WO2014147193A1 - Nouveaux motifs peptidiques pénétrant dans les cellules - Google Patents

Nouveaux motifs peptidiques pénétrant dans les cellules Download PDF

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Publication number
WO2014147193A1
WO2014147193A1 PCT/EP2014/055633 EP2014055633W WO2014147193A1 WO 2014147193 A1 WO2014147193 A1 WO 2014147193A1 EP 2014055633 W EP2014055633 W EP 2014055633W WO 2014147193 A1 WO2014147193 A1 WO 2014147193A1
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Prior art keywords
compound
cell
seq
sequence seq
compound according
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PCT/EP2014/055633
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English (en)
Inventor
May Catherine Morris
Gilles Divita
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Centre National De La Recherche Scientifique (Cnrs)
Universite Montpellier 2 Sciences Et Techniques
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Publication of WO2014147193A1 publication Critical patent/WO2014147193A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4739—Cyclin; Prad 1
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to new cell-penetrating peptide motifs which allow the intracellular delivery of products to which they are covalently linked or fused. It also relates to compounds comprising at least one of said cell-penetrating peptide motifs.
  • the present invention also relates to a process for intracellular delivery of compounds and for targeting biological functions.
  • the present invention furthermore relates to the use of said cell-penetrating peptide motifs for the intracellular delivery of compounds, in particular for detecting intracellular compounds, for interfering with biological function, for cellular imaging and for screening libraries of compounds.
  • Non- viral strategies that promote efficient delivery of bioactive macro molecules into living cells include lipid-based formulations, polycationic polymers and particles, carbon nanotubes and nanoparticles, as well as peptide-based formulations.
  • PTDs Protein transduction domains
  • CPPs cell-penetrating peptides or cell-penetrating peptide motifs
  • TAT the regulatory transactivating domain of HIV
  • Antennapaedia homeodomain protein also known as Penetratin (Derossi et al., 1994) and Transportan (Pooga et al., 1998)
  • calcitonin- derived peptides Herpes simplex viral protein VP22 (Elliot et al., 1997) polyarginine peptides (Wender et al., 2000) and polyproline sweet arrow peptide (Pujals et al., 2006).
  • CADY secondary amphipathic peptide
  • Kim et al. (2009) describes PEP 1 -CAT and PEP 1 -SOD fusion proteins. Shirong et al. (2007) describes a PEP1-P27mt fusion protein. Duk-Soo et al. (2012) describes a PEP 1 -pi 8 fusion protein.
  • WO 2007/108749 describes chimeric constructs between cancer-homing peptides and a PEP1 cell-penetrating peptide.
  • Kang Myung et al. (201 1) describes a PEP-1 peptide-modified liposomal nanocarrier system for intracellular drug delivery.
  • US 2006/035815 describes compounds comprising a 21 amino acid peptidic motif. These documents describe a « PEP1 » motif comprising 21 amino acids.
  • WO 02/061105 cites some peptides susceptible to be able to bind to a Permeability Transition Pore Complex.
  • Kurzawa et al. (2011) describes a polypeptide non-covalently complexed to a cell-penetrating peptide carrier.
  • the peptides differ from a peptide according to the invention.
  • PEPCOV Tryptophan-rich peptide
  • the inventors have now developed a Tryptophan-rich peptide, named PEPCOV, which promotes delivery into living cells and in vivo, of compounds which are covalently coupled to it or directly synthesized/or produced as a single fusion compound.
  • PEPCOV promotes rapid and efficient and homogeneous delivery of products such as peptide inhibitors and fluorescent biosensors, enzymes, cofactors into cells and leads to a related biological response.
  • This technology has been validated with respect to delivery both in cellulo and in vivo, and has proven to be a useful tool for diagnostic and therapeutic approaches based on delivery of peptide or protein biosensors into cells, as well as a useful laboratory tool.
  • These carriers can further be applied to high content, high throughput screening assays in the context of drug discovery programs, as well as in vivo for molecular imaging or for therapeutic strategies.
  • Each amino acid is herein represented according to the IUPAC amino-acid abbreviation, such as follows: Amino-acid or amino-acid residue Abbreviation Abbreviation
  • the present invention first relates to a cell-penetrating peptide motif comprising at least the amino acid sequence: WW/FXXWW/F (SEQ ID N°l). More preferably, the present invention relates a to a cell-penetrating peptide motif comprising at least an amino acid sequence chosen in the group consisting of: WWXXWW (SEQ ID N°2), WFXXWW (SEQ ID N°3), WWXXWF (SEQ ID N°4) and WFXXWF (SEQ ID N°5).
  • the invention relates to a compound comprising a cell-penetrating peptide motif, wherein said cell-penetrating peptide consists of an amino acid sequence chosen in the group consisting of: WWXXWW (SEQ ID N°2), WFXXWW (SEQ ID N°3), WWXXWF (SEQ ID N°4) and WFXXWF (SEQ ID N°5).
  • polypeptide polypeptide
  • peptide protein
  • domain refers to parts of the peptide structure constitutive of an entity exhibiting a particular characteristic.
  • a peptide comprising a cell-penetrating motif according to the present invention might also be referred as "PEPCOV”.
  • cell-penetrating peptide motif refers to a short peptide, for example comprising from 5 to 50 amino acids, which can readily cross biological membranes and is capable of facilitating the cellular uptake of various molecular cargos, in vitro and/or in vivo.
  • the "cell-penetrating peptide motif comprises a short polycationic or amphiphilic peptide.
  • the terms "cell-penetrating motif, "self cell-penetrating domain”, “cell-permeable peptide”, “protein-transduction domain”, and “peptide carrier” are equivalent.
  • the ability of a cell-penetrating peptide motif according to the invention to facilitate the intracellular uptake may be assessed, for example, by overlaying said peptide and its "molecular cargo" onto cells cultured to subconfluency, for example for 1 hour at 37°C.
