Linear peptides that inhibit CK2-mediated phosphorylation and compositions containing same - Patents.com

Abstract

A linear peptide that inhibits phosphorylation mediated by the enzyme casein kinase 2 (CK2), having an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 57 to SEQ ID NO: 62, and a polypeptide comprising said peptide and an intracellular penetrating peptide. A pharmaceutical composition comprising at least one of these linear peptides or polypeptides and a pharma- ceutically acceptable carrier. The present invention also discloses the use of said peptide or polypeptide for the manufacture of a medicament, and a method of treating solid or liquid tumors in which said medicament is administered.

Description

[Technical field]

[0001] The present invention relates to the field of molecular pharmacology and human medicine. In particular, the present invention relates to obtaining linear peptides that inhibit phosphorylation events mediated by the enzyme casein kinase 2 (CK2) through direct interaction with phosphoacceptor sites on substrates. Due to their biological activity, the peptides and polypeptides of the present invention are useful for the treatment of different types of solid tumors and of other origins. [Background technology]

[0002] CK2 is a serine / threonine enzyme involved in increased cell proliferation, and its subcellular localization is essentially nuclear during the process of malignant transformation (Tawfic S., et al., 2001, Histol. Histopathol. 16:573-582). Therefore, this enzyme is an attractive therapeutic target (Duncan JS, et al., 2008, Biochim. Biophys. Acta, 1784:33-47).

[0003] CK2 enzyme-mediated phosphorylation events have been manipulated in experimental oncology by using chemical compounds derived from 4,5,6,7-tetrabromobenzotriazole (TBB) (Pagano MA, et al., 2004, J. Med. Chem., 47:6239-6247), antisense oligonucleotides (Slaton JW, et al., 2004, Mol. Cancer Res. 2:712-720) and peptides that block the interaction between the catalytic and regulatory subunits of the enzyme (Laudet B., et al., 2007, Biochem. J., 408:363-373). The chemical compound CX-4945 inhibits the two catalytic subunits of the enzyme CK2 (Siddiqui-Jain A., et al., 2010, Cancer Res. 70:10288-10298) and is a potent cell proliferation inhibitor in vitro, with a mean maximum inhibitory concentration (IC) of 5.5 μM. 50). This is the only CK2 enzyme inhibitor that has been fully evaluated in humans, with favorable pharmacokinetic and safety parameters recorded in phase I clinical trials (Lim JKC, et al., 2010, Cancer Res. 70: 2763-2766). This is the only CK2 enzyme inhibitor that has been fully evaluated in humans, with favorable pharmacokinetic and safety parameters recorded in phase I clinical trials (Patent Claims WO03 / 054002; Perea SE, et al., 2004, Cancer Res., 64: 7127-7129).

[0004] The CIGB-300 peptide is a chimeric molecule that contains a cyclic peptide that interacts with the CK2 phosphoacceptor site on the substrate and a "cell-penetrating" peptide derived from the Tat1 protein of the human immunodeficiency virus (HIV), ensuring its internalization within the cell. CIGB-300 has shown antiproliferative and proapoptotic properties in vitro and in vivo in various lineages of solid tumors, such as lung, cervix, and larynx (Perera Y., et al., 2014, Mol Clin Oncol, 2(6):935-944), as well as in liquid tumors, such as chronic lymphocytic leukemia (Martins L., et al., 2013, Oncotarget, 5(1):258-263) and T-type acute lymphoblastic leukemia (Perera Y., et al., 2020, Cancers, 12, 1377). Furthermore, this peptide has shown signs of clinical benefit validated in Phase I / II clinical trials (Perea S., et al., 2011, Mol Cell Biochem., 356(1-2):45-50) (Sarduy M., et al., 2015, Br. J. Cancer, 112:1636-1643).

[0005] However, CIGB-300 is a 25 amino acid peptide that contains methionine and intramolecular disulfide bonds in its sequence. These are sources of intramolecular chemical instability and may be the source of inconsistencies at higher stages of product development, either in liquid or lyophilized formulations and in stability studies. (Frokjaer S. et al., 2000, Pharmaceutical formulation development of peptides and proteins. Taylor & Francis). The main impurities present in this peptide are due to oxidation of methionine and formation of aggregates (dimers and trimers) due to the formation of intermolecular disulfide bonds. (Garay H.,et al.,2018,J Pept Sci.24(6):e3081). On the other hand, the synthesis process of peptides containing cysteine ​​is more cumbersome (Albericio F.,et al.,2000,Fmoc solid phase peptide synthesis.A Practical Approach.Oxford University Press Inc,New York.Chapter 4,77-114), and the formation of intramolecular disulfide bonds introduces an additional step in the manufacturing process and increases the production costs. Therefore, it remains interesting to obtain inhibitors of phosphorylation mediated by the CK2 enzyme, of linear peptide type, of reduced size and structural complexity, which are simpler and cheaper to synthesize, for the development of treatments against various tumors. Summary of the Invention

[0006] The present invention solves the above problems by providing linear peptides that inhibit CK2-mediated phosphorylation and have an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 57 to SEQ ID NO: 62. These sequences are shown below: [Table 1]

[0007] These peptides differ from CIGB-300 in that they are linear, smaller in size and more chemically stable. They do not have methionine or cysteine ​​in their sequence, which is an advantage. In addition, from a production point of view, the acquisition is more feasible.

[0008] The linear peptides identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 57 to SEQ ID NO: 62 were selected for their ability to interact with the phosphoacceptor site of the CK2 enzyme on the E7 substrate of human papillomavirus (HPV)-16 (LNDSSEEEDEI) and to inhibit the CK2-mediated phosphorylation of this oncoprotein. As can be seen in the examples of the present invention, these linear peptides are able to inhibit the CK2-mediated phosphorylation of the E7 protein in a manner similar to that of peptide CIGB-300.

