ERK-derived peptides and uses thereof

An isolated peptide being no longer than 20 amino acids comprising a sequence at least 95% homologous to the sequence GQLNHILGILGX1PX2QED (SEQ ID NO: 4), wherein X1 and X2 are any amino acid, the peptide being capable of preventing extracellular signal-regulated kinase1/2 (ERK) translocation into the nucleus.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No. PCT/IL2014/050822 having International filing date of Sep. 15, 2014, which claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 61/878,633 filed on Sep. 17, 2013. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 65470SequenceListing.txt, created on Mar. 16, 2016, comprising 3,279 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to peptides derived from extracellular signal-regulated kinase1/2 (ERK) which may be used for treating cancer.

The extracellular signal-regulated kinase1/2 (ERK) cascade is an intracellular signaling pathway that regulates cellular processes, such as proliferation and differentiation. Being a central signaling component, its dysregulation is involved in various pathologies, particularly cancer. Indeed, inhibitors of both Rafs and MEK1/2 within the cascade were recently developed, but despite the widespread involvement of ERK in the induction and maintenance of cancers, these inhibitors were proven beneficial almost only in B-Raf mutated melanomas. In addition, most sensitive melanomas develop resistance to the Raf/MEK inhibitors within several months of treatment. The lack of effect in many cancer types, and the mechanisms of acquired resistance are now being investigated, and shown to often involve the inhibition of ERK-dependent negative feedback loops. Consequently, this inhibition allows hyperactivation of upstream signaling components that circumvent the inhibited ERK cascade. Hence, inhibiting the ERK cascade without affecting the feedback loops should result in a more general anti cancer drug.

One of the key steps in the transmission of extracellular signals is the nuclear translocation of ERK. In resting cells, most of ERK is localized in the cytoplasm due to anchoring to cytoplasmic proteins, but stimulation causes a rapid and massive nuclear translocation of a large portion of the ERK molecules. The molecular mechanism of translocation involves first TEY-phosphorylation-dependent conformational change, which results in the detachment of the ERK molecules from their anchors. This exposes the ERK to an additional phosphorylation on two Ser residues within a nuclear translocation signal (NTS). The phosphorylation of the NTS then allows the beta-like importin (Imp), Imp7, to bind it, and consequently, induce the nuclear translocation of the kinases. This rapid translocation allows the phosphorylation and activation of many nuclear proteins, which are important for the induction and regulation of cellular processes.

U.S. Patent Application Publication No. 20100099627 teaches 18 amino acid peptides that are capable of preventing ERK translocation into the nucleus.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an isolated peptide being no longer than 20 amino acids comprising a sequence at least 95% homologous to the sequence GQLNHILGILGX1PX2QED (SEQ ID NO: 4), wherein X1and X2are any amino acid, the peptide being capable of preventing extracellular signal-regulated kinase1/2 (ERK) translocation into the nucleus.

According to an aspect of some embodiments of the present invention there is provided an isolated peptide being 17 amino acids comprising the sequence GQLNHILGILGX1PX2QED (SEQ ID NO: 4), wherein X1and X2are any amino acid, the peptide being capable of preventing ERK translocation into the nucleus.

According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising the isolated peptide described herein, attached to a cell penetrating agent.

According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the peptide described herein, thereby treating the cancer.

According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of matter described herein, thereby treating the cancer.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the peptide described herein as an active agent and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the composition of matter described herein as the active agents and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the isolated peptide is 17 amino acids long.

According to some embodiments of the invention, X1and X2are each independently selected from the group consisting of glutamic acid, aspartic acid, alanine and serine.

According to some embodiments of the invention, X1and X2are each independently selected from the group consisting of glutamic acid and aspartic acid.

According to some embodiments of the invention, X1and X2are glutamic acid.

According to some embodiments of the invention, X1and X2are aspartic acid.

According to some embodiments of the invention, the isolated peptide is devoid of the amino acid sequence Leu-Aspartic acid.

According to some embodiments of the invention, neither X1nor X2is alanine.

According to some embodiments of the invention, the cell penetrating agent comprises myristic acid.

According to some embodiments of the invention, the myristic acid is attached to the N terminus of the peptide.

According to some embodiments of the invention, the cell penetrating agent is a cell penetrating peptide.

According to some embodiments of the invention, the cell penetrating peptide comprises an amino acid sequence which is attached to the N terminus of the isolated peptide described herein.

According to some embodiments of the invention, the cell penetrating peptide comprises an acid sequence as set forth in SEQ ID NO: 5.

According to some embodiments of the invention, the isolated peptide described herein is attached to the cell penetrating peptide via a peptide bond.