  • Methods for testing the ability of a peptide non-covalently linked to a molecular cargo to facilitate the intracellular uptake of said "molecular cargo” are described in Kurzawa et al. (2010).
  • a person skilled in the art may easily adapt said protocol to assess the delivering capacities of a cell-penetrating peptide motif according to the present invention.
  • the cell-penetrating peptide motif of a compound according to the invention is capable of facilitating cellular uptake of peptides of more than 5 amino- acids.
  • the cell-penetrating peptide motif of a compound according to the invention is capable of facilitating cellular uptake of a molecular cargo up to 500 kDa.
  • the inventors have found that the presence, within the amino acid sequence of a peptide, and from the N-terminal to the C-terminal extremity of said sequence, of two hydrophobic motifs, each of these hydrophobic motif comprising two hydrophobic amino acids being possibly two Tryptophan residues (WW) and/or a Tryptophan and a Phenylalanine (WF) residue, with said two motifs being separated by two amino acids, are associated with an ability for the peptide to cross the cellular membrane and to be detected within the cell. From these observations, the inventors have defined the consensus sequence: WW/FXXWW/F (SEQ ID N°l).
  • the present invention relates to a compound comprising a peptide comprising at least two Tryptophan residue, preferably three Tryptophan residue and more preferably four Tryptophane residue.
  • the present invention relates to a compound comprising a peptide comprising at least one cell-penetrating peptide motif having the consensus sequence: WW/FXXWW/F (SEQ ID N°l).
  • a compound of the invention may be prepared to allow its direct use in vitro, in cell extracts, in a cell, a cell culture, including tissue culture, or on animal and/or human tissues, originating for example from biopsies, or in living animal models.
  • a compound according to the invention allows the delivery of a molecular cargo into a cell, more particularly a eukaryotic cell, and even more particularly an animal cell, including a human cell.
  • a compound according to the invention allows the delivery of a molecular cargo into a healthy or non-healthy cell, and even more particularly it allows the delivery of an agent into a cancer cell.
  • a compound according to the invention allows the delivery of a molecular cargo to the nucleus, to the mitochondrial compartment, to the plasma membrane or to any other organelle.
  • a compound according to the invention comprises a peptide comprising a cell-penetrating peptide motif from 5 to 50 amino acids in length, preferably from 5 to 25 amino acids and more preferably from 6 to 13 amino acids. In a more particular embodiment, a compound according to the invention comprises a peptide comprising a cell-penetrating motif comprising 13 amino acids. In another particular embodiment, a compound according to the invention comprises a peptide comprising a cell-penetrating motif comprising 6 amino acids. In a more particular embodiment, a compound according to the invention comprises 6, 7, 8, 9, 10, 11, 12 or 13 amino acids. In an even more particular embodiment, a compound according to the invention consists of 6, 7, 8, 9, 10, 11, 12 or 13 amino acids.
  • the invention relates to a compound comprising a peptide wherein said cell-penetrating peptide motif comprises an amino acid sequence chosen in the group consisting of: the sequence SEQ ID N°2, the sequence SEQ ID N°3, the sequence SEQ ID N°4 and the sequence SEQ ID N°5.
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acids in position 3 and in position 4 of the sequence of said cell-penetrating motif sequence (SEQ ID N°l) are independently chosen in the group consisting of: K, R, E, D, T and G.
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acids in position 3 and in position 4 of the sequence of said cell-penetrating motif sequence chosen in the group consisting of: SEQ ID N°2, SEQ ID N°3, SEQ ID N°4 and SEQ ID N°5 are independently chosen in the group consisting of: K, R, E, D, T and G.
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acid in position 3 of the sequence of said cell-penetrating motif sequence is Glutamic acid (E).
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acid in position 4 of the sequence of said cell-penetrating motif sequence is Threonine (T).
  • T Threonine
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acids in position 3 and in position 4 of the sequence of said cell-penetrating motif sequence are respectively Glutamic acid (E) and Threonine (T).
  • the invention relates to a compound comprising a cell-penetrating peptide motif wherein the amino acid in position 3 and in position 4 of the sequence of said cell-penetrating motif sequence are Glycine (G).
  • the invention relates to a compound comprising a cell-penetrating peptide motif, wherein said cell-penetrating peptide motif consists of 6, 7, 8, 9 or 10 amino acids and comprises at least an amino acid sequence chosen in the group consisting of: WETWW (SEQ ID N°33), WFETWW (SEQ ID N°34), WETWF (SEQ ID N°35) and WFETWF (SEQ ID N°36).
  • the invention relates to a compound comprising a cell- penetrating peptide motif, wherein said cell-penetrating peptide consists of an amino acid sequence chosen in the group consisting of: WETWW (SEQ ID N°33), WFETWW (SEQ ID N°34), WETWF (SEQ ID N°35) and WFETWF (SEQ ID N°36).
  • the invention relates to a compound comprising a peptide wherein said cell-penetrating peptide motif comprises an amino acid sequence chosen in the group consisting of: the sequence SEQ ID N°7, the sequence SEQ ID N°8, the sequence SEQ ID N°9, the sequence SEQ ID N°10, the sequence SEQ ID N°l 1, the sequence SEQ ID N°12, the sequence SEQ ID N°13, the sequence SEQ ID N°14, the sequence SEQ ID N°15, the sequence SEQ ID N°16 and the sequence SEQ ID N°17.
  • the invention relates to a compound wherein said cell-penetrating peptide motif comprises an amino acid sequence chosen in the group consisting of: the sequence KETWWETWWTEK (SEQ ID N°7), the sequence KETWFETWFTEKK (SEQ ID N°8), the sequence KETWWETWFTEKK (SEQ ID N°9) and the sequence KETWFETWWTEKK (SEQ ID N°10).
