Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
  • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

• the present invention relates to the branch of molecular pharmacology and human medicine.
• the invention relates to obtaining linear peptides that inhibit the phosphorylation event mediated by the Casein Kinase 2 (CK2) enzyme, through direct interaction with the phospho-acceptor site on the substrate. Due to their biological activity, the peptides and polypeptides of the invention are useful for the treatment of different types of solid tumors and of other origins Prior state of the art
• CK2 is a serine/threonine enzyme that is involved in increasing cell proliferation, and its intracellular localization is fundamentally nuclear during the malignant transformation process (Tawfic S., et ai, 2001, Histol. Histopathol. 16:573- 582). Therefore, said enzyme constitutes an attractive therapeutic target (Duncan J.S., et al., 2008, Biochim. Biophys. Acta, 1784: 33-47).
• the phosphorylation event mediated by the CK2 enzyme has been manipulated in experimental oncology, through the use of chemical compounds derived from 4,5,6,7-tetrabromobenzotriazole (TBB) (Pagano MA, etai, 2004, J. Med. Chem. , 47: 6239-6247), antisense oligonucleotides (Slaton JW, etai, 2004, Mol. Cancer Res. 2:712-720) and peptides that block the interaction between the catalytic and regulatory subunits of the enzyme (Laudet B., etai , 2007, Biochem J., 408: 363-373).
• TAB 4,5,6,7-tetrabromobenzotriazole
• the chemical compound CX-4945 inhibits the two catalytic subunits of the CK2 enzyme (Siddiqui-Jain A., et ai, 2010, Cancer Res. 70: 10288-10298), and is a potent inhibitor of cell proliferation in vitro, with a mean maximum inhibitory concentration (IC50) of 5.5 mM.
• IC50 mean maximum inhibitory concentration
• high doses of said compound 25 and 75 mg/kg
• an aggressive administration regimen are required to inhibit tumor growth in vivo. Only this CK2 enzyme inhibitor has been successfully evaluated in humans, where favorable pharmacokinetic and safety parameters were reported in Phase I clinical studies (Lim JKC, et ai, 2010, Cancer Res. 70: 2763-2766).
• CIGB-300 Patent Application W003/054002; Perea SE, etai, 2004 , Cancer Res., 64: 7127-7129.
• the CIGB-300 peptide is a chimeric molecule, which contains a cyclic peptide that interacts with the CK2 phosphoacceptor site in the substrate and an "intracellular penetration" peptide, derived from the Tat1 protein of the Human Immunodeficiency Virus (HIV). , to guarantee its internalization in the cell.
• HIV Human Immunodeficiency Virus
• CIGB-300 has demonstrated antiproliferative and proapoptotic properties, in vitro and in vivo, in various lines of solid tumors such as lung, cervix, larynx (Perera Y., et al., 2014, Mol Clin Oncol, 2 (6): 935 -944) and in fluid 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 ai, 2020, Cancers, 12, 1377).
• solid tumors such as lung, cervix, larynx (Perera Y., et al., 2014, Mol Clin Oncol, 2 (6): 935 -944) and in fluid tumors such as chronic lymphocytic leukemia (Martins L, et al., 2013, Oncotarget, 5(1): 258-263) and T-type acute lymphoblastic leukemia
• CIGB-300 is a 25 amino acid peptide, containing methionine in its sequence and an intramolecular disulfide bond. These are sources of chemical instability in the molecule, and can be the cause of inconsistencies in the higher stages of product development, whether in the liquid or lyophilized formulation 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 the oxidation of methionine and the formation of aggregates (dimers and trimers), due to the formation of intermolecular disulfide bonds (Garay H., et ai., 2018, J Pept Sci.
• the present invention solves the aforementioned problem, by providing linear peptides that inhibit the phosphorylation mediated by the CK2 enzyme and that have an amino acid sequence that is 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:
• peptides differ from CIGB-300 in that they are linear, smaller in size, and chemically more stable. They do not present methionine or cysteine in their sequence, which represents an advantage. In addition, from the productive point of view, its obtaining is more feasible.
