocarditis, primary liver cirrhosis, Goodpasture's syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rhe
  • the peptide of the present invention is administered to the subject in a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the peptide of the present invention for reaching a therapeutic effect (e.g. treating cancer). It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific peptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the peptide of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Sterile injectable solutions are prepared by incorporating the Peptide at the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 A. Each amino acid in CID sequence was replaced by alanine (alanine scanning) and all constructs were co-transfected with SH3-PLCyl in HEK cells. For each co- transfection, luminescence was assessed and a ratio was calculated as follows: (luminescence for mutated construct/ luminescence for wild type CID) x 100).
  • CID-F2 and SH3-PLCyl-Fl evaluated by densitometric analysis of proteins as follows: ((densitometric value of mutated CID-F2 construct/ densitometric value of wild type CID-F2) x (densitometric value of co-transfected SFB-PLCyl-Fl/ densitometric value of SFB-PLCyl-Fl co-transfected with wild type CID)).
  • TCRKHR wild type CD95
  • TCAAHR double
  • TCAAHRA triple
  • FIG. 1 A. CID-CD95 (aa 175-210) or DD-CD95 (aa 210-303)-F2 were co- transfected into HEK cells with the indicated domains of PLCyl (Ca2+ response) or FADD (apoptosis) fused to Fl . Light emission indicates refolding of the luciferase and reconstitution of enzyme activity through protein/protein interactions. The inhibition of light emission for the indicated PPIs was assessed in cells incubated for the indicated times in the presence of TAT-CID (25 ⁇ ). Data represent means ⁇ SD of three independent experiments. B. Schematic representation of the different CID constructs and the peptide control. C.
  • CID-CD95-F2 was co-transfected into HEK cells with SH3-PLCyl-Fl . After 24 hours, cells were incubated for 4 hrs with indicated peptides (25 ⁇ ) and light emission was assessed. Data represent means ⁇ SD of the percentage of light emission inhibition of three independent experiments. Percentage of light emission inhibition was measured as follows: [100 - (light emission measured with peptide treatment / light emission measured without treatment (maximum)) x 100].
  • FIG. 3 A. Thl7 cells were pre-incubated for 1 h with TAT-control, TAT-minCID or minCID (1 ⁇ ) and then stimulated with cl-CD95L (100 ng/niL). [Ca2+]CYT was assessed in FuraPE3-AM (1 ⁇ )- ⁇ cells. Data represent means ⁇ the SD of 3 independent experiments.
  • CD95 triggers a DD-independent Ca 2+ response
  • Engagement of CD95 evoked a Ca 2+ response in activated T lymphocytes, resulting in transient inhibition of cellular apoptosis (Khadra et al, 2011) and cell migration (Malleter et al, 2013; Tauzin et al, 201 1).
  • Due to the instrumental role of the CD95-mediated apoptotic signal in anti-infectious and anti-tumor responses we assumed that the inhibition of the CD95 non-apoptotic responses while conserving the apoptotic signal could be an attractive therapeutic option to prevent Thl7 recruitment in inflamed organs without altering immune surveillance.
  • CD95 constructs devoid of the entire intracellular domain of CD95 (CD95 1 175 ), the DD (CD95 1-210 ), or the last 15 aa involved in FAP-1 protein tyrosine phosphatase recruitment(Sato et al., 1995) (CD95 1 303 ). These constructs were expressed in the CEM-IRC T-cell line, which was selected for its low CD95 expression (Beneteau et al, 2008).
  • CEM-IRC cells showed minimal cell death in response to a multimeric (dodecamer) and cytotoxic CD95L (IgCD95L); however, expression of CD95 1 303 or wild-type CD95 restored cell death levels to those observed for parental CEM cells. Contrarily, introduction of CD95 1-175 or CD95 1-210 failed to induce apoptosis and, as previously observed, these constructs behaved as dominant-negative receptors (Siegel et al, 2000). Furthermore, reconstituting CEM-IRC cells with wild-type CD95 or CD95 1 303 restored CD95-mediated Ca 2+ signaling.
  • CD95-DD deletion did not affect induction of Ca 2+ signaling.
  • CD95 construct devoid of the entire intracellular region CD95 1 175
  • Ca 2+ signaling is triggered by CD95 aa 175-210.
  • CD95 1-210 was capable of recruiting PLCyl .
  • GFP green fluorescent protein
  • CD95 175 210 /mCherry interacted with PLCyl and inhibited its recruitment to CD95, suggesting that interference with this juxtamembrane domain may prevent CD95-mediated Ca 2+ signaling.
  • TAT-CID cell-penetrating peptide, TAT-CID, by linking CID to the nine-aa human immunodeficiency virus (HIV)-TAT sequence, which serves as a carrier for protein translocation across the plasma membrane.
  • HIV human immunodeficiency virus
  • PBLs peripheral blood lymphocytes
  • TAT-CID impaired the recruitment of PLCyl and abolished the induction of CD95-mediated Ca 2+ signaling.
  • pre-incubation of Jurkat cells with TAT-CID inhibited PLCyl binding to CD95 and abrogated the CD95 -facilitated Ca 2+ response.
  • TAT-CID also inhibited Akt phosphorylation at serine 473 (a hallmark of PI3K signaling activation) in cl-CD95L-exposed PBLs. Nevertheless, although TAT-CID impeded CD95-mediated Ca 2+ and PI3K signaling, the peptide did not affect the apoptotic signaling pathway. Hence, we mapped a novel domain in CD95 receptor, namely CID, which recruits PLCyl and elicits Ca 2+ responses.
  • TAT-CID cell-penetrating TAT-CID peptide could inhibit the PLCyl -SH3/CD95-CID interaction. While TAT-control peptide did not alter luciferase activity in cells co-expressing PLCyl-SH3-Fl and CD95-CID-F2, TAT-CID efficiently blocked this activity in a dose- dependent manner, supporting the hypothesis that CD95 directly associates with PLCyl through the CD95-CID domain. Intriguingly, SH3 domains primary bind to peptides containing a consensus PxxP sequence that is not present in CID; however, many examples of unconventional SH3-binding peptides have previously been described (Saksela and Permi, 2012).
  • CD95 CID might interact with PLCyl-SFB
  • two computational peptide screenings were performed (Deng et al, 2005).
  • a protein-peptide docking approach was carried out by using each aforementioned CID heptapeptide alternatively docked within the PLCyl-SFB domain.
  • the canonical PxxP motif-containing peptides could be docked in two opposite orientations regarding the relative positioning of a positively charged residue (+xxPxxP or xPxxPx+) interacting with a negatively charged cleft on the SH3 surface (Saksela and Permi, 2012).
  • E189 could contribute to the positioning of the minimal CD95-CID peptide in PLCyl SH3 domain.
  • TAT-CID selectively inhibits CD95-mediated Ca 2+ signaling.
  • TCR T-cell receptor
  • RMSD RMSD vs energy Waals Contact Contact vs I V WO (kcal/mol) energy Surface Surface I V WO backbone
  • the minimum CID is a cell-penetrating peptide
  • minCID minimum CID
  • TCRKHR amino acids 182 to 188
  • minCID amino acids 182 to 188
  • TAT-minCID and minCID abrogated the CD95-mediated Ca2+ response in Thl 7 cells (Fig.3 A) and thereby prevented Thl 7 trafficking across endothelial cells (Fig.3B).
  • Adachi M., Watanabe-Fukunaga, R., and Nagata, S. (1993). Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice. Proc Natl Acad Sci U S A 90, 1756-1760.
  • Thl7 the third member of the effector T cell trilogy. Curr Opin Immunol 19, 652-657.
  • Promiscuity as a functional trait intrinsically disordered regions as central players of interactomes. Biochem J 454, 361-369.
  • CD95 triggers Orail -mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) beta2, and prevents death- inducing signaling complex formation.
  • PKC protein kinase C
  • Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.
  • DISC death-inducing signaling complex
  • Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Nat Med.
  • SH3 domain ligand binding What's the consensus and where's the specificity? FEBS Lett 586, 2609-2614.
  • FAP-1 a protein tyrosine phosphatase that associates with Fas. Science 268, 411-415.