  • the invention relates to a compound wherein said cell-penetrating peptide motif has an amino acid sequence chosen in the group consisting of: the sequence KETWWETWWTEKK (SEQ ID N°7), the sequence KETWFETWWTEKK (SEQ ID N°8), the sequence KETWWETWFTEKK (SEQ ID N°9) and the sequence KETWFETWFTEKK (SEQ ID N°10).
  • the invention relates to a compound wherein said cell-penetrating peptide motif comprises the amino acid sequence SEQ ID N°7 (PEP1).
  • the invention relates to a compound wherein said cell- penetrating peptide motif comprises an amino acid sequence having at least 80% identity, and preferably at least 90% identity with a sequence chosen in the group consisting of: SEQ ID N° l to SEQ ID N° 17, said compound comprising the amino acid sequence WW/FXXWW/F (SEQ ID N° 1).
  • the term “identical” means that two peptide sequences, when optimally aligned, share 100 %> sequence identity.
  • identity herein means that two amino acid sequences are identical (i.e. at the amino acid by amino acid basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage of sequence identity of an amino acid sequence can also be calculated using BLAST software with the default or user defined parameter.
  • Single amino acid substitutions, deletions, or insertions can be used, provided that the consensus motif is present. Substitutions may be conservative, regarding certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity), as known by a person skilled in the art biochemistry.
  • the substituting amino acids are not limited to those naturally occurring in proteins, such as L-a-amino acids, or their D- isomers.
  • the peptides can be substituted with a variety of moieties such as amino acid mimetics well known to those of skill in the art.
  • the invention relates to a compound comprising a peptide comprising two or more cell-penetrating peptide motifs according to the invention.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding for at least one cell- penetrating peptide motif having an amino acid sequence chosen in the group consisting of: the sequence SEQ ID N°2, the sequence SEQ ID N°3, the sequence SEQ ID N°4, the sequence SEQ ID N°5, the sequence SEQ ID N°7, the sequence SEQ ID N°8, the sequence SEQ ID N°9, the sequence SEQ ID N°10, the sequence SEQ ID N°l l, the sequence SEQ ID N°12, the sequence SEQ ID N°13, the sequence SEQ ID N°14, the sequence SEQ ID N°15, the sequence SEQ ID N°16 and the sequence SEQ ID N°17.
  • the present invention relates to a nucleic acid vector containing a nucleic acid molecule according to the invention, said vector being preferably an expression vector comprising all means for the expression of polypeptides encoded by nucleic acid molecules.
  • the invention relates to a host cell transformed by a vector according to the invention.
  • said host cell is chosen among eukaryotic cells, such as yeasts, or among prokaryotic cells, such as bacteria, and even more particularly said host cell is a bacteria adapted for the production of heterologous polypeptides, well known by a person skilled in the art.
  • the methods classically used in molecular biology are well known to those skilled in the art and are fully described in the literature (Maniatis T. et al, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y, Edition 1999).
  • the invention relates to a compound wherein said at least one cell-penetrating peptide motif is covalently linked or fused to at least one compound to be delivered into a cell.
  • the terms "compound to be delivered into a cell” and "molecular cargo” refer to a molecule or a macromolecule which can be natural or synthetic, organic or inorganic, and which are chosen in the list consisting of: peptides, lipids, glucids, nucleic acids and macro molecules comprising thereof.
  • the cell-penetrating motif and the compound to be delivered into a cell are associated through covalent bonds, wherein the cell-penetrating peptide motif and the compound to be delivered can be associated in any order respectively to the peptide chain comprising the cell-penetrating peptide motif.
  • the molecular cargo is located at the C-terminal extremity of the peptide chain comprising the at least one cell-penetrating peptide motif.
  • a compound according to the invention is a peptide comprising, from its N-terminal extremity to its C-terminal extremity, a cell-penetrating peptide motif and a molecular cargo.
  • At least one cell-penetrating peptide motif is linked or fused directly to a molecular cargo.
  • the cell-penetrating peptide motif is linked or fused to a molecular cargo via a linker.
  • the invention relates to a compound wherein said molecular cargo is a peptide chosen in the group consisting of: antibodies, enzymes, antigens, receptor ligands, and analogs, fragments and derivatives thereof.
  • said analogs, fragments and derivatives exhibit the functional activity of the original molecule.
  • the invention relates to a compound comprising a cell-penetrating motif and a molecular cargo, wherein said molecular cargo is able to recognize and bind at least one CDK/Cyclin complex.
  • said compound comprises an amino acid sequence chosen in the group consisting of: the sequence SEQ ID N°20, the sequence SEQ ID N°21 , the sequence SEQ ID N°22, the sequence SEQ ID N°23 and the sequence SEQ ID N°24.
  • K D an equilibrium dissociation constant, K D , is commonly used in biological sciences to measure the affinity and to characterize the binding of a molecule for another molecule. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. Typically, a smaller K D means a greater affinity.
  • the specific binding of a compound according to the invention for at least one CDK can be characterized by a K D comprised between about 100 nM and about 500 nM, and preferably of about 150 nM. It is intended herein that the term “about” is equivalent to "+/- 10%".
  • the specific binding of a compound according to the invention for at least one cyclin can be characterized by a K D comprised between about 100 nM and about 500 nM, and preferably of about 150 nM.
  • Table 2 summarizes the description of peptides according to the invention and their amino acid sequences. Amino acids in bold belong to the cell-penetrating peptide motif of amino-acid sequences.
  • the invention relates to a compound wherein said peptide comprises an amino acid sequence chosen in the group consisting of the sequence SEQ ID N°19, the sequence SEQ ID N°20, the sequence SEQ ID N°21, the sequence SEQ ID N°22, the sequence SEQ ID N°23, the sequence SEQ ID N°24, the sequence SEQ ID N°31 and the sequence SEQ ID N°32.
  • the present invention relates to a compound comprising a peptide comprising a cell-penetrating peptide motif, a molecular cargo and a reporter molecule.
