Synthetic anticancer lipopeptide and application thereof

Abstract

The invention provides a synthetic anti-cancer lipopeptide and application thereof, and particularly provides a non-natural polypeptide comprising domains X1, X2 and X3, in which X1 is selected from C6-C20 fatty acid residues, X2 is selected from C6-C20 fatty acid residues, X3 is selected from C6-C20 fatty acid residues, and X3 is selected from C6-C20 fatty acid residues; x2 is selected from a fragment of a cell penetrating peptide (CPPs); x3 is selected from fragments of peptides that specifically bind to protein kinase 2 (CK2). Compared with the prior art, the polypeptide can more effectively inhibit CK2, has higher targeting property on RPS6 protein, has better tumor inhibition activity and anti-metastasis effect and higher safety, and can realize large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment, and in particular to a synthetic anti-cancer lipopeptide and application thereof. Background Art

[0002] Protein kinase CK2 has become an important molecular target in the field of protein kinases and plays a key role in cancer cell biology (Buontempo et al, 2018; et al, 2020; Klink et al, 2021). Increasing evidence shows that CK2 plays an important role in the study of human malignancies.

[0003] Preclinical studies have explored a variety of strategies to inhibit CK2 activity. However, only two compounds, the ATP competitive inhibitor CX-4945 and the CIGB-300 peptide, have entered clinical trials in cancer patients as anti-CK2 drugs (Zakharia et al, 2019; Perea et al, 2018). The CIGB-300 peptide is a peptide chimera that contains a cyclic peptide that inhibits CK2-mediated phosphorylation and a cell-penetrating peptide Tat that promotes its delivery to cells. It is a "first-in-class" synthetic peptide that has entered clinical trials. It inhibits CK2-mediated phosphorylation through a dual mechanism of targeting the substrate phospho-receptor domain and the CK2α subunit (Perea et al, 2004; Perera et al, 2020). Proteomic analysis of the CIGB-300 peptide in non-small cell lung cancer (NSCLC) cells found that the CIGB-300 peptide can regulate multiple proteins involved in apoptosis, cell proliferation, ribosome biogenesis, anticancer drug resistance, cell metastasis, and angiogenesis (Rodriguez-Ulloa et al, 2010). Similarly, a study of the phosphoproteomic profile of CIGB-300 peptide in NSCLC cells revealed a group of CK2 substrates that were significantly inhibited as cellular processes were perturbed (Perea et al, 2020). Thus, CIGB-300 peptide not only induces apoptosis and controls cell proliferation, but also induces anti-angiogenic / anti-metastatic effects in mouse cancer models (Benavent et al, 2011; Benavent et al, 2016). CIGB-300 peptide has a synergistic effect in combination with paclitaxel, cisplatin, cytarabine (AraC), and epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and nimotuzumab (Perea et al, 2015; Perea et al, 2018). In mouse cancer models, the antitumor activity of CIGB-300 peptide has been validated by local intratumoral injection (Perea et al, 2004) and systemic administration routes (Perea et al, 2008). Currently, CIGB-300 peptide is in the clinical research stage, and its safety, tolerability, pharmacokinetics and efficacy data have been confirmed in phase I and phase II clinical trials in patients with tumors of different origins (Soriano et al, 2013; Sarduy et al, 2015; Batista et al, 2018).

[0004] However, some tumor cells are intrinsically resistant to the antiproliferative and proapoptotic effects of CIGB-300 peptide, possibly due to the peptide internalization rate and nucleolar localization (Perera et al, 2012; Benavent et al, 2014). Summary of the invention

[0005] The present invention describes a synthetic anticancer lipopeptide (SEQ ID NO: 1, referred to as "lipopeptide"), which has a better anti-tumor effect than the CIGB-300 peptide, and has a 4-11-fold increase in sensitivity to tumor cells. In addition, the lipopeptide of the present invention has a low manufacturing cost and can be mass-produced.

[0006] Specifically, in a first aspect, the present application provides a non-natural polypeptide comprising domains X1, X2 and X3, wherein:

[0007] X1 is selected from C6-C 20 Fatty acid residues;

[0008] X2 is selected from fragments of cell penetrating peptides (CPPs);

[0009] X3 is selected from fragments of peptides that specifically bind to protein kinase 2 (CK2).

[0010] In a second aspect, the present application provides a pharmaceutical composition comprising the non-natural polypeptide described in any one of the first aspects and a pharmaceutically acceptable excipient.

[0011] In a third aspect, the present application provides a drug kit comprising the non-natural polypeptide described in any one of the first aspect or the pharmaceutical composition described in any one of the second aspect, and an optional second therapeutic agent.

[0012] In another aspect, the present application provides the use of the non-natural polypeptide according to any one of the first aspect, the pharmaceutical composition according to any one of the second aspect, or the drug kit according to any one of the third aspect in the preparation of a CK2 modulator, or in the preparation of a drug, wherein the drug is used for:

[0013] (1) Inhibit tumor cell proliferation or metastasis, or promote tumor cell apoptosis;

[0014] (2) inhibiting angiogenesis, tumor growth or metastasis;

[0015] (3) Treatment of diseases related to CK2 activity.

[0016] In another aspect, the present application provides a method for regulating CK2 activity in a cell, the method comprising contacting the cell with the non-natural polypeptide according to any one of the first aspect, the pharmaceutical composition according to any one of the second aspect, or the drug kit according to any one of the third aspect.

[0017] In another aspect, the present application provides a method for inhibiting tumor cell proliferation or metastasis, or promoting tumor cell apoptosis, the method comprising administering to the subject an effective amount of the non-natural polypeptide described in any one of the first aspect, the pharmaceutical composition described in any one of the second aspect, or the drug kit described in any one of the third aspect.