According to some embodiments of the invention, the isolated peptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 6 and 7.

According to some embodiments of the invention, the isolated peptide attached to the cell penetrating peptide is no longer than 30 amino acids.

According to some embodiments of the invention, the peptide is attached to a cell penetrating agent.

According to some embodiments of the invention, the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, prostate cancer and cervical cancer.

According to some embodiments of the invention, the cancer is melanoma.

According to some embodiments of the invention, the melanoma comprises B-Raf melanoma.

According to some embodiments of the invention, isolated peptide is for use in treating cancer.

According to some embodiments of the invention, the composition of matter is for use in treating cancer.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to peptides derived from extracellular signal-regulated kinase1/2 (ERK) which may be used for treating cancer.

ERK1/2 signaling plays a crucial role in the induction of proliferation, as well as cancer development and progression. Inhibitors of the ERK cascade (e.g. vemurafenib and trametinib) serve as anti-cancer drugs. However, the majority of these agents have only a limited effect on human malignancies, and even the most effective inhibitors affect only a limited number of cancers. In addition, these inhibitors may have serious side effects including the induction of skin cancer, and the treated cancers (i.e. melanoma) develop resistance to the drugs within 6-8 months. Much of the shortcomings of the current inhibitors are probably mediated by reduced negative feedback loops.

The present inventors sought to prevent the nuclear translocation of ERK1/2, thus preventing ERK-dependent proliferation but not the negative feedback loops induced by it. The present inventors synthesized numerous peptides based on the nuclear translocation signal of ERK and showed that they were able to efficiently and specifically inhibit the interaction of ERK with Imp7, thereby preventing the nuclear translocation of ERK, without changing AKT activity that is usually enhanced by inhibition of the ERK-related negative feedback loops.

The EPE based peptide was shown to inhibit the stimulated nuclear translocation of ERK in all the cell lines examined; however, its effect on cell proliferation varied in different cell lines. The most notable effect of the peptide was on B-Raf melanoma cells, which underwent apoptosis a few hours following treatment (FIGS. 5A-D).

The application of the peptide to cultured cells induced apoptosis of melanoma cells, while inhibiting the proliferation/survival of other cancer cells, including PLX4032 and U0126-resistant melanoma cells (FIGS. 4A-B); however, it had no effect on the proliferation of immortalized cells (FIGS. 3A-B). When used in xenograft models, systemic application of the EPE peptide inhibited the growth of breast, colon and melanoma-derived tumors, and eradicated the growth of low-passage melanoma xenografts (FIGS. 6A-Band10A-B).

Thus, according to one aspect of the present invention there is provided an isolated peptide being no longer than 20 amino acids comprising a sequence at least 95% homologous to the sequence GQLNHILGILGX1PX2QED (SEQ ID NO: 4), wherein X1and X2are any amino acid, the peptide being capable of preventing extracellular signal-regulated kinase1/2 (ERK) translocation into the nucleus.

The phrase “being capable of preventing extracellular signal-regulated kinase1/2 (ERK) translocation into the nucleus” refers to the ability to down-regulate the amount of ERK from translocating from the cytoplasm into the nucleus by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Methods of detecting whether a peptide is capable of preventing ERK translocation are described in the Examples section, herein below.

The peptide of this aspect of the present invention may be 16, 17, 18, 19 or 20 amino acids long.

The peptide has an amino acid sequence which is typically at least 94% homologous or identical to the sequence as set forth in SEQ ID NO: 4, 95% homologous or identical to the sequence as set forth in SEQ ID NO: 4, 96% homologous or identical to the sequence as set forth in SEQ ID NO: 4, at least 97% homologous or identical to the sequence as set forth in SEQ ID NO: 4, at least 98% homologous or identical to the sequence as set forth in SEQ ID NO: 4, at least 99% homologous or identical to the sequence as set forth in SEQ ID NO: 4 or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 4 as determined using the Standard protein-protein BLAST [blastp] software of the NCBI.

As mentioned, X1and X2in SEQ ID NO: 4 may be any amino acid (as specified herein below). According to one embodiment, X1and X2are each independently selected from the group consisting of glutamic acid, aspartic acid, alanine and serine. For example, the X1and X2may both be glutamic acid. For example, the X1and X2may both be aspartic acid. For example, the X1and X2may both be serine. For example, X1may be glutamic acid and X2may be aspartic acid or X1may be aspartic acid and X2may be glutamic acid. According to another embodiment, neither X1nor X2is alanine.