[0009] To achieve intracellular action on endogenous CK2 substrates, the peptides of the invention can be chemically conjugated to "cellularly penetrating" peptides belonging to proteins such as the Tat1 protein of HIV (Schwarze SR, et al., 2000, Trends Pharmacol, 21:45-48), la proteina VP22 del Virus Herpes Simples (Lindgreen M., et al., 2000, Trends Pharmacol Sci, 21:99-103), Penetratin and Transportan (Gariepy J., et al., 2001, Trends Biotech 19:21-28), among others.

[0010] Thus, the present invention also provides a polypeptide comprising a linear peptide having an amino acid sequence selected from the sequences identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 57 to SEQ ID NO: 62, and one cell-penetrating peptide.

[0011] In one embodiment of the invention, the cell-penetrating peptide in these polypeptides is a peptide derived from the HIV Tat1 protein, in one embodiment of which the cell-penetrating peptide has the amino acid sequence identified as either SEQ ID NO:65 or SEQ ID NO:66.

[0012] In certain embodiments, the polypeptide of the invention has an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 51 to SEQ ID NO: 56. These sequences are shown below: [Table 2]

[0013] Most of these polypeptides were IC in various cancer cell lines (lung, bladder, laryngeal, pancreatic, and leukemia). 50 The polypeptide was able to produce a dose-dependent effect on the proliferation of cancer cells. Furthermore, the polypeptide identified as SEQ ID NO: 35 had an IC value similar to that of CIGB-300 in these cell lines. 50 However, it is believed to be superior because of its smaller size, more chemically stable linear amino acid sequence, and manufacturability advantages that will facilitate the development of therapeutic products.

[0014] To identify the peptides described in the present invention, a combinatorial library of linear peptides of the type "one-bead-one-compound" containing more than 16 million different peptide sequences was synthesized and screened using synthetic peptides containing the 28-38 region of the E7 oncoprotein modified with biotin. Mass spectrometry was used to assign the sequences of peptides contained in the nine beads that were positive in the screening. The peptides identified as SEQ ID NO: 1 to SEQ ID NO: 9 were chemically synthesized and confirmed to be capable of inhibiting CK2-mediated phosphorylation in the E7 oncoprotein of HPV-16. To achieve intracellular action against endogenous CK2 substrates, a polypeptide containing nine peptides was designed and synthesized, and an intracellular penetrating peptide corresponding to the HIV Tat1 protein was added to the N-terminus (GRKKRRQRRRPPQ, identified as SEQ ID NO: 65). Next, the effect of these nine polypeptides on cell proliferation of the NCI-H125 strain (non-small cell lung cancer) was evaluated. None of the polypeptides reached the same inhibitory value as CIGB-300 in the range of concentrations evaluated. Of these, only three, identified as SEQ ID NO:18 to SEQ ID NO:20, inhibited cell proliferation by 20% to 30% at the highest concentration tested, and therefore their sequences were modified to improve their biological activity.

[0015] To evaluate the effect of Trp incorporation on the biological activity of the polypeptides identified as SEQ ID NO:18 to SEQ ID NO:20, a Trp positional scanning library was designed and obtained. Next, the anti-proliferative effects of 24 polypeptides (identified as SEQ ID NO:21 to SEQ ID NO:44) generated in the Trp library in NCI-H125 cell line. Of the 24 polypeptides, six identified as SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:43 showed similar cell proliferation inhibition rates as peptide CIGB-300 at the highest concentration evaluated. The IC values ​​of the polypeptide identified as SEQ ID NO:35 and CIGB-300 obtained in NCI-H125 and AsPC-1 (pancreatic adenocarcinoma) cell lines were compared. 50In particular, the polypeptide identified as SEQ ID NO: 35 is believed to be superior to CIGB-300 because it is a linear sequence of smaller size, is more chemically stable, and has manufacturing advantages.

[0016] Two other linear polypeptides more active than the CIGB-300 peptide, identified in the present invention as SEQ ID NO: 47 and SEQ ID NO: 48, are the result of testing the introduction of a second Trp residue at position 3 of the polypeptide identified as SEQ ID NO: 35. The polypeptides identified as SEQ ID NO: 47 and SEQ ID NO: 48 were shown to be more potent than peptide CIGB-300 in their ability to inhibit cell proliferation in pancreatic cancer (AsPC-1, PanC-1) and acute myeloid leukemia (OCI-AML3) cell lines, and similar to CIGB-300 in the lung cancer cell line used (NCI-H125).

[0017] The introduction of unnatural amino acids into the sequences identified as SEQ ID NO: 47 and SEQ ID NO: 48 at the sites susceptible to enzymatic degradation resulted in six new linear polypeptides identified as SEQ ID NO: 51 to SEQ ID NO: 56, which were more active than CIGB-300. Antiproliferative assays were performed in NCI-H125, Hep2C (larynx cancer) cell lines and two bladder cancer lines (MGH-U3 and MGH-U4). The IC of peptides identified as SEQ ID NO: 51 to SEQ ID NO: 56 obtained in NCI-H125, Hep2C, MGH-U3 and MGH-U4 cell lines were 50 The IC values ​​were lower than those obtained with CIGB-300, indicating that they are more potent. For bladder cancer lines (MGH-U3 and MGH-U4), IC 50 An order of magnitude lower value is very attractive because it is a little explored niche, is not degraded, and has a major impact on health. The introduction of unnatural amino acids was performed to increase stability against proteases. Surprisingly, it was also possible to enhance the ability of these polypeptides to inhibit CK2-mediated phosphorylation.

