Synthesized Anti-cancer lipopeptide and use thereof

Abstract

Provided are a synthesized anti-cancer lipopeptide and the use thereof. Specifically, the anti-cancer lipopeptide comprises a domain X1, X2 and X3, wherein: X1 is selected from C6-C20 fatty acid residues; X2 is selected from fragments of cell-penetrating peptides (CPPs); and X3 is selected from fragments of peptides that specifically bind to protein kinase 2 (CK2). Compared with CIGB-300, the polypeptide can more effectively inhibit CK2, has a higher RPS6 protein-targeting property, has better tumor inhibitory activity, anti-metastatic effect and safety, and can enable large-scale production.

Description

A synthetic anticancer lipopeptide and its application

[0001] This application is based on the application with CN application number 202311352929.6 and application date October 18, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application relates to the field of cancer treatment, and in particular to a synthetic anticancer lipopeptide and its application. Background Art

[0003] 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). Accumulating evidence indicates that CK2 plays an important role in the study of human malignancies.

[0004] Preclinical studies have explored various strategies for inhibiting 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 agents (Zakharia et al., 2019; Perea et al., 2018). CIGB-300 peptide is a peptide chimera consisting of a cyclic peptide that inhibits CK2-mediated phosphorylation and a cell-penetrating peptide, Tat, that facilitates its delivery into 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 CIGB-300 peptide in non-small cell lung cancer (NSCLC) cells revealed that it regulates multiple proteins involved in apoptosis, cell proliferation, ribosome biogenesis, anticancer drug resistance, cell metastasis, and angiogenesis (Rodriguez-Ulloa et al., 2010). Similarly, studies of the phosphoproteomic profile of CIGB-300 peptide in NSCLC cells revealed a panel of CK2 substrates that were significantly inhibited following perturbations in cellular processes (Perea et al., 2020). Consequently, CIGB-300 peptide not only induces apoptosis and controls cell proliferation but also induces antiangiogenic / antimetastatic effects in mouse cancer models (Benavent et al., 2011; Benavent et al., 2016). CIGB-300 peptide exhibits synergistic effects 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). The antitumor activity of CIGB-300 peptide has been validated in mouse cancer models by both local intratumoral injection (Perea et al., 2004) and systemic administration (Perea et al., 2008). Currently, the 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).

[0005] However, some tumor cells are intrinsically resistant to the antiproliferative and proapoptotic effects of CIGB-300 peptide, possibly due to peptide internalization rate and nucleolar localization (Perera et al, 2012; Benavent et al, 2014).

[0006] Summary of the Invention

[0007] This invention describes a synthetic anticancer lipopeptide (SEQ ID NO: 1, referred to as "lipopeptide") that exhibits superior anti-tumor activity compared to the CIGB-300 peptide, with a 4- to 11-fold increase in tumor cell sensitivity. Furthermore, the lipopeptide is low-cost to manufacture, enabling large-scale production.

[0008] Specifically, in a first aspect, the present application provides a non-natural polypeptide comprising domains X1, X2 and X3, wherein:

[0009] X1 is selected from C6-C 20 fatty acid residues;

[0010] X2 is selected from fragments of cell-penetrating peptides (CPPs);

[0011] X3 is selected from fragments of a peptide that specifically binds to protein kinase 2 (CK2).

[0012] 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.

[0013] In a third aspect, the present application provides a drug kit comprising the non-natural polypeptide according to any one of the first aspect or the pharmaceutical composition according to any one of the second aspect, and an optional second therapeutic agent.

[0014] In another aspect, the present application provides 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 preparing a CK2 modulator, or in preparing a medicament, wherein the medicament is used for:

[0015] (1) Inhibit tumor cell proliferation or metastasis, or promote tumor cell apoptosis;

[0016] (2) inhibit angiogenesis, tumor growth or metastasis;

[0017] (3) Treatment of diseases related to CK2 activity.

[0018] In another aspect, the present application provides a method for regulating CK2 activity in a cell, 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.

[0019] 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 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.

[0020] In another aspect, the present application provides a method for inhibiting angiogenesis, or inhibiting tumor growth or metastasis, 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.

[0021] 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 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0023] Figure 1: MS spectrum of the lipopeptide.

