Patent Grant
9439975
The invention relates to small cell penetrating peptides (CPP) derived from the scorpion toxin maurocalcine and to their use as vectors for the intracellular delivery of various drugs and agents.
Cell-penetrating peptides (CPP), also called protein transduction domains (PTDs), membrane translocation sequences (MTS) or translocating peptides are capable of crossing the plasma membrane of cells and of carrying cell-impermeable compounds across the plasma membrane, efficiently, rapidly, at low concentration, in vitro and in vivo, into various cell types. The 60 amino-acid long homeodomain of the Drosophila transcription factor Antennapedia (ANTP) was the first CPP discovered and shown to serve as a signal for the internalization of other polypeptides. Its penetration and translocation properties were further restricted to a peptide of 16 residues (penetratine or Pen). Now along with Pen, multiple CPPs including protein transduction domains (PTD) found in proteins such as HIV-1 Tat and HSV-1 VP22, synthetic 7-9 homoarginine peptides and chimera peptides such as transportan, are intensively studied. Little structural resemblances have been found between the different families of CPP. The only characteristic common to all these peptides is that they are unusually enriched in basic amino acids resulting in a high positive net charge. Various molecules or particles of different sizes such as oligonucleotides, peptide nucleic acids (PNAs), siRNAs, cDNA, plasmids, peptides, proteins, antibodies, pharmacologicaly active drugs, imaging agents, liposomes and nanoparticles have been successfully delivered into cells when attached to a CPP. Thus, CPPs represent powerful tools for the delivery of various cargoes to their site of action in a cell, in particular the cytosol and the nucleus. These peptides have opened a new avenue in medicine and research, allowing otherwise impermeable agents of therapeutic, diagnostic and technical value to enter cells and induce biological responses.
Maurocalcine (MCa) is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. The toxin is a 33-mer peptide (SEQ ID NO: 1) that was initially isolated from the venom of a Tunisian chactid scorpion, Scorpio maurus palmatus (Fajloun et al., 2000). Maurocalcine belongs to a family of peptide that folds according to an Inhibitor Cystine Knot (ICK motif), and thus contains three disulfide bridges with a Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 connecting pattern (Moshbah et al., 2000). The solution structure, as defined by 1H-NMR, illustrates that MCa contains three β-strands (strand 1 from amino acid residues 9 to 11, strand 2 from 20 to 23, and strand 3 from 30 to 33). One distinctiveness of MCa is the fact that it is greatly enriched in basic amino acid residues. Out of the 33 amino acids that compose MCa, twelve of them are basic, most of them represented by Lys residues. Interestingly, the β-strands of MCa encompass most of the basic domains (
First, it is an exquisite pharmacological activator of the ryanodine receptor type 1 (RyR1) from skeletal muscle since it promotes high Po gating modes and long-lasting subconductance states of the ion channel (Chen et al., 2003; Lukacs et al., 2008). On myotubes, application of MCa rapidly induces Ca2+ release from the sarcoplasmic reticulum (SR) (Estève et al., 2003), a result further confirmed by positive effect of MCa on the release of Ca2+ from purified SR vesicles (Chen et al., 2003; Estève et al., 2003). The interaction of MCa with RyR1 has been witnessed by increased [3H]-ryanodine binding onto purified RyR1 (Chen et al., 2003; Estève et al., 2003). The binding site for MCa on RyR1 has also been mapped and shown to correspond to domain(s) that have a predicted localization within the cytoplasm (Altafaj et al., 2005).
Second, MCa has a unique sequence homology with the II-III loop of the L-type calcium channel Cavil subunit over a domain that is slightly larger than the second β-strand of MCa (
Third, MCa has been shown to act as a cell penetrating peptide (CPP) (Estève et al., 2005; International PCT Application WO 2006/051224 and corresponding US Patent Application US 2009/0142266). This discovery stemmed from earlier criticisms that MCa may not be an activator of RyR1 because peptide toxins were not known to cross the plasma membrane, which would be required here to bind to RyR1. Studies that were undertaken to demonstrate the ability of MCa to reach its target showed that i) MCa triggers Ca2+ release from the sarcoplasmic reticulum a few seconds after its application in the extracellular medium (Estève et al., 2003), and ii) intracellular accumulation of fluorescent-streptavidine occurs if it incubated first with biotinylated MCa (Estève et al., 2005; International PCT Application WO 2006/0512249 and corresponding US Patent Application US 2009/0142266). Since these pioneering studies, MCa or full-length 33 amino acid analogues thereof proved powerful vectors for the cell entry of proteins, peptides (Ram et al., 2008), nanoparticles, or drugs such as doxorubicine (Aroui et al., Cancer Lett., 2009; Aroui et al., Apoptosis, 2009; Aroui et al., Pharm. Res., 2009). Although the mode of cell penetration of MCa may vary according to cargo nature, cell type or chemical linkage employed, the data gathered so far suggest that the peptide may enter cells according to two priming steps onto the plasma membrane: first an interaction with proteoglycans with an affinity in the micromolar range, followed by a second interaction with negatively charged lipids which occurs with greater affinity (Boisseau et al., 2006; Ram et al., 2008). The mode of cell entry of MCa is not altered by the absence of proteoglycans, but simply reduced quantitatively, suggesting that proteoglycans do not orient the mode of cell penetration. Two modes seem to concur to MCa cell entry, as far as observed, one related to macropinocytosis and another to membrane translocation. The balance between both modes of entry was found correlated to cargo nature and the type of MCa analogue used.
It is of great interest to pursue the study of MCa as CPP in spite of the wealth of new CPP sequences that are discovered yearly. Among the competitive advantage of MCa over other CPP sequences are the facts that it has almost no associated toxicity in vitro and in vivo, penetrates into cells at very low concentrations, and is extremely stable in vivo upon intravenous injection (over 24 hrs). While MCa appears as an elaborate and efficient CPP, its pharmacological properties represent a serious hindrance while envisioning in vitro and in vivo applications. In addition, because of its length (33 amino acid residues) and the presence of three internal disulfide bridges, MCa is a relatively difficult to synthesize CPP, comparatively to other CPP. Several attempts were made in the past to try to design MCa analogues which are less complex than MCa, lack the pharmacological effects of wild-type MCa but preserve or enhance its cell penetration efficiencies.
