Anti-cancer leucin-rich peptides and uses thereof

Abstract

To provide a novel anticancer agent having improved selectivity for cancer cells so as not to damage healthy tissues.SOLUTION: A peptide having a sequence comprising the motif GLLxLLxLLLxAAG, wherein x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D), or glutamic acid (E); a pharmaceutically acceptable composition comprising the peptide; a kit comprising the composition; nucleotides encoding the peptide; and vectors expressing the peptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a family of anti-cancer peptides (ACPs) that can be used in the treatment of cancer. [Background technology]

[0002] Background of the Invention Tumors are heterogeneous at the cellular level and are composed of different subtypes of cancer cells. Among these subtypes, cancer stem cells (CSCs) are increasingly recognized as a major obstacle to conventional drug treatment with current anticancer drugs. Breast cancer is the second most common cancer worldwide, and most cases occur in women. Several studies have shown that breast cancer stem cells can acquire resistance to conventional anticancer drugs and can survive, self-renew, differentiate, and recur. 1-6 CSCs readily acquire resistance to anticancer drugs, and chemotherapy for solid tumors typically results in a significant increase in the proportion of drug-resistant CSCs in patients. This can lead to recurrence and metastasis formation. Furthermore, breast tumors can vary within the same patient and may be resistant to conventional anticancer drugs. 7-9 Commonly used anticancer drugs such as doxorubicin are generally highly toxic to healthy tissue and cause acute damage to organs such as the liver, kidneys, and heart, making treatment difficult at high doses. 10-12 Therefore, there is an urgent and unmet need to develop new anticancer drugs that have improved selectivity for cancer cells and can kill most cancers and CSCs in solid tumors at doses sufficient to spare healthy tissue. Summary of the Invention

[0003] SUMMARY OF THE INVENTION In a first aspect of the present invention there is provided a pharmaceutically acceptable composition for use in treating cancer, the composition comprising one or more peptides having a sequence comprising the motif GLLxLLxLLLxAAG, wherein each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E), and one or more pharmaceutically acceptable excipients. [Brief explanation of the drawings]

[0004] Detailed Description of the Invention The invention will now be described in detail, by way of example only, with reference to the following figures:

[0005] [Figure 1] Figure 1 shows the design of a combinatorial leucine-rich peptide library and its comparison with other pore-forming and cancer-targeting membrane-active peptides. A) The sequences of the combinatorial peptide library are shown along with their projection onto a helical wheel, which is the predicted membrane-active conformation. B) The isoelectric points and hydrophobicity of the library peptides were compared with those of other pore-forming and cancer-targeting membrane-active peptides. These peptides are 26 amino acids long and belong to the Antimicrobial Peptide Database (APD), melittin and its analogs (gain- and loss-of-function analogs), pH-dependent melittin, and the cancer-targeting low-pH insertion peptide (pHLIP).

[0006] [Figure 2]Figure 2 shows the results of in vitro cytotoxicity screening of a library of 36 combinatorial peptides (SEQ ID NOs: 1-36) against different human cell lines derived from both cancerous and healthy tissues. Also shown are in vitro cytotoxicity screening results for selected D-peptides and the clinically used anticancer drugs salinomycin and doxorubicin. Cytotoxicity was assessed in different human cell lines and quantified using half-maximal inhibitory concentrations (IC50s) for A) HMLER vs. MCF-10A, B) HMLER-shEcad vs. MCF-10A, C) HMLER vs. HMLER-shEcad, D) HMLER vs. HEK293T, E) HMLER-shEcad vs. HEK293T, and F) U2OS vs. HEK293T.

[0007] [Figure 3A-1] Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3A-2] Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3B-1]Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3B-2] Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3C-1] Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3C-2] Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line). [Figure 3D]Figure 3 shows the in vitro cytotoxicity dose response of two clinically used anticancer drugs, doxorubicin and salinomycin, two selected D-form anticancer peptides (D-DEK and D-EEK), and 36 leucine-rich anticancer peptides against different human cell lines, e.g., HMLER (triangles), HMLER-shEcad (diamonds), MCF-10A (solid line), U2OS (squares), and HEK293T (dotted line).

[0008] [Figure 4AB] Figure 4 shows the in vitro cytotoxicity and dose-response of doxorubicin (filled squares), salinomycin (filled triangles), and the leucine-rich anticancer peptides L-EEE (squares), L-DEK (circles), L-EEK (gray circles), and D-EEK (black circles) on tumorspheres (HMLER-shEcad cells). A) Cell viability was measured to quantify the efficacy of anticancer drugs on tumor cell (HMLER-shEcad) mammospheres. B) Mammosphere populations after treatment with selected anticancer compounds. The dashed line indicates the expected negative control without treatment. [Figure 4C] C) Measurements of IC50 (gray bars) and IC90 (black bars) for each anticancer drug and light microscopy images of mammospheres at the specified concentrations. Scale bar is 100 μm.

[0009] [Figure 5AB] Figure 5 shows the in vitro cytotoxicity and dose-response of doxorubicin (filled squares), salinomycin (filled triangles), and the leucine-rich anticancer peptides L-EEE (squares), L-DEK (circles), L-EEK (gray circles), and D-EEK (black circles) on mammospheres (MCA-10A cells). Cell viability was measured to quantify the efficacy of anticancer drugs on healthy human breast endothelial cell (MCA-10A) mammospheres. A) Mammosphere populations after treatment with selected anticancer compounds. The dashed line indicates the negative control without any treatment. [Figure 5C]C) Measured IC50 (solid bars) and IC90 (bars) for each anticancer drug and optical microscope images of mammospheres at specific concentrations. Scale bar is 100 μm.

[0010] [Figure 6] Figure 6 shows the in vitro cytotoxicity and dose-response profiles of doxorubicin, salinomycin, L-EEK, and D-EEK against different human cell lines: HMLER (circles), HMLER-shEcad (gray filled circles), U2OS (squares), MCF-10A (black filled circles), and HEK293T (triangles). The shaded areas indicate the ideal compound concentrations that are selective for cancer cell lines and have minimal effect on normal cell lines (MCF-10A and HEK293T).