[0018] The present invention also provides a pharmaceutical composition comprising at least one linear peptide having an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 57 to SEQ ID NO: 62, or at least one polypeptide having an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 51 to SEQ ID NO: 56, and a pharma- ceutically acceptable excipient.

[0019] Excipients that can form part of the pharmaceutical composition of the present invention are known to those skilled in the art. In one embodiment of the present invention, the pharmaceutical composition is formulated for administration by systemic, intratumoral, oral, or mucosal routes. In one embodiment of the present invention, the pharmaceutical composition is useful for the treatment of solid or liquid tumors in an individual in need thereof.

[0020] The object of the present invention is the use of a linear peptide having a sequence selected from the group consisting of the sequences specified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 57 to SEQ ID NO: 62, or a polypeptide having an amino acid sequence selected from the group consisting of the sequences specified as SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 51 to SEQ ID NO: 56, for the manufacture of a drug.

[0021] In one embodiment of the invention, the medicament is used for the treatment of solid or liquid tumors.

[0022] In another aspect, the present invention discloses a method of treating a solid or liquid tumor in an individual in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one linear peptide having an amino acid sequence selected from the group consisting of sequences identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 57 to SEQ ID NO: 62, or at least one polypeptide having an amino acid sequence selected from the group consisting of sequences identified as SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 51 to SEQ ID NO: 56. In one embodiment of the present invention, the solid tumor treated is a tumor of the lung, cervix, larynx, pancreas, or bladder. In another embodiment of the present invention, the liquid tumor treated is a chronic lymphocytic leukemia, T-type acute lymphoblastic leukemia, or acute myeloid leukemia. [Brief description of the drawings]

[0023] [Figure 1] Effect of synthetic peptides SEQ ID NO: 1 to SEQ ID NO: 9 on CK2-mediated phosphorylation of HPV-16 E7 oncoprotein. Two peptides (identified as SEQ ID NO: 10 and SEQ ID NO: 11) that do not interact with the phosphoacceptor site of the CK2 enzyme. Counts per minute (cpm) are plotted on the Y-axis. [Diagram 2] Inhibition rate (%I) of cell proliferation in NCI-H125 cell line of nine polypeptides identified as SEQ ID NO: 12 to SEQ ID NO: 20. Peptides CIGB-300 and Tat were used as positive and negative controls of the assay, respectively. Polypeptides showing inhibition rate of cell proliferation of 20% or more were selected. [Diagram 3] Dose-effect curves of polypeptides F11P19, F11P20 and F11P21 (SEQ ID NO: 18 to SEQ ID NO: 20) in NCI-H125 cell line. Included is the dose-effect curve of CIGB-300 peptide used as positive control in the assay. % I: Inhibition of cell proliferation C: Concentration [Figure 4]Percentage of inhibition of cell proliferation in NCI-H125 cell line (% I) for polypeptides generated by positional sweep of Trp residues in charge (SEQ ID NO:21 to SEQ ID NO:44) from sequences identified as SEQ ID NO:18 to SEQ ID NO:20. A. Percentage of inhibition achieved by polypeptides identified as SEQ ID NO:21 to SEQ ID NO:44 at two concentrations. Precursor peptides identified as SEQ ID NO:18 to SEQ ID NO:20 and CIGB-300 as positive control. The dashed box outlines the six polypeptides and CIGB-300 that showed more than 60% cell proliferation inhibition at 200 μM. B. Percentage of inhibition achieved by various concentrations from 12.5 μM to 200 μM for the six most active polypeptides. [Diagram 5] Dose-effect curves of polypeptides A14P27 (SEQ ID NO:21), A14P31 (SEQ ID NO:25), A14P33 (SEQ ID NO:27), A14P43 (SEQ ID NO:32), A14P46 (SEQ ID NO:35), A14P59 (SEQ ID NO:43) in NCI-H125 cell line. Included is the dose-effect curve of CIGB-300 peptide used as control peptide. % I: Inhibition of cell proliferation C: Concentration [Figure 6] Dose-effect curves of polypeptide A14P46 (SEQ ID NO: 35) and CIGB-300 peptide in AsPC-1 cell line. % I: inhibition rate of cell proliferation. C: concentration [Figure 7] Dose-effect curves of polypeptides A15P35, A15P36 and A15P37 (SEQ ID NO: 46 to SEQ ID NO: 48) in NCI-H125 cell line. Dose-effect curves including precursor polypeptide A14P46 (SEQ ID NO: 35) and CIGB-300 peptide used as control. % I: inhibition of cell proliferation. C: concentration. [Figure 8] Dose-effect curves of polypeptides E17P01, E17P02 and E17P03 (SEQ ID NO: 51 to SEQ ID NO: 53) in NCI-H125 cell line. Dose-effect curves of precursor polypeptide A15P36 (SEQ ID NO: 47) and CIGB-300 used as control. % I: inhibition of cell proliferation. C: concentration [Figure 9]Dose-effect curves of polypeptides D17P78, D17P79 and D17P80 (SEQ ID NO: 54 to SEQ ID NO: 56) in NCI-H125 cell line. Dose-effect curves including precursor polypeptide A15P37 (SEQ ID NO: 48) and CIGB-300 used as control. % I: inhibition of cell proliferation. C: concentration. [Figure 10] Antitumor effect of E17P01 (SEQ ID NO: 51), a polypeptide inhibitor of CK2 phosphorylation, in a human bladder tumor model implanted in athymic mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0024] Example 1. Selection of a linear peptide that interacts with the phosphoacceptor site of the CK2 enzyme in the E7 protein of HPV-16. To identify bioactive linear peptides that interact with the phosphoacceptor site of the CK2 enzyme, a combinatorial library of synthetic linear peptides, named BCP-1 by the type of "one-bead-one-compound," containing more than 16 million different peptide sequences, was screened. The library was designed to be compatible with electrospray ionization mass spectrometry (ESI-MS) sequence analysis (Masforrol Y., et al., 2012, ACS Combinatorial Science, 14(3):145-9). Screening was performed with a biotinylated synthetic peptide, called BIOT, that contains the phosphoacceptor site of the CK2 enzyme in the E7 substrate of HPV-16 (LNDSSEEEDEI, identified as SEQ ID NO: 63). Streptavidin-alkaline phosphatase conjugate was used as the recognition molecule, and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) was used as the growth substrate. The blue color, compared to the light pale yellow color of the TentaGel resin in the BCP-1 library, facilitates visual identification of beads containing the bioactive sequence recognized by the BIOT peptide. The BIOT peptide was synthesized on 4-methylbenzhydrylamine (MBHA) resin using Fmoc / tBu chemistry (Field GB and Noble RL. Int. J. Peptide Protein Res. 1990;35(3):161-214). It was purified by reversed-phase chromatography (RP-HPLC) and its identity confirmed by ESI-MS. It was obtained in greater than 95% purity, and ESI-MS analysis confirmed its molecular weight (MM) to be 1574.6 Da.