Thus, according to this aspect of the present invention the peptide is at least 94% homologous or identical to the sequence as set forth in SEQ ID NO: 2, 95% homologous or identical to the sequence as set forth in SEQ ID NO: 2, 96% homologous or identical to the sequence as set forth in SEQ ID NO: 2, at least 97% homologous or identical to the sequence as set forth in SEQ ID NO: 2, at least 98% homologous or identical to the sequence as set forth in SEQ ID NO: 2, at least 99% homologous or identical to the sequence as set forth in SEQ ID NO: 2 or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 2 as determined using the Standard protein-protein BLAST [blastp] software of the NCBI, wherein the serine in position X1and X2is not replaceable by another amino acid.

Thus, according to this aspect of the present invention the peptide is at least 94% homologous or identical to the sequence as set forth in SEQ ID NO: 3, 95% homologous or identical to the sequence as set forth in SEQ ID NO: 3, 96% homologous or identical to the sequence as set forth in SEQ ID NO: 3, at least 97% homologous or identical to the sequence as set forth in SEQ ID NO: 3, at least 98% homologous or identical to the sequence as set forth in SEQ ID NO: 3, at least 99% homologous or identical to the sequence as set forth in SEQ ID NO: 3 or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 3 as determined using the Standard protein-protein BLAST [blastp] software of the NCBI, wherein the alanine in position X1and X2is not replaceable by another amino acid.

Thus, according to this aspect of the present invention the peptide is at least 94% homologous or identical to the sequence as set forth in SEQ ID NO: 6, 95% homologous or identical to the sequence as set forth in SEQ ID NO: 6, 96% homologous or identical to the sequence as set forth in SEQ ID NO: 6, at least 97% homologous or identical to the sequence as set forth in SEQ ID NO: 6, at least 98% homologous or identical to the sequence as set forth in SEQ ID NO: 6, at least 99% homologous or identical to the sequence as set forth in SEQ ID NO: 6 or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 6 as determined using the Standard protein-protein BLAST [blastp] software of the NCBI, wherein the glutamic acid in position X1and X2is not replaceable by another amino acid.

Thus, according to this aspect of the present invention the peptide is at least 94% homologous or identical to the sequence as set forth in SEQ ID NO: 7 (GQLNHILGILGDPDQED, 95% homologous or identical to the sequence as set forth in SEQ ID NO: 7, 96% homologous or identical to the sequence as set forth in SEQ ID NO: 7, at least 97% homologous or identical to the sequence as set forth in SEQ ID NO: 7, at least 98% homologous or identical to the sequence as set forth in SEQ ID NO: 7, at least 99% homologous or identical to the sequence as set forth in SEQ ID NO: 7 or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 7 as determined using the Standard protein-protein BLAST [blastp] software of the NCBI, wherein the aspartic acid in position X1and X2is not replaceable by another amino acid.

Peptides which are not 100% homologous to the sequences disclosed herein may comprise either conservative or non-conservative substitutions, deletions or additions.

The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered conservative substitutions.

For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acids is well documented in the literature known to the skilled practitioner.

When effecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The phrase “non-conservative substitutions” as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or —NH—CH[(—CH2)5—COOH]—CO— for aspartic acid. Those non-conservative substitutions which fall within the scope of the present invention are those which still constitute a polypeptide being able to prevent ERK translocation into the nucleus.

Preferably, the peptides of the present invention are typically devoid of the sequence Leu-Aspartic acid.

Further, preferably the N terminal amino acid of the peptide is glycine.

The term “peptide” as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided herein under.

Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds (—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds (—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g., methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds (—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic double bonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally present on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) bonds at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.

The peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2), which can be used with some embodiments of the invention.

The peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.

Since the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form, the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.

Further contemplated modifications of the peptides of the present invention include C-terminal amidation.

In order to improve the bioavailability of the ERK peptides, a single, a portion or even all the amino acids in the peptide can be D amino acids which are not susceptible to enzymatic proteolytic activity and can improve altogether the use of the peptides of the invention as pharmaceuticals. The peptides of the present invention may be attached (either covalently or non-covalently) to a penetrating agent.

As used herein the phrase “penetrating agent” refers to an agent which enhances translocation of any of the attached peptide across a cell membrane.

According to one embodiment, the penetrating agent is a peptide and is attached to the ERK (either directly or non-directly) via a peptide bond. Preferably, the penetrating agent is attached to the N terminus of the ERK derived peptide.

Typically, peptide penetrating agents have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.

By way of a non-limiting example, cell penetrating peptide (CPP) sequences may be used in order to enhance intracellular penetration. CPPs may include short and long versions of the protein transduction domain (PTD) of HIV TAT protein, such as for example, YARAAARQARA (SEQ ID NO: 5), YGRKKRR (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), or RRQRR (SEQ ID NO: 10)]. However, the disclosure is not so limited, and any suitable penetrating agent may be used, as known by those of skill in the art.