[0018] In another aspect, the present application provides a method for inhibiting angiogenesis, or inhibiting tumor growth or metastasis, the method comprising administering to the subject an effective amount of the non-natural polypeptide described in any one of the first aspect, the pharmaceutical composition described in any one of the second aspect, or the drug kit described in any one of the third aspect.

[0019] In another aspect, the present application provides a method for treating a disease associated with CK2 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of the non-natural polypeptide of any one of the first aspect, the pharmaceutical composition of any one of the second aspect, or the drug kit of any one of the third aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The illustrative embodiments and their descriptions herein are used to explain the present invention and do not constitute improper limitations of the present invention. The accompanying drawings and their descriptions are used to further understand the present invention. In the accompanying drawings:

[0021] Figure 1 : The lipopeptide MS spectrum.

[0022] Figure 2A : The lipopeptide and CIGB-300 peptide induced apoptosis in NCI-H226 cells, and the apoptosis was quantified by flow cytometry after annexin V staining.

[0023] Figure 2B : The lipopeptide and CIGB-300 peptide induced apoptosis in SK-MES1 cells, and the apoptosis was quantified by flow cytometry after annexin V staining.

[0024] Figure 3 : In vivo toxicity of the lipopeptides and CIGB-300 peptide, animal body weight was recorded as a marker of treatment toxicity.

[0025] Figure 4 : The inhibitory effects of the lipopeptide and CIGB-300 peptide on phosphorylation and CK2-related proteins such as cdc37 in the NCI-H226 cell line were analyzed by Western blotting experiments.

[0026] Figure 5: The targeting effect of the lipopeptide and CIGB-300 peptide on RPS6 protein in NCI-H226 cell line, the specific binding of the synthetic anticancer lipopeptide to RPS6 protein was analyzed by Western blotting experiments and further quantified by densitometry.

[0027] Definition of terms

[0028] In this document, unless otherwise specified, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and other operating procedures used in this document are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0029] As used herein, the singular forms "a", "an" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, the terms "includes", "comprising", "having", "containing" or variations thereof are open ended and are not exclusive or exhaustive. Furthermore, the terms "herein", "preamble" and "hereinafter" or words of similar meaning shall refer to this application as a whole and not to any particular parts.

[0030] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). In some embodiments, the amino acids used may be common amino acids, such as the 20 protein L-amino acids. In some embodiments, the amino acids used may be non-natural amino acids. The "non-natural amino acids" described herein may include any amino acid other than the 20 proteinogenic amino acids in the L-configuration. Such amino acids may include amino acids with non-classical side chains, D-amino acids, β-amino acids, etc. Unless otherwise specified, the amino acids used herein are in the L-configuration.

[0031] As used herein, the terms "subject", "patient" or "individual" include mammals and non-mammals. Mammals can be any member of the class Mammalia, including but not limited to humans; non-human primates, such as chimpanzees, apes or other monkeys; farm animals, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs (or canines), cats; experimental animals, including rodents, such as rats, mice and guinea pigs; and the like. Non-mammals can include birds, fish, etc. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be a male. In some cases, the subject can be a female. In some cases, the subject is diagnosed with or suspected of having a disease. In some cases, the disease is cancer. The subject can be a patient or an individual. In some cases, the subject, patient or individual can be used interchangeably.

[0032] As used herein, the terms "treat," "treat," "improve," or "alleviate" include alleviating or relieving the symptoms of a disease, inhibiting a disease, such as arresting the development of a disease, relieving a disease, causing regression of a disease, alleviating symptoms caused by a disease, or stopping symptoms of a disease. The terms "treat," "treat," "improve," or "alleviate" may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the disease being treated. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the disease being treated, resulting in an observable improvement in the subject, although in some embodiments, the subject may still be afflicted with the underlying disease.

[0033] As used herein, the term "effective amount", "therapeutically effective amount" refers to a sufficient amount of the drug administered, which will at least partially alleviate the symptoms of the disease being treated. The dosage regimen can be adjusted to provide the best required response. For example, a single bolus can be administered, or several divided doses can be administered over time, or the dosage can be reduced or increased in proportion to the treatment situation. It should be noted that the dosage value can vary with the type and severity of the disease to be alleviated, and can include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to individual needs and drug instructions or clinical physician professional judgment. In general, the effective dose is about 0.0001 to about 50 mg per kg body weight per day, for example, about 0.01 to about 10 mg / kg / day (single or divided administration). For a 70 kg person, this will add up to about 0.007 mg / day to about 3500 mg / day, for example, about 0.7 mg / day to about 700 mg / day. In some cases, dosage levels no higher than the lower limit of the aforementioned range may be sufficient, while in other cases, larger doses may still be employed without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0034] As used herein, the terms "peptide" and "polypeptide" are used interchangeably to encompass naturally occurring and non-naturally occurring proteins and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. In addition, a polypeptide may comprise a plurality of different domains, each domain having one or more different activities. A "polypeptide" may be any length greater than two amino acids. In some cases, a peptide may have a positive charge overall. In some cases, a peptide may have a negative charge overall. In some cases, a peptide may have a neutral charge overall. A peptide may also exist in a zwitterionic form.