[0024] FIG2A : Apoptosis induced by the lipopeptides and CIGB-300 peptide in NCI-H226 cells, and apoptosis was quantified by flow cytometry using annexin V staining.

[0025] FIG2B : Apoptosis induced by the lipopeptides and CIGB-300 peptide in SK-MES1 cells, and apoptosis was quantified by flow cytometry using annexin V staining.

[0026] Figure 3: In vivo toxicity of the lipopeptides and CIGB-300 peptide. Animal body weight was recorded as a marker of treatment toxicity.

[0027] FIG4 : 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.

[0028] FIG5 : Targeting effect of the lipopeptides and CIGB-300 peptide on RPS6 protein in NCI-H226 cell line. The specific binding of the synthetic anticancer lipopeptides to RPS6 protein was analyzed by Western blotting and further quantified by densitometry.

[0029] Definition of terms

[0030] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0031] 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. Furthermore, the terms "include," "comprising," "having," "containing," and variations thereof are intended to be open-ended and not exclusive or exhaustive. Furthermore, the terms "herein," "hereinbefore," and "hereafter," or words of similar meaning, shall refer to this application as a whole and not to any particular portions.

[0032] As used herein, 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 can be common amino acids, such as the 20 protein L-amino acids. In some embodiments, the amino acids used can be unnatural amino acids. As used herein, "unnatural amino acids" 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, and the like. Unless otherwise specified, amino acids used herein are in the L-configuration.

[0033] As used herein, the terms "subject," "patient," or "individual" include mammals and non-mammals. A mammal 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, and pigs; domestic animals such as rabbits, dogs (or canines), and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and the like. Non-mammals can include birds, fish, and the like. 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 male. In some cases, the subject can be 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, subject, patient, or individual are used interchangeably.

[0034] 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 the 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 suffering from the underlying disease.

[0035] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to a sufficient amount of the drug administered that will at least partially alleviate the symptoms of the disease being treated. The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus injection can be administered, or several divided doses can be administered over time, or the dosage can be proportionally reduced or increased according 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, a 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 would 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 to be administered throughout the day.

[0036] 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. Polypeptides can be monomeric or polymeric. In addition, a polypeptide can comprise a plurality of different domains, each domain having one or more different activities. A "polypeptide" can be any length greater than two amino acids. In some cases, a peptide can have a positive charge overall. In some cases, a peptide can have a negative charge overall. In some cases, a peptide can have a neutral charge overall. Peptides can also exist in zwitterionic form.

[0037] Peptides as described herein can be salts thereof. In some embodiments, even if not explicitly mentioned, the quotations of the terms "peptide" or "polypeptide" should be interpreted as including salts thereof. In some embodiments, the salts can include carboxylates (e.g., formate, acetate, trifluoroacetate, trichloroacetate, propionate, isobutyrate, enanthate, decanoate, decanoate, caprylate, stearate, acrylate, hexanoate, propionate, ascorbate, citrate, glucuronate, glutamate, glycolate, α-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, pamoate, nicotinate, isonicotinate, cinnamon) or the like. The salts may be halides (e.g., oxalates, malonates, succinates, suberates, sebacates, fumarates, malates, maleates, hydroxymaleates, hippurates, phthalates, or terephthalates); halides (e.g., chlorides, bromides, or iodides); sulfonates (e.g., benzenesulfonates, methyl, bromo, or chlorobenzenesulfonates, xylenesulfonates, methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, propanesulfonates, isethionates, 1- or 2-naphthalenesulfonates, or 1,5-naphthalenedisulfonates); sulfates; pyrosulfates; bisulfates; sulfites; bisulfites; phosphates; monohydrogenphosphates; dihydrogenphosphates; metaphosphates; pyrophosphates; nitrates; 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.

[0038] As used herein, the term "identity" refers to the match 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 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using 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.

[0039] As used herein, the terms "co-administered," "combined administration," include administering a selected therapeutic agent (i.e., the second therapeutic agent herein) to a subject, and may include administering the agent by the same or different routes of administration or at the same or different times. In some embodiments, polypeptides described herein may be administered in combination with other therapeutic agents. Including administering two or more medicaments to a subject so that the medicaments and / or their metabolites are present in the subject's body simultaneously. They may include administering simultaneously, administering at different times, and / or administering in a composition in which 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.