When the peptides of the present invention are attached to cell penetrating peptides, it is contemplated that the full length peptide is no greater than 25 amino acids, no greater than 26 amino acids, no greater than 27 amino acids, no greater than 28 amino acids, no greater than 29 amino acids, or no greater than 30 amino acids.

Another method of enhancing cell penetration is via N-terminal myristoilation. In this protein modification, a myristoyl group (derived from myristic acid) is covalently attached via an amide bond to the alpha-amino group of an N-terminal amino acid of the peptide.

The peptides of some embodiments of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide and so forth. Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505.

A preferred method of preparing the peptide compounds of some embodiments of the invention involves solid phase peptide synthesis.

Large scale peptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.

Since the peptides of the present invention are able to specifically inhibit the nuclear activities of ERK without modulating its cytoplasmic activities, these peptides may be used to inhibit ERK nuclear activities (e.g. proliferation) without harming other ERK-related cytoplasmic activities in the cells. Therefore, the peptides of this aspect of the present invention may serve as therapeutic agent for hyperproliferative diseases such as cancer without having the side-effects of other ERK inhibitors.

Thus, according to another aspect of the present invention there is provided a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the peptides disclosed herein, thereby treating the cancer.

According to a specific embodiment, the cancer is melanoma, breast cancer, lung cancer, prostate cancer or cervical cancer.

According to another embodiment, the melanoma is PLX4032 and/or U0126-resistant melanoma.

Herein the term “active ingredient” refers to the peptides disclosed herein accountable for the biological effect.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (peptide) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

Dosage amount and interval may be adjusted individually to ensure blood or tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

As mentioned, various animal models may be used to test the efficacy of the peptides of the present invention. A transgenic mouse model for cancer (e.g., breast cancer) such as the erb model (Shah N., et al., 1999, Cancer Lett. 146: 15-2; Weistein E J., et al., 2000, Mol. Med. 6: 4-16) or MTV/myc model (Stewart T A et al., 1984, Cell, 38: 627-637), the c-myc model (Leder A., et al., 1986, Cell, 45:485-495), v-Ha-ras or c-neu model (Elson A and Leder P, 1995, J. Biol. Chem. 270: 26116-22) can be used to test the ability of the peptides of the present invention to suppress tumor growth in vivo.

For the formation of solid tumors, athymic mice can be injected with human or animal (e.g., mouse) cancerous cells such as those derived from breast cancer, ovarian cancer, prostate cancer or thyroid cancer, and following the formation of cancerous tumors the mice can be subjected to intra-tumor and/or systemic administration of the peptides.

The following cell lines (provided with their ATCC Accession numbers) can be used for each type of cancer model:

For breast cancer:

For ovarian cancer:

For prostate cancer:

For thyroid cancer:

For lung cancer:

Mouse lung carcinoma LL/2 (LLCI) cells (Lewis lung carcinoma)—These cells are derived from a mouse bearing a tumor resulting from an implantation of primary Lewis lung carcinoma. The cells are tumorigenic in C57BL mice, express H-2b antigen and are widely used as a model for metastasis and for studying the mechanisms of cancer chemotherapeutic agents (Bertram J S, et al., 1980, Cancer Lett. 11: 63-73; Sharma S, et al. 1999, J. Immunol. 163: 5020-5028).

For melanoma:

The cancerous cells can be cultured in a tissue culture medium such as the DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, supplemented with 10% fetal calf serum (FCS), according to known procedures (e.g., as described in the ATCC protocols).

Tumor formation in animal models by administration of cancerous cells—Athymic nu/nu mice (e.g., female mice) can be purchased from the Jackson Laboratory (Bar Harbor, Me.). Tumors can be formed by subcutaneous (s.c.) injection of cancerous cells (e.g., 2×106cells in 100 μl of PBS per mouse). Tumors are then allowed to grow in vivo for several days (e.g., 6-14 days) until they reach a detectable size of about 0.5 cm in diameter. It will be appreciated that injection of cancerous cells to an animal model can be at the organ from which the cell line is derived (e.g., mammary tissue for breast cancer, ovary for ovarian cancer) or can be injected at an irrelevant tissue such as the rear leg of the mouse.

To test the effect of the peptides of the present invention on inhibition of tumor growth, the agents may be administered to the animal model bearing the tumor either locally at the site of tumor or systemically, by intravenous injection of infusion, via, e.g., the tail vein. The time of administration may vary from immediately following injection of the cancerous cell line (e.g., by systemic administration) or at predetermined time periods following the appearance of the solid tumor (e.g., to the site of tumor formation, every 3 days for 3-10 times as described in Ugen K E et al., Cancer Gene Ther. 2006 Jun. 9; [Epub ahead of print]).