[0035] Peptides as described herein can be salts thereof. In some embodiments, even if not explicitly mentioned, the reference to the term "peptide" or "polypeptide" should be interpreted as including salts thereof. In some embodiments, the salt can include carboxylates (e.g., formate, acetate, trifluoroacetate, trichloroacetate, propionate, isobutyrate, enanthate, decanoate, decanoate, caprylate, stearate, acrylate, caproate, propionate, ascorbate, citrate, glucuronate, glutamate, glycolate, alpha-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, pamoate, nicotinate, isonicotinate, cinnamon, hydroxybenzoate ... The salt may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a pharmaceutically acceptable salt such as a halogen salt (e.g., a chloride, bromide or iodide salt); a sulfonate (e.g., a benzenesulfonate, a methyl, bromo or chlorobenzenesulfonate, a xylenesulfonate, a methanesulfonate, a trifluoromethanesulfonate, an ethanesulfonate, a propanesulfonate, a hydroxyethanesulfonate, a 1- or 2-naphthalenesulfonate or a 1,5-naphthalenedisulfonate); a sulfate; a pyrosulfate; a hydrogensulfate; a sulfite; a hydrogensulfite; a phosphate; a monohydrogenphosphate; a dihydrogenphosphate; a metaphosphate; a pyrophosphate; a nitrate; and the like. In some embodiments, the salt may be a pharmaceutically acceptable salt. A pharmaceutically acceptable salt may be a salt described in Berge et al., J. Pharm. Sci, 1977.

[0036] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0037] As used herein, the terms "co-administered", "co-administered" include administering a selected therapeutic agent (i.e., the second therapeutic agent described herein) to a subject, and may include a treatment regimen of administering an agent by the same or different routes of administration or at the same or different times. In some embodiments, the polypeptide described herein may be co-administered with other therapeutic agents. Including administering two or more agents to a subject so that the agents and / or their metabolites are present in the subject simultaneously. They may include administering simultaneously, administering at different times, and / or administering in a composition where both agents are present. Therefore, in some embodiments, the peptide and the second therapeutic agent may be administered in a single composition. In some embodiments, the peptide and the second therapeutic agent may be mixed in a composition. In some embodiments, the same peptide or therapeutic agent may be administered by a combination of different routes of administration. In some embodiments, each therapeutic agent administered may be a therapeutically effective amount.

[0038] Anticancer lipopeptides

[0039] The present application provides a non-natural polypeptide, which comprises a cyclic peptide targeting CK2 and a cell-penetrating peptide, wherein the non-natural polypeptide comprises a lipophilic carboxylic acid modification at the N-terminus. Compared with CIGB-300, the polypeptide can more effectively inhibit CK2, has a higher targeting to the RPS6 protein, has better tumor suppression activity and anti-metastasis effect, and has higher safety.

[0040] In some embodiments, the present application provides a non-natural polypeptide comprising domains X1, X2 and X3, wherein:

[0041] X1 is selected from C6-C 20 Fatty acid residues;

[0042] X2 is selected from fragments of cell penetrating peptides (CPPs);

[0043] X3 is selected from fragments of peptides that specifically bind to protein kinase 2 (CK2).

[0044] In some embodiments, X1 is selected from C6-C 12 Fatty acid residues, such as C6, C7, C8, C9, C 10 , C 11 or C 12 Fatty acid residue. In some embodiments, X1 is decanoyl.

[0045] In some embodiments, X2 comprises a fragment of a peptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to GRKKRRQRRRPPQ (SEQ ID NO:2).

[0046] In some embodiments, X3 has CK2 inhibitory activity, such as inhibiting CK2 phosphorylation.

[0047] In some embodiments, X3 comprises a fragment of a peptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to CWMSPRHLGTC (SEQ ID NO:3, with a disulfide bond formed between the cysteine ​​at position 1 and the cysteine ​​at position 11).

[0048] In some embodiments, there is an amino acid or peptide linker between two adjacent domains among X1, X2 and X3.

[0049] In some embodiments, X1, X2, and X3 are linked in any order.

[0050] In some embodiments, X1, X2 and X3 are arranged in sequence from N-terminus to C-terminus.

[0051] In some embodiments, the polypeptides are linked from N-terminus to C-terminus as X1-X2-β-Ala-X3.

[0052] In some embodiments, the C-terminus of the non-natural polypeptide is amidated.

[0053] In some embodiments, the non-natural polypeptide comprises or consists of the following amino acid sequence:

[0054] X1-Gly 1 -Arg 2 -Lys 3 -Lys 4 -Arg 5 -Arg 6 -Gln 7 -Arg 8 -Arg 9 -Arg 10 -Pro 11 -Pro 12 -Gln 13 -β-Ala 14 -Cys 15 -Trp 16 -Met 17 -Ser 18 -Pro 19 -Arg 20 -His 21 -Leu 22 -Gly 23 -Thr 24 -Cys 25-NH2 (SEQ ID NO: 1), wherein

[0055] X1 is decanoyl;

[0056] Cys 15 and Cys 25 Disulfide bonds are formed between them.

[0057] In some specific embodiments, the chemical name and structure of the lipopeptide described herein are as follows:

[0058] (15→25) (Cyclic dithio)decanoic acid-glycyl-L-histidyl-L-lysyl-L-lysyl-L-arginyl-L-arginyl-L-glutaminyl-L-arginyl-L-arginyl-L-arginyl-L-arginyl-L-prolyl-L-prolyl-L-glutaminyl-β-alanyl-L-cysteinyl-L-tryptophanyl-L-methionyl-L-seryl-L-prolyl-L-arginyl-L-histidyl-L-leucyl-glycyl-L-threonyl-L-cysteinamide.

[0059] Synthesis of anticancer lipopeptides

[0060] Those skilled in the art are aware of a variety of suitable methods for synthesizing peptides. In some embodiments, the polypeptides described herein can be synthesized in a liquid phase system or a solid phase system using techniques known in the art (see, e.g., Atherton, E., Sheppard, RC (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press; Stewart, JM, Young, JD (1984). Solid phase peptide synthesis (2nd edition). Rockford: Pierce Chemical Company).

[0061] Pharmaceutical compositions and kits

[0062] In another aspect, the present application provides a pharmaceutical composition comprising the non-natural polypeptide described in any one of the above and a pharmaceutically acceptable excipient.

