Patent Application
20240317827
The present invention relates to the field of molecular pharmacology and human medicine. In particular, the invention relates to obtaining linear peptides that inhibit the phosphorylation event mediated by the enzyme Casein Kinase 2 (CK2), through direct interaction with the phosphoacceptor 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.
CK2 is a serine/threonine enzyme that is involved in increasing cell proliferation, and its intracellular localization is fundamentally nuclear during the process of malignant transformation (Tawfic S., et al., 2001, Histol. Histopathol. 16:573-582). Therefore, this enzyme constitutes an attractive therapeutic target. (Duncan J. S., et al., 2008, Biochim. Biophys. Acta, 1784: 33-47).
The CK2 enzyme-mediated phosphorylation event has been manipulated in experimental oncology by using chemical compounds derived from 4,5,6,7-tetrabromobenzotriazole (TBB) (Pagano M. A., et al., 2004, J. Med. Chem., 47: 6239-6247), antisense oligonucleotides (Slaton J. W., 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 inhibitor of cell proliferation in vitro, with a mean maximum inhibitory concentration (IC50) of 5.5 μM. Only this CK2 enzyme inhibitor has been satisfactorily evaluated in humans, where favorable pharmacokinetic and safety parameters were recorded in Phase I clinical studies (Lim J. K. C., et al., 2010, Cancer Res. 70: 2763-2766 Only this CK2 enzyme inhibitor has been satisfactorily evaluated in humans, where favorable pharmacokinetic and safety parameters were recorded in Phase I clinical studies. (Patent request WO03/054002; Perea S. E., et al., 2004, Cancer Res., 64: 7127-7129). The CIGB-300 peptide is a chimeric molecule that contains a cyclic peptide that interacts with the CK2 phosphoacceptor site on the substrate and an “intracellular penetration” peptide from the Tat1 protein of the Human Immunodeficiency Virus (HIV), to guarantee its internalization in the cell. 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 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). In addition, this peptide showed signs of clinical benefit, verified in Phase I/II clinical studies (Perea S., et al., 2011, Mol Cell Biochem., 356 (1-2): 45-50) (Sarduy M., et al., 2015, Br. J. Cancer, 112: 1636-1643). However, 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 al., 2018, J Pept Sci. 24(6): e3081). On the other hand, the synthesis processes of peptides containing cysteines are 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 the intramolecular disulfide bond introduces an extra step in the production processes, which increases production costs. Therefore, it is still of interest to obtain inhibitors of phosphorylation mediated by the CK2 enzyme, of the linear peptide type, of smaller size and structural complexity, whose synthesis is simpler and cheaper, for the development of therapies against different types of tumors.
The present invention solves the aforementioned problem by providing linear peptides that inhibit the CK2-mediated phosphorylation 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: 57-SEQ ID NO: 62. These sequences are shown below:
These peptides differ from CIGB-300 in that they are linear, smaller in size, and chemically more stable. They do not have methionine or cysteine in their sequence, which is an advantage. In addition, from the productive point of view, the obtention is more feasible.
The 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 Papillomavirus (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 of the E7 protein in a similar manner to peptide CIGB-300.
To achieve intracellular action on endogenous CK2 substrates, the peptides of the invention can be chemically conjugated with peptides of “intracellular penetration”, belonging to proteins such as the Tat1 protein of HIV (Schwarze S. R., et al., 2000, Trends Pharmacol, 21: 45-48), la proteína VP22 del Virus Herpes Simples (Lindgreen M., et al., 2000, Trends Pharmacol Sci, 21: 99-103), the Penetratin and the Transportan (Gariepy J., et al., 2001, Trends Biotech 19: 21-28), among others.
Therefore, the invention also provides polypeptides comprising linear peptides that have an amino acid sequence that is 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 one cell penetrating peptide.
In one embodiment of the invention, the intracellularly penetrating peptide in these polypeptides is a peptide from the Tat1 protein of HIV. In one embodiment thereof, the intracellularly penetrating peptide has an amino acid sequence that is identified as SEQ ID NO: 65 either SEQ ID NO: 66.
In a particular embodiment, the 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: 56. These sequences are shown below:
Most of these polypeptides are more active than peptide CIGB-300, as they have a lower IC50 in various 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. Besides, 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.
For the identification of the peptides described in the invention, a combinatorial library of linear peptides by type “one-bead-one-compound” was synthesized, containing more than 16 million of different peptide sequences, and the screening was carried out with a synthetic peptide comprising the 28-38 region of the E7 oncoprotein modified with Biotin. With the use of mass spectrometry, the sequences of the peptides contained in the nine beads that were positive to the screening were assigned. The peptides identified a SEQ ID NO: 1-SEQ ID NO: 9 were chemically synthesized, and it was confirmed that they were capable of inhibiting CK2-mediated phosphorylation in the E7 oncoprotein of HPV-16. In order to achieve intracellular action on endogenous CK2 substrates, polypeptides containing the nine peptides were designed and synthesized, in which the intracellular penetration peptide corresponding to the HIV Tat1 protein was added at the N-terminus (GRKKRRQRRRPPQ, identified as SEQ ID NO: 65). Next, the effect of these nine polypeptides on cell growth of the NCI-H125 line (non-small cell lung cancer) was evaluated. None of the polypeptides reached inhibition values similar to CIGB-300, in the range of the concentrations evaluated. Only three of them, identified as SEQ ID NO: 18-SEQ ID NO: 20, inhibited between 20% and 30% cell proliferation at the highest concentration studied, so modifications were made to their sequences to improve their biological activity.