  • reporter molecule relates a molecule able to emit a signal or to allow its recovery, and therefore allowing the detection, and possibly the isolation, of a compound to which it is covalently or non-covalently bound.
  • This includes a recoverable label such as a biotinyl moiety that can be recovered by avidin or streptavidin.
  • Recoverable labels can include covalently linked polynucleotide sequences that can be recovered by hybridization to a complementary sequence polynucleotide.
  • labels include, but are not limited to, the following: radioisotopes (e.g., H, C, S, I, I), fluorescent or phosphorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase), biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags). Labels can also be attached by spacer arms of various lengths, e.g., to reduce potential steric hindrance.
  • radioisotopes e.g., H, C, S, I, I
  • fluorescent or phosphorescent labels e.g., FITC, rhodamine,
  • the reporter molecule is a fluorophore.
  • fluorophore or fluorescent probe, it is herein meant a molecule capable of re-emitting light upon light excitation, or other electromagnetic light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed light.
  • a compound according to the invention comprises an environmentally- sensitive dye or couples of fluorescent dyes capable of FRET.
  • solvatochromic dye By environment-sensitive dye, it is herein meant a fluorophore the properties of which change, for example intensity, half-life, and excitation or emission spectra, in a measureable manner upon a change in the fluorophore environment.
  • environment-sensitive dye it is herein meant a fluorophore the intensity or emission spectrum of which changes together with a change in its environment.
  • the change in the fluorophore environment may be due to at least one of a variety of different environmental factors, such as polarity or hydrophobicity. Environment-sensitive dyes have been reviewed in Loving et al. (2010).
  • the invention relates to a compound comprising a polypeptide and a unique fluorophore, wherein said fluorophore is coupled to a unique residue within the amino acid sequence of said polypeptide.
  • the fluorophore is coupled to specific functional groups, for example specific functional groups of amino-acid residues, such as amino, carboxyl, thiol or azide groups. Coupling the fluorophore to an amino acid functional group is a technique well known to the skilled person.
  • the invention relates to a compound according to the invention comprising at least one molecular cargo and at least one detectable moiety.
  • a compound according to the invention bears at least one site for coupling to a probe.
  • a compound according to the invention bears a unique cysteine for coupling of a fluorescent probe.
  • a compound according to the invention comprises at least one cell-penetrating peptide motif, a compound to be delivered into a cell and at least one fluorophore, wherein said at least one fluorophore being coupled to an amino acid of said compound to be delivered.
  • the present invention relates to a compound comprising an amino acid sequence SEQ ID N°20 (PEP1-CDKQUANT1) and a fluorophore.
  • a compound according to the present invention comprises a peptide having an amino acid sequence SEQ ID N°20 (PEP1-CDKQUANT1) and a fluorophore FITC which is coupled to the unique cysteine residue of the peptide.
  • the present invention relates to a compound comprising an amino acid sequence SEQ ID N°20 (PEP1-CDKQUANT1) and a fluorophore.
  • a compound according to the present invention comprises a peptide having an amino acid sequence SEQ ID N°20 (PEP1-CDKQUANT1) and a fluorophore Cy3 which is coupled to the unique cysteine residue of the peptide.
  • the invention relates to a compound comprising at least one cell-penetrating peptide motif and a targeting motif.
  • targeting motif is intended a motif designed to target a defined compartment or molecule within the cell.
  • said targeting motif could allow the targeting to a subcellular or extracellular compartment, or to a cell surface receptor or an antigen.
  • a motif designed to target a defined compartment or molecule within the cell comprises, is able to bind specifically to a ligand, being in particular a receptor or an antigen, within the cell.
  • the invention relates to a compound comprising at least one cell-penetrating peptide motif and a protein tag.
  • a motif defined as a "peptide-Tag” or a "protein-Tag” (or tag) may be added, for example, to allow an easier purification of a protein to which it is linked, as an example, a polypeptide according to the invention may comprise a Glutathione-S-Transferase (GST) sequence.
  • GST Glutathione-S-Transferase
  • Protein Tags are well known by the skilled person and may for example be chosen in the list consisting of Isopeptag, BCCP, Myc-tag, Calmodulin-tag, FLAG-tag, HA-tag, His-tag, Maltose binding protein-tag, Nus-tag, Glutathione-S-transferase-tag, Green Fluorescent Protein-tag, Red Fluorescent Protein (RFP) tag and other genetically encoded auto fluorescent proteins, Thioredoxin-tag, S-tag, Softag 1, Softag 3, Strep-tag, SBP-tag, Ty tag, V5 tag or TC tag.
  • a compound according to the invention may comprise an "mRFP sequence" which provides an intramolecular protein fluorophore which allows to visualize cellular internalization through fluorescence microscopy or by FACS.
  • the mRFP or any similar auto fluorescent protein may also serve as a scaffold as well as an inert intramolecular fluorophore linked or fused to another molecular cargo of interest, or to a biosensor which bears an environmentally sensitive probe. This is of particular interest for imaging applications and high content screening where the RFP may be employed for standardized ratiometric quantification .
  • the invention relates to a compound comprising at least one cell-penetrating peptide motif, a targeting motif and a protein tag.
  • the compound of the invention may have such attributes as being non-hydrolyzable, thereby increasing the stability against proteases or other physiological conditions which degrade the corresponding peptide.
  • peptide analogs can be generated using benzodiazepines, substituted ⁇ - lactam rings, C7 mimics, ⁇ -turn dipeptides cores, ⁇ -aminoalocohols, diaminoketones, and methylene amino-modified.
  • surrogates of the amide bond including in the group of trans-olefms, fluoroalkylene, methyleneamino, phosphonamides or sulfonamides can be used in order to increase the half-life of the polypeptide.
  • the invention in another aspect, relates to a process for preparing a compound comprising at least a cell-penetrating peptide motif of the invention.
  • a compound of the invention may be obtained by standard methods known in the art.