[0063] The excipients may be those described in Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavoring agents, sweeteners, coloring agents.

[0064] In some embodiments, suitable buffering agents include calcium bicarbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts, etc. or combinations thereof.

[0065] In some embodiments, suitable preservatives include antioxidants such as alpha-tocopherol and ascorbate, and antimicrobial agents such as parabens, chlorobutanol and phenol. Antioxidants may further include EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N-acetyl cysteine, etc.

[0066] In some embodiments, suitable binders include starches such as potato starch, corn starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinyl pyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water, or combinations thereof.

[0067] In some embodiments, suitable lubricants include metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal dodecyl sulfate (e.g., sodium dodecyl sulfate, magnesium dodecyl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0068] In some embodiments, disintegrants can be non-effervescent disintegrants. Suitable non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginate, sodium starch glycolate, gums, such as agar, guar fat, locust bean fat, karaya gum, pectin (pecitin) and tragacanth. In some embodiments, disintegrants can be effervescent disintegrants. Suitable effervescent disintegrants include sodium bicarbonate combined with citric acid and sodium bicarbonate combined with tartaric acid.

[0069] In some embodiments, suitable flavoring agents may be selected from cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; anise oil; eucalyptus oil; vanilla; citrus oils, such as lemon oil, orange oil, grape and grapefruit oil; and fruit flavors, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot flavors.

[0070] In some embodiments, suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as the sodium salt; dipeptide sweeteners, such as aspartame; the dihydrochalcone compound, glycyrrhizin; Stevia Rebaudiana (stevioside); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol (sylitol), and the like.

[0071] In some embodiments, suitable colorants include food, drug, and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C).

[0072] In some embodiments, suitable chelating agents include ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid Monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid; 1,3-diaminopropane-N,N,N',N'-tetraacetic acid; ethylenediamine-N,N'-diacetic acid; ethylenediamine-N,N'-dipropionic acid dihydrochloride; ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N , N', N'-triacetic acid; ethylenediamine-N, N, N', N'-tetra(methylenephosphonic acid); O, O'-bis(2-aminoethyl)ethylenediol-N, N, N', N'-tetraacetic acid; N, N-bis(2-hydroxybenzyl)ethylenediamine-N, N-diacetic acid; 1,6-hexanediamine-N, N, N', N'-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2- Diaminopropane-N,N,N',N'-tetraacetic acid; nitrilotriacetic acid; nitrilotripropionic acid; trisodium salt of nitrilotris(methylenephosphoric acid); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid, etc.

[0073] In some embodiments, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0074] In some embodiments, suitable surfactants include polysorbates, sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, sugar esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0075] In some embodiments, the pharmaceutical composition may further include a second active pharmaceutical ingredient. In some embodiments, the second active pharmaceutical ingredient is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab.

[0076] The polypeptide of the present application can be formulated into a variety of formulations, and is used in a variety of different ways, such as oral, rectal or parenteral administration.Term "parenteral" used herein can include intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravenous, intravitreal, epidural, subcutaneous, suction, transdermal, through mucosa, sublingual, cheek and local (including epidermis, dermis, enema, eye drops, ear drops, intranasal, vaginal) administration.In some exemplary embodiments, route of administration can be by injection, such as intramuscular, intravenous, subcutaneous or intraperitoneal injection.Preparations for oral administration can include capsules, tablets, caplets, pills, lozenges, lozenges, powders and granules etc.

[0077] In another aspect, the present application provides a drug kit, comprising any of the above-described non-natural polypeptides or pharmaceutical compositions, and an optional second therapeutic agent.

[0078] In some embodiments, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab. In some embodiments, the non-natural polypeptide or pharmaceutical composition and the second therapeutic agent are placed in the same or different formulation units.

[0079] In some embodiments, the kit is used for:

[0080] (1) inhibiting tumor cell proliferation or metastasis, or promoting tumor cell apoptosis; preferably, the tumor cells are selected from prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, colon cancer cells, small cell lung cancer (SCLC) cells, non-small cell lung cancer (NSCLC) cells, breast cancer cells, renal cancer cells, melanoma cancer cells, multiple myeloma cells, glioblastoma cells, Burkitt's lymphoma cells, leukemia cells, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma) cells, adenocarcinoma (e.g., vulvar adenocarcinoma) cells, liver cancer and cervical cancer cells;

[0081] (2) inhibiting angiogenesis, tumor growth or metastasis;

[0082] (3) Treating diseases associated with CK2 activity (e.g., tumors).

[0083] In some embodiments, the tumor is a refractory cancer or a cancer insensitive to the CIGB-300 peptide, preferably, the tumor is selected from prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, kidney cancer, melanoma cancer, multiple myeloma, glioblastoma, Burkitt's lymphoma, leukemia, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma), adenocarcinoma (e.g., vulvar adenocarcinoma), liver cancer and cervical cancer.

[0084] application

[0085] Cell lines from different sources were used to detect the effect of the polypeptides described herein on promoting tumor cell death. According to literature reports, cell apoptosis was determined using methods such as sulforhodamine, alamar blue or crystal violet, and expressed as half inhibitory concentration (IC50) (Boyd et al, 1995). The IC50 value refers to the peptide dose that causes 50% cell apoptosis obtained by the dose-response curve. The effect of the polypeptides at a dose of 0-200 μM on each tumor cell line was detected. In some embodiments, compared with the CIGB-300 peptide, the potency of the lipopeptide on tumor cells is increased by at least 4 times, indicating that the lipopeptide can achieve the same tumor cell killing effect with only a quarter of the CIGB-300 peptide dose, which can significantly reduce the drug dose and improve safety.