The first strategy, based on single point mutations spanning MCa sequence, preserved the disulfide bridges and the 3D structure of the analogues. The study of the MCa mutants demonstrated that the molecular determinant of MCa implicated in pharmacology and cell penetration overlap partially. In addition, this work confirmed that the main requirement for an efficient cell penetration of MCa is the presence of a basic surface. Many of the amino acids involved in RyR1 binding and pharmacology were located within the cluster of basic amino acids that presented sequence homology with the L-type Cav1.1 channel. Some of these residues, but not all, were also important for cell penetration properties. Using this approach, full-length MCa analogues with reduced or complete loss of pharmacological effects were defined (Mabrouk et al., 2007). Nevertheless, none of the analogues totally preserved the cell penetration efficiency of MCa. One analogue only had lost entirely its pharmacological action (MCa R24A). However, its cell penetration efficiency was decreased. Some other analogues were better that MCa itself for cell penetration. However, their affinity for RyR1 was unchanged or increased (MCa E12A). Combining a pair of mutations, one aiming at disrupting pharmacology and one at improving penetration, may thus be used in the future to define still better CPP analogues derived of full-length MCa.
The second strategy was based on the chemical synthesis of D-MCa, a full-length analogue entirely based on the use of D-amino acids. This peptide is a mirror image of the natural L-MCa but, like other D-CPP, preserves its cell penetration properties, while losing entirely its ability to interac with RyR1 (Poillot et al., 2010). This CPP analogue has several advantages. It no longer is sensitive to proteases which may be an additional advantage for in vivo experiments where the half-life of the circulating peptide matters. In these two strategies while being effective, one may argue that i) the peptides are still among the longest CPP known to date, implying increased costs of production, and ii) the yield of production of these peptides is hampered by the folding process. Also, the use of peptides with internal disulfide bridges, despite having advantageous features in term of stability in vivo, makes chemical coupling of these CPP to cargoes more complicated (difficulty to add extra Cys residues to the peptides for instance without interfering with the correct folding process).
The third strategy that was used to circumvent one of this criticism was the chemical synthesis of a full-length MCa analogue in which all internal Cys residues where replaced by isosteric 2-aminobutyric acid residues (Ram et al., J. Biol. Chem., 2008). The resulting peptide was still 33-mer long but one step in production was saved by avoiding the folding process. In addition, an extra-Cys residue could be added to the N-terminus of the peptide in order to favor simplified cargo grafting on this CPP analogue. This peptide, termed here C-MCaUF1-33 (C for extra-Cys, UF for unfolded, and 1-33 for its length,
The inventors have developed several truncated Mca peptides that have highly potent cell penetration capabilities, while losing pharmacological activity, preserving lack of cell toxicity, and with facilitated cargo grafting. Here, the inventors demonstrate that several efficient CPP can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and a sequence truncation down to peptides of up to 7 residues in length. A surprising finding is that all the truncated maurocalcine analogues possessed cell penetrating properties indicating that the maurocalcine is a highly specialized CPP. Many of the unfolded truncated MCa peptides are better CPP than unfolded MCa itself.
More surprisingly, the inventors have also found out that poorly charged MCa peptides (net positive charge of 0 to +3) can behave as efficient CPP. This is the case for MCaUF1-9, MCaUF3-9, MCaUF1-9(W3), HadUF1-11 and HadUF3-11 which are ones of the best performing CPPs, especially when low concentration of use is a quality of importance.
Interestingly, the truncated MCa peptides differ somewhat in their mode of cell penetration (direct membrane translocation versus endocytosis), some being more prone to enter cells by macropinocytosis than others. Various peptides were even insensitive to amiloride application suggesting that macropinocytosis did not contribute at all to their entry.
The inventors have also demonstrated that cargo coupling can occur at the N-terminus as well as the C-terminus of the peptide, enhancing the flexibility of cargo coupling to these CPPs.
The CPP potential of these peptides can be optimized by reintroducing one disulphide bond to restore some of the secondary structures that confer a competitive advantage to MCa for cell penetration. Further optimization of these CPPs is obtained by mutagenesis of the region Lys11-Ser18 including the mutation of E12 and D15 as well as further mutagenesis of negatively charged residues including D2 and E29.
The inventors have identified several interesting lead CPP based on unfolded MCa (MCaUF) truncation strategy. This is the case for MCaUF18-33 (macropinocytosis entry-independent), MCaUF1-9 (penetrates better at low concentration), and MCaUF14-25 (yields the greatest cell entry of the dye). These peptides are easy to produce, yield good cell penetration, and their cell penetrating characteristics can be further optimized by mutagenesis or by reintroducing one disulfide bridge to restore some of the secondary structures. This new generation of MCa analogues is predicted to have bright futures for CPP applications in vitro and in vivo.
In the following description, the standard one letter amino acid code is used. In addition, non-natural amino acids and groups of particular amino acids are referred to using the following one letter code:
One aspect of the present invention relates to the use of a peptide as a vector for the intracellular delivery of a molecular cargo, wherein said peptide is a maurocalcine derived cell penetrating peptide consisting of a sequence selected from the group consisting of:
wherein:
wherein:
The peptide according to the present invention is a cell penetrating peptide or CPP. Therefore, it is capable of crossing the plasma membrane of cells and of carrying small and large non-permeant molecular cargoes across the plasma membrane, efficiently, rapidly, at low concentration, in vitro and in vivo, into various cell types. Non-permeant molecular cargoes include but are not limited to peptides, proteins including antibodies, small (oligonucleotides, PNAs) and large nucleic acids, small and large chemical compounds, nanoparticles and liposomes. These properties can be readily verified by technique known to those skilled in the art such as those described in the examples of the present application.