It will be appreciated that administration may also be effected using a nucleic acid construct designed to express the peptide agent (e.g., a viral vector), naked pDNA and/or liposomes.

Tumor sizes are measured two to three times a week. Tumor volumes are calculated using the length and width of the tumor (in millimeters). The effect of the treatment can be evaluated by comparing the tumor volume using statistical analyses such as Student's t test. In addition, histological analyses can be performed using markers typical for each type of cancer.

According to another embodiment, the agents of the present invention are co-administered or co-formulated with other known chemotherapeutic agents and/or anti-inflammatory agents. In addition, they may be administered with other known therapies, including but not limited to chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy.

The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.

As used herein the term “about” refers to ±10%.

The term “consisting of” means “including and limited to”.

EXAMPLES

Materials and Methods

Reagents and Antibodies: Tetradecanoyl phorbol acetate (TPA), EGF, Avidin-FITC and TGF-diamino-2-phenylindole (DAPI), 3,3′-Diaminobenzidine (DAB) were obtained from Sigma (St Louis, Mich.). Anti general Elk1 (gElk1) and gRSK1 Abs were from Santa Cruz Biotechnology (CA, USA). Anti pElk-1 (Ser383), PARP-1, pAKT (Ser473) and pRSK (Ser381) Abs were from Cell Signaling Technology (Beverly, Mass., USA). Anti Imp7 Ab was from Abnova (Taipei, Taiwan). Anti pERK (pTEY-ERK), gERK, gAKT, gp38, pp38 (TGY), gJNK and pJNK (TPY) Abs were from Sigma (Rehovot, Israel). Polyclonal and monoclonal anti phospho SPS-ERK Abs were produced in the Biological Service Unit of the Weizmann Institute of Science (Rehovot, Israel). Secondary fluorescent Ab conjugates were from Jackson Immunoresearch (West Grove, Pa.). Secondary Ab conjugated to peroxidase (Simple Stained Max PO) was from Nichirei Biosciences (Japan).

Fluorescence Microscopy: Cells were fixed in 3% paraformaldehyde in PBS (20 min, 23° C.), and then permeabilized and blocked with 0.2% Triton X-100 in PBS-BSA (2%) for 20 min at 23° C. The fixed cells were sequentially incubated with appropriated Abs, (diluted in 10 μg/ml BSA/PBS) for 1 h, followed by either Cy-2 or rhodamine-conjugated secondary Abs and DAPI (diluted in BSA/PBS, 1:200) for 1 h. To follow the subcellular localization of the biotin-conjugated peptides, cells were incubated with Avidin-FITC (diluted in BSA/PBS 1:400) and DAPI. Slides were analyzed and photographed by a fluorescent microscope (Nikon, Japan, 600× magnification).

Morphology and apoptosis (TUNEL) assays: Cells were seeded on glass cover slips in a 12-wells plate at 25% confluence with medium containing 1% of FCS. Peptides (EPE or Scr), DMSO, or U0126 in a final concentration of 10 μM, were added after 4 h (considered as time “0”). TUNEL staining was performed at 24 h after the treatment according to manufacturer instruction (Roche Applied Science, Nutley, N.J., USA). Briefly, the medium was removed and cells were fixed with 3% of paraformaldehyde (1.5 h, 23° C.). Cells were rinsed twice with PBS and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. Cells were rinsed twice with PBS and 28 μl of TUNEL reaction mixture (25 μl of TUNEL label, 2.5 μl of TUNEL enzyme and 0.1 mg/ml of DAPI) was added directly on top of the slide, cells were incubated for 16 h in humid box at 37° C. Cover slips were rinsed three times with PBS and mounted on microscope slides. The slides were dried and then subjected to image acquisition by a florescence microscope. For morphology assay, the cells were seeded at approximately 25% confluence in 6 cm plates with medium containing 1% FCS. Peptides (EPE and Scr) or DMSO or U0126 in a final concentration of 10 μM, were added after 4 h (considered to be time 0), 24 h and 48 h. Images were obtained by a light microscope (Olympus BX51) after 72 h.