• linear peptides identified as SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 57 - SEQ ID NO: 62 were selected for their ability to interact with the phosphoacceptor site of the CK2 enzyme. on the E7 substrate of the Human Papilloma Virus (HPV)-16 (LNDSSEEEDEI), and also for its ability to inhibit CK2-mediated phosphorylation of this oncoprotein. As seen in the examples of the present invention, these linear peptides are capable of inhibiting CK2-mediated phosphorylation on the E7 protein in a manner similar to the CIGB-300 peptide.
• HPV Human Papilloma Virus
• the peptides of the invention can be chemically conjugated with "intracellular penetrating" peptides, belonging to proteins such as the HIV Tat1 protein (Schwarze S.R., et al., 2000, Trends Pharmacol, 21: 45-48), Herpes Simplex Virus VP22 protein (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.
• HIV Tat1 protein Korean S.R., et al., 2000, Trends Pharmacol, 21: 45-48
• Herpes Simplex Virus VP22 protein Lidgreen 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.
• polypeptides comprising linear peptides 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-SEQ ID NO: 62 and an intracellular penetration peptide.
• the intracellular penetration peptide in these polypeptides is a peptide derived from the HIV Tat1 protein.
• the intracellular penetration peptide has an amino acid sequence that is identified as SEQ ID NO: 65 or SEQ ID NO: 66.
• polypeptide of the invention has an amino acid sequence that is 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 -SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 51 -SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 51 -SEQ ID
• polypeptides are more active than the CIGB-300 peptide, as they have a lower IC50 in several cancer cell lines (lung, bladder, larynx, pancreas, and leukemia).
• the polypeptides were able to produce a dose-dependent effect on the proliferation of cancer cells.
• the polypeptide identified as SEQ ID NO: 35 shows IC50 values similar to those of CIGB-300 in these cell lines. However, it is considered superior because it has a linear amino acid sequence, smaller in size and more chemically stable, with productive advantages that accelerate the development of the therapeutic product.
• a combinatorial library of linear peptides of the "one-pearl-one-compound" type containing more than 16 million different peptide sequences was synthesized and the screening was carried out with a synthetic peptide comprising the 28-38 region of the E7 oncoprotein modified with Biotin.
• a synthetic peptide comprising the 28-38 region of the E7 oncoprotein modified with Biotin.
• Masses were assigned to the sequences of the peptides contained in the nine pearls that were positive to the screening.
• the peptides identified as SEQ ID NO: 1- SEQ ID NO: 9 were chemically synthesized, and it was confirmed that they were capable of inhibiting CK2-mediated phosphorylation of the E7 oncoprotein of HPV-16.
• polypeptides containing the nine peptides were designed and synthesized, in which the intracellular penetration peptide corresponding to the HIV Tat1 protein (GRKKRRQRRRPPQ) was added at the N-terminus. , identified as SEQ ID NO: 65).
• SEQ ID NO: 65 the intracellular penetration peptide corresponding to the HIV Tat1 protein
• a Trp position scanning library was designed and obtained to evaluate the impact of Trp incorporation on the biological activity of the polypeptides identified as SEQ ID NO: 18 - SEQ ID NO: 20. Next, the antiproliferative effect was evaluated. of the 24 polypeptides generated in the Trp library (identified as SEQ ID NO: 21 - SEQ ID NO: 44) in the NCI-H125 cell line. Six of the 24 polypeptides, 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 percentage inhibition of the cell proliferation similar to the CIGB-300 peptide at the maximum concentration evaluated.
• the IC50 values of the polypeptide identified as SEQ ID NO: 35 and of CIGB-300, obtained in the cell lines NCI-H125 and AsPC-1 (pancreatic adenocarcinoma) are comparable.
• the polypeptide identified as SEQ ID NO: 35 is considered superior to CIGB-300, because it is a smaller linear sequence, more chemically stable and with productive advantages.
• Two other linear polypeptides more active than the CIGB-300 peptide, identified in the invention as SEQ ID NO: 47 and SEQ ID NO: 48, are the result of a study where a second Trp residue was introduced in three positions of the polypeptide identified as SEQ ID NO: 35.
• polypeptides identified as SEQ ID NO: 47 and SEQ ID NO: 48 were shown to be more potent than the CIGB-300 peptide, in their ability to inhibit cell proliferation in pancreatic cancer (AsPC-1, PanC-1) and acute myeloid leukemia (OCI-AML3) cell lines, and were similar to CIGB-300 in the lung cancer cell line used. (NCI-H125).