The peptide of the invention provides an efficient carrier or vector for the delivery of various drugs and agents of therapeutic, diagnostic and technological value to their site of action in a cell, in particular the cytosol and the nucleus. Therefore, the peptide of the invention can be used for various in vivo applications including therapy, diagnosis, medical imaging and research.
“cargo”, “molecular cargo”, ‘impermeant cargo’ refers to a substance that can be transported across the plasma membrane of cells and delivered into cells by using a cell penetrating peptide as a delivery vehicle. The cargo may be a molecule such as a small molecule or a macromolecule or a particle such as a nanoparticle or a liposome.
The peptide consisting of the sequence (I) is also named peptide (I) or peptide I and the peptide consisting of the sequence (II) is also named peptide (II) or peptide II. “Peptide” refers to both peptides (I) and (II).
The peptide (I) which consists of 7 to 20 amino acids is derived from the maurocalcine peptide 1 to 20 (MCa1-20). The peptide MCa1-20 which has the amino acid sequence SEQ ID NO: 2 comprises the sequence X1 to X7 in positions 3 to 9. The peptide MCa3-9 has the sequence SEQ ID NO: 3, where X1 is C, X2 is L, X3 is P, X4 is H, X5 is L, X6 is K and X7 is L. Furthermore, X7 is R in Opi/IpTx3-9 (SEQ ID NO: 6); X2 is I, X3 is K, X6 is Q, and X7 is R in Hadru5-11 (SEQ ID NO: 9). Z is derived from the peptide MCa1-2 (GD) and Z′ is derived from the peptide MCa10-20 (SEQ ID NO: 21). Preferably, X6, X3 and X7, X6 and X7, X3 and X7 or X6 are basic amino acids, and/or X1 or X7, or both, are hydrophobic amino acids. In preferred embodiments, all the hydrophobic amino acids of said peptide (I) are J1. Preferably, X2 and X5 are L or X2 is I and X5 is L.
Preferably, the sequence X1-X2-X3-X4-X5-X6-X7 is chosen from the group consisting of SEQ ID NO: 3 to 20 and the sequences wherein one, two, three, four, five, six or seven amino acids of SEQ ID NO: 3 to 20 have been substituted with a different amino acid. In preferred embodiments, at least the cysteine (C) in position 1 (X1), the histidine (H) in position 4 (X4) and/or the lysine (K) in position 6 (X6) of SEQ ID NO: 3 to 20 have been substituted with a different amino acid. In some embodiments, the cysteine (C) in position 1 is substituted with 2-amino butyric acid. In some preferred embodiments, the sequence X1-X2-X3-X4-X5-X6-X7 is chosen from the group consisting of SEQ ID NO: 3, 9, 12 and 18.
Preferably, Z consists of a sequence of 1 to 4 amino acids. In some embodiments Z consists of 2, 3 or 4 amino acids. Preferably, Z comprises one or two acidic amino acids (D, E). In some embodiments, Z is chosen from GD, GA, KD and SEKD.
Preferably, Z′ is no amino acid or consists of the sequence
wherein:
wherein Z′ does not comprise any internal deletion other than the deletion of Z′2 to Z9′, Z′3 to Z6′, or one or more of Z′3, Z′6 and Z′9.
In preferred embodiments, Z′ is chosen from the group consisting of:
In preferred embodiments, the peptide (I) has a net charge of zero, +1 or +2. Preferably, X1 to X7 and Z together have a net charge of zero.
The peptide (II) which consists of 8 to 20 amino acids is derived from the maurocalcine peptides 6 to 25 and 18 to 33. The peptide MCa6-25 has the amino acid sequence of SEQ ID NO: 106. The peptide MCa18-33 has the amino acid sequence of SEQ ID NO: 107. The peptide MCa18-25 has the sequence SEQ ID NO: 108. U is derived from the peptide MCa6-17 (SEQ ID NO: 109) and U′ is derived from the peptide MCa26-33 (SEQ ID NO: 110).
In some embodiments Xa is chosen from S, G, T and A. In other embodiments, Xa is chosen from J, Q and N.
Preferably, the central sequence XaBBJBBBXb is chosen from: SKKCKRR and GKKCKRR (SEQ ID NO: 108 and 111), and the sequences wherein one, two, three, four, five, six or seven amino acids of SEQ ID NO: 108 and 111 have been substituted with a different amino acid.
Preferably, U is no amino acid or consists of the sequence
wherein:
In preferred embodiments, U is chosen from:
Preferably, U′ is no amino acid or consists of the sequence U′1-U′2-U′3-U′4-U′5-U′6-U′7-U′8,
wherein:
In preferred embodiments, U′ is chosen from:
In preferred embodiments of the invention, the peptide (I) has a sequence selected from the group consisting of: SEQ ID NO: 2, 3 and 36 to 43 and the sequences which have at least 60% similarity to the full length sequence of any of SEQ ID NO: 2, 3 and 36 to 43. In some embodiments, the sequences have at least 70%, 80% or 90% similarity with said full-length sequences. In some embodiments, said sequences have also at least 40% identity, preferably at least 50%, 60%, 70% or 80% identity with said full-length sequences.
In preferred embodiments of the invention, the peptide (II) has a sequence selected from the group consisting of: SEQ ID NO: 106, 107, 108 and 119 and the sequences which have at least 60% similarity to the full length sequence of any of SEQ ID NO: 106, 107, 108 and 119. In some embodiments, the sequences have at least 70%, 80% or 90% similarity with said full-length sequences. In some embodiments, said sequences have also at least 40% identity, preferably at least 50%, 60%, 70% or 80% identity with said full-length sequences.
Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequences (referred to by SEQ ID NO: X) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.