[0011] [Figure 7] Figure 7 shows the results of a tryptophan fluorescence binding assay. The lipid concentrations at which 50% of the peptide bound to either single-lipid species POPC liposomes (circles) or mixed-lipid species POPC:POPG (3:1 ratio, squares) liposomes are shown. Briefly, 50 μM peptide was immobilized and incubated with titrated POPC vesicles (black) or 3 POPC / POPG vesicles (gray) at concentrations of 0, 12.5, 25, 50, 100, 250, 500, 1000, 2500, and 5000 μM in phosphate-buffered saline (1X, pH 7.4). The lipid concentration resulting in 50% peptide binding was determined using a tryptophan fluorescence binding assay, and the values ​​are expressed as lipids per peptide. This data demonstrates that the peptides of the present invention can distinguish between neutral (POPC) and charged (POPC / POPG) vesicles, the latter of which serve as a model for cancer cells (Warburg effect).

[0012] [Figure 8A]Figure 8 shows the peptide concentration that causes 50% leakage of ANTS / DPX dye from liposomes. Briefly, 0.5 mM POPC vesicles (gray) or POPC:POPG vesicles (3:1 ratio, black) were incubated with peptide concentrations of 0, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25, 2.5, 5, 10, and 20 μM. A) Hydrochloric acid-adjusted phosphate-buffered saline (1X, pH 4.8) and [Figure 8B] B) Phosphate-buffered saline (1X, pH 7.4). The intensity of dye leakage by peptides is reported as the number of lipids per peptide (higher values ​​indicate more potent peptides at disrupting lipid membranes).

[0013] [Figure 9] Figure 9 shows the mechanism of action of leucine-rich ACPs. A) Hemolytic activity of L-EEK (black triangles) and D-EEK (gray triangles) against human erythrocytes. B) Entry of a high-affinity nucleic acid stain (SYTOX green) into HeLa cell lines by peptides at titrating peptide concentrations: L-EEK (black triangles), D-EEK (gray triangles), and melittin (squares) as a positive control. C) Cell viability of HMLER-shEcad (human mammary endothelial carcinoma stem cells) co-incubated with necrostatin (a necroptosis inhibitor) and ZVAD-FMK (an apoptosis inhibitor) in the presence of L-EEK (black circles) and D-EEK (gray circles). D) Viability of HMLER-shEcad cells treated with doxorubicin (circles), and the combination of doxorubicin and 5 μM of the capase inhibitor z-VAD-FMK (squares), and the combination of doxorubicin and 20 μM of necrostatin-1 (triangles).

[0014] [Figure 10] FIG. 10 shows the synthetic strategy for the conjugation of the ACP of the present invention with copper-based small molecule anticancer drugs.

[0015] [Figure 11]Figure 11 shows the pore structure and membrane perforation mechanism of ACPs in atomic detail. Molecular dynamics simulations demonstrate a) spontaneous adsorption of ACPs into the membrane, b) insertion, and c) pore formation (showing a large, heterogeneous, completely water-filled EEK pore). d) e) The bound peptide forms an ensemble of transient pores of 2–16 peptides (top) that conduct both water (middle) and ions (bottom) across the membrane. DETAILED DESCRIPTION OF THE INVENTION

[0016] The inventors surprisingly discovered that a family of peptides conforming to the claimed formulations exhibits enhanced selectivity for cancer cells, leaving healthy tissue intact at doses sufficient to kill most cancers and all CSCs in solid tumors. Unlike many conventional anticancer drugs, the pore-forming membrane-active peptides developed in this invention target and disrupt cell membranes to kill cancer cells. This avoids the cumbersome task of transporting drugs into the cytoplasm, thus improving tumor penetration compared to traditional chemotherapy drugs. The claimed peptides selectively act on the cell membranes of cancer cells, forming pores therein, thereby short-circuiting their electrochemical gradient and killing them. Without wishing to be bound by theory, it is believed that the peptides can directly target the lipid composition and chemical microenvironment of cancer cell membranes. As a result, the peptides are less likely to induce resistance (similar to the difficulty cells have in developing resistance to detergents) because tumor cells have difficulty modifying their lipid composition. 13-15 .

[0017] Some of the disclosed peptides have nanomolar activity against most cancers and CSCs, comparable to currently approved anticancer drugs such as salinomycin. Furthermore, in the mammosphere model, one of the best in vitro breast cancer models to date, which mimics actual solid tumors by growing cells into spherical masses, some of the peptides disclosed herein exhibit excellent activity against cancer cells while minimizing toxicity to normal, healthy cells.

[0018] The peptides work in both L- and D-amino acid forms (the latter having the major advantage of in vivo stability against protease degradation) and selectively eliminate cancer cells grown in two dimensions as well as in three-dimensional cultures (spheroids) at very low micromolar and, in some cases, nanomolar concentrations. Concentrations 3-200 times higher are required to damage non-cancerous human breast and kidney cells.

[0019] Peptides are inexpensive and easy to synthesize, amenable to modification and high-throughput screening, and offer a chemical and structural repertoire for specifically targeting cancer cells.

[0020] The claimed peptides were designed de novo and have no known natural analogs, as can be confirmed by comparison with existing peptide databases. Short, flexible peptides of this type will have low immunogenicity and are therefore suitable for pharmaceutical applications.

[0021] As used herein, the term "peptide" refers to any peptide comprising amino acids joined to one another by peptide bonds or modified peptide bonds, i.e., peptide isosteres. The peptide will generally comprise naturally occurring amino acids but may include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques well known to those skilled in the art. Such modifications are well described in basic textbooks. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains, and its amino or carboxy termini. It will be understood that the same type of modification can be present in the same or varying degrees at several sites in a given peptide. Also, a given peptide may contain many types of modifications.

[0022] Preferably, the peptide is an isolated peptide. The term "isolated" means that the peptide is removed from its original environment. For example, a peptide present in a living animal is not isolated, but the same peptide, or a fragment of such a peptide, separated from some or all of the coexisting substances in nature is isolated. Such a peptide can be part of a vector and / or a peptide can be part of a composition, but such a vector or part of a composition is still isolated in that it is not part of its natural environment.

[0023] Pharmaceutical compositions containing peptides can be for human or animal use in human and veterinary medicine and will typically contain one or more suitable additives. Acceptable additives for therapeutic use are well known in the pharmaceutical industry and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed. 1985). The choice of pharmaceutical additive can be selected taking into account the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain, as or in addition to additives, any suitable binder, lubricant, suspending agent, coating agent or solubilizer.