[0086] Using the biomarker of apoptosis, annexin-V, as a cell death indicator, a short-term time course comparison experiment was carried out, and the lipopeptide also showed obvious advantages after flow cytometric analysis. In some embodiments, the lipopeptide can induce higher early apoptosis compared with the CIGB-300 peptide based on the percentage of apoptotic cells. In other embodiments, after treatment with the lipopeptide, the level of reactive oxygen species (ROS) is higher. And non-tumorigenic normal-like cells in mice are more resistant to the lipopeptide.

[0087] The anti-tumor effect of the polypeptide was verified by a head-to-head experimental in vivo metastasis test. In some embodiments, C57BL6 mice were intravenously injected with mouse Lewis lung cancer 3LL cells, and 72 hours after tumor cell attack, the lipopeptide and CIGB-300 peptide were intravenously injected for 5 consecutive days, and the lung cell metastasis formation was quantified after 30 days. The results showed that even with one-tenth of the CIGB-300 peptide dose, the lipopeptide still had an effective anti-metastatic effect, and no signs of systemic toxicity were found.

[0088] In addition, the targeting effect of the polypeptide on CK2 signal transduction was detected. In some embodiments, after incubating the tumor cell line with the IC50 dose of the lipopeptide and CIGB-300 peptide, the endogenous CK2 enzyme activity in the cell extract was detected. The results showed that the inhibition rate of the lipopeptide on the CK2 enzyme activity in all tumor cells was more than 50%. Western blotting (WB) experiments showed that compared with the CIGB-300 peptide, the lipopeptide had a stronger inhibitory effect on phosphorylation and CK2-related proteins such as cdc37. These results indicate that the interaction between the lipopeptide and the RPS6 protein is stronger than that of the CIGB-300 peptide, and can more effectively inhibit CK2-mediated phosphorylation and induce a higher internalization rate in tumor cells.

[0089] Based on the above, the peptides and pharmaceutical compositions described herein can be administered to subjects to at least partially improve the disease. In some embodiments, the subject may have been previously diagnosed with a disease described herein, or may be at risk of developing a disease described herein.

[0090] In one aspect, the present application provides a use of the non-natural polypeptide, pharmaceutical composition or kit described in any of the above in the preparation of a CK2 modulator (e.g., an inhibitor), or in the preparation of a medicament, wherein the medicament is used for:

[0091] (1) Inhibit tumor cell proliferation or metastasis, or promote tumor cell apoptosis;

[0092] (2) inhibiting angiogenesis, tumor growth or metastasis;

[0093] (3) Treating diseases associated with CK2 activity (e.g., tumors).

[0094] In another aspect, the present application provides a method for regulating (eg, inhibiting) CK2 activity in a cell, the method comprising contacting the cell with any of the above-described non-natural polypeptides, pharmaceutical compositions or kits.

[0095] In another aspect, the present application provides a method for inhibiting tumor cell proliferation or metastasis, or promoting tumor cell apoptosis, comprising administering to the subject an effective amount of any of the above-described non-natural polypeptides, pharmaceutical compositions or kits.

[0096] In another aspect, the present application provides a method for inhibiting angiogenesis, or inhibiting tumor growth or metastasis, the method comprising administering to the subject an effective amount of any of the above-described non-natural polypeptides, pharmaceutical compositions or kits.

[0097] In another aspect, the present application provides a method for treating a disease associated with CK2 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the above non-natural polypeptides, pharmaceutical compositions or kits.

[0098] In any of the above aspects, the cell is a tumor cell. In some embodiments, the tumor cell is a cell line or a cell from a subject. In some embodiments, the tumor cell is selected from prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, colon cancer cells, small cell lung cancer (SCLC) cells, non-small cell lung cancer (NSCLC) cells, breast cancer cells, renal cancer cells, melanoma cancer cells, multiple myeloma cells, glioblastoma cells, Burkitt's lymphoma cells, leukemia cells, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma) cells, adenocarcinoma (e.g., vulvar adenocarcinoma) cells, liver cancer and cervical cancer cells.

[0099] In any of the above aspects, the disease associated with CK2 activity is a tumor. In some embodiments, the tumor is a refractory cancer or a cancer that is insensitive to CIGB-300 peptide. In some embodiments, the tumor is selected from prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, kidney cancer, melanoma cancer, multiple myeloma, glioblastoma, Burkitt's lymphoma, leukemia, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma), adenocarcinoma (e.g., vulvar adenocarcinoma), liver cancer and cervical cancer.

[0100] In any of the above methods, the subject is also administered a therapeutically effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab. In some embodiments, the non-natural polypeptide or pharmaceutical composition is administered simultaneously or sequentially with the second therapeutic agent.

[0101] Beneficial technical effects

[0102] The lipopeptide described in this article contains decanoic acid chemically fused to the N-terminus of the CIGB-300 peptide. It is not only a new anticancer compound, but also has more effective anticancer activity than the CIGB-300 peptide. In vitro experiments on tumor cell groups have verified the advantages of the lipopeptide, and the dose required to kill 50% of the cells is at least 4 times lower than the dose required for the CIGB-300 peptide. The lipopeptide can achieve an effective anti-metastatic effect at one-tenth the dose of the CIGB-300 peptide. From a molecular level, the advantage of the lipopeptide compared to the CIGB-300 peptide is that it can more effectively inhibit the activity of the CK2 enzyme. At the same time, the lipopeptide has a stronger targeting effect on the RPS6 protein.

[0103] The lipopeptide described herein is obtained by fusing decanoic acid to the end of CIGB-300 peptide through simple chemical modification, which overcomes the baseline or inherent resistance defect of tumor cells to CIGB-300 peptide. Therefore, the lipopeptide described herein is a more effective new anticancer compound that can reduce the pharmacological dose without increasing toxicity.