• SEQ ID NO: 47 and SEQ ID NO: 48 in sites prone to enzymatic degradation, six new linear polypeptides more active than CIGB-300 were obtained, which were identified as SEQ ID NO: 51 - SEQ ID NO: 56.
• the antiproliferative assays were performed on the NCI-H125, Hep2C (larynx carcinoma) cell lines and on two bladder cancer lines (MGH-U3 and MGH-U4 ).
• the Clso values of the peptides identified as SEQ ID NO: 51 - SEQ ID NO: 56, obtained in the NCI-H125, Hep2C, MGH-U3 and MGH-U4 cell lines, are lower than those obtained for CIGB-300. , which shows that they are more powerful.
• the Clso values are one order of magnitude lower than the CIGB-300, which is very attractive because it is a little-explored niche, not solved and with a high health incidence.
• the introduction of the unnatural amino acids was done to increase stability to proteases. Surprisingly, however, it was also possible to enhance the ability of said polypeptides to inhibit CK2-mediated phosphorylation.
• the invention also provides pharmaceutical compositions that comprise at least one linear peptide that has an amino acid sequence that is 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 - SEQ ID NO: 62, or at least one polypeptide having an amino acid sequence that is 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 - SEQ ID NO: 56, and a pharmaceutically acceptable excipient.
• the excipients that can form part of the pharmaceutical compositions of the invention are known to those skilled in this field of technology.
• the pharmaceutical composition is formulated for administration by systemic, intratumoral, oral, or mucosal routes.
• the pharmaceutical compositions are useful for the treatment of solid or liquid tumors in an individual who requires it.
• the object of the invention is the use of linear peptides that have a sequence that is 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 - SEQ ID NO: 62, or polypeptides having an amino acid sequence selected from the group consisting of the identified sequences such as SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 51 - SEQ ID NO: 56, for manufacturing a medicament.
• said drug is used in the treatment of solid or liquid tumors.
• the invention discloses a method of treating solid or liquid tumors in an individual in need thereof, comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising at least one linear peptide having an amino acid sequence that is 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 - SEQ ID NO: 62, or at least one polypeptide that has a sequence of amino acids that is 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 - SEQ ID NO: 56.
• the solid tumor being treated is a tumor of the lung, cervix, larynx, pancreas, or bladder.
• the fluid tumor being treated is chronic lymphocytic leukemia, T-type acute lymphoblastic leukemia, or acute myeloid leukemia.
• Figure 1 Effect of synthetic peptides from SEQ ID NO: 1 - SEQ ID NO: 9 on CK2-mediated phosphorylation of HPV-16 E7 protein.
• Two peptides that do not interact with the phosphoacceptor site of the CK2 enzyme (identified as SEQ ID NO: 10 and SEQ ID NO: 11) were used as controls.
• the Y-axis plots counts per minute (cpm).
• FIG. 1 Percentage inhibition of cell proliferation (% I) in the NCI-H125 cell line for the nine polypeptides identified as SEQ ID NO: 12 - SEQ ID NO: 20. Peptides CIGB-300 and Tat were used as control positive and negative test, respectively. Polypeptides showing a percentage inhibition of cell proliferation ⁇ 20% were selected.
• the precursor peptides identified as SEQ ID NO: 18 - SEQ ID NO: 20 and the CIGB-300 were included as a positive control.
• the dashed box frames the six polypeptides that showed greater than 60% inhibition of cell proliferation at 200 mM and CIGB-300.
• the dose-effect curve of the CIGB-300 peptide used as control peptide is included.
• % I Percentage inhibition of cell proliferation.
• C concentration.
• the dose-effect curves of the precursor polypeptide A 15P36 (SEQ ID NO: 47) and the CIGB-300 that was used as control are included.
• % I Percentage inhibition of cell proliferation.
• C concentration.
• Figure 9 Dose-effect curves of the D17P78, D17P79 and D17P80 polypeptides (SEQ ID NO: 54 - SEQ ID NO: 56) in the NCI-H125 cell line.
• the dose-effect curves of the precursor polypeptide A15P37 (SEQ ID NO: 48) and of the CIGB-300 that was used as control are included.
• % I Percentage inhibition of cell proliferation.
• C concentration.