[0024] Preservatives, stabilizers, and dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0025] The pharmaceutical composition may also contain tolerance-promoting adjuvants and / or tolerance-promoting cells. Tolerance-promoting adjuvants include IL-10, recombinant cholera toxin B subunit (rCTB), ligands for Toll-like receptor 2, and biologics and monoclonal antibodies that modulate immune responses, such as anti-CD3 and costimulatory blockers, which may be co-administered with the peptide. Tolerance-promoting cells include immature dendritic cells and dendritic cells treated with vitamin D3 (1α,25-dihydroxyvitamin D3) or its analogs.

[0026] When cancer is "treated," this means that one or more clinical symptoms of the cancer are improved. It does not mean that the symptoms of the cancer are completely cured and no longer present in the patient, although in some methods this may be the case. "Treatment" results in one or more symptoms of the cancer being less severe than they were before treatment. For example, a tumor may be reduced in size or completely eradicated.

[0027] A second aspect of the present invention relates to a pharmaceutically acceptable composition for use in the manufacture of a medicament for the treatment of cancer, the composition comprising one or more peptides having a sequence comprising the motif GLLxLLxLLLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E) and one or more pharmaceutically acceptable excipients.

[0028] In one embodiment, the peptide may comprise a sequence selected from any one or combination of SEQ ID NOs: 1-36. In a further embodiment, the peptide may consist of any one of SEQ ID NOs: 1-36.

[0029] In one embodiment, the pharmaceutically acceptable composition comprises a peptide having a sequence comprising the motif GLLxLLELLLxAAG, where x is selected from arginine (R), histidine (H), lysine (K), aspartic acid (D), or glutamic acid (E), and combinations thereof. The inventors have surprisingly discovered that peptides of this sequence have superior cancer cell selectivity.

[0030] In one embodiment, the pharmaceutically acceptable composition comprises a peptide having a sequence comprising the motif GLLxLLxLLLxAAG, where x is selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E) and combinations thereof, wherein the sequence does not include SEQ ID NO:29 or SEQ ID NO:33.

[0031] In one embodiment, the pharmaceutically acceptable composition comprises a sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:25 or SEQ ID NO:26, and combinations thereof. More preferably, the pharmaceutically acceptable composition comprises a sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:25 or SEQ ID NO:26, and combinations thereof. Even more preferably, the pharmaceutically acceptable composition comprises a sequence selected from SEQ ID NO:25 and / or SEQ ID NO:26. The inventors have discovered that these sequences are particularly selective for cancer cells.

[0032] The pharmaceutically acceptable compositions of the present invention can be used to treat any type of cancer, such as skin cancer, lung cancer, breast cancer, prostate cancer, colon cancer, bladder cancer, lymphoma, kidney cancer, pancreatic cancer, or endometrial cancer, however, in certain embodiments of the present invention, the cancer is breast cancer.

[0033] In one embodiment, the pharmaceutically acceptable composition comprises a peptide and further comprises a tryptophan residue (W) at the C-terminus of the motif, which aids in accurate concentration measurement and precise dosing.

[0034] The N- and C-termini of the peptide sequence or motif may be any termini known to those skilled in the art, and may be, for example, NH2, NH3 + , COOH and COO - may include:

[0035] In one embodiment, the pharmaceutically acceptable composition comprises a peptide, wherein the peptide sequence consists of the motif GLLxLLxLLLxAAG.

[0036] In one embodiment of the present invention, the composition is intended for use in combination with a chemotherapeutic agent. The inventors have discovered that the pore-forming properties of the claimed peptides facilitate access of standard chemotherapeutic agents to target cancer cells. The chemotherapeutic agent is selected from cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, docetaxel, bleomycin, vinblastine, dacarbazine, mustine, vincristine, procarbazine, prednisolone, etoposide, cisplatin, epirubicin, methotrexate, capecitabine, vinorelbine, folic acid, oxaliplatin, and combinations thereof. Preferably, the chemotherapeutic agent is doxorubicin. Figure 10 provides an example of a means of conjugating the peptide to a chemotherapeutic agent.

[0037] Depending on the delivery system selected, different composition / formulation requirements may apply to the pharmaceutical composition. For example, the pharmaceutical compositions of the present invention may be formulated for parenteral delivery, where the composition is formulated in an injectable form for delivery via, for example, intravenous, intradermal, intramuscular, subcutaneous, or intraperitoneal routes. For parenteral administration, the composition may best be used in the form of a sterile aqueous solution that may contain other substances, such as sufficient salts or monosaccharides to make the solution isotonic with blood. Intradermal administration routes include any intradermal access means, such as microneedle-based injection and infusion systems (or other means for precisely targeting the intradermal space), needleless or needle-free ballistic injection of fluids or powders into the intradermal space, Mantoux-type intradermal injection, microdevice-assisted iontophoresis, and direct application of liquid, solid, or other dosage forms to the skin, including the use of patches to apply the composition to the skin. The compositions may also be formulated for oral or topical administration, including intranasal, oral, or epicutaneous administration. Preferably, the compositions are formulated for intravenous delivery.

[0038] The amount or dose of the disclosed anti-cancer peptides administered should be sufficient to effectively target cancer cells in vivo, which will be determined by the efficacy of the particular formulation and the location of the tumor in the subject, as well as the body weight of the subject being treated.

[0039] The dosage of the disclosed anti-cancer peptides will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular formulation. Typically, a physician will determine the dosage of peptide to treat an individual subject, taking into account various factors, such as age, body weight, general health, diet, sex, the compound / formulation administered, the route of administration, and the severity of the condition being treated. Approximate dosages can be determined by those skilled in the art. As non-limiting examples, the total dosage of the anti-cancer peptides of the present invention can be about 0.001 to about 1,000 mg / kg of body weight, about 0.01 to about 100 mg / kg of body weight, about 0.1 mg / kg to about 10 mg / kg of body weight, and about 0.5 mg to about 5 mg / kg of body weight of the subject being treated. In another embodiment, the total dosage of the peptide can be at a concentration of about 1 nM to about 10,000 nM, preferably about 10 nM to about 5,000 nM, and more preferably about 100 nM to about 500 nM.

[0040] In a preferred embodiment, the composition comprising the peptide of the present invention is administered four times, at least once a month, preferably once every 1 to 4 weeks.