  • a compound according to the invention is prepared by chemical synthesis, such as solid-phase peptide synthesis, without being limited to this particular method.
  • a compound according to the invention may be chemically synthesized as a whole or as parts which are subsequently covalently linked or coupled.
  • a compound according to the invention is prepared by recombinant technology.
  • a compound according to the invention may be partly produced by recombinant technology, then covalently coupled to another part of the compound.
  • a nucleic acid encoding for at least one polypeptide comprising a cell- penetrating motif is used for transforming a suitable host, then, by assessing suitable culture conditions, performing the production of a recombinant polypeptide comprising at least a cell-penetrating motif. Said polypeptide may then be coupled by covalent link to a compound to be delivered into a cell.
  • a compound according to the invention may optionally be prepared as a fusion protein by recombinant technology, using a nucleic acid molecule encoding for, at least, a polypeptide comprising a cell-penetrating motif and optionally for a molecular cargo to be delivered into a cell.
  • polypeptides associated with, for example, an mRFP or a GST sequence may be prepared by genetic engineering, as fusion proteins.
  • Production of the polypeptide according to the invention may be performed for example in expression systems derived from bacteria, yeast, baculovirus, insect, and mammalian cells, or in cell- free expression systems, reviewed in Higgins et al. (1999), Baneyx et al. (2004) and in Atherton et al. (1989).
  • a chemical synthesis or a recombinant preparation process of a compound according to the invention includes all needed purification steps.
  • the invention relates to a process for preparing a compound according to the invention, said process comprising the steps of:
  • the invention relates to a process for preparing a compound according to the invention, said process comprising the steps of: a) providing a nucleic acid comprising at least a nucleic acid sequence encoding for a cell-penetrating peptide motif having a sequence chosen in the group consisting of: the sequence SEQ ID N°2, the sequence SEQ ID N°3, the sequence SEQ ID N°4, the sequence SEQ ID N°5, the sequence SEQ ID N°7, the sequence SEQ ID N°8, the sequence SEQ ID N°9, the sequence SEQ ID N°10, the sequence SEQ ID N°l l, the sequence SEQ ID N°12, the sequence SEQ ID N°13, the sequence SEQ ID N°14, the sequence SEQ ID N°15, the sequence SEQ ID N°16 and the sequence SEQ ID N°17, and a nucleic acid sequence encoding for a compound to be delivered into a cell,
  • the present invention also relates to a compound such as directly obtained by a process according to the invention.
  • the invention relates to a composition
  • a composition comprising at least one compound according to the invention and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency or listed in a generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the compound of the invention may be solubilized in a buffer or water or incorporated in emulsions and microemulsions.
  • Suitable buffers include, but are not limited to, phosphate buffered saline Ca++/Mg++ free (PBS), phosphate buffered saline (PBS), normal saline (150 mM NaCl in water), Tris buffer and surfactants.
  • composition according to the invention further comprises stabilizers.
  • Stabilizers according to the invention include cyclodextrine and derivatives thereof (see for reference US5730969). Suitable preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran and glycerin can also be added to stabilize the final formulation. A stabilizer selected from ionic and non-ionic surfactants, D-glucose, D- galactose, D-xylose, D-galacturonic acid, trehalose, dextrans, hydroxyethyl starches, and mixtures thereof may be added to the formulation. Addition of alkali metal salt or magnesium chloride may stabilize the compound according to the invention.
  • the peptide may also be stabilized by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulphuric acid, starch, glycogen, dextrin, and alginic acid salt.
  • a saccharide selected from the group consisting of dextran, chondroitin sulphuric acid, starch, glycogen, dextrin, and alginic acid salt.
  • Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (E.g., glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol).
  • Polyols may stabilize a peptide, and are water- miscible or water-soluble.
  • Suitable polyols may be polyhydroxy alcohols, monosaccharides and disaccharides including mannitol, glycrol, ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and polymers thereof.
  • Various excipients may also stabilize peptides, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinyl pyrrolidone, hydrolysed gelatin, and ammonium sulfate.
  • composition of the invention may be formulated according to standard pharmaceutical practice.
  • the composition may be formulated in a form suitable for oral, enteral or parenteral administration, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, respiratory and topical routes of administration,
  • Ways to penetrate the cellular membrane may involve specific formulations of the compound according to the invention, preferentially formulations suitable for administration to cells or animal and/or human tissues.
  • the compound of the invention may be encapsulated in liposomes to form pharmaceutical preparations suitable for administration to cells and animal and/or human tissues.
  • lipid aggregates may be used to formulate the compound of the invention.
  • Such aggregates include liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, having particle sizes in the nanometer to micrometer range. Methods of making lipid aggregates are by now well-known in the art.
  • composition of the present invention is for in vivo diagnosis or for in vivo medical imaging.
  • the invention relates to a method for in vitro delivering a molecular cargo into a cell of a sample, comprising the steps of:
  • contacting the compound of the invention with a cell in a sample is performed for example by contacting the compound of the invention directly with a sample.
  • a sample might be living cells, preferably living cells in an in vitro culture, animal tissues, preferably animal tissues in an in vitro culture or in mouse models with tumour xenografts.
  • cells from the sample might be living or fixed on a support.
  • Contacting said compound with said cell is performed in conditions allowing the delivery of the compound into the cell.
  • the invention relates to a method for detecting the presence of an intracellular compound in a sample, said method comprising the steps of:
  • step d) comparing the signal of step c) with a reference signal
  • step e) determining from the comparison of step d) if said intracellular product is present in said sample.
  • the reference signal is a predetermined measure obtained from a biological sample with a known presence and/or known quantification of the intracellular element to be detected. This presence may be determined by conventional techniques known by the skilled person.