[0104] Compared with CIGB-300 peptide, the lipopeptide described herein achieves more effective anti-tumor effect at a lower dose, and has a high reference value for clinical medication for cancer patients. Since the synthesis method disclosed in this application is consistent with the synthesis method of CIGB-300 peptide obtained in clinical batches, large-scale production can be achieved. DETAILED DESCRIPTION

[0105] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0106] Sequence Information Table

[0107]

[0108] Example 1 Peptide Preparation Method

[0109] The CIGB-300 peptide was synthesized by a solid-phase method based on Fmoc / tBu chemistry, purified by reverse-phase HPLC with a purity higher than 95% under acetonitrile / H2O-trifluoroacetic acid gradient conditions, and then verified by ion spray mass spectrometry.

[0110] The lipophilic carboxylic acid (decanoic acid, C 10 H 20 O2) was inserted into the N-terminus of the CIGB-300 peptide to obtain the lipopeptide. The MS detection results are shown in Figure 1: Average mass = 3213.9089, single isotopic mass = 3211.7607.

[0111] Example 2 Direct comparison of the anti-tumor cell effects of lipopeptides and CIGB-300 peptides

[0112] The human tumor cells used in this application were all from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 medium (Gibco) containing 10% (v / v) fetal bovine serum and gentamicin (50 μg / ml) under 5% CO2 and 37°C. The effects of the lipopeptides and CIGB-300 peptide on tumor cell viability were detected using Alamar Blue reagent (Life Technologies, Carlsbad, CA, USA). The cells were seeded in flat-bottom 96-well plates (2×10 5 After 24 hours of incubation, 100-3.12 μM of the lipopeptide and CIGB-300 peptide were added, serially diluted at a ratio of 1:2. After 48 hours of incubation, 10% (v / v) Alamar blue was added, and the cell suspension was further incubated for 4 hours. Fluorescence in the microplate was detected, and the IC was evaluated using CalcuSyn software (version 2.1) (Biosoft, Cambridge, UK). 50 value.

[0113] The data in Table 1 show that the lipopeptide has at least 4-fold higher potency than the CIGB-300 peptide against all tumor cells. However, benign tumor cells appear to be more resistant to cell death induced by the lipopeptide. For the same benign tumor cells, the cell death rate induced by the lipopeptide is no more than 1.5 times that of the CIGB-300 peptide.

[0114] Table 1: Effects of lipopeptides and CIGB-300 peptide on in vitro tumor cell viability

[0115]

[0116]

[0117] Note: SCLC refers to small cell lung cancer; NSCLC refers to non-small cell lung cancer

[0118] Example 3 Time course experiment of lipopeptide and CIGB-300 peptide inducing cell apoptosis and reactive oxygen species

[0119] The Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Jose, CA, USA) was used to detect apoptosis induced by lipopeptides and CIGB-300 peptide in five tumor cell lines.

[0120] The specific experimental steps were as follows: cells were incubated with equivalent doses of the lipopeptide and CIGB-300 peptide for 0.5 h, 1 h, 3 h, 6 h and 24 h, then washed twice with ice-cold PBS solution, and then incubated with 1×10 5 The cells were resuspended in 1x binding buffer at a final concentration of cells / mL. Subsequently, 5 μL of Annexin V-FITC apoptosis detection reagent and propidium iodide (PI) were added, and the cell suspension was incubated at room temperature in the dark for 15 minutes. Flow cytometry analysis of the stained cells was performed using a Partec CyFlow Space device, and data analysis and visualization were performed using FlowJo software (version 7.6.1) (BD, Ashland, OR, USA). Figure 2A and 2B The percentages of apoptotic cells induced in NCI-H226 cells and SK-MES1 cells are shown, respectively, and the lipopeptide induced a higher apoptotic rate compared with the CIGB-300 peptide.

[0121] Compared with CIGB-300 peptide, the lipopeptide can not only induce higher levels of apoptosis, but also induce higher levels of ROS after incubation with A549 cells for 2 hours under the action of N-acetyl-L-cysteine ​​(NAC) antioxidant, as shown by flow cytometry analysis (Table 2). As the level of ROS increases, the lipopeptide can affect cell viability to a greater extent.

[0122] Table 2: ROS levels induced by lipopeptides and CIGB-300 peptide in A549 cells

[0123]

[0124]

[0125] Example 4 Head-to-head comparative experiment to evaluate the anti-metastatic effect of the lipopeptide and CIGB-300 peptide

[0126] The specific experimental steps were to inject 2x10 5 Lewis rat lung cancer 3LL cells were used to induce experimental metastasis for lung colonization experiments. For 5 consecutive days, 0.1, 1, 5 and 10 mg / kg of the lipopeptide or CIGB-300 peptide were injected intravenously every day, with 10 mice in each group. The control group was injected with PBS intravenously. The animals were sacrificed on the 30th day, and the lungs were removed and fixed in Bonn's fixative. Metastatic lung nodules were counted using a dissecting microscope. In addition, the weight and signs of toxicity of the animals were monitored throughout the experiment.

[0127] The data in Table 3 show that the lipopeptide significantly reduced lung colonization at all dose levels compared to the CIGB-300 peptide, which had an anti-metastatic effect only at the highest dose (10 mg / kg).

[0128] This head-to-head comparison demonstrated that the lipopeptide induced significant anti-metastatic effects at one-twentieth the dose of CIGB-300 peptide. 10mg / kg of the lipopeptide induced 100% of the anti-metastatic effects compared to CIGB-300. In the treatment group that received the highest dose (10mg / kg), no signs of toxicity were found, and the animals gained weight during the trial ( Figure 3 ).

[0129] Table 3: Anti-metastatic effects of lipopeptides and CIGB-300 peptide in direct animal comparison experiments

[0130]

[0131]

[0132] Note: Bilateral lung nodules were counted for each animal, and the mean values ​​of 10 animals in each group are shown. *(p<0.05)**(p<0.01)***(p<0.001) Mann-Whitney U test.