[0041] The peptides can be present in either the D- or L-form. In one embodiment, the pharmaceutically acceptable composition comprises the peptide in the L-form. Surprisingly, the inventors have discovered that the peptides provided herein are more selective for cancer cells when in the L-form.

[0042] In one embodiment, the pharmaceutically acceptable composition comprises a peptide that forms an alpha-helical assembly. Preferably, the peptide forms a pore in the cancer cell membrane. It is believed that the peptide directly targets the lipid composition and chemical microenvironment of the cancer cell membrane, forming a pore therein and shunting their electrochemical gradient, thereby killing the cancer cells.

[0043] A third aspect of the present invention relates to a method of treating cancer, in which a pharmaceutically acceptable composition of the present invention is administered to a patient with cancer, hi one embodiment, the cancer is breast cancer.

[0044] A fourth aspect of the invention relates to a peptide having a sequence comprising the motif GLLxLLELLLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E).

[0045] A fifth aspect of the invention relates to a peptide having a sequence comprising the motif GLLxLLxLLLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E), and which sequence does not include SEQ ID NO:29 or SEQ ID NO:33.

[0046] A sixth aspect of the present invention relates to a kit for treating cancer comprising a pharmaceutically acceptable composition of the present invention. In a preferred embodiment, the kit is for treating breast cancer. The kit further comprises a chemotherapeutic agent.

[0047] A seventh aspect of the present invention relates to a nucleotide sequence encoding a peptide comprising any one of SEQ ID NOs: 1 to 36.

[0048] An eighth aspect of the present invention relates to a vector that expresses a peptide comprising any one of SEQ ID NOs: 1 to 36 and a combination thereof.

[0049] The vector can be any suitable vector for expressing the peptide of the present invention, including viral and non-viral vectors. Viral vectors include parvovirus, adenovirus, retrovirus, lentivirus, or herpes simplex virus. The parvovirus can be adenovirus-associated virus (AAV). The vector is preferably a recombinant adeno-associated virus (rAAV) vector or a lentivirus vector. More preferably, the vector is a rAAV vector.

[0050] The vector according to the present invention may be a gene delivery vector. Such a gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector.

[0051] Thus, the present invention provides gene delivery vectors and components thereof (e.g., animal parvovirus genomes) based on animal parvoviruses, particularly dependoviruses such as infectious human or simian AAV, for use as vectors for introducing and / or expressing the peptides of the present invention into mammalian cells. The term "parvovirus," as used herein, therefore, encompasses any type of dependovirus, such as AAV.

[0052] Those skilled in the art will understand that all aspects of the present invention, whether they relate to, for example, pharmaceutically acceptable compositions, peptides, their uses, or methods of treatment, are equally applicable to all other aspects of the present invention. In particular, aspects such as pharmaceutically acceptable compositions may be described in more detail than other aspects of the present invention, such as the peptides themselves. However, those skilled in the art will understand that when more detailed information is given about a particular aspect of the present invention, this information is equally applicable generally to other aspects of the present invention. [Example]

[0053] Example 1 material and method Peptide synthesis and purification The peptides were synthesized on a solid phase and purified to 98% purity. The purity and identity of the peptides were confirmed by HPLC and ESI mass spectrometry. The N-terminus was a free amino group, and the C-terminus was either a free carboxyl group or amidated.

[0054] Liposome preparation Lipids, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), were purchased from Avanti Polar Lipids and dissolved in chloroform. Large unilamellar vesicles (LUVs) were prepared by extrusion through a 100 nm pore filter using an extruder and filters purchased from Avanti Polar Lipids.

[0055] Cell lines and cell culture conditions HMLER (human mammary endothelial carcinoma cells), HMLER-shEcad (human mammary endothelial carcinoma stem cells), and MCF-10A (healthy human mammary endothelial) cells were maintained in mammary epithelial cell growth medium (MEGM) containing supplements and growth factors: bovine pituitary extract (BPE), hydrocortisone, human epidermal growth factor (hEGF), insulin, and gentamicin / amphotericin-B. HEK293T (human embryonic kidney cells) and U2OS (Homo sapiens osteosarcoma) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Cells were cultured in T75 flasks at 310°C in a humidified atmosphere containing 5% CO2.

[0056] Cytotoxicity assay To measure the toxicity of anticancer peptides and conventional anticancer drugs, a colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used. 3Cells were seeded into each well of a 96-well microplate. The cells were incubated overnight. High concentrations of compounds (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 μM) were added and incubated for 72 hours in a total volume of 200 μL. Compound stock solutions were prepared as 5 mM solutions in DMSO and diluted using culture medium or pure water. The final concentration of DMSO in each well was either 0.5% or 0%, the same amount present in the untreated control. After 72 hours, 20 μL of a 4 mg / mL MTT solution in PBS was added to each well, and the plate was incubated for an additional 4 hours. The MEGM / MTT mixture was aspirated, and 100 μL of DMSO was added to dissolve the resulting purple formazarin crystals. The absorbance of the solution in each well was read at a wavelength of 550 nm. Absorbance values ​​were normalized to control wells containing either DMSO or no DMSO and plotted as test compound concentration versus % cell viability. 50 Values ​​were interpolated from the dose-response curves obtained. Reported IC 50 Values ​​are the average of two independent experiments with six replicates per concentration level (n=12 overall). IC of 36 leucine-rich based peptides 50 Values ​​were the mean of two independent experiments (n=2 overall).

[0057] Tumorsphere formation and viability assays HMLER-shEcad cells (5x10 3 ) were seeded into low-binding 96-well plates (Corning) and incubated for 5 days in MEGM supplemented with B27 (Invitrogen), 20 ng / mL EGF, and 4 μg / mL heparin (Sigma). Studies were performed in the absence and presence of anticancer peptides, doxorubicin, and salinomycin. Mammospheres treated with anticancer peptides, doxorubicin, and salinomycin were counted and imaged using the inverted-based reagent TOX8 (Sigma). After 16 hours of incubation, the fluorescence of the solution was measured at 590 nm (λ exThe fluorescence was read at a wavelength of 560 nm (IC = 560 nm). Viable mammospheres showed a decrease in the amount of oxidized TOX8 with a concomitant increase in the amount of fluorescent TOX8 intermediates, indicating the degree of mammosphere cytotoxicity caused by the test compound. Fluorescence values ​​were normalized to DMSO-containing or DMSO-free controls and plotted as test compound concentration versus % mammosphere viability. IC 50 Values ​​were interpolated from the dose-response curves obtained. Reported IC 50 Values ​​are the mean of two independent experiments with two replicates for each concentration level (n=4 overall).