Proliferation assay: All cells, except of MCF-10A, were seeded into 12-well cell plates in 1% FBS medium. MCF-10A cells were seeded in their complete medium diluted 5 fold. DMSO, Scr peptide, EPE peptide, or U0126 (final concentration of 10 μM each) were added to the appropriated wells four hours later. Every day medium was changed to fresh one containing the same agents. The number of viable cells was measured by Methylene Blue assay at 72h after cell seeding. Shortly, cells were fixed with 4% Formaldehyde for 2 h at 23° C., washed once with 0.1M Borate Buffer pH 8.5 and stained with of 1% Methylene Blue in 0.1M Borate Buffer for 10 min. Color was extracted by adding 0.1M HCl for 3 h at 23° C., and examined at 595 nm. For time course experiments, viable cells were measured at 0, 24, 48, 72 and 96 h after cell seeding. For dose response experiments we treaded the cells with 0.1, 1, 3, 10, or 30 μM of peptides for 72 h, as described above.

Animal studies: All animal experiments were approved by the Animal Care and Use Committee of the Weizmann Institute of Science (Rehovot, Israel). Female CD-1 nude mice (Harlan), 5-6 weeks of age, were inoculated s.c. into the flank region with 2×106MDA-MB-231, LOXIMVI, or HCT-116 cells in 150 μl PBS. Female SCID mice (Harlan), 5-6 weeks of age, were inoculated s.c. into the flank region with 107A2352 cells in 150 μl mixture of PBS with matrigel (2:1). Tumors were allowed to develop to the size of 5-6.5 mm in diameter (50˜110 mm3volume) and then the animals were randomly allocated to different treatment groups. The peptides (100 mM stock in DMSO), were diluted to the necessary concentration in PBS and boiled for 5 minutes. Then, DMSO, Scr or EPE peptides were administered by i.v. injection into the tail vein (150 μl/mouse, 3 times a week). Tumor dimensions were measured with a digital sliding caliper. Tumor volume was calculated using the formula: V=D1×D2×D3×π/6, where D1, D2, D3—represent the three mutually orthogonal growth diameters. To assess any signs of systemic toxicity, body weight was monitored, and recorded at the end of the experiment.

Histology and immunohistochemistry: Tumor xenografts, lungs, livers, kidneys and hearts of animals from different treatment groups were removed and subjected for histological analysis by staining of 5 μm paraffin embedded tissue slides with H&E, and examination by light microscope. MDA-MB-231 and LOXIMVI tumor xenografts were subjected to immunohistochemical analysis using αgERK Abs. Briefly, paraffin embedded blocks of tumors after different treatments were cut at 4 μm thickness and stained with ERK Abs, followed by second antibodies conjugated to peroxidase and DAB staining. Representative fields of each specimen were photographed at ×20 and ×40 magnifications.

Statistical analysis: Digital images were processed with Adobe Photoshop 7.0 software. The statistical differences were analyzed using two-tailed t-Students test.

RESULTS

The EPE-peptide inhibits ERK's translocation: The nuclear translocation of ERK is a key step in mediating cellular proliferation, while having only minor influence on other cellular processes. It was found that the stimulated translocation of ERK requires the binding of its phosphorylated NTS with Imp7. To prevent this interaction, the present inventors used an NTS-derived peptide (GQLNHILGILGSPSQED, SPS—SEQ ID NO: 2) that could compete with the binding. To be effective, the peptide would need to rapidly penetrate through the cell membrane and remain in the cytoplasm for a certain amount of time. To reach this goal, the present inventors examined two known ways to allow peptide penetration: modified viral TAT sequence22or myristic acid (Myr;23), both in the N-terminus of the peptide. Using biotinylated peptides with each of the leaders, it was found that both of them induced an efficient penetration into HeLa cells, but the Myr peptide remained in the cytoplasm longer than the peptide with the TAT sequence (FIGS. 7A-B).

The present inventors then undertook to examine the effect of the peptide on the nuclear translocation of ERK1/2. Treatment of HeLa cells with the SPS peptide prevented TPA-induced nuclear translocation of ERK, which was similar to the inhibition by the MEK inhibitor U0126 (FIG. 1A). Next, the efficacy of the peptide was compared to similar peptides in which the SPS motif was replaced with either phosphomimetic (EPE) or nonphosphorylatable (APA) residues. The inhibitory effect of the EPE peptide was stronger than that of the other two (FIGS. 8A-B), probably because the EPE peptide better mimics the Imp7-bound ERK; The study was therefore continued with this peptide only. Repetition of the experiments with T47D, MDA-MB-231, A2352 cells and two immortalized non-transformed cells: melanocytes (NHEM-Ad) and breast (HB2) revealed a similar effect (FIGS. 1A-F), pointing to the generality of the effect. The same trend of inhibition was observed with subcellular fractionation as well (FIGS. 12A-D). No significant differences between the inhibition of ERK1 and ERK2 were observed (FIGS. 13A-C), In addition, the effect was specific to ERK, as the peptide affected neither the translocation of other MAPKs (FIGS. 9A-B), nor that of AKT (data not shown).