• Figure 10 Antitumor effect of the CK2 phosphorylation inhibitory polypeptide E17P01 (SEQ ID NO: 51) in a human bladder tumor model implanted in nude mice. Detailed exposition of embodiments / Examples of embodiment
• Example 1 Selection of linear peptides that interact with the phosphoacceptor site of the CK2 enzyme in the E7 protein of HPV-16.
• BCP-1 a combinatorial library of "one-pearl-one-compound" synthetic linear peptides named BCP-1, containing more than 16 million of different peptide sequences.
• This library was designed to be compatible with sequence analysis by electrospray ionization mass spectrometry (ESI-MS) (Masforrol Y, et al., 2012, ACS Combinatorial Science, 14(3): 145-9).
• BIOT biotinylated synthetic peptide
• LNDSSEEEDEI the E7 substrate of HPV-16
• the streptavidin-alkaline phosphatase conjugate was used as the recognition molecule, and the p-toluidine salt of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) was used as the substrate for development.
• BCIP 5-bromo-4-chloro-3-indolyl-phosphate
• 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 reverse phase chromatography (RP-HPLC) and its identity was confirmed by ESI-MS. It was obtained with more than 95% purity, and ESI-MS analysis confirmed its molecular mass (MM) of 1574.6 Da.
• RP-HPLC reverse phase chromatography
• the BCP-1 library was placed in a polypropylene reactor with a vacuum filtration system and mechanical agitation. Washed with purified water and phosphate buffered saline (PBS) pH 7.4; and blocked with 1% bovine serum albumin (BSA) in PBS pH 7.4 for one hour. Solvents and reagents were removed by vacuum filtration. The library was incubated with 80 mL of a 150 pg/mL BIOT peptide solution in 1% BSA in PBS pH 7.4, for 16h at 4°C.
• PBS phosphate buffered saline
• BSA bovine serum albumin
• T-PBS Tween-PBS
• streptavidin-alkaline phosphatase conjugate 0.4 pg/mL
• BCIP BCIP
• the positive beads were separated from the rest by visual exploration, with the aid of an ocular lens, they were transferred to a reactor with a frit, and they were regenerated. Subsequently, tests were carried out to rule out non-specific recognition by the streptavidin-alkaline phosphatase conjugate and by BCIP. A search was carried out under more stringent conditions, to select the sequences with the highest affinity and reduce the number of positive beads, for which the concentration of the BIOT peptide was reduced to 120 pg/mL. The positive beads were regenerated and the sequence identification was carried out by nanoESI-MS/MS, according to the previously described procedure (Masforrol Y., et al, 2012, ACS Combinatorial Science, 14(3): 145-9).
• Peptide Protein Res., 35(3): 161-214 An enzymatic reaction was developed on the peptide coupled to the resin, with the BIOT peptide at the concentration of 120 pg/mL.
• the streptavidin-alkaline phosphatase conjugate was used as a recognition molecule, and BCIP was used as a substrate for development.
• Nine reactors containing 15 mg of the encoded peptides (P1-P9) coupled to resin were placed in a vacuum filtration system with mechanical stirring.
• Two control peptides coupled to the resin were used (P10 identified as SEQ ID NO: 10 and P11 identified as SEQ ID NO: 11) that do not interact with the CK2 phosphoacceptor site in the E7 protein.
• 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). Peptides were purified by RP-HPLC and their identity was confirmed by ESI-MS. For the synthesis, they were designated as F11P04 - F11P12, and their amino acid sequences correspond to the sequences identified as SEQ ID NO: 1- SEQ ID NO: 9. As observed in Table 2, all were obtained with more than 95 % purity and the analysis by ESI-MS corroborated its identity. Table 2. Characterization of the synthetic peptides that contain the sequences identified after the screening of the BCP-1 library with BIOT.
• the assay was performed according to the procedure previously described (Perea SE, et al., 2004, Cancer Res., 64: 7127-7129). The results are observed in Figure 1, and indicate that the peptides identified as SEQ ID NO: 1- SEQ ID NO: 9 are capable of inhibiting CK2-mediated phosphorylation of the E7 protein of HPV-16.
• Example 3 Effect of the synthetic polypeptides identified as SEQ ID NO: 12 - SEQ ID NO: 20 on cell growth in the non-small cell lung cancer line NCI-H125.