[0058] Tryptophan fluorescence binding assay Peptide (50 μM) and POPC / POPG LUVs (600 μM) were prepared in 10 mM phosphate buffer (pH 7.0). The solution was incubated and measured after 60 minutes. The excitation light was fixed at 280 nm (slit 9 nm), and fluorescence was collected between 300 and 450 nm (slit 9 nm). BioTek's Synergy H1 Hybrid Multimode Reader (Figure 3A) and Cytation (商標) Spectra were recorded using a 5 cell imaging multimode reader (Figure 2) and averaged over three scans.

[0059] Liposome leakage assay 5 mM ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt) and 12.5 mM DPX (p-xylene-bis-pyridinium bromide) were encapsulated in extruded lipid-containing vesicles with a diameter of 0.1 μm. Gel filtration chromatography using Sephadex G-100 (GE Healthcare Life Sciences Inc.) was used to remove external free ANTS / DPX from the encapsulated LUVs. LUVs were diluted to 0.5 mM and used to measure leakage activity by adding an aliquot of the peptide. Leakage was measured after 3 hours of incubation. 10% Triton was used as a positive control to determine the maximum leakage of the vesicles. Fluorescence emission spectra were recorded using a BioTek Synergy H1 Hybrid Multimode Reader with an excitation wavelength of 350 nm and an emission wavelength of 510 nm for ANTS / DPX.

[0060] Homolysis assay Peptides were serially diluted in PBS starting at a concentration of 100 μM. The final volume of peptide in each well was 50 μL. Each well contained 2×10 8 50 μL of RBCs in PBS at 1000x g was added at 1000x g / mL. 1% Triton was used as a positive lysis control. The mixture was incubated at 37°C for 1 hour and then centrifuged at 1000x g for 5 minutes. After centrifugation, 10 μL of the supernatant was transferred to 90 μL of DI H2O in a new 96-well plate. The absorbance of released hemoglobin at 410 nm was recorded, and fractional hemolysis was calculated based on the 100% and 0% lysis controls.

[0061] Cytotoxicity measurement against HeLa cells using Sytox Green assay Hela cells were grown to confluence in T-75 flasks in complete DMEM (10% FBS). The day before the cytotoxicity experiment, cells were trypsinized, detached from the flask, and pelleted at 1300 rpm. The trypsin and spent medium were discarded, and the cells were resuspended in complete DMEM. Cell counts were obtained using a cell counter. The cells were then seeded at a density of 10,000 cells / well in 96-well tissue culture plates. The next day, peptides were serially diluted in complete DMEM (10% FBS) and 0.1% Sytox green in another 96-well plate, starting with concentrations of 100 μM (first), 67 μM (second), followed by a 2:3 serial dilution. The final volume of peptide in each well was 100 μL. To perform the cytotoxicity assay, the medium was removed from the wells and replaced with the peptide / DMEM / Sytox green solution. No peptide was used as a negative control, and 20 μM MelP5 was used as a positive control. Fluorescence readings were taken every 5 minutes for 1 hour at excitation and emission wavelengths of 504 and 523 nm, respectively. Cytotoxicity was calculated based on the 100% and 0% lysis controls, based on Sytox green entering the cells due to cell wall destabilization.

[0062] Molecular Dynamics Simulation and Analysis Unbiased all-atom MD simulations were performed using GROMACS 2018.3 ( www.gromacs.org ), Hippo BETA ( http: / / www.biowerkzeug.com ), and VMD ( http: / / www.ks.uiuc.edu / Research / vmd / ).

[0063] Extended peptide structures were generated using Hippo BETA. These initial structures were relaxed through 200 Monte Carlo steps, with water implicitly treated using Generalized Born solvent. After relaxation, the peptides were placed in an atomic-level peptide / lipid / water system containing a model membrane containing 100 mM K and Cl ions using CHARMM-GUI (http: / / www.charmm-gui.org / ). Protein folding simulations were equilibrated for 10 ns with positional constraints on the peptide. For pore formation simulations, a single peptide was allowed to fold on the bilayer membrane for up to 600 ns. Once a stable surface state was obtained, the system was multiplied 4x4 in the x and y directions (excluding the z direction) to form a system consisting of 16 peptides. Starting with peptides on both sides of the membrane, the initial structure had one peptide in the top and one in the bottom leaflet. A larger system was then constructed by multiplying 3x3, resulting in a simulation box consisting of 18 peptides. MD simulations were performed in GROMACS 2018.3 using the CHARMM36 force field with the TIP3P water model. Electrostatic interactions were calculated using PME, and a 10 Å cutoff was used for van der Waals interactions. The integration time step was 2 fs, and the neighbor list was updated every 5 steps. All simulations were performed in the NPT ensemble without any restraints or bias potentials. Water and protein were coupled separately to a thermal bath with a time constant τ = 0.5 ps using velocity-rescaled temperature coupling. Atmospheric pressure of 1 bar corresponds to a compressibility factor κ = κ xy =4.6 10 -5 bar -1 , maintained using a weak semi-isotropic pressure coupling with a time constant τP = 1 ps.

[0064] Oligomer population analysis To identify the most abundant pore assemblies during the simulation, a complete list of all oligomers was generated for each trajectory frame. An n-oligomer was defined as a set of n peptides in contact with each other, with a minimum heavy atom (N, C, O) distance of <3.5 Å. This definition often overcounts oligomeric states due to the large number of transient, surface-bound (S-state) peptides that are only loosely bound to the transmembrane peptides that make up the oligomer's core. These S-state peptides frequently change position and move in and out of the stable portion of the pore. To focus the analysis on longer-lived TM pores, a cutoff criterion of 75° for the peptide tilt angle τ was introduced. Peptides with τ ≥ 75° were considered to be in the S-state and were excluded from the oligomer analysis. This strategy significantly reduced noise in the oligomer clustering algorithm by focusing on true long-lived pore structures. Next, a population plot was generated by multiplying the occupancy of oligomer n by the number of peptides in it, n. This revealed how much of the peptide mass was concentrated in which oligomeric state during the simulation time.