[0040] Anticancer lipopeptides

[0041] The present application provides a non-natural peptide comprising a cyclic peptide targeting CK2 and a cell-penetrating peptide, wherein the non-natural peptide comprises a lipophilic carboxylic acid modification at the N-terminus. Compared to CIGB-300, the peptide can more effectively inhibit CK2, has higher targeting for the RPS6 protein, exhibits better tumor suppressive activity and anti-metastatic effects, and has a higher safety profile.

[0042] In some embodiments, the present application provides a non-natural polypeptide comprising domains X1, X2, and X3, wherein:

[0043] X1 is selected from C6-C 20 fatty acid residues;

[0044] X2 is selected from fragments of cell-penetrating peptides (CPPs);

[0045] X3 is selected from fragments of a peptide that specifically binds to protein kinase 2 (CK2).

[0046] 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.

[0047] In some embodiments, X2 comprises a fragment of a peptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to GRKKRRQRRRPPQ (SEQ ID NO: 2).

[0048] In some embodiments, X3 has CK2 inhibitory activity, such as inhibiting CK2 phosphorylation.

[0049] 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).

[0050] In some embodiments, an amino acid or peptide linker exists between two adjacent domains of X1, X2, and X3.

[0051] In some embodiments, X1, X2, and X3 are linked in any order.

[0052] In some embodiments, X1, X2, and X3 are arranged in sequence from N-terminus to C-terminus.

[0053] In some embodiments, the polypeptides are linked from N-terminus to C-terminus in the order X1-X2-β-Ala-X3.

[0054] In some embodiments, the C-terminus of the non-natural polypeptide is amidated.

[0055] In some embodiments, the non-natural polypeptide comprises or consists 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 -C ys 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

[0056] X1 is a decanoyl group;

[0057] Cys 15 and Cys 25 Disulfide bonds are formed between them.

[0058] In some specific embodiments, the chemical name and structure of the lipopeptide described herein are as follows:

[0059] (15→25)(Cyclic disulfide)decanoic acid-glycyl-L-histidyl-L-lysyl-L-lysyl-L-arginyl-L-arginyl-L-glutaminyl-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.

[0060] Synthesis of anticancer lipopeptides

[0061] 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 ed.). Rockford: Pierce Chemical Company).

[0062] Pharmaceutical compositions and kits

[0063] In another aspect, the present application provides a pharmaceutical composition comprising the non-natural polypeptide described in any one of the above items and a pharmaceutically acceptable excipient.

[0064] 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, flavorings, sweeteners, coloring agents.

[0065] 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.

[0066] In some embodiments, suitable preservatives include antioxidants such as α-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.

[0067] 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 methylcellulose, 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.

[0068] 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 lauryl sulfate (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0069] 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, alginates, 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 in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0070] In some embodiments, suitable flavoring agents may be selected from cinnamon oil; oil of wintergreen; 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.

[0071] 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.

[0072] 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).

[0073] 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'-tetrakis(methylenephosphonic acid); O, O'-bis(2-aminoethyl)ethylene glycol-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.

[0074] In some embodiments, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0075] 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.

[0076] 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.

[0077] The polypeptide of the present application can be formulated into various formulations, and is used in a variety of different ways, such as oral, rectal or parenteral administration.Term used herein " parenteral " 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, corium, enema, eye drops, ear drops, intranasal, vagina) use.In some exemplary embodiments, route of administration can be by injection, such as intramuscular, intravenous, subcutaneous or intraperitoneal injection.Preparation for oral administration can include capsule, tablet, caplet, pill, lozenge, lozenge, powder and granule etc.

[0078] In another aspect, the present application provides a drug kit comprising any of the above non-natural polypeptides or pharmaceutical compositions, and optionally a second therapeutic agent.

[0079] 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 dosage units.

[0080] In some embodiments, the kit is for:

[0081] (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 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;

[0082] (2) inhibit angiogenesis, tumor growth or metastasis;

[0083] (3) Treatment of diseases associated with CK2 activity (e.g., tumors).