  • the invention relates to a method for detecting the presence of an intracellular compound in a sample wherein said reporter molecule is a fluorescent probe. More preferably, said fluorescent probe is coupled to an amino acid of the peptide which is not an amino acid of the cell-penetrating peptide motif. Even more preferably, said fluorescent probe is coupled to the molecular cargo, and in particular is coupled to a unique site on the molecular cargo.
  • the invention relates to a method for determining the relative quantity of an intracellular compound in at least two different samples, said method comprising the steps of:
  • step d) comparing the fluorescent signal of step d) with a reference fluorescent signal
  • step f) determining from the comparison of step e) the relative quantity of said intracellular product is said at least two different samples.
  • determining the fluorescence in step d) can be achieved by any technique and using any appropriate apparatus known in the art. Any device adapted to measure the properties of emitted light, preferably fluorescence light may be used to determine the fluorescence of step d). The skilled person will easily adapt the intensity and wavelength of the excitation light of step c). As used herein, “determining the fluorescence emitted” refers to measuring the properties of the emitted fluorescence, such as for example measuring the wavelength spectrum, intensity or half-life of the emitted fluorescence.
  • comparing the fluorescence in step e) means comparing the properties of the emitted fluorescence of step d) and the properties of the fluorescence reference, said fluorescence reference being a predetermined measurement of fluorescence obtained from a reference biological sample.
  • a fluorescently labeled compound according to the invention emits a fluorescence that changes, for example in intensity or wavelength, depending on binding of the compound to its target.
  • the invention relates to a method for selecting a molecule able to affect the presence and/or the level of an intracellular compound within a sample, said method comprising the steps of:
  • step d) comparing the signal of step c) with a reference signal, e) selecting, from the comparison of step d), a molecule able to affect the presence and/or the level of said intracellular compound.
  • said molecule may belong to a library of molecules.
  • a method for selecting a molecule comprises the detection of the presence and/or the level of an intracellular compound and may be performed in the context of a high throughput and/or high content screening of a library of molecules.
  • the reference signal is a predetermined measure obtained from a biological sample with a known presence and/or known quantification of the intracellular element to be detected.
  • Said reference signal may be a signal obtained in the presence of a molecule with a known ability to affect the presence and/or the level of said intracellular compound. This presence may be determined by conventional techniques known by the skilled person.
  • the invention relates to a method for selecting a molecule able to affect the activity of an intracellular compound within a sample, said method comprising the steps of:
  • step d) comparing the signal of step c) with a reference signal
  • step d selecting, from the comparison of step d), a molecule able to affect the activity and/or the level of said intracellular compound.
  • test molecule may belong to a library of molecules.
  • a method for selecting a molecule according to the invention comprises the detection of the activity of an intracellular compound and may be performed in the context of a high throughput and/or high content screening of a library of molecules.
  • the invention relates to a method for inhibiting the growth of a cell of a sample, said method comprising the steps of:
  • said method comprises the measuring of cell growth and the detection of growth inhibition, in particular the inhibition of cell-proliferation and of signaling pathways.
  • the invention in another aspect, relates to a method for detecting the presence of an intracellular product in an organ.
  • determining the “relative quantity”, the “level”, the “amount”, the “level of expression” or the “concentration” of an intracellular product it is meant that the intensity of the signal emitted by the reporter molecule is compared between at least two samples, and the relative difference between the signals emitted is expressed as a percentage.
  • the invention relates to a method for detecting the level of an intracellular product in living or fixed cells.
  • the invention relates to a process wherein a compound according to the invention is contacted with a cell which is present within a tissue or an organ.
  • the invention also relates to a method for medical imaging, especially endoscopic imaging, said method comprising the steps of:
  • subject and “host” are used herein interchangeably and refer to any subject for whom diagnosis is desired, particularly humans.
  • Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like.
  • the subject is a human.
  • biological sample refers to biological material from a subject.
  • the sample assayed by the present invention is not limited to any particular type. Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supematants and/or extracts of any of the foregoing.
  • tissue removed for biopsy examples include tissue removed for biopsy, tissue removed during resection, blood, serum, plasma, sputum, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, skin, saliva, gastric secretions, semen, seminal fluid, tears, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal central nervous system tissue and stool samples.
  • sample used will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
  • the present invention relates to the use of a compound according to the invention for the intracellular delivery of a molecular cargo.
  • the present invention relates to the use of compounds according to the invention for detecting an intracellular compound in a sample.
  • the invention relates to the use of a compound according to the invention for in vitro diagnostic of a disease characterized by the altered presence of the level of an intracellular ligand of said compound.
  • the invention relates to the use of a compound according to the invention for medical imaging.
  • the invention relates to the use of a compound according to the invention for high throughput and/or high content screening of libraries of compounds within cell samples.
  • the invention relates to the use of a compound according to the invention for modulating a biological function.
  • the invention relates to the use of a compound according to the invention for activating, or enhancing, a biological function.
  • the invention relates to the use of a compound according to the invention for stopping or inhibiting a biological function.
  • the invention relates to the use of a compound according to the invention for determining the relative quantity of at least one intracellular component in at least two different samples.
  • the invention relates to the use of a compound according to the invention for screening a plurality of compounds for their ability to affect the presence and/or the quantity of a given a intracellular component, or to affect a given biological function.
  • the invention relates to the use of a compound according to the invention for the in vitro diagnosis of a condition characterized by the presence or the absence of an intracellular component, or by the level of expression of a given intracellular component.
  • the invention relates to the use of a compound according to the invention for the monitoring of the efficacy of a therapeutic treatment.
  • the invention also relates to the use of at least one compound and/or a composition of the invention for medical imaging.
  • the invention also relates to the use of a compound and/or a composition of the invention for medical imaging, preferably endoscopic imaging.
  • the invention also relates to the use of at least one compound and/or a composition of the invention for in vitro imaging.
  • a compound according to the invention will be used in an effective amount, according to the nature of said use.