• the intracellular penetration peptide Tat corresponding to the protein, was added to the sequences identified as SEQ ID NO: 1 - SEQ ID NO: 9. HIV Tat1, (GRKKRRQRRRPPQ). A betaAlanine (bA) residue was introduced as a spacer between the peptide of interest and the Tat peptide.
• Polypeptides were coded as F11P13-F11P21 for synthesis, and correspond to sequences identified as SEQ ID NO: 12 - SEQ ID NO: 20, as shown in Table 3.
• 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 was performed as shown in Example 2. As seen in Table 3, all were obtained with greater than 95% purity and ESI-MS analysis confirmed its identity.
• the evaluation of the antiproliferative activity of the F11P13-F11P21 polypeptides was carried out in the NCI-H125 cell line.
• the line cell exhibits high sensitivity to low concentrations of the drug under test (HU L.-Y., etal, 2018, European Review for Medical and Pharmacological Sciences, 22: 4551-4556).
• the antiproliferative effect of the CIGB-300 peptide has been widely characterized in this line (Cirigliano SM, et al., 2017, Cáncer Cell International, 17, 42), for which it was selected as a positive control.
• the Tat peptide was used as a negative control.
• Polypeptides were evaluated at concentrations of 25, 50, 100, and 200 mM.
• the cell proliferation assay was performed by a previously described procedure (Perera Y., et al., 2012, J. Pept. Sci, 18 (4):215-23).
• Figure 2 shows the result of the antiproliferative assay in NCI-H125 cells. Each bar corresponds to the values of the percentage inhibition of cell proliferation (% I) achieved for the concentrations of the polypeptides evaluated (25, 50, 100 and 200 mM).
• the polypeptides that showed a value of inhibition of cell proliferation greater than or equal to 20% were selected, since this percentage value is within the coefficient of variation of the assay. Only the F11P19, F11P20 and F11P21 polypeptides inhibited cell proliferation between 20% and 30%, at the concentration of 200 pM. Only 50% inhibition of proliferation was achieved with the CIGB-300 peptide, used as control.
• Figure 3 shows the dose-effect curves of the F11P19, F11P20 and F11P21 polypeptides, in comparison with the CIGB-300 peptide.
• the IC50 values for polypeptides F11P19, F11P20, F11P21, and for CIGB-300 were 336 pM, 305 pM, 668 pM, and 64 pM, respectively.
• the IC50 values of the F11P19, F11P20, and F11P21 polypeptides are still far from the IC50 value obtained for CIGB-300, so it is necessary to optimize these primary structures to improve their biological activity.
• Example 4 Optimization of the sequences identified as SEQ ID NO:18- SEQ ID NO: 20 by means of a Tryptophan scan in the cargo.
• Trp amino acid was excluded in the synthesis of the BCP-1 library by design (Masforrol Y., et al, 2012, ACS Combinatorial Science, 14(3): 145-9). For this reason, Trp position scanning libraries were designed for the sequences identified as SEQ ID NO: 18 - SEQ ID NO: 20, in which the punctual substitution of each amino acid residue was made by the Trp in the cargo (Table 4). This allowed studying the contribution of Trp at each position of the cargo sequence.
• Polypeptides from the Trp position scanning library were synthesized on MBHA resin, using Fmoc/tBu chemistry (Field GB and Noble RL. Int. J. Peptide Protein Res. 1990; 35(3):161-214), RP-HPLC were purified and identity confirmed. by ESI-MS. As can be seen in Table 4, which shows the amino acid sequences, all were obtained with more than 95% purity and the ESI-MS analysis confirmed the identity with the designed molecule.
• the 24 polypeptides identified as SEQ ID NO: 21- SEQ ID NO: 44 maintained the ability to inhibit the phosphorylation of the E7 protein of HPV-16, mediated by CK2, since phosphorylation inhibition values between 60-85% were obtained. CIGB-300 showed 87% inhibition of phosphorylation.
• Figure 5 shows the dose-effect curves of the A14P27, A14P31, A14P33, A14P43, A14P46, A14P59 and CIGB-300 polypeptides.
• Table 5 shows that the IC50 values for these six polypeptides are lower than the obtained for its precursor polypeptides F11P19 (336 mM), F11P20 (305 mM) and F11P21 (668 pM).