[0025] The BCP-1 library was placed in a polypropylene reaction vessel equipped with a vacuum filtration system and mechanical stirring. It was washed with purified water and saline phosphate buffer (PBS) pH 7.4 and blocked with 1% bovine serum albumin (BSA) in PBS pH 7.4 for 1 h. Solvent and reagents were removed by vacuum filtration. The library was incubated with 80 mL of BIOT peptide solution at 150 μg / mL in 1% BSA in PBS pH 7.4 for 16 h at 4° C. It was washed with Tween-PBS (T-PBS) pH 7.4 and incubated with streptavidin-alkaline phosphatase conjugate (0.4 μg / mL) in a solution of 1% BSA in PBS pH 7.4. After 2 h, it was washed with T-PBS pH 7.4 and incubated with BCIP (0.5 mg / mL) in substrate solution for 30 min. When a blue color appeared, the reaction was stopped by filtration and extensive washing with PBS pH 7.4.

[0026] The positive beads were separated from the rest by visual inspection using an eyepiece, transferred to a fritted reactor and regenerated. Subsequently, tests were performed to exclude non-specific recognition by streptavidin-alkaline phosphatase conjugate and BCIP. The screening was performed under higher stringency conditions to select sequences with higher affinity and reduce the number of positive beads, and thus the concentration of BIOT peptide was reduced to 120 μg / mL. Following the procedure described above, the positive beads were regenerated and the sequences were identified by nanoESI-MS / MS (Masforrol Y., et al, 2012, ACS Combinatorial Science, 14(3):145-9).

[0027] Initial screening with 150 μg / mL of peptide BIOT resulted in the selection of 56 positive beads. After a second screening with a lower concentration of BIOT (120 μg / mL), 9 positive beads were selected from the first screening that showed stronger blue coloration than the rest, proving a greater affinity of these peptides to the CK2 phosphoacceptor site. The nanoESI-MS / MS spectra obtained from the 9 positive beads were analyzed in parallel by manual de novo sequencing and by automated sequencing using the Mascot program to search the BCP-1 database. Table 1 shows the sequences of the 9 peptides identified (SEQ ID NO: 1 to SEQ ID NO: 9). Analysis of the sequences assigned to each peptide yielded a perfect correspondence of m / z signal assignments between manual identification and automated sequencing, which allowed accurate identification of the peptide sequence in each case. [Table 3]

[0028] Similarity was observed in the amino acid composition of the nine peptides shown in Table 1. This is characterized by the predominance of basic amino acids (K, R) and the presence of hydrophobic amino acids (L, A, V, F, Y).

[0029] To confirm that the nine sequences identified by nanoESI-MS / MS interact with the phosphoacceptor site of the CK2 enzyme in the E7 protein, peptides were synthesized directly on Tentagel resin using Fmoc / tBu chemistry (Field GB et al., 1990, Int. J. Peptide Protein Res., 35(3):161-214). Enzymatic reactions were performed on the resin-bound peptides using BIOT peptide at a concentration of 120 μg / mL. Streptavidin-alkaline phosphatase conjugate was used as the recognition molecule and BCIP was used as the substrate for growth. Nine reactors containing 15 mg of encoded peptides (P1-P9) bound to the resin were placed in a vacuum filtration system with mechanical stirring. Two control peptides (P10 identified as SEQ ID NO:10 and P11 identified as SEQ ID NO:11) that do not interact with the resin-bound CK2 phosphoacceptor site on the E7 protein were used, which were bound to the resin. The procedure was adjusted to reproduce the BCP-1 library screening assay with a working volume of 500 μL for each reactor. The results showed that nine peptides coupled to the resin (P1-P9) generated an intense blue color after the enzymatic assay, demonstrating the ability of the nine identified linear peptides to interact with the phosphoacceptor site of the CK2 enzyme in the substrate E7 model. In contrast, the resin-coupled control peptides (P10 and P11) did not generate blue coloration.