[0065] Permutational Cluster Analysis All oligomers of the same order n were conformationally clustered using a clustering algorithm with a cutoff criterion of 4 Å for backbone RMSD similarity. Because each oligomer may consist of different peptides, and even the same peptides may be ordered differently, clustering was performed by comparing n! permutations of the peptide arrangement of one oligomer with another. Permutations were generated using Heap's algorithm. The final RMSD value for conformational similarity was considered to be the lowest RMSD value obtained from the n! permutation comparisons. The clustering results were generally flat, indicating that the structures are highly ephemeral and dynamic.

[0066] Membrane flux The water and ion fluxes through the membrane pores were calculated by measuring the sum of the instantaneous fluxes through the entire bilayer patch. Two planes perpendicular to the membrane normal were considered as z = -7 Å and z = +7 Å, and all transition events crossing these planes were counted. The number of transitions was then divided by the area of ​​the membrane patch and the elapsed time of each trajectory frame to obtain the flux. The curves were then smoothed by averaging 1000 frames.

[0067] Example 2 Peptide Rationale Table 1 below includes 36 peptides that fall within the scope of this disclosure.

[0068] [Table 1-1] [Table 1-2] [Table 1-3]

[0069] The interfacial binding free energy is a measure of how well a peptide binds to a membrane, and the hydrophobic moment is a measure of how evenly hydrophobic residues are distributed around the peptide surface in a helical, membrane-inserted, conformation.

[0070] To quantify the peptide concentration, an additional tryptophan was introduced at the C-terminus. To further enhance membrane permeability, a charged carboxyl group (-CO2 -) was changed to a neutral amide group (-NH2). This peptide was designed so that the charged residues are located on the same polar face of the helical structure. Therefore, the distribution of charges can affect the peptide's hydrophobic moment, pKa, binding strength to cancer cell membranes, and ultimately the structure of the peptide assembly within the cancer cell membrane (Figure 1A). Many pH-dependent peptides with cancer-targeting biomedical applications have a pKa of ~4.0. This may be due to the slightly acidic microenvironment of cancer cells, which is caused by the Warburg effect. Therefore, it is believed that cancer cell membranes can protonate the negatively charged amino acids of this invention, resulting in pH-triggered membrane activity (Figure 1B and Table 1). 16-19 .

[0071] All 36 leucine-rich peptide sequences were synthesized as L-forms. 界面 represents the binding free energy of peptide partition between the water and membrane interface. ΔG 界面 The hydrophobic moment was estimated using the Wimley-White hydrophobicity scale with MPEx software. The binding free energy is the energy released when the peptide binds to the membrane. A value of 0 indicates that the peptide is 50% in water and 50% on the membrane; negative indicates preferential insertion; positive indicates a preference for the aqueous phase. The hydrophobic moment is a measure of how the hydrophobic residues are arranged around the helical ring; a large moment indicates that they are all clustered on one side, while a small moment indicates that they are evenly distributed around the ring. A large moment favors surface binding (i.e., the hydrophobic face is submerged in the bilayer, and the hydrophilic face faces the water surface).

[0072] Example 3 Cytotoxicity and efficacy The peptides were screened against several different human cell lines to measure their cytotoxicity. The cell lines used included MCF-10A (human breast epithelial cells), HMLER (human breast cancer bulk cells), HMLER-shEcad (human breast cancer stem cells), HEK293T (human embryonic kidney cells), and U2OS (human osteosarcoma). The peptides were as potent as conventional anticancer drugs, eliminating cancer cells at low micromolar concentrations, and many were highly selective for cancer cell lines (Figure 2 and Table 1). While both doxorubicin and salinomycin also showed selectivity for transformed HMLER over healthy MCF-10A cells, they both showed significant toxicity against HEK293T cells. Furthermore, both drugs were much less effective at eliminating cancer cells grown as three-dimensional mammospheres, currently considered a more accurate in vitro model of solid tumors. Half-maximal inhibitory concentrations (IC) of doxorubicin and salinomycin against HMLER-shEcad in two dimensions 50 ) are 2.5 ± 0.3 nM and 370 ± 0.5 nM, respectively. However, in the more realistic three-dimensional cell culture model of mammospheres, which is much closer to in vivo conditions, these values ​​drop to 43 ± 6 μM and 22 ± 5 μM, respectively, representing a 1700-fold decrease in doxorubicin activity and a 63-fold decrease in salinomycin activity. See Table 2 below and Figure 3. In contrast, EEK (GLLELLLKAAGW) and its D-isomer peptides exhibit nano- to low-micromolar activity in two-dimensional cultures of HMLER, HMLER-shEcad, and U2OS cells, and 7-13 μM activity in mammospheres, demonstrating efficacy in both two-dimensional and three-dimensional mammosphere tumor models. See Figures 4 through 6.

[0073] All data points were performed in duplicate. Selected D-peptides, conventional anticancer drugs, EEK peptide, and 25B2 peptide were repeated six times. †N-terminus is free, C-terminus: -WNH2.

[0074] [Table 2-1] [Table 2-2]

[0075] Example 4 Tryptophan binding assay and liposome leakage assay The peptides of the present disclosure are mostly neutral or anionic and do not contain many positive charges in the sequence (Table 1). We have demonstrated high selectivity for cancer cell lines, including MCF-10A (IC 50 We identified six sequences: EEE, KEE, EHE, EEH, DEK, and EEK, that have negligible effects on cytotoxicity (≥100 μM) and relatively low cytotoxicity against HEK293T cells (Figure 2 and Table 2). Their net charges range from -2 to 0, with pKa values ​​ranging from 3.85 to 7.96. These sequences contain either one positive charge (a positively charged N-terminus) or two positive charges (one positively charged N-terminus and one lysine at the 4th or 11th position). Several studies have shown that cancer cell membranes can have negatively charged membrane surfaces. 20,21 Ishikawa et al. found that, like HMLER, the breast cancer cell line MCF-7 contains small amounts of negatively charged sialic acid on its membrane surface. 20 This suggests that the anticancer activity and cell selectivity of this leucine-rich peptide cannot be explained solely by electrostatic interactions, but may be related to charge distribution in the cancer cell microenvironment due to the Warburg effect. To confirm this hypothesis, we performed tryptophan binding assays (see Table 3 and Figure 7 below) and ANTS / DPX liposome leakage assays (see Table 4 and Figure 8 below) using two different lipid model vesicles (zwitterionic POPC and anionic 3POPC / 1POPG mixture) at pH 7.4 (physiological conditions) and pH 4.8 (weakly acidic), respectively.