[0084] In some embodiments, the tumor is a refractory cancer or a cancer that is 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.

[0085] application

[0086] The effects of the polypeptides described herein on promoting tumor cell death were tested using cell lines from various sources. Cell apoptosis was measured using methods such as sulforhodamine, alamar blue, or crystal violet, as reported in the literature, and expressed as the half-inhibitory concentration (IC50) (Boyd et al, 1995). The IC50 value refers to the peptide dose that results in 50% cell apoptosis, as determined by a dose-response curve. The effects of the polypeptides on various tumor cell lines were tested at doses ranging from 0 to 200 μM. In some embodiments, the lipopeptides exhibited at least a 4-fold increase in tumor cell potency compared to the CIGB-300 peptide, indicating that the lipopeptides can achieve the same tumor cell-killing effect at only one-quarter the CIGB-300 peptide dose, significantly reducing drug dosage and improving safety.

[0087] Short-term comparative experiments using Annexin-V, a biomarker of apoptosis, as an indicator of cell death were conducted. Flow cytometric analysis revealed that the lipopeptide also demonstrated significant advantages. In some embodiments, the lipopeptide induced a higher percentage of early apoptotic cells compared to the CIGB-300 peptide. In other embodiments, treatment with the lipopeptide resulted in higher levels of reactive oxygen species (ROS). Furthermore, non-tumorigenic normal-like cells in mice were more resistant to the lipopeptide.

[0088] The anti-tumor effects of the polypeptides were validated in a head-to-head experimental in vivo metastasis assay. In some embodiments, C57BL6 mice were intravenously injected with Lewis lung carcinoma 3LL cells. 72 hours after tumor cell challenge, the lipopeptide and CIGB-300 peptide were intravenously administered for five consecutive days. Lung metastasis formation was quantified 30 days later. The results showed that even at a dose one-tenth that of CIGB-300 peptide, the lipopeptide exhibited potent anti-metastatic effects, with no signs of systemic toxicity.

[0089] In addition, the CK2 signal transduction targeting effect of the polypeptide was tested. 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 lipopeptide inhibited the CK2 enzyme activity by more than 50% in all tumor cells. 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 it can more effectively inhibit CK2-mediated phosphorylation and induce a higher internalization rate in tumor cells.

[0090] Based on the above, the peptides and pharmaceutical compositions described herein can be administered to a subject to at least partially improve the disease. In some embodiments, the subject may have previously been diagnosed with a disease described herein, or may be at risk of developing a disease described herein.

[0091] In one aspect, the present application provides a use of any of the above non-natural polypeptides, pharmaceutical compositions, or kits in the preparation of a CK2 modulator (e.g., an inhibitor), or in the preparation of a medicament, wherein the medicament is used for:

[0092] (1) Inhibit tumor cell proliferation or metastasis, or promote tumor cell apoptosis;

[0093] (2) inhibit angiogenesis, tumor growth or metastasis;

[0094] (3) Treatment of diseases associated with CK2 activity (e.g., tumors).

[0095] In another aspect, the present application provides a method for regulating (eg, inhibiting) CK2 activity in a cell, comprising contacting the cell with any one of the aforementioned non-natural polypeptides, pharmaceutical compositions, or kits.

[0096] 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 foregoing non-natural polypeptides, pharmaceutical compositions, or kits.

[0097] In another aspect, the present application provides a method for inhibiting angiogenesis, or inhibiting tumor growth or metastasis, comprising administering to the subject an effective amount of any of the foregoing non-natural polypeptides, pharmaceutical compositions, or kits.

[0098] In another aspect, the present application provides a method for treating a disease associated with CK2 activity in a subject, comprising administering to the subject a therapeutically effective amount of any of the above non-natural polypeptides, pharmaceutical compositions, or kits.

[0099] 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.

[0100] 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 the 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, 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.

[0101] In any of the above methods, the present invention further comprises administering to the subject a therapeutically effective amount of a second therapeutic agent in combination. 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.