[0030] Example 2. Effect of peptides identified as SEQ ID NO:1 to SEQ ID NO:9 on CK2-mediated phosphorylation of HPV-16 E7 oncoprotein. The peptides were synthesized on MBHA resin using Fmoc / tBu chemistry (Field GB and Noble RL. Int. J. Peptide Protein Res. 1990;35(3):161-214). The peptides were purified by RP-HPLC and their identity was confirmed by ESI-MS. For synthesis, they were designated F11P04 to F11P12 and their amino acid sequences correspond to the sequences specified as SEQ ID NO:1 to SEQ ID NO:9. As can be seen in Table 2, all were obtained with a purity of over 95% and their identity was confirmed by ESI-MS analysis. [Table 4]

[0031] The assay to measure the effect of these peptides on the CK2-mediated phosphorylation of the HPV-16 E7 oncoprotein was based on an in vitro phosphorylation reaction, in which the E7 oncoprotein of HPV-16 expressed in E. coli was used as a substrate. This oncoprotein was obtained as a fusion protein to glutathione S-transferase. The assay was carried out according to a previously described procedure. (Perea SE, et al., 2004, Cancer Res., 64:7127-7129). The results are shown in Figure 1 and show that the peptides identified as SEQ ID NO:1 to SEQ ID NO:9 are able to inhibit the CK2-mediated phosphorylation of the E7 protein of HPV-16.

[0032] Example 3. Effect of synthetic polypeptides identified as SEQ ID NO:12 to SEQ ID NO:20 on cell proliferation in non-small cell lung cancer line NCI-H125. To achieve intracellular action on endogenous CK2 substrates, the intracellular penetrating peptide Tat was added to the N-terminus with the sequences identified as SEQ ID NO: 1 to SEQ ID NO: 9, corresponding to the Tat1 protein of HIV (GRKKRRQRRRPPQ). A beta-alanine (βA) residue was introduced as a spacer between the peptide of interest and the Tat peptide. The polypeptides were coded as F11P13 to F11P21 for synthesis and correspond to the sequences identified as SEQ ID NO: 12 to SEQ ID NO: 20, as shown in Table 3. The peptide CIGB-300 was identified as SEQ ID NO: 64, and the Tat peptide was identified as SEQ ID NO: 65. The synthesis, purification and identity analysis of the peptides were performed as shown in Example 2. As shown in Table 3, all of them were obtained with a purity of more than 95% and their identity was confirmed by ESI-MS analysis. [Table 5]

[0033] The evaluation of the antiproliferative activity of polypeptides F11P13 to F11P21 (SEQ ID NO: 12 to SEQ ID NO: 20) was carried out on the NCI-H125 cell line. The cell line shows high sensitivity to low concentrations of the drug under evaluation (HU L.-Y., et al., 2018, European Review for Medical and Pharmacological Sciences, 22: 4551-4556). The antiproliferative effect of the CIGB-300 peptide has been extensively characterized in this line (Cirigliano SM, et al., 2017, Cancer Cell International, 17, 42), and therefore it was selected as a positive control. The Tat peptide was used as a negative control. The polypeptides were evaluated at concentrations of 25, 50, 100 and 200 μM. The cell proliferation assay was performed by a procedure previously described (Perera Y., et al., 2012, J. Pept. Sci, 18(4): 215-23).

[0034] Figure 2 shows data from the antiproliferative assay in NCI-H125 cells. Each bar corresponds to the value of cell growth inhibition (% I) reached at the evaluated concentrations of the polypeptide (25, 50, 100 and 200 μM). Polypeptides that showed cell growth inhibition values ​​of 20% or more were selected since this percentage value was within the range of the coefficient of variation of the assay. Only polypeptides F11P19, F11P20 and F11P21 inhibited cell growth by 20% to 30% at a concentration of 200 μM. With peptide CIGB-300, used as a control, only 50% growth inhibition was achieved.

[0035] Figure 3 shows the dose-effect curves of the polypeptides F11P19, F11P20 and F11P21 compared to the peptide CIGB-300. 50 The IC values ​​were 336 μM, 305 μM, 668 μM and 64 μM, respectively. 50 The IC values ​​obtained for CIGB-300 50 As these are still far from the expected value, it is necessary to optimize said primary structures in order to improve their biological activity.

[0036] Example 4. Optimization of sequences identified as SEQ ID NO:18 to SEQ ID NO:20 by tryptophan scan on charge. Trp amino acids were excluded from the BCP-1 library synthesis by design (Masforrol Y., et al, 2012, ACS Combinatorial Science, 14(3):145-9). To this end, a Trp positional scanning library was designed for the sequences identified as SEQ ID NO:18 to SEQ ID NO:20, in which the substitution of charge to each amino acid residue of Trp was performed in time (Table 4). This allowed us to investigate the contribution of Trp at each position of the cargo sequence.

[0037] The Trp positional scanning library polypeptides were synthesized on MBHA resin using Fmoc / tBu chemistry (Field GB y Noble RL. Int. J. Peptide Protein Res. 1990;35(3):161-214), purified by RP-HPLC, and confirmed by ESI-MS. As can be seen in Table 4 showing the amino acid sequences, they were all obtained with a purity of more than 95% and ESI-MS analysis confirmed their identity to the designed molecules. [Table 6]

[0038] To demonstrate that modification with Trp does not affect the ability of the polypeptides to interact with the phosphoacceptor site of the CK2 enzyme in the E7 protein, the procedure described in Example 1 was followed. The 24 polypeptides bound to the resin showed a strong blue color after the enzyme assay, proving the interaction of the model E7 substrate with the phosphoacceptor site of the CK2 enzyme. On the other hand, to demonstrate that modification with Trp does not affect the effect of the polypeptides on the phosphorylation of the E7 protein by CK2, the procedure described in Example 2 was followed. The 24 polypeptides identified as SEQ ID NO:21 to SEQ ID NO:44 maintained their ability to inhibit the phosphorylation of the E7 protein of HPV-16 mediated by CK2, since phosphorylation inhibition values ​​of 60 to 85% were obtained. CIGB-300 showed 87% inhibition of phosphorylation.