• the Clso of the A14P46 polypeptide (72 pM) is comparable to the Clso of the CIGB-300 reference peptide (64 pM)
• A14P46 polypeptide showed biological activity results similar to CIGB-300 in the NCI-H125 cell line, the behavior of the same polypeptide in the pancreatic adenocarcinoma tumor line (AsPC-1), in comparison with CIGB-300.
• the cell proliferation assay was performed following the previously described procedure (Perera Y, et al., 2012, J. Pept. Sci, 18 (4):215-23). As observed in Figure 6, both molecules are capable of producing a dose-dependent effect on the proliferation of AsPC-1 cells. IC50 values of 125 mM were obtained for A14P46 and 102 mM for CIGB-300, which shows that both have similar potency in this cell line.
• A14P46 polypeptide (SEQ ID NO: 35) is comparable to CIGB-300 in its ability to inhibit cell proliferation in NCI- cell lines.
• Example 5 Optimization of the polypeptide identified as SEQ ID NO: 35 by introducing a second Trp residue in its sequence.
• the A15P35, A15P36 and A15P37 polypeptides were capable of producing a dose-dependent effect on the proliferation of NCI-H125 cells.
• the three polypeptides also showed a dose-response pattern.
• Table 7 summarizes the Clso values of the A15P35, A15P36 and A15P37 polypeptides obtained in the NCI-H125, AsPC-1, PanC-1 and OCI-AML3 cell lines, in comparison with their precursor A14P46 and with the control peptide. CIGB-300.
• the Clso values of the A15P35 polypeptide are similar to those obtained for its precursor A14P46 and for CIGB-300, in the four lines studied, which shows that the introduction of the Trp in the X1 position of the sequence is irrelevant. On the contrary, the Clso values obtained for the A15P36 and A15P37 polypeptides were lower than those obtained for the A14P46 polypeptide and the CIGB-300 peptide, especially in pancreatic cancer cell lines (AsPC-1, PanC-1). and acute myeloid leukemia (OCI-AML3).
• Table 7 Clso values of the polypeptides identified as SEQ ID NO: 46 - SEQ ID NO: 48 in the cell proliferation assay with the lines NCI-H125, AsPC-1, PanC-1 and OCI-AML3.
• control peptides A14P46 and CIGB-300 are included.
• RKRSWYWP both peptides were synthesized without the Tat or bA sequence, with the use of the synthesis procedure described in this example.
• Peptides D15P105 (SEQ ID NO: 49) and D15P106 (SEQ ID NO: 50) were obtained with more than 95% purity, and ESI-MS analysis confirmed their molecular masses of 1245.7 Da and 1176.6 Da. , respectively.
• the phosphorylation assay was 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 the protein.
• results of this example show that the introduction of a second Trp at positions X4 and X5 of the A14P46 polypeptide sequence (SEQ ID NO: 35), favors the interaction of the peptide with the phosphoacceptor site, and therefore its ability to inhibit phosphorylation by CK2.
• A15P36 and A15P37 polypeptides are 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 that they are smaller linear sequences, chemically more stable, since they do not present sensitive residues such as methionine and cysteine. In addition, they are more attractive from the productive point of view.
• Example 6 Optimization of the polypeptides identified as SEQ ID NO: 47 and SEQ ID NO: 48 by introduction of unnatural amino acids (D- and L-N-methyl amino acids).
• A15P36 SEQ ID NO: 47
• A15P37 SEQ ID NO: 48
• non-natural amino acids were introduced in those sequence sites most prone to degradation by trypsin and chymotrypsin.
• the most sensitive sites for proteolysis were predicted with the help of the Expasy site (http://www.expasy.org) (Gasteiger E., et al., 2003, NucleicAcids Research. 31(13): 3784-3788).
• D-Arg was incorporated into the Rs of the Tat and the protection of K16, R17 and Y20 was evaluated, with the introduction of D-Lys, D-Arg and NMe-Tyr, respectively.
• the N-terminus of each polypeptide was acetylated to protect it from N-peptidases.
• the polypeptides E17P01, E17P02 and E17P03 are derivatives of A15P36 and the polypeptides D17P78, D17P79 and D17P80 (SEQ ID NO: 54 - 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 can be seen in Table 8, all were obtained with more of 95% purity and analysis by ESI-MS corroborated the identity of the molecule.