Molecular effects of the EPE peptide: Next, the present inventors undertook to identify the mechanism by which the EPE peptide prevents the nuclear translocation of ERK. As expected from the origin of the EPE-peptide, it was found that its addition to HeLa cells indeed prevented the interaction of Imp7 with ERK when examined by coimmunoprecipitation with anti Imp7 Abs (FIG. 2A). The effect of the peptide on the intracellular signaling of four distinct cell lines was examined. As expected, no effect of the peptide on cytoplasmic activities was detected, including either activatory ERK-TEY phosphorylation or the downstream activity of RSK (FIG. 2B). Moreover, even the NTS phosphorylation by CK2, which occurs in the cytosol, was only slightly affected despite the consensus CK2-phosphorylation site within the peptide. This lack of effect may suggest that the binding sites of CK2 and Imp7 to the NTS are not identical, and strongly support the specificity of the peptide to ERK-Imp7 interaction. Importantly, the peptide had no effect on the basal or stimulated phosphorylation of AKT, either shortly after stimulation, or in longer time periods after treatment (FIGS. 2E,F), indicating that the negative feedback loops of the cells were not affected by the peptide. Finally, although the peptide had no cytoplasmic effects, it did inhibit the phosphorylation of the transcription factor Elk1, which is a nuclear target of ERK1/2 (FIG. 2B), the phosphorylation of c-Myc (FIG. 2C) and, to a lesser extent, the expression and phosphorylation of c-Fos (FIGS. 14A-B). This effect on nuclear targets varied among the cell lines, (20-45% in Elk1,FIG. 2D), and was not so pronounced for c-Fos, probably due to the involvement of other, ERK1/2-independent, signaling components in some cells.

The EPE peptide effects in cultured cells: Given that the nuclear activity of ERK1/2 is critical for cell proliferation, the present inventors then examined the effect of the EPE peptide on proliferation/survival of different cancer-derived and immortalized cell lines. First, the optimal administration conditions of the EPE peptide (in which it presented the maximal effect on HeLa and T47D cells compared to a scrambled (Scr) peptide control) was found to be 10 μM, administered every 24 h in fresh medium (FIGS. 9A-B). Next, the present inventors examined the effect of the peptide on proliferation of different cell lines measured 72 h after peptide administration (FIG. 3A). Interestingly, the response of the different cells to the peptide can be categorized into four types. The first one was a profound reduction in cell number, which was observed in melanoma cells with oncogenic B-Raf (B-Raf melanoma). In the second group, including breast, prostate and cervical cancer-derived cells, the peptide prevented cell growth, but did not reduce the number of initial cells. The third group that included other melanomas, prostate and lung cancer-derived cells presented a small decrease in cell growth as compared to a peptide control, and a fourth group that included immortalized, non-transformed cells, did not respond to the peptide at all. Further comparison between the effects of EPE peptide and PLX4032 on the viability of some of the cell lines (FIG. 3B), revealed that in non-transformed melanocytes (NHEM-Ad) the EPE peptide does not have any significant effect despite the strong inhibitory effect of PLX4032. On the other hand, the EPE peptide was able to reduce the viability of N-Ras transformed melanoma cells LOXIMVI that are not sensitive to PLX4032 as was previously reported. In all other transformed cell lines examined, the effect of the EPE-peptide was at least as good, or even better, than that of PLX4032. Together, these results demonstrate the superior effects of the EPE peptide in treating various cancers without affecting the non-transformed cells.

EPE peptide effect on resistant melanoma cells: The major problem with the use of B-Raf and MEK inhibitors in the clinic is the development of drug-resistance after 6 to 8 months. Since the point of influence of the EPE peptide is downstream to the other two drugs, the present inventors examined whether it might affect melanoma cells resistant to the Raf and MEK inhibitors. For this purpose PLX-4032 and U0126 resistant cells were generated by adding the inhibitor to the A2352 melanoma line for 6 weeks. The surviving cells proliferated slower in the presence of the inhibitors, but regained normal growth when the inhibitors were removed. Using these cells, it was found that the EPE peptide was able to reduce cell growth, although this effect was not as impressive as observed in the non-resistant ones (FIG. 4A).

Similar effects were seen in two low-passage melanoma cells from vemurafenib-resistant patients (FIG. 4B). As expected, the EPE peptide reduced the amount of nuclear ERK1/2 both before and after stimulation (FIGS. 15A-B). No significant effects of the peptides were detected on the activation of AKT were detected (data not shown), indicating that the effects is not through the cytosolic negative feedback loops. Thus, the present results indicate that the resistant melanoma cells are highly responsive to the EPE peptide.