  • the term "effective amount" of a composition means the amount which is sufficient to allow for measurement of the fluorescence in the subject, particularly. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, the nature of the disease or condition being investigated, and the nature of the effect desired. The effective amount can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
  • kits of parts comprising at least one compound according to the invention and an acceptable solvent.
  • a kit according to the invention comprises at least one compound according to the invention and at least one component chosen among the group consisting of: buffer, positive control, phospholipids, cells to be trans fected and instructions for use.
  • a kit according to the invention comprises a compound provided either in an aqueous or a lyophilized stock.
  • FIGS. 1A to ID Internalization of C4 peptide into HeLa cells.
  • the C4 peptide is an inhibitor of CDK2/Cyclin A that blocks cancer cells proliferation (Gondeau et ah, 2005).
  • Figure 1A shows the internalization of TAT-C4 biosensor (SEQ ID N°26) into HeLa cells.
  • Figure IB shows the internalization of PEP1-C4 biosensor (SEQ ID N°19) into HeLa cells.
  • Figure 1C shows the internalization of PEP2b-C4 biosensor (SEQ ID N°31) into HeLa cells.
  • Figure ID shows the internalization of PEP3b-C4 biosensor (SEQ ID N°32) into HeLa cells.
  • Peptides labeled with FITC were directly applied onto cultured HeLa cells, they penetrate readily and distribute homogeneously throughout the cytoplasm within less than 1 hour.
  • FIGS. 2A to 2D Internalization of PEP1-CDKQUANT into HeLa cells.
  • Figure 2B represents the internalization of CDKSENS1 (SEQ ID N°29) into Hela cells when CDKSENS1 is labeled with Cy3 and is non covalently associated with CADY2 peptide (SEQ ID N°28).
  • Figure 2D represents the internalization of PEP1- CDKQUANT1 (SEQ ID N°20) into Hela Cells, wherein PEP1-CDKQUANT1 is labeled with Cy3. Peptides were directly applied onto cultured HeLa cells.
  • Figures 2 A and 2C represent the respective Hoechst staining of Hela cells (control staining).
  • Figures 3 A to 3D Quantification of CDK/Cyclin levels in HeLa and HS68 cells through ratiometric quantification of PEP1-CDKQUANT1-Cy3/Ctrl-Cy5 fluorescence versus CADY2 formulations of CDKSENS1-Cy3/Ctrl-Cy5
  • the histograms represent the comparison of the fluorescence ratio of PEP1-CDKQUANT-Cy3 (SEQ ID N°20) /PEPl-control-Cy5 (SEQ ID N°25) in HeLa (left histogram) and in HS68 fibroblasts (right histogram), after normalization of cell extracts.
  • the histograms represent the comparison of fluorescence ratio CADY2- CDKSENS1-Cy3 (SEQ ID N°29) /control-Cy5 (SEQ ID N°30) in HeLa (left histogram) and in HS68 fibroblasts (right histogram).
  • Figure 3C shows a schematic representation of PEP1-CDKQUANT1.
  • Figure 3D shows a schematic representation of the non-covalent formulation of CDKSENS1/ CADY2.
  • Figure 4 Biodistribution of PEPl-CDKQUANTl-CyS in mouse tissues and organs
  • Figure 4 is a histogram representation of the distribution of PEP1-CDKQUANT1 through different tissues and organs, for each organ, respectively, after intratracheal/nebullisation (left column), intravenous (central column) and intraperitoneal (right column) administration.
  • Figures 5A and 5B Ratiometric quantification of PEP1-CDKQUANT-Cy5/Ctrl- Alexa750 in tumour xenografts treated or not with si RNA targeting cyclin B.
  • Figure 5 A represents the imaging of a mouse bearing a tumour and treated with PEP1- CDKQUANT 1 -Cy5 and PEP 1 -Ctrl- Alexa750.
  • Figure 5B represents a histogram showing the differences in the fluorescence intensity ratio of PEP1/CDKQUANT1-Cy5 / PEP1-Ctrl-Alexa755 measured in ten mice treated with siRNA targeting cyclin B (left histogram) and ten mock-treated mice (right histogram).
  • Figures 6A to 6D Application of PEP1-CDKQUANT1 to high content / high throughput screening.
  • Figure 6A Schematic representation of PEP1-CDKQUANT expressed as a fusion protein with mRFP, so that an intramolecular fluorescence ratio could be used for quantification of CDK/Cyclin levels.
  • Figure 6B Internalization of PEPl-RFP- CDKQUANT-Cy5 in HeLa cells (left panel) and in HS68 (right panel).
  • Figure 6C Example of multiparametric high content screen of a small chemical compound library to identify compounds that affect CDK/Cyclin levels. The higher panel represents cytotoxicity, the middle panel represents PEP1-CDKQUANT fluorescence and the lower panel represents effect on cell cycle progression.
  • Figure 6D Histogram representation of the fluorescence distribution profiles of PEP1-CDKQUANT-Cy5 fluorescence/RFP from a field of 2000 HeLa cells either mock treated or treated with 50uM roscovitine for 7h.
  • PEP1-C4 biosensor bears a unique cysteine for coupling of an environmentally sensitive fluorescent probe.
  • PEP1-C4 compound was synthesized by solid-phase Fmoc strategy.
  • PEP1-C4 was labeled on its unique cysteine with fluoresceinisothiocyanate (FITC) and further purified on NAP-5 columns (GE Healthcare).
  • FITC fluoresceinisothiocyanate
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS Fetal Calf Serum
  • Cell extracts were prepared in lysis buffer containing 50 mM TrisHCl, pH 7.4, 150 mM NaCl, 0.1% NP40, 0.1% Deoxycholate, 2 mM EDTA, 1 mM phenylmethlsulfonyl fluoride (PMSF), CompleteTM protease inhibitors (Roche), 50 mM NaF, 40 mM ⁇ -Glycero-phosphate, 1 mM Na3V04 and normalized following spectrophometric dosage at 280nm.