[0133] Effects of the lipopeptides and CIGB-300 peptide described in Example 5 on CK2 enzyme activity and CK2 signal transduction

[0134] CK2 radioactivity analysis was performed in increasing concentrations of the lipopeptide solution (final volume of 18 μL), with CIGB-300 peptide solution or an equal amount of dimethyl sulfoxide (DMSO) as a control group. Prepare 3 μL of recombinant CK2α (28 ng) and a mixture containing 1 mM specific CK2 peptide substrate "RRREEETEEE" (Promega), 10 mM MgCl2, 1 μCi 32P-γATP (6000 Ci / mmol), and 100 μM ATP. Incubate at room temperature for 5 minutes, then add 60 μL of 4% trichloroacetic acid to terminate the assay. Subsequently, add 5 μL of the above-obtained reaction solution to Whatman PE-81 filter paper, and then wash the filter paper four times with 10 mM H3PO4. Finally, the radioactivity on the filter paper was quantified, and CPM represents CK2 enzyme activity.

[0135] The data in Table 4 show that, compared with the CIGB-300 peptide, the lipopeptide can more effectively inhibit the CK2 enzyme activity, which indicates that the lipopeptide of the present invention has a more effective target interaction.

[0136] Table 4: Head-to-head comparison of CK2 enzyme activity inhibition experiments

[0137]

[0138]

[0139] Based on the CK2 enzyme activity data, a head-to-head comparison experiment was conducted between the lipopeptide and the CIGB-300 peptide using the lung squamous cell carcinoma cell line NCI-H226 to detect the ability of the lipopeptide to inhibit other CK2 signaling substrates such as cdc37 protein. Figure 4 The WB experimental results analysis confirmed that compared with the CIGB-300 peptide, the lipopeptide could more effectively inhibit the phosphorylation and cdc37 protein level after 3 hours and 6 hours of incubation with the NCI-H226 cell line lipopeptide.

[0140] In order to explore the targeting effect of the lipopeptide and CIGB-300 peptide on CK2 signaling, RPS6 protein pull-down experiments were performed. The specific steps included inoculating 3,600,000 NCI-H226 cells in a T-75 flask, collecting and washing the cells after 24 hours, and using 1 mM DTT (Sigma, St. Louis, MO, USA), Triton X-100 (1%) and cOmplete TM Cells were lysed with 1x PBS solution of protease inhibitors (Roche, Basel, Switzerland). Cell lysates were then cleared by centrifugation, and 300 μg of total protein was incubated with 100 μM biotin-labeled lipopeptides, CIGB-300 peptides or culture medium at room temperature for 30 minutes and then added to 30 μL of pre-equilibrated streptavidin-agarose matrix (GE Healthcare, Chicago, IL, USA). After treatment at 4 ° C for 1 hour, the matrix was collected by centrifugation and then thoroughly washed with ice PBS solution containing 1 mM DTT. The pulled RPS6 protein was eluted, separated in SDS-PAGE gel, analyzed by WB, and specific signals were quantified by densitometry. Figure 5 The data showed that the lipopeptide had a better targeting effect on RPS6 protein (PD3) than CIGB-300 peptide (PD1).

[0141] Using the lung squamous cell carcinoma cell line NCI-H226, a head-to-head comparison experiment was conducted on the proteins targeted by the lipopeptide and the CIGB-300 peptide. We performed pull-down experiments and mass spectrometry analysis. The data in Table 5 reveal the proteins targeted by the lipopeptide, and the data in Table 6 reveal the proteins that the lipopeptide targets more effectively than the CIGB-300 peptide.

[0142] Table 5 Proteins targeted by the lipopeptides in the NCI-H226 cell line

[0143]

[0144] Table 6 Proteins targeted more effectively by the lipopeptides than the CIGB-300 peptide in the NCI-H226 cell line

[0145]

[0146] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A non-natural polypeptide comprising domains X1, X2 and X3, wherein: X1 is selected from C6-C 20 Fatty acid residues; X2 is selected from fragments of cell penetrating peptides (CPPs); X3 is selected from fragments of peptides that specifically bind to protein kinase 2 (CK2).

2. The non-natural polypeptide according to claim 1, wherein X1 is selected from C6-C 12 Fatty acid residues (e.g. C6, C7, C8, C9, C 10 , C 11 or C 12 fatty acid residues); Preferably, X1 is decanoyl.

3. The non-natural polypeptide according to claim 1 or 2, wherein X2 comprises a fragment of a peptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to GRKKRRQRRRPPQ (SEQ ID NO:2).

4. The non-natural polypeptide according to any one of claims 1 to 3, wherein X3 has CK2 inhibitory activity, such as inhibiting CK2 phosphorylation; Preferably, X3 comprises a fragment of a peptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identity to CWMSPRHLGTC (SEQ ID NO:3, with a disulfide bond formed between cysteine ​​at position 1 and cysteine ​​at position 11).

5. The non-natural polypeptide according to any one of claims 1 to 4, wherein an amino acid or peptide linker exists between two adjacent domains in X1, X2 and X3.

6. The non-natural polypeptide according to any one of claims 1 to 5, wherein X1, X2 and X3 are connected in any order; Preferably, X1, X2 and X3 are arranged in sequence from the N-terminus to the C-terminus; Preferably, the polypeptides are linked from N-terminus to C-terminus in the order of X1-X2-β-Ala-X3.