Since inhibition of the MAPK cascade often results in activation of negative feedback loops and stimulation of the PI3K/AKT pathway, this pathway was examined in the resistant cells as well. The results demonstrate no significant change in the influence of the PI3K inhibitor, indicating that the resistance was probably not due to the PI3K, and the EPE peptide operated via a distinct pathway. It was also demonstrated that the effect is not due to a change in the multidrug resistance system, as it was found that Taxol was as effective in non-resistant, as resistant cells. Thus, the present results strongly indicate that the EPE peptide is able to affect the resistant cells via inhibition of downstream machinery.

The EPE peptide induces apoptosis of melanoma: The effect of EPE peptide on cell morphology was visualized using light microscopy. While no effect was observed in most cell lines examined, the peptide did change the appearance of B-Raf melanoma cells by inducing cell-break already 24 hours after treatment (FIG. 5A). A similar appearance, albeit a weaker one, was observed with the MEK inhibitor U0126 as well, indicating that this effect is likely to be MEK/ERK-dependent. This change of morphology resembled cell death, which was previously reported to occur upon inhibition of the ERK cascade in B-Raf melanoma. Indeed, using TUNEL (FIGS. 5B,C) or PARP-1 (FIG. 5D) as marker for apoptosis, it was found that the morphology change correlated with an enhanced apoptosis. This apoptotic effect was specific to B-Raf mutated melanomas and, in these cells, was as strong as the apoptosis induced by Taxol. No apoptosis was detected in the other cell lines examined, despite their clear ability to undergo a Taxol or H2O2-induced cell death.

The EPE peptide effect on cancer xenografts: The present inventors then examined the effect of the EPE peptide on the growth of tumors in xenograft models (FIG. 6A). For this purpose, the tumors were allowed to grow to a size of ˜60 mm3and only then the peptide was systemically administrated by injecting it in a proper formulation into the tail vein of the mice. Using such xenograft models in nude mice, dose dependent inhibition of the growth of MDA-MB-231, LOXIMVI was noted, and to some extent, also on HCT-116. Remarkably, an even stronger effect of the peptide was seen with a xenograft of the low passage A2352 B-Raf melanoma in SCID mice. In this model, the peptide completely irradiated the melanoma within 2 weeks of tail vein administration. None of the animal treated exhibited any significant change in weight, organ morphology or other toxicity-related effects. Moreover, the treatment did not affect the size or structure of the kidneys, livers and hearts that were inspected at the end of the experiment. Interestingly, the structure of the lungs was not affected as well, although metastatic foci lungs in vehicle- and Scr peptide-treated were found, but not EPE peptide-treated mice (not shown).

In order to verify that the EPE peptide indeed operated by preventing the nuclear translocation, sections of xenograft tumors were excised from the treated animals at the end of the experiments, and stained with anti ERK antibody (Ab). As expected, it was found that EPE peptide did prevent such translocation in the treated MDA-MB-231 and LOXIMVI xenografts. In samples from the control treated xenografts, ERK was found all over the cells, with some preference to the nucleus, while in the EPE peptide-treated xenografts, ERK was localized almost exclusively in the cytoplasm (FIGS. 10A-B). These findings support the notion that the cytoplasmatic retention of ERK is the cause for the specific effect of the EPE peptide. Therefore, the prevention of the nuclear translocation of ERK1/2, which do not affect the cytoplasmic activity of the cascade, may serve as a good tool to prevent cancer growth, with less side-effects than the currently used inhibitors of the ERK1/2 cascade.

The major problem with the use of B-Raf and MEK inhibitors in the clinic is the development of resistance after 6 to 8 months, which results in tumor and metastasis recurrence. In order to study the recurrence of the disease after EPE peptide in comparison to PLX4032 treatment, mice bearing A2352 xenografts were treated with both reagents. Both treatments were proven beneficial in reducing the size of the initial ˜80 mm3 tumors. Treatment of the EPE peptide resulted in a complete disappearance of the tumors of all mice within 10-23 days (FIG. 6B), while the PLX4032 treatment resulted in a complete disappearance in three mice (13-23 days after treatment), and two mice with small tumors. Following last treatment administration mice were kept for further follow up and evaluation of melanoma condition for up to 11 weeks. None of the EPE-peptide-treated mouse (N=7) showed any tumor recurrence, and all of them, as well as 5 other animals in a repeating experiment, remained healthy up to 11 weeks after treatment. On the other hand, as expected, in some (3 out of 5) of the PLX4032-treated mice, the tumor did recur, and one of them appeared to develop resistance within 13 days of treatment and exhibited a massive tumor growth thereafter. These results indicate that the EPE peptide treatment may prevent resistance and tumor recurrence better than that of PLX4032.

Bibliography