[0102] Beneficial technical effects

[0103] The lipopeptide described herein, comprising decanoic acid chemically fused to the N-terminus of the CIGB-300 peptide, is not only a novel anticancer compound but also exhibits more potent anticancer activity than the CIGB-300 peptide. In vitro experiments on tumor cell lines demonstrated the advantages of this lipopeptide, demonstrating that the dose required to kill 50% of cells was at least four times lower than that required for the CIGB-300 peptide. The lipopeptide achieved effective anti-metastatic effects at one-tenth the dose of the CIGB-300 peptide. At a molecular level, this lipopeptide's advantage over the CIGB-300 peptide lies in its more potent inhibition of CK2 enzyme activity. Furthermore, it exhibits stronger targeting of the RPS6 protein.

[0104] The lipopeptide described herein is derived by fusing a decanoic acid moiety to the terminus of the CIGB-300 peptide through a simple chemical modification, overcoming the baseline or inherent resistance of tumor cells to the CIGB-300 peptide. Therefore, the lipopeptide described herein is a more potent novel anticancer compound that can reduce pharmacological doses without increasing toxicity.

[0105] Compared to the CIGB-300 peptide, the lipopeptide described herein achieves more effective anti-tumor effects at a lower dose, providing a valuable reference for clinical use in cancer patients. Given that the synthesis method disclosed in this application is consistent with the synthesis method used to obtain clinical batches of the CIGB-300 peptide, large-scale production is feasible. DETAILED DESCRIPTION

[0106] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0107] Sequence Information Table

[0108] Example 1 Peptide Preparation Method

[0109] The CIGB-300 peptide was synthesized using a solid-phase method based on Fmoc / tBu chemistry and purified to a purity greater than 95% by reverse-phase high-performance liquid chromatography 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, monoisotopic mass = 3211.7607.

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

[0112] The human tumor cells used in this application were obtained 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) at 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, the lipopeptide and CIGB-300 peptide were added at a concentration of 100-3.12 μM at a 1:2 ratio. After 48 hours of incubation, 10% (v / v) Alamar Blue was added and the cell suspension was incubated for a further 4 hours. Fluorescence in the microplate was detected and the IC was estimated using CalcuSyn software (version 2.1) (Biosoft, Cambridge, UK). 50 value.

[0113] The data in Table 1 demonstrate that the lipopeptide exhibited at least a 4-fold greater potency against all tumor cell lines compared to the CIGB-300 peptide. However, benign tumor cells appeared to be more resistant to lipopeptide-induced cell death. The lipopeptide-induced cell death rate was no more than 1.5-fold greater than that of the CIGB-300 peptide against the same benign tumor cells.

[0114] Table 1: Effects of lipopeptides and CIGB-300 peptide on tumor cell viability in vitro Note: SCLC refers to small cell lung cancer; NSCLC refers to non-small cell lung cancer

[0115] Example 3 Time course experiment of lipopeptide and CIGB-300 peptide inducing cell apoptosis and reactive oxygen species

[0116] 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.

[0117] 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, and then washed twice with ice-cold PBS solution. 5The cells were resuspended in a 1x concentration of 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 in the dark at room temperature for 15 minutes. The stained cells were analyzed by flow cytometry using a Partec CyFlow Space device, and data were analyzed and visualized using FlowJo software (version 7.6.1) (BD, Ashland, OR, USA). Figures 2A and 2B show the percentage of apoptotic cells induced in NCI-H226 cells and SK-MES1 cells, respectively. Compared with the CIGB-300 peptide, the lipopeptide induced a higher rate of cell apoptosis.

[0118] Compared to the CIGB-300 peptide, the lipopeptide not only induced higher levels of apoptosis but also, after incubation with A549 cells for 2 hours in the presence of the antioxidant N-acetyl-L-cysteine ​​(NAC), flow cytometry analysis showed that it induced higher levels of ROS (Table 2). With the increase in ROS levels, the lipopeptide was able to affect cell viability to a greater extent.

[0119] Table 2: ROS levels induced by lipopeptides and CIGB-300 peptide in A549 cells

[0120] Example 4 Head-to-head comparative experiment to evaluate the anti-metastatic effects of the lipopeptide and CIGB-300 peptide

[0121] The specific experimental steps were to inject 2x10 5 Lewis rat lung carcinoma 3LL cells were used to induce experimental metastasis and perform lung colonization studies. Ten mice were injected intravenously daily for five consecutive days with 0.1, 1, 5, and 10 mg / kg of the lipopeptide or CIGB-300 peptide. A control group received an intravenous injection of PBS. On day 30, the animals were sacrificed, and the lungs were excised and fixed in Bonn's fixative. Metastatic lung nodules were counted using a dissecting microscope. Additionally, animal body weight and signs of toxicity were monitored throughout the study.