Percentage (%) sequence similarity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequences (referred to by SEQ ID NO: X) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity and considering any conservative substitutions as part of the sequence identity. Sequence similarity is preferably calculated over the entire length of the respective sequences.
Alignment for purposes of determining percent amino acid sequence identity/similarity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.
In some embodiments, the peptides according to the present invention comprise conservative amino acid replacements which, for example, may be between amino acids within the following groups: G, A, S and T; D and E; R, H and K; N and Q; I, L and V; F, Y and W; C and 2-aminobutyric acid.
In some embodiments, the peptide comprises a unique free cysteine (cysteine not linked to another cysteine via a disulfide bond). Preferably, said unique free cysteine is at the N- or C-terminus of the peptide. Preferably, the other cysteine residues of maurocalcine which are not linked via a disulfide bond are replaced with 2-amino butyric acid. In preferred embodiments of the present invention, said unique free cysteine is used for coupling the cargo covalently to the peptide by a disulphide, thioether or thiol-maleimide linkage.
In other embodiments, the peptide comprises two cysteines which are linked via a disulfide bond (intramolecular disulphide bond). Preferably, the peptide (I) comprises at least the residues Z′1 to Z′8 of the sequence Z′, with or without the residue(s) Z′3, Z6′, and/or Z′9, Z′2 to Z′9, or Z′3 to Z6′, and X1 and Z′8 or X1 and Z′1 are cysteine residues which are linked via a disulfide bond. Preferably, the peptide (11) comprises at least the residues U5 to U12 of the sequence U and U5 and the residue J of the central sequence of said peptide II are cysteine residues which are linked via a disulfide bond. The presence or absence of disulphide bonds can be advantageous to improve the cell penetration efficiency and/or the conformational stability of the peptides according to the present invention.
The peptide according to the invention may consist of L-amino acids, D-amino acids or mixtures thereof. Preferably, the peptides comprise D-amino acids. In preferred embodiments of the present invention, the peptide consists of D-amino acids. Peptides comprising D-amino acids have the advantage of being more stable in vivo due to their increased resistance to proteolytic cleavage.
Preferred peptides (I) according to the present invention have a sequence selected from the group consisting of: SEQ ID NO: 2 to 105, 148, 150, 152, 154, 158, 160, 162, 164, 166, 168 and 170. More preferred peptides (I) are selected from the group consisting of SEQ ID NO: 2, 3, 9, 12, 18, 36, 37, 44, 46, 66, 150, 152 and 154.
Preferred peptides (II) according to the present invention have a sequence selected from the group consisting of: SEQ ID NO 106 to 108, 111 and 119 to 133.
In preferred embodiments, the peptide is modified. The modifications include esterification, glycosylation, acylation such as acetylation or linking myristic acid, amidation, phosphorylation, biotinylation, PEGylation, coupling of famesyl and similar modifications which are well-known in the art. Modifications can be introduced at the the N-terminus, the C-terminus of the peptide or if deemed suitable, also to any amino acid other than the terminal amino acids (e.g. famesyl coupling to a cysteine side chain). Conversion of the acid function on the C-terminus into an aldehyde and alkylation of the thiol function of a cysteine residue are used for chemoselective ligation or the formation of reduced peptide bonds.
Preferably, the peptide is used as a complex comprising at least the peptide and a molecular cargo.
The use according to the present invention comprises contacting the complex comprising the peptide and the cargo with cells, in vitro or in vivo, wherein the complex crosses the plasma membrane of the cells and the cargo is delivered into the cells, in vitro or in vivo. Preferably, the cargo is delivered into the cytoplasm and/or the nucleus of the cells.
The cargo can be a small molecule, a macromolecule or a particle. In a preferred embodiment, the cargo is selected from the group consisting of: oligonucleotides including antisense oligonucleotides, peptide nucleic acids (PNAs), small interfering RNAs, locked nucleic acids (LNAs), phosphorodiamidate morpholino oligonucleotides (PMOs) and decoy DNA molecules; plasmids; cDNAs; aptamers including DNA, RNA or peptide aptamers; peptides; proteins including antibodies; small and large chemical compounds including bioactive substances like drugs for the treatment of human, animal or plant diseases; labels such as fluorescent or radioactive molecules; imaging agents; liposomes, micelles and nanoparticles including liposomes, micelles and nanoparticles carrying an active agent such as nanocarriers. For example, the active agent can be encapsulated into the particles or grafted onto said particles by means well-known in the art. It is within the present invention that the complex comprises more than one peptide according to the present invention, i.e., a plurality of such peptides, whereby the plurality of the peptides may comprise a plurality of the same or of different peptides. Also, the complex according to the present invention may also comprise more than one cargo molecule, whereby the plurality of the cargo molecules may comprise a plurality of the same or of different cargo molecules.
In a preferred embodiment, the cargo is covalently or non-covalently bound to the peptide of the invention.
The cargo may be coupled to the peptide, directly or indirectly. Indirect coupling of the cargo to the peptide may be through a linker that is attached to the peptide of the invention. Linkers, also named spacers, that can be used to physically separate the peptide of the invention to the cargo are known in the art and include a peptide bond, an amino acid, a peptide of appropriate length or a different molecule providing the desired feature. The linker may be attached to the the N-terminus, the C-terminus of the peptide or if deemed suitable, also to any amino acid other than the terminal amino acids.