  • Example 2 Detection and quantification of CDK/cyclin level in different cell lines using PEP1-CDKQUANT1-Cy3 sensor.
  • Antibodies against Cyclin A H432, sc-751), Cyclin Bl (GNS1, sc-245), Cyclin Dl (C20, sc-717), Cdkl (CI 9, sc-954) Cdk2 (M2, sc-163), and Cdk4 (C22, sc-260) were purchased from Tebu-Bio (Santa-Cruz), anti-actin from Sigma (A2668), and used at 1 : 1000 dilution for Western blotting, except for anti-cyclin Bl at 1 :500 dilution, 1 : 100 for indirect imuno fluorescence. Secondary antibodies labelled with Alexa-488 were used for indirect immunofluorescence.
  • 3uM PEP1-CDKQUANT1-Cy3 and Ctrl Peptide-Cy5 were overlaid onto cells for 1 hour then imaged by fluorescence microscopy. Live-cell imaging acquisitions were substracted for background signal corresponding to minimal fluorescence levels using Metamorph. Image J was then used for analysis and quantification of fluorescence values, as described previously (Kurzawa et al., 2010).
  • PEP1-CDKQUANT1-Cy3 was applied to probe CDK/cyclin levels in living cells through ratiometric quantification of its fluorescence over that of a control peptide which is equally capable of penetrating cells without a carrier but which does not bear any CDK or cyclin-binding sequence and labeled with Cy5. Both self-penetrating peptides (PEP1-CDKQUANT1 and PEP- 1 -CTRL) were overlaid onto cells, and live- cell imaging of cells was performed to acquire PEP1-CDKQUANT1-Cy3 and PEP1- Ctrl-Cy5 fluorescence.
  • Ratiometric quantification strategy provides a means of standardizing PEP1-CDKQUANT1-Cy3 fluorescence with respect to the CTRL-Cy5 peptide ( Figure 3B). Ratiometric quantification experiments were performed between normal diploid fibroblasts HS68 and HeLa cells. 45% difference in the PEP1- CDKQUANT1-Cy3/CTRL-Cy5 ratio was determined between HeLa cells and HS68 fibroblasts.
  • CADY2 and CDKSENS1-Cy3, or CADY2 and control peptide-Cy5 were overlaid onto Hela and HS68 cells, and live-cell imaging of cells was performed to acquire Cy3 and Cy5 fluorescence.
  • Established ratiometric quantification strategy provides a means of standardizing Cy3 fluorescence with respect to Cy5 fluorescence ( Figure 3 A). Ratiometric quantification experiments were performed between normal diploid fibroblasts HS68 and HeLa cells. 15% difference in the CADY2/CDKSENS- Cy3/CTRL-Cy5 ratio was determined between HeLa cells and HS68 fibroblasts.
  • PEP1-CDKQUANT1 (SEQ ID N°20) compound was synthesized by solid-phase Fmoc strategy.
  • PEP1-CDKQUANT1 was labeled on its unique cysteine with Cy3 and further purified on NAP-5 columns (GE Healthcare).
  • PEP1-CDKQUANT1 labeled with Cy3 was injected into mice xenografted with tumoral cells and its biodistribution and clearance were characterized over time ( Figure 4).
  • PEP1-CDKQUANT1 biosensors can be applied in vivo, and observed to distribute throughout different tissues and organs, including tumors, following intravenous, intraperitoneal or intratracheal administration.
  • Example 4 Monitoring of a response to siRNA treatment using PEP1- CDKQUANT1 biosensor
  • siRNA targeting Cyclin Bl was a Smart Pool TM M003206-02 purchased from Dharmacon. siRNA transfections were performed for 72h with the cell-penetrating siRNA carrier CADY as described in Crombez et al (2009).
  • mice xenografted with tumoral cells were exposed to siRNA-mediated knockdown of cyclin Bl in tumour xenografts in mice.
  • PEP1-CDKQUANT1-Cy5 and PEPl-Ctrl- Alexa750 were administered in vivo in mice, by intraveinous, intraperitoneal and intratracheal administration.
  • CDK/Cyclin relative abundance in mice treated with siRNA targeting cyclin B compared to mock-treated mice was assessed through quantification of PEP1-CDKQUANT1-Cy5 / PEP 1 -Ctrl- Alexa750 fluorescence by in vivo fluorescence imaging ( Figures 5A and 5B).

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Abstract

La présente invention concerne de nouveaux motifs peptidiques pénétrant dans les cellules qui permettent l'administration intracellulaire de produits auxquels ils sont liés de façon covalente ou fusionnés. L'invention concerne également des composés comprenant au moins l'un desdits motifs peptidiques pénétrant dans les cellules. La présente invention concerne également un procédé d'administration intracellulaire de composés et de ciblage de fonctions biologiques. La présente invention concerne en outre l'utilisation desdits motifs peptidiques pénétrant dans les cellules pour l'administration intracellulaire de produits, en particulier pour la détection de composés intracellulaires, pour interférer avec une fonction biologique, pour l'imagerie cellulaire et pour le criblage de banques de composés à administrer dans une cellule.
PCT/EP2014/055633 2013-03-20 2014-03-20 Nouveaux motifs peptidiques pénétrant dans les cellules WO2014147193A1 (fr)

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WO2018156892A1 (fr) 2017-02-23 2018-08-30 Adrx, Inc. Inhibiteurs peptidiques de l'agrégation du facteur de transcription
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WO2018226992A1 (fr) 2017-06-07 2018-12-13 Adrx, Inc. Inhibiteur d'agrégation de tau
WO2019036725A2 (fr) 2017-08-18 2019-02-21 Adrx, Inc. Inhibiteurs peptidiques d'agrégation de tau
JP2019043946A (ja) * 2017-08-31 2019-03-22 国立大学法人埼玉大学 分子精製用リガンド、分子精製用タグペプチド及びこれらを用いた分子精製方法

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