[0039] The synthetic polypeptides (SEQ ID NO: 21 to SEQ ID NO: 44) generated by positional scanning of Trp were evaluated for their antiproliferative activity against the NCI-H125 cell line. The test was performed according to a previously described procedure. (Perera Y., et al., 2012, J. Pept. Sci, 18(4):215-23). ​​Precursor polypeptides (F11P19, F11P20, F11P21) and peptide CIGB-300 were included as positive controls. Polypeptides were evaluated at concentrations of 12.5, 25, 50, 100 and 200 μM. Figure 4A shows the results of the percentage inhibition of cell proliferation at polypeptide concentrations of 100 and 200 μM. The results show that six of the 24 polypeptides evaluated (A14P27 (SEQ ID NO: 21), A14P31 (SEQ ID NO: 25), A14P33 (SEQ ID NO: 27), A14P43 (SEQ ID NO: 32), A14P46 (SEQ ID NO: 35), A14P59 (SEQ ID NO: 43)) showed more than 60% inhibition of cell proliferation at 200 μM, similar to the effect seen with peptide CIGB-300. Figure 4B shows the percent inhibition values ​​of cell proliferation obtained for the six selected polypeptides at the concentrations evaluated. As can be seen, the A14P46 polypeptide inhibits more than 50% of cell proliferation at concentrations above 100 and 200 μM, similar to the behavior of CIGB-300. At lower concentrations, the A14P46 polypeptide showed a better dose-effect correlation compared to the performance of the CIGB-300 peptide.

[0040] Figure 5 shows the dose-effect curves of the polypeptides A14P27, A14P31, A14P33, A14P43, A14P46, A14P59 and CIGB-300. For its part, Table 5 shows the IC 50 The IC values ​​for the A14P46 polypeptide (72 μM) are lower than those obtained for their precursor polypeptides F11P19 (336 μM), F11P20 (305 μM) and F11P21 (668 μM). 50 is the IC of the CIGB-300 reference peptide (64 μM) 50 is equivalent to.

[0041] Structural studies carried out on the peptide sequence X1X2X3X4X5X6X7X8 allowed to conclude that the change of D to W at position X7 was essential to enhance the biological activity (A14P46>>A14P33>A14P59), with the highest response for the polypeptide A14P46. Furthermore, it was concluded that the position X4>X5>X1 also showed significant results in terms of biological activity, suggesting the introduction of a second amino acid W in the substitution at said position. [Table 7]

[0042] To demonstrate that the biological activity of polypeptide A14P46 (SEQ ID NO: 35) is solely due to their charge position (RKRSRYWP), a similar peptide was synthesized without the Tat and βA sequences using the synthesis procedure described in this example. Peptide D14P165, identified as SEQ ID NO: 45, was obtained with 95% purity and its MW was demonstrated to be 1146.7 Da by analysis by ESI-MS. Phosphorylation inhibition assay was performed as described in Example 2, and 85% inhibition was obtained, confirming the ability of the peptide to inhibit CK2-mediated phosphorylation of HPV-16 E7 protein.

[0043] Considering that the polypeptide A14P46 showed similar biological activity results as CIGB-300 in NCI-H125 cell line, the behavior of such a polypeptide in a pancreatic adenocarcinoma tumor line (AsPC-1) was tested in comparison with CIGB-300. Cell proliferation assays were performed according to a procedure previously described (Perera Y., et al., 2012, J. Pept. Sci, 18(4):215-23). ​​As shown in Figure 6, both molecules can produce a dose-dependent effect on the proliferation of AsPC-1 cells. An IC of 125 μM for A14P46 50 value and IC of 102 μM for CIGB-300 50 Values ​​were obtained indicating that both peptides have similar potency in this cell line.

[0044] Polypeptide A14P46 (SEQ ID NO: 35) is shown to be comparable to CIGB-300 in its ability to inhibit cell proliferation in NCI-H125 and AsPC-1 cell lines (Figures 5 and 6), but is superior because it is a linear polypeptide of smaller size, is more chemically stable and does not contain either methionine or cysteine ​​in its sequence, making it more attractive from a production standpoint.

[0045] Example 5. Optimization of the polypeptide identified as SEQ ID NO: 35 by introducing a second Trp residue into the sequence. Based on the results obtained in Example 4, additional Trp residues were introduced into the A14P46 polypeptide at positions X1, X4 and X5 and their effect on biological activity was evaluated. The polypeptides were synthesized on MBHA resin using Fmoc / tBu chemistry (Field GB y Noble RL. Int. J. Peptide Protein Res. 1990; 35(3): 161-214), purified by RP-HPLC and confirmed identity by ESI-MS. As shown in Table 6, three peptides were obtained with a purity of more than 95% and the identity of the molecules was confirmed by ESI-MS analysis. [Table 8]

[0046] The evaluation of the antiproliferative activity of the synthetic polypeptides A15P35, A15P36 and A15P37 (SEQ ID NO: 46 to SEQ ID NO: 48) was carried out on NCI-H125, AsPC-1, PanC-1 (pancreatic cancer) and OCI-AML3 (acute myeloid leukemia-3) cell lines. The tests were carried out according to the procedure previously described (Perera Y, et al., 2012, J. Pept. Sci, 18(4):215-23). ​​Figure 7 shows the dose-effect curves of the three polypeptides in the NCI-H125 cell line compared to their precursor A14P46 and the control peptide CIGB-300. The polypeptides A15P35, A15P36 and A15P37 were able to produce a dose-dependent effect on the proliferation of NCI-H125 cells. In the AsPC-1, PanC-1 and OCI-AML3 lines, the three polypeptides also showed a dose-response pattern.