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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des peptides et des utilisations de ces derniers pour diminuer la motilité cellulaire induite par le CD95. Les inventeurs ont identifié un peptide capable de traverser la membrane plasmatique, de se lier à PLCyl et de déstabiliser l'interaction CD95/PLCyl souhaitée. En particulier, la présente invention concerne un peptide comprenant une séquence d'acides aminés de formule X182-X183-RK-X186-X187-K (SEQ ID NO : l), où X182 représente un acide aminé sélectionné dans le groupe constitué de A, T, C et K, X183 représente un acide aminé sélectionné dans le groupe constitué de A et C, X186 représente un acide aminé sélectionné dans le groupe constitué de A, H, N et R, et X187 représente un acide aminé sélectionné dans le groupe constitué par A, K et L.
PCT/EP2017/054771 2016-03-02 2017-03-01 Peptides et utilisations de ces derniers pour diminuer la motilité cellulaire induite par le cd95 Ceased WO2017149012A1 (fr)

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EP16305242 2016-03-02
EP16305242.6 2016-03-02

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WO2017149012A1 true WO2017149012A1 (fr) 2017-09-08

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1424343A1 (fr) * 2002-11-29 2004-06-02 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugué peptidique pour l'imagerie moléculaire des noyeux cellulaires et la thérapie des tumeurs
WO2007070372A2 (fr) * 2005-12-09 2007-06-21 Entremed, Inc. Compositions et procedes d'inhibition de la proliferation cellulaire
WO2015158810A1 (fr) * 2014-04-17 2015-10-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides et leurs utilisations pour réduire la motilité cellulaire médiée par cd95
WO2016061133A1 (fr) * 2014-10-14 2016-04-21 Riptide Bioscience, Inc. Peptides ayant des propriétés anti-inflammatoires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1424343A1 (fr) * 2002-11-29 2004-06-02 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugué peptidique pour l'imagerie moléculaire des noyeux cellulaires et la thérapie des tumeurs
WO2007070372A2 (fr) * 2005-12-09 2007-06-21 Entremed, Inc. Compositions et procedes d'inhibition de la proliferation cellulaire
WO2015158810A1 (fr) * 2014-04-17 2015-10-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides et leurs utilisations pour réduire la motilité cellulaire médiée par cd95
WO2016061133A1 (fr) * 2014-10-14 2016-04-21 Riptide Bioscience, Inc. Peptides ayant des propriétés anti-inflammatoires

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
S B SCHRIER ET AL.: "Prediction of calcite morphology from computational and experimental studies of mutations of a de-novo designed peptides", LANGMUIR, vol. 27, 2011, American Chemical Society, pages 11520 - 11527, XP002758620, ISSN: 0743-7463 *
Z YE ET AL.: "Structural requirements of human tissue factor pathway inhibitor (TFPI) and heparin for TFPI-heparin interaction", THROMBOSIS RESEARCH, vol. 89, 1998, TARRYTOWN, NY, pages 263 - 270, XP002758621, ISSN: 0049-3848 *

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