The peptide of the invention can be chemically linked to the cargo by covalent bonds using standard conjugation techniques. The cargo can be linked to the N-terminus, the C-terminus of the peptide, or if applicable, to any amino acid other than the terminal amino acids. Functional groups, modifications also called derivatizations or a linker may also be introduced into the peptide for conjugating the peptide to the cargo. Such covalent bonds are preferably formed between either a suitable reactive group of the peptide and the cargo and more preferably between a terminus of the peptide according to the present invention and the cargo molecule(s). Depending on the chemical nature of the cargo molecules, the moiety, group or radical with which such covalent bond is formed varies and it is within the skills of a person of the art to create such bond. Chemical linkage may be via a disulphide bond, thioether, thiol-maleimide or amide linkage. Other ways of linking the peptide to the cargo include use of a C-terminal aldehyde to form an oxime, use of a click reaction or formation of a morpholino linkage with a basic amino acid on the peptide. For coupling the peptide to the cargo using click chemistry, an alcyne or azido function may be added to the peptide using the N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-propargylglycine, (S)-2-(Fmoc-amino)-4-pentynoic acid or N-alpha-(9-Fluorenylmethyloxycarbonyl)-4-azido-L-homoalanine or (S)-2-(9-Fluorenylmethyloxycarbonylamino)-4-azidobutanoic acid reagents. This type of construct is produced by well-known peptide chemical synthesis methods, preferably by solid phase synthesis.
In addition, when the cargo is a peptide or a protein including an antibody, the complex may be a fusion protein/peptide in which the cargo is fused to the N-terminus or the C-terminus of the peptide of the invention, directly or via a peptide spacer. This complex is produced by making a fusion in frame of a nucleotide sequence encoding the peptide of the invention to a nucleotide sequence encoding the peptide/protein cargo, and expressing the resulting chimeric gene using standard recombinant DNA techniques. The resulting fusion protein/peptide is of heterologous origin, i.e., it is different from naturally occurring peptides or proteins such as maurocalcine or other toxins of the same family.
The peptide can also be bound to the cargo (molecule or particle carrying the molecule) via non-covalent bounds such as ionic bonds, hydrogen bonds or hydrophobic interactions or a combination of such bonds. Non-limitative examples include streptavidine-biotin interactions between a biotinylated peptide and a cargo (for example nanoparticles like Quantum dots) that is conjugated to streptavidine or a biotinylated cargo and a peptide that is conjugated to streptavidine.
In some embodiments, the complex further comprises a targeting moiety, for example a targeting peptide for targeting the complex to specific cell types.
In a preferred embodiment, the cargo is linked covalently to the peptide of the invention via a cysteine linker that is attached to the N-terminus or the C-terminus of the peptide of the invention. Preferably, the cysteine residues of the peptide are replaced with 2-amino butyric acids, so that the cysteine linker is the unique cysteine of the peptide which is used for coupling the cargo covalently to the peptide by a linkage such a disulphide, thioether or thiol-maleimide linkage.
Examples of such peptides are SEQ ID NO: 134 to 139, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171. Preferred peptides (I) are selected from the group consisting of: SEQ ID NO: 134-136, 151, 153, 155 and 157.
The invention provides a peptide according to the present invention. Peptides according to the present invention may be provided in isolated or purified form, with or without the cargo.
The invention provides a complex comprising a peptide of the invention and a cargo.
The invention provides a pharmaceutical composition comprising (1) a complex comprising a peptide of the invention and an active agent, and (2) a pharmaceutically acceptable carrier.
The pharmaceutical composition comprises a therapeutically effective amount of the complex, e.g., sufficient to show benefit to the individual to whom it is administered. The pharmaceutical composition is formulated for administration by a number of routes, including but not limited to oral, parenteral and nasal.
The invention provides a method of treatment of a patient or subject in need for treatment for a disease condition, comprising the step of administering a therapeutically effective amount of a complex comprising a peptide of the invention and an active agent to the patient or subject.
The invention provides also a complex comprising a peptide of the invention and an active agent for treating a disease.
The active agent is a pharmaceutical agent or therapeutic capable of preventing, treating or ameliorating a disease in humans or animals. The active agent may be a protein including an antibody, an oligonucleotide including an antisense oligonucleotide, peptide nucleic acid (PNA), small interfering RNA, locked nucleic acids (LNA), phosphorodiamidate morpholino oligonucleotides (PMO) and decoy DNA molecule, a plasmid, an aptamer including DNA, RNA or peptide aptamer, a small or large chemical drug, or mixtures thereof. In particular, the active agent may be a chemotherapeutic drug used for treating cancer. Coupling anti-tumoral drugs to the cell penetrating peptides represent a valuable strategy to overcome drug resistance. The active agent is also an antigen or a nucleic acid molecule encoding said antigen (DNA vaccine) for use as a vaccine for the prevention or the treatment of an infectious disease or a cancer. The complex may comprise particles like nanoparticles, micelles or liposomes carrying both the peptide(s) and the active(s) agent(s).
Diseases to be prevented, treated or ameliorated may include any disease where improved penetration of the plasma and/or nuclear membrane by a pharmaceutical or therapeutical molecule may lead to an improved therapeutic effect. Diseases to be treated may include cancer, genetic, neurological, cardiovascular, metabolic, inflammatory, auto-immune and infectious diseases.
The invention provides a detection reagent, for example a diagnostic reagent, comprising a complex comprising a peptide of the invention and a detection agent. Preferably, the detection agent is covalently or non-covalently bound to the peptide. Detection agents are known in the art and include but are not limited to antibodies and oligonucleotide probes that are used for the detection (qualitative or quantitative detection) of an intracellular target, for example a protein or a nucleic acid target. When the intracellular target is associated with a disease (e.g., diagnostic marker), the detection agent is a diagnostic agent. According to a preferred embodiment, the complex further comprises a detectable moiety that produces a detectable signal when the intracellular target reacts with the detection agent. For example, the detectable moiety can be a fluorescent reporter system.
The invention provides a method of detecting an intracellular target, comprising:
The invention provides a method of diagnosis a disease, comprising:
The invention provides an imaging reagent, comprising a complex comprising a peptide of the invention and a label or a contrast agent. Preferably, the label or contrast agent is covalently or non-covalently bound to the peptide.
In optical imaging methods, the imaging reagent comprises a complex comprising a peptide of the invention and a label. The label is any molecule that produces a signal that can be detected in situ in living cells or tissues. The label can be a fluorescent molecule. This imaging reagent can be applied in optical imaging methods for research or diagnostic purposes.