[0122] 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).

[0123] This head-to-head comparison demonstrated that the lipopeptide induced significant anti-metastatic effects at a dose one-twentieth that of CIGB-300. A 10 mg / kg dose of the lipopeptide induced 100% of the anti-metastatic effect compared to CIGB-300. No signs of toxicity were observed in the treatment group receiving the highest dose (10 mg / kg), and the animals showed weight gain during the trial (Figure 3).

[0124] Table 3: Anti-metastatic effects of lipopeptides and CIGB-300 peptide in direct comparison experiments in animals Note: Nodules were counted in both lungs of 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.

[0125] Effects of the lipopeptides and CIGB-300 peptide described in Example 5 on CK2 enzyme activity and CK2 signaling

[0126] CK2 radioactivity assay was performed in increasing concentrations of the lipopeptide solution (final volume 18 μL), with CIGB-300 peptide solution or an equal amount of dimethyl sulfoxide (DMSO) as a control. Prepare 3 μL of recombinant CK2α (28 ng) and a mixture containing 1 mM specific CK2 peptide substrate "RRREEETEEE" (Promega), 10 mM MgCl2, 1 μCi32P-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.

[0127] The data in Table 4 show that the lipopeptide can more effectively inhibit CK2 enzyme activity compared with the CIGB-300 peptide, which indicates that the lipopeptide of the present invention has more effective target interaction.

[0128] Table 4: Head-to-head comparison of CK2 enzyme activity inhibition experiments

[0129] Based on the CK2 enzyme activity data, a head-to-head comparison of the lipopeptide and the CIGB-300 peptide was conducted in the NCI-H226 squamous cell lung cancer cell line to test the lipopeptide's ability to inhibit other CK2 signaling substrates, such as cdc37 protein. Western blotting analysis in Figure 4 demonstrates that, compared to the CIGB-300 peptide, the lipopeptide more effectively inhibits phosphorylation and cdc37 protein levels in NCI-H226 cell lines after 3 and 6 hours of incubation.

[0130] To explore the targeting effect of the lipopeptide and CIGB-300 peptide on CK2 signaling, an RPS6 protein pull-down experiment was performed. The specific steps included seeding 3,600,000 NCI-H226 cells in a T-75 flask, harvesting and washing the cells 24 hours later, and then using a 5-well plate containing 1 mM DTT (Sigma, St. Louis, MO, USA), Triton X-100 (1%), and cOmplete TM The cells were lysed with a 1x concentration of PBS solution containing protease inhibitors (Roche, Basel, Switzerland). The cell lysate was then cleared by centrifugation, and 300 μg of total protein was incubated with 100 μM biotin-labeled lipopeptides, CIGB-300 peptide, 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-cold PBS solution containing 1 mM DTT. The pulled-down RPS6 protein was eluted, separated in an SDS-PAGE gel, subjected to WB analysis, and the specific signal was quantified by optical density determination. The data in Figure 5 show that the lipopeptide has a better targeting effect on the RPS6 protein (PD3) than the CIGB-300 peptide (PD1).

[0131] The lipopeptide was compared head-to-head with the CIGB-300 peptide in terms of protein targeting using the NCI-H226 squamous cell lung cancer cell line. Pull-down assays and mass spectrometry analysis were performed. The data in Table 5 reveal the proteins targeted by the lipopeptide, and the data in Table 6 reveal the proteins that the lipopeptide more effectively targets compared to the CIGB-300 peptide.

[0132] Table 5 Proteins targeted by the lipopeptides in the NCI-H226 cell line

[0133] Table 6 Proteins targeted more effectively by the lipopeptides than the CIGB-300 peptide in the NCI-H226 cell line

[0134] 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 all within the scope of protection of the present invention. The full scope of the present invention is given by the appended 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 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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