[0076] Table 3 shows the 50% peptide binding to liposomes as a function of lipid concentration. 50 μM peptide was immobilized and incubated with titrated lipids (POPC vesicles or 3POPC / POPG vesicles) at concentrations of 0, 12.5, 25, 50, 100, 250, 500, 1000, 2500, and 5000 μM in phosphate-buffered saline (1X, pH 7.4). The lipid concentration resulting in 50% peptide binding was determined using a tryptophan fluorescence binding assay, and values ​​are expressed as lipids per peptide. †N-terminus is free; C-terminus: -W-NH2.

[0077] Table 4 shows the leakage of 50% ANTS / DPX liposomes as a function of peptide concentration. 0.5 mM POPC and 3POPC / 1POPG vesicles were immobilized and incubated with titrated peptide concentrations (0, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25, 2.5, 5, 10, and 20 μM) in phosphate-buffered saline (1X, pH 7.4) and HCl-adjusted phosphate-buffered saline (1X, pH 4.8). Values ​​are expressed as lipid per peptide. †N-terminus is free; C-terminus: -W-NH2.

[0078] [Table 3-1] [Table 3-2]

[0079] [Table 4-1] [Table 4-2]

[0080] The results showed that the cell-selective peptides did not have any significant binding selectivity or peptide-induced liposome leakage between zwitterionic and anionic vesicles at neutral pH, whereas four of the six membrane-selective peptides (EHE, EEH, DEK, and EEK) had relatively high liposome leakage activity from anionic vesicles at pH 4.8. This suggests that these four peptides are environmentally triggered membrane-active peptides that depend on both lipid composition and pH conditions; however, the mechanisms of the other two membrane-selective peptides (EEE and KEE) remain unclear.

[0081] Example 5 Mechanism of action of leucine-rich peptides Figure 9 shows that the L-form of EEK causes minimal lysis at concentrations below 90 μM, well below the therapeutic concentration of ~10 μM. D-form of EEK is more soluble. Comparing the concentration-dependent transduction of SYTOX green, a high-affinity nucleic acid stain, into HeLa cells showed that L- and D-forms of EEK behave similarly to melittin, a potent pore-forming peptide. Collectively, these results demonstrate selective pore formation in cancer cell membranes as a mechanism of action.

[0082] Figure 9C shows that the cell viability of HMLER-shEcad cells treated with L- or D-EEK could not be improved by co-incubation with the necroptosis inhibitor necrostatin or the apoptosis inhibitor z-VAD-FMK, suggesting that ACPs induce necrosis by forming pores in the cell membrane. On the other hand, Figure 9D shows that the cell viability of HMLER-shEcad cells treated with doxorubicin could be dramatically improved by co-incubation with either z-VAD-FMK or necrostatin.

[0083] Taken together, these results suggest that the primary mechanism of ACP's anticancer effect is through selective formation of pores in the cell membrane of cancer cells, resulting in necrosis.

[0084] Example 6 Structure and function of the APC pore Membrane-spanning peptides typically form transient pores, which cannot be experimentally determined using current techniques. To clarify the molecular mechanisms underlying membrane perforation pores, we studied the folding-partitioning and pore formation of EEK using unbiased, long-timescale, atomistically detailed molecular dynamics simulations. ACPs rapidly absorb and fold at the membrane interface (Figure 14a). Then, over a timescale of tens of microseconds, ACPs cooperatively insert and translocate through the lipid bilayer, filling both membrane interfaces (Figure 14b), forming an ensemble of pores (Figure 14d). Structural analysis revealed that the pore structure is highly heterogeneous, consisting mostly of 6–10 peptides, which constantly form and disassemble within the membrane (Figure 14e). The pore conducts both water and ions (Figure 14d), and leakage is dominated by a larger, stable pore consisting of 10–12 peptides that form a large aqueous channel lined with polar and charged side chains (Figure 14c).

[0085] [Table 5-1] [Table 5-2]

[0086] References All patent and scientific literature cited herein is incorporated by reference in its entirety. 1. Nguyen, LV; Vanner, R.; Dirks, P.; Eaves, CJ, Cancer stem cells: an evolving concept. Nat Rev Cancer 2012, 12 (2), 133-43. 2. Plaks, V.; Kong, N.; Werb, Z., The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells? Cell Stem Cell 2015, 16 (3), 225-38. 3. Dean, M.; Fojo, T.; Bates, S., Tumour stem cells and drug resistance. Nat Rev Cancer 2005, 5 (4), 275-84. 4. Marx, J., Molecular biology. Cancer's perpetual source? Science 2007, 317 (5841), 1029-31. 5. Kaiser, J., The cancer stem cell gamble. Science 2015, 347 (6219), 226-9. 6. Pattabiraman, D. R.; Weinberg, R. A., Tackling the cancer stem cells - what challenges do they pose? Nat Rev Drug Discov 2014, 13 (7), 497-512. 7. Gupta, P. B.; Onder, T. T.; Jiang, G.; Tao, K.; Kuperwasser, C.; Weinberg, R. A.; Lander, E. S., Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009, 138 (4), 645-659. 8. Bao, S.; Wu, Q.; McLendon, R. E.; Hao, Y.; Shi, Q.; Hjelmeland, A. B.; Dewhirst, M. W.; Bigner, D. D.; Rich, J. N., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444 (7120), 756-60. 9. Papaccio, F.; Paino, F.; Regad, T.; Papaccio, G.; Desiderio, V.; Tirino, V., Concise Review: Cancer Cells, Cancer Stem Cells, and Mesenchymal Stem Cells: Influence in Cancer Development. Stem Cells Transl Med 2017, 6 (12), 2115-2125. 10. Izzedine, H.; Perazella, M. A., Anticancer Drug-Induced Acute Kidney Injury. Kidney Int Rep 2017, 2 (4), 504-514. 11. Rosner, M. H.; Perazella, M. A., Acute Kidney Injury in Patients with Cancer. N Engl J Med 2017, 377 (5), 500-501. 12. Rosner, M. H.; Capasso, G.; Perazella, M. A., Acute kidney injury and electrolyte disorders in the critically ill patient with cancer. Curr Opin Crit Care 2017, 23 (6), 475-483. 13. Wimley, W. C.; Hristova, K., Antimicrobial peptides: successes, challenges and unanswered questions. J Membr Biol 2011, 239 (1-2), 27-34. 14. Shai, Y., Mode of action of membrane active antimicrobial peptides. Biopolymers 2002, 66 (4), 236-48. 15. Gaspar, D.; Veiga, A. S.; Castanho, M. A., From antimicrobial to anticancer peptides. A review. Front Microbiol 2013, 4, 294. 16. Andreev, O. A.; Engelman, D. M.; Reshetnyak, Y. K., pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents. Mol Membr Biol 2010, 27 (7), 341-52. 17. Andreev, O. A.; Karabadzhak, A. G.; Weerakkody, D.; Andreev, G. O.; Engelman, D. M.; Reshetnyak, Y. K., pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path. Proc Natl Acad Sci U S A 2010, 107 (9), 4081-6. 18. An, M.; Wijesinghe, D.; Andreev, O. A.; Reshetnyak, Y. K.; Engelman, D. M., pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation. Proc Natl Acad Sci U S A 2010, 107 (47), 20246-50. 19. Wyatt, L. C.; Moshnikova, A.; Crawford, T.; Engelman, D. M.; Andreev, O. A.; Reshetnyak, Y. K., Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. Proc Natl Acad Sci U S A 2018, 115 (12), E2811-E2818. 20. Ishikawa, K.; Medina, S. H.; Schneider, J. P.; Klar, A. J. S., Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide. Cell Chem Biol 2017, 24 (2), 149-158. 21. Freire, J. M.; Gaspar, D.; Veiga, A. S.; Castanho, M. A., Shifting gear in antimicrobial and anticancer peptides biophysical studies: from vesicles to cells. J Pept Sci 2015, 21 (3), 178-85.