In magnetic resonance imaging (MRI) the imaging reagent comprises a complex comprising a peptide of the invention and a contrast agent, usually a paramagnetic contrast agent (usually a gadolinium compound) or a superparamagnetic contrast agent (iron oxide nanoparticles). MRI was primarily used in medical imaging to demonstrate pathological or other physiological alteration of living tissues.
In positron emission tomography (PET), the imaging reagent comprises a complex comprising a peptide of the invention and a short-lived radioactive tracer isotope which has been chemically incorporated into a metabolically active molecule (usually a sugar, fluorodeoxyglucose, FDG) and which decays by emitting a positron. PET is a nuclear medicine imaging technique which produces a three-dimensional image or map of functional processes in the body.
The invention provides a nucleic acid molecule (DNA, RNA) comprising a nucleotide sequence encoding the peptide or the fusion peptide/protein according to the present invention.
The invention provides a nucleic acid recombinant vector comprising said nucleic acid molecule. Preferably, said recombinant vector is an expression vector comprising a regulatory sequence (promoter) operably linked to said nucleotide sequence, wherein the vector is capable of expressing the peptide or fusion peptide/protein of the invention when transfected or transformed into a host cell (mammalian, bacterial or fungal). Recombinant vectors include usual vectors used in genetic engineering and gene therapy including for example plasmids and viral vectors.
The invention provides a cell transformed with said recombinant vector.
The nucleic acid molecule, vector, cell of the invention are useful for the production of the peptide or fusion peptide/protein of the invention using well-known recombinant DNA techniques.
The invention provides a kit comprising one or more of:
For a better understanding of the invention and to show how the same may be carried into effect, there will now be shown by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:
1) Reagents
N-α-Fmoc-L-aminoacid, Wang-Tentagel resin and reagents used for peptide syntheses were obtained from Iris Biotech. Solvents were analytical grade products from Acros Organics. Cy5 maleimide mono-reactive dye was purchased from GE Healthcare.
2) Solid-Phase Peptide Syntheses
Chemical syntheses of MCa analogues were performed as previously described (Poillot et al., 2010). Briefly, analogues of MCa were chemically synthesized by the solid-phase method (Merrifield, R. B., 1969) using an automated peptide synthesizer (CEM© Liberty). Peptide chains were assembled stepwise on 0.24 mEq of Fmoc-D-Arg-Pbf-Wang-Tentagel resin using 0.24 mmol of N-α-fluorenylmethyloxycarbonyl (Fmoc) L-amino-acid derivatives. The side-chain protecting groups were: Trityl for Cys and Asn, tert-butyl for Ser, Thr, Glu and Asp, Pbf for Arg and tert-butylcarbonyl for Lys. Reagents were at the following concentrations: Fmoc-amino-acids (0.2 M Fmoc-AA-OH in dimethylformamide (DMF)), activator (0.5 M 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF), activator base (2M diisopropylethylamine in N-methyl-pyrrolidone (NMP)) and deprotecting agent (5% piperazine/0.1 M 1-hydroxybenzotriazole in DMF), as advised by PepDriver (CEM©). After peptide chain assembly, resins were treated 4 hrs at room temperature with a mixture of trifluoroacetic acid/water/triisopropylsilan (TIS)/dithiothreitol (DTT) (92.5/2.5/2.5/2.5). The peptide mixtures were then filtered and the filtrates were precipitated by adding cold t-butylmethyl ether. The crude peptides were pelleted by centrifugation (10.000×g, 15 min) and the supernatants were discarded. MCa analogues were purified by HPLC using a Vydac C18 column (218TP1010, 25×10 cm). Elutions of the peptides were performed with a 10-60% acetonitrile linear Gradient containing 0.1% trifluoroacetic acid. The purified fractions were analyzed by analytical RP-HPLC (Vydac C18 column 218TP104, 25×4.6 cm). All analogues were characterized by MALDI-TOF mass spectrometry.
3) Labeling of Peptide with Cy5
Each peptide was labeled with Cy5 according to the manufacturer's protocol (GE HEALTHCARE). Peptides were dissolved at 1 mg/ml in 0.1 M Na2CO3 buffer, pH 9.3. 300 μl of the solubilized peptides were added to Cy5-maleimide containing tubes. The mixtures were incubated during 2 hrs at room temperature and then purified by HPLC using an analytical Vydac C18 column. Elution of the Cy5-labeled peptides was performed with a 10-60% acetonitrile linear gradient containing 0.1% trifluoroacetic acid. The pure peak fractions were lyophilized and peptides quantified by UV spectrophotometer at 649 nm.
4) Cell Culture
Chinese hamster ovary (CHO) and F98 rat glioma cell lines (from ATCC) were maintained at 37° C. in 5% CO2 in F-12K nutrient medium (INVITROGEN) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (INVITROGEN) and 10,000 units/ml streptomycine and penicillin (INVITROGEN) for the CHO cells, and 2% heat-inactivated fetal bovine serum and 100 units/ml streptomycine and penicillin for the F98 cells
5) MTT Assay
Cells were seeded into 96-well micro plates at a density of approximately 8×104 cells/well. After 2 days of culture, the cells were incubated for 24 hrs at 37° C. with MCa analogues at a concentration of 10 μM. Control wells containing cell culture medium alone or with cells, both without peptide addition, were included in each experiment. 0.1% saponin was used as toxic agent for comparison. The cells were then incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) for 30 min. Conversion of MTT into purple colored MTT formazan by the living cells indicates the extent of cell viability. The crystals were dissolved with dimethyl sulfoxide (DMSO) and the optical density was measured at 540 nm using a microplate reader (Biotek ELx-800, MANDEL SCIENTIFIC INC.) for quantification of cell viability. All assays were run in triplicates.