[0047] Table 7 shows the IC values ​​of A15P35, A15P36 and A15P37 polypeptides obtained in NCI-H125, AsPC-1, PanC-1 and OCI-AML3 cell lines. 50 The values ​​are summarized relative to their precursor A14P46 and the control peptide CIGB-300. 50 The IC values ​​are similar to those obtained for their precursors A14P46 and CIGB-300 in the four lines tested, indicating that the introduction of Trp at position X1 of the sequence is irrelevant. In contrast, the IC values ​​obtained for the polypeptides A15P36 and A15P37 50 The values ​​were lower than those obtained for the polypeptide A14P46 and peptide CIGB-300, especially in pancreatic cancer cell lines (AsPC-1, PanC-1) and acute myeloid leukemia (OCI-AML3). [Table 9]

[0048] To demonstrate that the biological activity of A15P36 polypeptide (SEQ ID NO: 47) and A15P37 (SEQ ID NO: 48) is solely due to the charges RKRWRYWP and RKRSWYWP, both peptides were synthesized without Tat or βA sequences using the synthesis procedure described in this example. Peptides D15P105 (SEQ ID NO: 49) and D15P106 (SEQ ID NO: 50) were obtained with a purity of more than 95% and ESI-MS analysis confirmed their molecular weights of 1245.7Da and 1176.6Da, respectively. Phosphorylation assays were performed as described in Example 2, and phosphorylation inhibition values ​​of 87% and 82%, respectively, were obtained, confirming the ability of both peptides to inhibit CK2-mediated phosphorylation of HPV-16 E7 protein.

[0049] The results of this example show that the introduction of a second Trp at positions X4 and X5 of the polypeptide sequence A14P46 (SEQ ID NO: 35) supports its ability to inhibit the interaction of the peptide with the phosphoacceptor site and thus phosphorylation by CK2. The A15P36 and A15P37 polypeptides have been shown to be more potent than CIGB-300 in their ability to inhibit cell proliferation in pancreatic cancer (AsPC-1, PanC-1) and acute myeloid leukemia (OCI-AML3) cell lines. Both polypeptides have the additional advantage of being smaller sized, more chemically stable linear sequences, since they do not have sensitive residues such as methionine and cysteine. In addition, they are more attractive from a production standpoint.

[0050] Example 6. Optimization of the polypeptides identified as SEQ ID NO: 47 and SEQ ID NO: 48 by introducing unnatural amino acids (D- and LN-methyl amino acids). To protect the polypeptides A15P36 (SEQ ID NO: 47) and A15P37 (SEQ ID NO: 48) from the action of endopeptidases in body fluids, unnatural amino acids were introduced into the sites of the sequence that are more susceptible to degradation by trypsin and chymotrypsin. The most susceptible sites to proteolysis were predicted with the help of the Expasy site (http: / / www.expasy.org) (Gasteiger E., et al., 2003, Nucleic Acids Research. 31(13):3784-3788). D-Arg was incorporated into R8 of Tat, and protection of K16, R17 and Y20 was evaluated by introducing D-Lys, D-Arg and NMe-Tyr, respectively. The N-terminus of each polypeptide was acetylated to protect against N-peptidases. Polypeptides E17P01, E17P02 and E17P03 (SEQ ID NO:51 to SEQ ID NO:53) are derivatives of A15P36, and polypeptides D17P78, D17P79 and D17P80 (SEQ ID NO:54 to SEQ ID NO:56) are derivatives of A15P37. Polypeptides were synthesized on MBHA resin using Fmoc / tBu chemistry, purified by RP-HPLC and identity confirmed by ESI-MS. As shown in Table 8, all were obtained with a purity of >95% and ESI-MS analysis confirmed the identity of the molecules. [Table 10]

[0051] To demonstrate that the introduction of unnatural amino acids does not affect the ability of the polypeptides to interact with the phosphoacceptor site of the CK2 enzyme in the E7 protein, the procedure described in Example 1 was followed. Six polypeptides bound to the resin appeared a deep blue color after the enzyme assay, proving the interaction of the model E7 substrate with the phosphoacceptor site of the CK2 enzyme. On the other hand, to demonstrate that the modifications do not affect the effect of the peptides on phosphorylation at the E7 protein by the enzyme CK2, the procedure described in Example 2 was followed. Polypeptides E17P01-E17P03 and D17P78-D17P80, identified as SEQ ID NO:51-SEQ ID NO:56, maintained their ability to inhibit CK2-mediated phosphorylation at the E7 protein of HPV-16, with phosphorylation inhibition values ​​of 83-90%.

[0052] Antiproliferative assays were performed on NCI-H125, Hep2C (laryngeal cancer) cell lines, and two bladder cancer lines (MGH-U3 and MGH-U4). The tests were performed according to a previously described procedure (Perera Y., et al, 2012, Pept. Sci, 18(4):215-23).

[0053] Figures 8 and 9 show the dose-effect curves of polypeptides E17P01, E17P02 and E17P03 (SEQ ID NO:51 to SEQ ID NO:53) and D17P78, D17P79 and D17P80 (SEQ ID NO:54 to SEQ ID NO:56), respectively, in NCI-H125 cell line. The control CIGB-300 peptide and the precursor polypeptides A15P36 and A15P37, respectively, were included. The six polypeptides could produce a dose-dependent effect on the proliferation of NCI-H125 cell line. In Hep2C, MGH-U3 and MGH-U4 cell lines, all six polypeptides also showed a dose-response pattern.