7. The non-natural polypeptide according to any one of claims 1 to 6, comprising or consisting of the following amino acid sequence: X1-Gly 1 -Arg 2 -Lys 3 -Lys 4 -Arg 5 -Arg 6 -Gln 7 -Arg 8 -Arg 9 -Arg 10 -Pro 11 -Pro 12 -Gln 13 -β-Ala 14 -Cys 15 -Trp 16 -Met 17 -Ser 18 -Pro 19 -Arg 20 -His 21 -Leu 22 -Gly 23 -Thr 24 -Cys 25 -NH2 (SEQ ID NO:1), wherein, X1 is decanoyl; Cys 15 and Cys 25 Disulfide bonds are formed between them.

8. A pharmaceutical composition comprising the non-natural polypeptide according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A kit comprising the non-natural polypeptide of any one of claims 1 to 7 or the pharmaceutical composition of claim 8, and optionally a second therapeutic agent; Preferably, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor, and further preferably, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab; Preferably, the non-natural polypeptide or pharmaceutical composition and the second therapeutic agent are placed in the same or different formulation units; Preferably, the kit is used for: (1) inhibiting tumor cell proliferation or metastasis, or promoting tumor cell apoptosis; preferably, the tumor cells are selected from prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, colon cancer cells, small cell lung cancer (SCLC) cells, non-small cell lung cancer (NSCLC) cells, breast cancer cells, renal cancer cells, melanoma cancer cells, multiple myeloma cells, glioblastoma cells, Burkitt's lymphoma cells, leukemia cells, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma) cells, adenocarcinoma (e.g., vulvar adenocarcinoma) cells, liver cancer and cervical cancer cells; (2) inhibiting angiogenesis, tumor growth or metastasis; (3) Treatment of diseases associated with CK2 activity (e.g., tumors); Preferably, the tumor is a refractory cancer or a cancer insensitive to the CIGB-300 peptide, preferably, the tumor is selected from prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, kidney cancer, melanoma cancer, multiple myeloma, glioblastoma, Burkitt's lymphoma, leukemia, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma), adenocarcinoma (e.g., vulvar adenocarcinoma), liver cancer and cervical cancer.

10. Use of the non-natural polypeptide according to any one of claims 1 to 7, the pharmaceutical composition according to claim 8 or the kit according to claim 9 in the preparation of a CK2 modulator (e.g., an inhibitor), or in the preparation of a medicament, wherein the medicament is used for: (1) Inhibit tumor cell proliferation or metastasis, or promote tumor cell apoptosis; (2) inhibiting angiogenesis, tumor growth or metastasis; (3) Treatment of diseases associated with CK2 activity (e.g., tumors); Preferably, the tumor is a refractory cancer or a cancer insensitive to the CIGB-300 peptide, preferably, the tumor is selected from prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, kidney cancer, melanoma cancer, multiple myeloma, glioblastoma, Burkitt's lymphoma, leukemia, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma), adenocarcinoma (e.g., vulvar adenocarcinoma), liver cancer and cervical cancer.

11. A method for regulating (e.g., inhibiting) CK2 activity in a cell, the method comprising contacting the cell with the non-natural polypeptide of any one of claims 1 to 7, the pharmaceutical composition of claim 8, or the kit of claim 9; Preferably, the cells are tumor cells.

12. A method for inhibiting tumor cell proliferation or metastasis, or promoting tumor cell apoptosis, the method comprising administering to the subject an effective amount of the non-natural polypeptide according to any one of claims 1 to 7, the pharmaceutical composition according to claim 8, or the drug kit according to claim 9; Preferably, the method further comprises co-administering to the subject an effective amount of a second therapeutic agent; Preferably, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor, and further preferably, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab; Preferably, the non-natural polypeptide or pharmaceutical composition is administered simultaneously or sequentially with the second therapeutic agent.

13. A method for inhibiting angiogenesis, or inhibiting tumor growth or metastasis, the method comprising administering to the subject an effective amount of the non-natural polypeptide according to any one of claims 1 to 7, the pharmaceutical composition according to claim 8, or the drug kit according to claim 9; Preferably, the method further comprises co-administering to the subject an effective amount of a second therapeutic agent; Preferably, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor, and further preferably, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab; Preferably, the non-natural polypeptide or pharmaceutical composition is administered simultaneously or sequentially with the second therapeutic agent.

14. The use according to claim 10, or the method according to any one of claims 11 to 13, wherein the tumor cell is a cell line or a cell from a subject; Preferably, the tumor cell is selected from prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, colon cancer cells, small cell lung cancer (SCLC) cells, non-small cell lung cancer (NSCLC) cells, breast cancer cells, renal cancer cells, melanoma cancer cells, multiple myeloma cells, glioblastoma cells, Burkitt's lymphoma cells, leukemia cells, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma) cells, adenocarcinoma (e.g., vulvar adenocarcinoma) cells, liver cancer and cervical cancer cells.

15. A method for treating a disease associated with CK2 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of the non-natural polypeptide of any one of claims 1 to 7, the pharmaceutical composition of claim 8, or the kit of claim 9; Preferably, the method further comprises co-administering to the subject a therapeutically effective amount of a second therapeutic agent; Preferably, the second therapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor, and further preferably, the EGFR inhibitor is selected from paclitaxel, cisplatin, cytarabine (AraC), erlotinib and nimotuzumab; Preferably, the non-natural polypeptide or pharmaceutical composition is administered simultaneously or sequentially with the second therapeutic agent; Preferably, the disease associated with CK2 activity is a tumor, preferably, the tumor is a refractory cancer or a cancer insensitive to the CIGB-300 peptide, preferably, the tumor is selected from prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, kidney cancer, melanoma cancer, multiple myeloma, glioblastoma, Burkitt's lymphoma, leukemia, squamous cell carcinoma (e.g., laryngeal squamous cell carcinoma), adenocarcinoma (e.g., vulvar adenocarcinoma), liver cancer and cervical cancer.

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