6) Confocal Microscopy
For analysis of the subcellular localization of MCa-Cy5 analogues in living cells, cell cultures were incubated with the fluorescent peptides for 2 hrs, and then washed with phosphate-buffered saline (PBS) alone. The plasma membrane was stained with 5 μg/ml rhodamine-conjugated concanavalin A (MOLECULAR PROBES) for 5 min. Cells were washed once more. Live cells were then immediately analyzed by confocal laser scanning microscopy using a Leica TCS-SPE operating system. Rhodamine (580 nm) and Cy5 (670 nm) were sequentially excited and emission fluorescence were collected in z-confocal planes of 10-15 nm steps.
7) Flow Cytometry
CHO and F98 cells were incubated with various concentrations of Cy5-labeled peptides in F-12K culture medium without serum at 37° C. for 2 hrs. The cells were then washed with PBS to remove excess extracellular peptide and treated with 1 mg/ml trypsin (INVITROGEN) for 5 min at 37° C. to detach cells from the surface, and centrifuged at 200 g before suspension in PBS. For experiments with the macropinocytosis inhibitor, amiloride, CHO cells were initially washed with F-12K and preincubated for 30 min at 37° C. with 1 mM amiloride (SIGMA). The cells were then incubated for 2 hrs at 37° C. with 1 μM of the Cy5-MCa analogues. For all these experimental conditions, flow cytometry analyses were performed with live cells using a Becton Dickinson flow cytometer LSR II or an Accuri® flow cytometer (BD BIOSCIENCES). Data were obtained and analyzed using FCS express software (DE NOVO) or Accuri® proprietary software CFlow sampler. Live cells were gated by forward/side scattering from a total of 10,000 events.
8) Preparation of Heavy SR Vesicles
Heavy SR vesicles were prepared following the method of Kim et al., J. Biol. Chem., 1983. Protein concentration was measured by the Biuret method. [3H]-Ryanodine binding assay—Heavy SR vesicles (1 mg/ml) were incubated at 37° C. for 2 hrs in an assay buffer composed of 10 nM [3H]-ryanodine, 150 mM KCl, 2 mM EGTA, 2 mM CaCl2 (pCa=5), and 20 mM MOPS, pH 7.4. Truncated MCa analogues were added prior to the addition of heavy SR vesicles. [3H]-ryanodine bound to heavy SR vesicles was measured by filtration through Whatman GF/B glass filters followed by three washes with 5 ml of ice-cold washing buffer composed of 150 mM NaCl, 20 mM HEPES, pH 7.4. [3H]-ryanodine retained on the filters was measured by liquid scintillation. Non-specific binding was measured in the presence of 80 μM unlabeled ryanodine. The data are presented as mean±S.E. Each experiment was performed in triplicate.
9) Statistical Analyses
All data are given as mean±SD for n number of observations, and statistical significance (p) was calculated using Student's t test.
While all truncated derivatives of MCaUF1-33 show cell penetration properties according to the flow cytometry analyses, the inventors examined whether there were differences in intracellular distribution among these peptides. This question was investigated by confocal microscopy after 2 hrs of peptide accumulation into CHO cells. Interestingly, all peptides showed very resembling intracellular distributions, although the degree of accumulated cell fluorescence varied somewhat with peptide sequences. In confirmation of the flow cytometry results, the peptide that appeared to penetrate the least was the full length unfolded MCa, MCaUF1-33-C-Cy5. The vast majority of the fluorescence appears in punctuate dots within the cells. In many cases, these dots appear at higher concentrations within one pole of the cell (MCaUF8-33-C-Cy5, MCaUF11-33-C-Cy5, MCaUF25-33-C-Cy5, and MCaUF1-9-C-Cy5 for instance). On various occasions also, all of the peptides tend to present a sub-plasma membrane distribution, forming a rim of smaller circumference than the concanavalin A labeling itself. This sub-plasma membrane rim localization was more evident for CHO cells labeled with MCaUF14-25-C-Cy5. Finally, more rarely, a direct plasma membrane labeling by the peptide-cargo complex was observable. This type of labeling could be observed with N-terminal truncated vectors exclusively and was most evident or MCaUF22-33-C-Cy5. The staining of the plasma membrane was always diffuse in contrast to intracellular staining which was mainly punctuated. Diffuse membrane labeling was also observed for MCaUF25-33-C-Cy5 and MCaUF20-33-C-Cy5, two peptides that differ from 2 to 3 amino acids with MCaUF22-33-C-Cy5. It was difficult to evidence for the other vector/cargo complexes. The inventors propose that this staining coincides with an alteration of the duration of peptide plasma membrane residency for these truncated MCaUF analogues. The lower occurrence of this diffuse staining for the other truncated variants may reflect faster internalization by endocytosis and/or membrane translocation. Globally, these effects reflect cell entry and distribution tendencies that were hard to quantify and they should therefore be interpreted with caution. In an attempt to better apprehend peptide behavior at the plasma membrane, the inventors quantified the extent of Cy5/rhodamine staining colocalization. Rhodamine-positive staining was also Cy5-positive for 63% to 86% of the pixels (best performing peptides were MCaUF14-33-C-Cy5, MCaUF18-33-C-Cy5, MCaUF20-33-C-Cy5 and MCaUF22-33-C-Cy5). This finding indicates that the peptides invade large membrane areas and that membrane interaction is not limited to small specialized surface areas. In contrast, Cy5-positive pixels were rhodamine-positive to far more variable extents (
In earlier studies, the inventors have demonstrated that the cell entry of MCaUF1-33 was largely sensitive to amiloride, suggesting a predominant macropinocytosis mechanism for its cell penetration (Ram et al., J. Biol. Chem., 2008, 283, 24274-24284). However, it was likely that such a predominant reliance on macropinocytosis was also conferred by the cargo type transported (streptavidine in that report). The inventors therefore conducted an in depth analysis of the amiloride-sensitivity of the various truncated MCaUF peptides with Cy5 as cargo and quantified by flow cytometry the degree of cell penetration inhibition in CHO cells.