[0054] Table 9 shows the IC of polypeptides E17P01, E17P02, E17P03, D17P78, D17P79 and D17P80 (SEQ ID NO: 51 to SEQ ID NO: 56) obtained in cell lines NCI-H125, Hep2C, MGH-U3 and MGH-U4. 50Values ​​are summarized relative to their precursors and to the control peptide CIGB-300.

[0055] Table 9 shows the IC of the polypeptides identified as SEQ ID NO:51 to SEQ ID NO:56. 50 The IC values ​​were lower than those obtained with peptide CIGB-300 in NCI-H125, Hep2C, MGH-U3 and MGH-U4 cell lines, indicating that they are more potent in terms of their antiproliferative effect. Surprisingly, in the case of bladder cancer lines (MGH-U3 and MGH-U4), the IC 50 The value is an order of magnitude lower compared to CIGB-300, which is very attractive because it is a little explored niche, is not degraded, and has a major impact on health. Modifications to the A15P36 and A15P37 polypeptides were introduced to increase their stability against proteases. However, surprisingly, their ability to inhibit CK2 phosphorylation was also enhanced. [Table 11]

[0056] To demonstrate that the biological activity of the peptides identified as SEQ ID NO:51 to SEQ ID NO:56 is due solely to their charge, peptides were synthesized without the Tat and βA sequences using the synthesis procedure described in this same example. Peptides D17P81 to D17P86 (SEQ ID NO:57 to SEQ ID NO:62) were obtained with a purity of more than 95% and ESI-MS analysis confirmed the identity of the molecules obtained as designed (Table 10). [Table 12]

[0057] Phosphorylation assays were performed as described in Example 2 and phosphorylation inhibition values ​​of 83-90% were obtained, confirming the ability of these peptides to inhibit CK2-mediated phosphorylation of HPV-16 E7 protein.

[0058] In summary, the polypeptides identified as SEQ ID NO:51 to SEQ ID NO:56 are more potent than peptide CIGB-300 in terms of their ability to inhibit cell proliferation, particularly in terms of their inhibitory activity in bladder cancer lines (MGH-U3 and MGH-U4). Furthermore, these peptides are linear, smaller in size and more chemically stable, providing obvious advantages from a manufacturing standpoint.

[0059] Example 7. Antitumor efficacy of polypeptide inhibitors of CK2 phosphorylation in a human bladder tumor model implanted in athymic mice. For these studies, athymic female BalbC mice aged 6-8 weeks were used. MGH-U3 cells derived from bladder cell carcinoma were used. For tumor implantation in this model, the cells were suspended in PBS at a concentration of 1 million cells per milliliter. The cell suspension was inoculated subcutaneously into the peritoneal cavity of the animals. Administration of E17P01 polypeptide (identified as SEQ ID NO: 51) or CIGB-300 was started 5 days after inoculation of the tumor cells and continued intraperitoneally for 5 consecutive days. The polypeptides and peptides were dissolved in PBS. The dose was 20 μg, corresponding to 1 mg / kg body weight. At different time points, tumor volume (mm ) was measured as a parameter to evaluate the antitumor effect of the polypeptides. 3 ) was measured. As seen in Figure 10, the E17P01 polypeptide demonstrated its ability to inhibit tumor progression that was greater than that observed with CIGB-300. These results demonstrated the anti-tumor effect of the polypeptide identified as SEQ ID NO:51 in a model of human tumors implanted in experimental animals.

Claims

1. A linear peptide that inhibits phosphorylation mediated by the enzyme casein kinase 2 (CK2), characterized by an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:57 to SEQ ID NO:

62.

2. A polypeptide comprising a linear peptide that inhibits phosphorylation mediated by the enzyme casein kinase 2 (CK2) according to claim 1 and a cell-penetrating peptide.

3. The polypeptide described in claim 2, wherein the intracellularly penetrating peptide is a peptide derived from the Tat1 protein of human immunodeficiency virus.

4. The polypeptide described in claim 2, wherein the intracellularly penetrating peptide has an amino acid sequence identified as SEQ ID NO: 65 or SEQ ID NO:

66.

5. 5. The polypeptide of claim 4 having an amino acid sequence selected from the group consisting of the sequences identified as SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:51 to SEQ ID NO:

56.

6. A pharmaceutical composition comprising at least one linear peptide that inhibits casein kinase 2 (CK2)-mediated phosphorylation according to claim 1 or a polypeptide according to any one of claims 2 to 5, and a pharma- ceutically acceptable excipient.

7. 7. The pharmaceutical composition of claim 6, formulated for administration by systemic, intratumoral, oral, or mucosal routes.

8. The pharmaceutical composition according to claim 6 for treating solid or liquid tumors.

9. Use of a linear peptide that inhibits phosphorylation mediated by casein kinase 2 (CK2) according to claim 1 or a polypeptide according to any one of claims 2 to 5 for the manufacture of a medicament.

10. The use according to claim 9, wherein the medicament is used for the treatment of a solid tumor or a liquid tumor.

11. The pharmaceutical composition of claim 6, wherein a therapeutically effective amount of the pharmaceutical composition is administered to an individual in need of treatment for a solid or liquid tumor.

12. The pharmaceutical composition of claim 11, wherein the solid tumor is in the lung, cervix, larynx, pancreas or bladder.

13. The pharmaceutical composition described in claim 11, wherein the liquid tumor is chronic lymphocytic leukemia, T-type acute lymphoblastic leukemia, or acute myeloid leukemia.

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