Claims

1. 1. A pharmaceutically acceptable composition for use in the treatment of cancer, said composition comprising one or more peptides having a sequence comprising the motif GLLxLLxLLLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E), and one or more pharmaceutically acceptable excipients.

2. 1. A pharmaceutically acceptable composition for use in the manufacture of a medicament for the treatment of cancer, said composition comprising one or more peptides having a sequence comprising the motif GLLxLLxLLLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (E), and one or more pharmaceutically acceptable excipients.

3. 10. The pharmaceutically acceptable composition of any one of the preceding claims, wherein the motif is GLLxLLELLLxAAG.

4. 10. The pharmaceutically acceptable composition of claim 1, wherein the sequence does not include SEQ ID NO: 29 or SEQ ID NO:

33.

5. 3. The pharmaceutically acceptable composition of claim 1 or claim 2, wherein the sequence comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 25 or SEQ ID NO: 26 and combinations thereof.

6. 4. The pharmaceutically acceptable composition of claim 3, wherein the sequence comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 25 or SEQ ID NO: 26 and combinations thereof.

7. 4. The pharmaceutically acceptable composition of claim 3, wherein the sequence comprises a sequence selected from SEQ ID NO: 25 and / or SEQ ID NO:

26.

8. 10. The pharmaceutically acceptable composition of any one of the preceding claims, wherein the cancer is breast cancer.

9. 10. The pharmaceutically acceptable composition of claim 1, wherein the motif further comprises a tryptophan residue (W) at the C-terminus.

10. 10. A pharmaceutically acceptable composition according to any one of the preceding claims, wherein the peptide sequence consists of the motif GLLxLLxLLLxAAG.

11. 10. A pharmaceutically acceptable composition according to any one of the preceding claims, wherein the composition is for use in combination with a chemotherapeutic agent.

12. 10. A pharmaceutically acceptable composition according to any one of the preceding claims, wherein the composition is for intravenous administration.

13. 10. The pharmaceutically acceptable composition of any one of the preceding claims, wherein the composition is for administration at a dosage ranging from 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.

14. 10. The pharmaceutically acceptable composition of claim 1, wherein the peptide is in the L-form.

15. 10. The pharmaceutically acceptable composition of claim 1, wherein the peptide forms an alpha helix assembly.

16. 10. The pharmaceutically acceptable composition of claim 1, wherein the peptide forms a pore in a cancer cell membrane.

17. A method of treating cancer comprising administering to a cancer patient a pharmaceutically acceptable composition according to any one of the preceding claims, wherein preferably said cancer is breast cancer.

18. A peptide having a sequence comprising the motif GLLxLLELLxAAG, where each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D), or glutamic acid (E).

19. A peptide having a sequence comprising the motif GLLxLLxLLLxAAG, wherein each x is independently selected from arginine (R), histidine (H), lysine (K), aspartic acid (D), or glutamic acid (E), and said sequence does not include SEQ ID NO: 29 or SEQ ID NO:

33.

20. 20. The peptide of claim 19, wherein the sequence comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 25 or SEQ ID NO: 26 and combinations thereof.

21. 19. The peptide of claim 18, wherein the sequence comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 25 or SEQ ID NO: 26 and combinations thereof.

22. 22. The peptide of claim 21, wherein the sequence comprises a sequence selected from SEQ ID NO: 25 and / or SEQ ID NO:

26.

23. 23. The peptide of any one of claims 18 to 22, wherein the motif further comprises a tryptophan residue (W) at the C-terminus.

24. 24. A peptide according to any one of claims 18 to 23, wherein the peptide sequence consists of the motif GLLxLLxLLLxAAG.

25. 25. The peptide of any one of claims 18 to 24, wherein the peptide is in the L-form.

26. 26. The peptide of any one of claims 18 to 25, wherein the peptide forms an alpha helical assembly.

27. 27. The peptide of any one of claims 18 to 26, wherein the peptide forms a pore in a cancer cell membrane.

28. 17. A kit for treating or preventing cancer, comprising a pharmaceutically acceptable composition according to any one of claims 1 to 16.

29. 29. The kit of claim 28, wherein the cancer is breast cancer.

30. 30. The kit of claim 28 or claim 29, further comprising a chemotherapeutic agent.

31. A nucleotide sequence encoding a peptide comprising any one of SEQ ID NOs: 1 to 36.

32. A vector for expressing a peptide comprising any one of SEQ ID NOs: 1 to 36 and combinations thereof.

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