While the inventors compared the properties of cell penetration of truncated peptides at rather mild concentrations, the inventors also aimed at comparing the dose-dependence of cell penetration of these peptides by flow cytometry (
An earlier report has shown that MCaUF1-33 is unable to interact with MCa's target, the ryanodine receptor RyR1 (Ram et al., J. Biol. Chem., 2008). This is due to the loss of secondary structures owing to the lack of internal disulfide bridging. The inventors did therefore expect that truncated analogues of MCaUF should also be pharmacologically inert. This hypothesis was challenged by testing the ability of the Cy5-free peptides to stimulate [3H]-ryanodine binding (
The peptide is synthesized by the solid phase method as described in example 1. The peptide bound to the resin is labeled with N-Succinimidyl-S-acetylthioacetate (SATA) to introduce a protected thiol group at the N-terminus of the peptide.
Labeling of Peptide with SATA
SATA (76 mg in 60 μl DMF) and then phosphate buffer saline (PBS), pH 7.4 (540 μl) were added to the peptide bound to the resin (10 mg in 600 μl of PBS, pH 7.4) and the mixture was agitated for 1 hour at room temperature, filtered and washed three times with methanol. The peptide was then deprotected and cleaved from the resin as follows. The preceding mixture was treated 4 hrs at room temperature with a mixture of TFA (Trifluoroacétique acid; 9.25 ml), triisopropylsilane (250 μl), water (250 μl), and Dithiothreitol (250 mg), filtered, and the filtrate was precipitated by adding cold diethyl ether (40 ml) The crude peptide was pelleted by centrifugation (10,000 g, 15 min). The pellet was washed three times by resuspension in diethyl ether (10 ml) and centrifugation (10,000 g, 15 min). The supernatant was discarded and the peptide was air-dried.
Disulphide Bond Formation 4,4′-Dipyridyl disulfide (114 μL of a 10 mM solution in methanol) was added to the labeled peptide (2 mg in 2 ml of acetonitrile (50%), water (50%) and TFA (0.1% TFA) and the mixture was agitated at room temperature for 2 hrs. The solvent was evaporated using a rotavapor. The oxydised peptide (containing an intramolecular disulfide bond) was solubilized in TFA (2 ml of a 0.1% solution in water), purified by HPLC using a JUPITER™ 4 μm PROTEO 90A column (250 mm×10 mm; PHENOMENEX) and a 10-60% acetonitrile gradient (40 minutes, 4 ml/min rate), and lyophilized.
Deprotection of the Thiol Group
The peptide (2 mg in 1 ml of PBS, pH 7.4) was treated with the deacetylation solution (100 μl of 0.5 M hydroxylamine, 25 mM EDTA in PBS, pH 7.4) for 2 hrs at room temperature, purified by HLPC as described above, and lyophilized.
Cy5-labeled peptides derived from MCaUF1-9 were synthesized and assayed by confocal microscopy or flow cytometry as described in example 1. The results are presented in
The analysis of the MCaUF1-9 truncated variant (MCaUF3-9(Abu3)) shows that a peptide of 7 residues derived from MCaUF1-9 is an efficient CPP (
The analysis of the MCaUF1-9 variants with point mutations, including Imperatoxin and Hadrucalcine derived peptides, show that the first residues of the MCaUF1-9 derived CPP may have various sequences including for example GA (MCaUF1-9(A2-Abu3);
The residue in position 3 of the MCaUF1-9 derived CPP may be, either a hydrophobic amino acid such as a cysteine analog (Abu; MCaUF1-9(Abu3);
The residue in position 4 of the MCaUF1-9 derived CPP is a hydrophobic amino acid, preferably a leucine (MCaU1-9(Abu3);
The residue in position 5 of the MCaUF1-9 derived CPP is a basic amino acid chosen from K (HadUF1-11(Abu5) and HadUF3-9(Abu5);
The residue in position 6 of the MCaUF1-9 derived CPP is either an amino acid different from S, T, D and E, such as H (MCaUF1-9(Abu3), HadUF1-11(Abu5) and HadUF3-9(Abu5),
The residue in position 7 of the MCaUF1-9 derived CPP is a hydrophobic amino acid, for example a leucine (MCaUF1-9(Abu3), HadUF1-11(Abu5), HadUF3-9(Abu5);
The residue in position 8 of the MCaUF1-9 derived CPP is a basic amino acid, for example a lysine (MCaUF1-9(Abu3),
The residue in position 9 of the MCaUF1-9 derived CPP is a hydrophobic amino acid, for example a leucine (MCaUF1-9(Abu3),
Interestingly, the hadrucalcine derived peptides HadUF1-11(Abu5) and HadUF3-9(Abu5) perform better as CPP than the maurocalcine derived peptide MCaUF1-9(Abu3) (
Comparison of MCa and Had derived peptides to a classical CPP (Tat basic peptide: GRKKRRQRRR-C; SEQ ID NO: 172) shows that 3 μM MCaUF1-9(Abu3) or 1 μM MCaUF3-9(Abu3), MCaUF1-9(W3), MCaUF1-9(Abu3-W4) or HadUF1-11(Abu5) proved better CPP than Tat at 3 μM (
| Number | Date | Country | Kind |
|---|---|---|---|
| 11170595 | Jun 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2012/053120 | 6/20/2012 | WO | 00 | 1/2/2014 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/176138 | 12/27/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6051429 | Hawley-Nelson et al. | Apr 2000 | A |
| 20050277181 | Berthet et al. | Dec 2005 | A1 |
| 20080234182 | Karginov | Sep 2008 | A1 |
| 20090142266 | Ronjat et al. | Jun 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 1849798 | Oct 2007 | EP |
| WO 0220769 | Mar 2002 | WO |
| WO-2006051224 | May 2006 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20140235557 A1 | Aug 2014 | US |