• polypeptides E17P01-E17P03 and D17P78-D17P80 identified with SEQ ID NO: 51 - SEQ ID NO: 56 maintain the ability to inhibit CK2-mediated phosphorylation in the HPV-16 E7 protein, phosphorylation inhibition values between 83 and 90% were obtained.
• Antiproliferative assays were performed on the NCI-H125, Hep2C (larynx carcinoma) cell lines and on two bladder cancer lines (MGH-U3 and MGH-U4).
• the assay was carried out by the previously described procedure (Perera Y., et al, 2012, Pept. Sci, 18 (4):215-23).
• Figures 8 and 9 show the dose-effect curves in the cell line NCI- H125 of the polypeptides E17P01, E17P02 and E17P03 (SEQ ID NO: 51- SEQ ID NO: 53) and D17P78, D17P79 and D17P80 (SEQ ID NO: 54- SEQ ID NO: 56), respectively.
• the control peptide CIGB-300 and the precursor polypeptides A15P36 and A15P37, respectively are included.
• the six polypeptides were capable of producing a dose-dependent effect on the proliferation of cells from the NCI-H125 line.
• MGH-U3 and MGH-U4 cell lines the six polypeptides also showed a dose-response pattern.
• Table 9 summarizes the IC50 values of the polypeptides E17P01, E17P02, E17P03, D17P78, D17P79 and D17P80 (SEQ ID NO: 51- SEQ ID NO: 56) obtained in the cell lines NCI-H125, Hep2C, MGH- U3 and MGH-U4, compared to their precursors and the CIGB-300 control peptide.
• Table 9 IC50 values of the polypeptides identified as SEQ ID NO: 51 - SEQ ID NO: 56 in the NCI-H125, Hep2C, MGH-U3 and MGH-U4 cell lines. The values obtained for the control polypeptides A15P36, A15P37 and CIGB-300 are included.
• Table 9 shows that the IC50 values of the polypeptides identified as SEQ ID NO: 51 - SEQ ID NO: 56, in the NCI-H125, Hep2C, MGH-U3 and MGH-U4 cell lines, are lower than the obtained for the CIGB-300 peptide, which shows that they are more potent in terms of their antiproliferative effect.
• the IC50 values are one order of magnitude lower than the CIGB-300, which is very attractive, as it is a little-explored, unresolved niche with a high health incidence.
• Modifications in the A15P36 and A15P37 polypeptides were introduced to increase stability to proteases. Surprisingly, however, the ability to inhibit CK2 phosphorylation was also potentiated.
• the phosphorylation assay was performed as described in Example 2, and phosphorylation inhibition values between 83 and 90% were obtained, confirming the ability of these peptides to inhibit CK2-mediated phosphorylation of the HPV E7 protein. -16.
• the polypeptides identified as SEQ ID NO: 51 - SEQ ID NO: 56 are more potent than the CIGB-300 peptide, in terms of their ability to inhibit cell proliferation; in particular, their inhibitory activity was surprising in cell lines. bladder cancer (MGH-U3 and MGH-U4).
• these peptides are linear, smaller, and chemically more stable, which brings undoubted advantages from the productive point of view.
• Example 7 Antitumor effect of a polypeptide inhibitor of phosphorylation by CK2 in a human bladder tumor model implanted in athymic mice.
• mice Female athymic BalbC mice, 6 to 8 weeks old, were used for these assays. MGH-U3 cells from bladder cell carcinoma were used. For tumor implantation in this model, cells were resuspended in PBS, at a concentration of 1,000,000 cells per milliliter. The cell suspension was inoculated subcutaneously in the abdominal cavity of the animals. The administration of the E17P01 polypeptide (identified as SEQ ID NO: 51) or CIGB-300 began 5 days after the tumor cells were inoculated, and continued for 5 consecutive days, by intraperitoneal route. The polypeptide and the peptide were dissolved in PBS. The dose administered was 20 pg, which corresponds to 1 mg/kg of weight.
• the tumor volume (in mm 3 ) was measured as a parameter to evaluate the antitumor effect of the polypeptide.
• the E17P01 polypeptide showed its ability to inhibit tumor progression, which was greater than that observed for CIGB-300.

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