A Trp positional scanning library was designed and obtained to assess the impact of Trp incorporation on the biological activity of polypeptides identified as SEQ ID NO: 18-SEQ ID NO: 20. Next, the antiproliferative effect 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 percentages of inhibition of cell proliferation similar to peptide CIGB-300 at the highest concentration evaluated. The IC50 values of the polypeptide identified as SEQ ID NO: 35 and CIGB-300, obtained in the NCI-H125 and (pancreatic adenocarcinoma) cell lines are comparable. Particularly, the polypeptide identified as SEQ ID NO: 35 is considered superior to CIGB-300, because it is a linear sequence of smaller size, more chemically stable and with productive advantages. 20 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 at three positions in the polypeptide identified as SEQ ID NO: 35. It was shown that the polypeptides identified as SEQ ID NO: 47 and SEQ ID NO: 48 are 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 were similar to CIGB-300 in the lung cancer cell line used (NCI-H125).
As a result of the introduction of unnatural amino acids in the sequences identified as 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. Antiproliferative assays were performed on the NCI-H125, Hep2C (laryngeal carcinoma) cell lines, and on two bladder cancer lines (MGH-U3 and MGH-U4). The IC50 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 the CIGB-300, which shows that they are more potent. In the case of the bladder cancer lines (MGH-U3 and MGH-U4), the IC50 values are lower by an order of magnitude compared to the CIGB-300, which is very attractive as it is a little explored niche, not resolved and with a high impact on health. The introduction of the unnatural amino acids was done to increase the stability to proteases. Surprisingly, it was also possible to enhance the ability of these 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 that 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: 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 the art. In one embodiment of the invention, the pharmaceutical composition is formulated for administration by the systemic, intratumoral, oral, or mucosal route. In one embodiment of the invention, the pharmaceutical compositions are useful for the treatment of solid or liquid tumors in an individual in need thereof.
The object of the invention is the use of linear peptides that have a sequence that is selected from the group composed 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 that have 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, to manufacture a drug.
In an embodiment of the invention, said medicament is used in the treatment of solid or liquid tumors.
In another aspect, the invention discloses a method of treating solid or liquid tumors in an individual in need thereof comprising administering 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 composed of the sequences identified a 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 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. In one embodiment of the invention, the solid tumor being treated is a tumor of the lung, cervix, larynx, pancreas, or bladder. In another embodiment of the invention, the liquid tumor being treated is chronic lymphocytic leukemia, T-type acute lymphoblastic leukemia, or acute myeloid leukemia.
For the identification of bioactive linear peptides that interact with the phosphoacceptor site of the CK2 enzyme, a combinatorial library of synthetic linear peptides named BCP-1 by type of “one-bead-one-compound” was screened, containing more than 16 million of different peptide sequences. This 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, which contains the phosphoacceptor site of the CK2 enzyme in the E7 substrate of HPV-16 (LNDSSEEEDEI, identified as SEQ ID NO: 63). The streptavidin-alkaline phosphatase conjugate was used as recognition molecule, and p-toluidine salt of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) was used as substrate for development. The blue color facilitates the visual differentiation of the beads containing bioactive sequences recognized by the BIOT peptide, compared to the bright light yellow color of the TentaGel resin at the BCP-1 library. The BIOT peptide was synthesized on 4-methylbenzhydrylamine (MBHA) resin, using the 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.
The BCP-1 library was placed in a polypropylene reactor with a vacuum filtration system and mechanical agitation. It was washed with purified water and saline phosphate buffer solution (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 BIOT peptide solution at 150 g/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 the streptavidin-alkaline phosphatase conjugate (0.4 μg/mL) in a solution of 1% BSA in PBS pH 7.4. Two hours later, it was washed with T-PBS pH 7.4 and incubated with BCIP (0.5 mg/mL) in substrate solution for 30 min. When the blue color appeared, the reaction was stopped by filtration and abundant washings with PBS pH 7.4.
The positive beads were separated from the rest by visual examination, with the aid of an ocular lens, transferred to a reactor with a frit, and regenerated. Subsequently, tests were performed to rule out nonspecific recognition by the streptavidin-alkaline phosphatase conjugate and BCIP. Screening was carried out under conditions of greater astringency, to select the sequences of greater affinity and reduce the number of positive beads, for which the concentration of the BIOT peptide was reduced to 120 μg/mL. Positive beads were regenerated and sequence identification was carried out by nanoESI-MS/MS, according to the procedure described above (Masforrol Y., et al, 2012, ACS Combinatorial Science, 14(3): 145-9).
As a result of the initial screen with the peptide BIOT at 150 μg/mL, 56 positive beads were selected. After the second screening with a lower concentration of BIOT (120 μg/mL), nine positive beads were selected that showed a more intense blue coloration than the rest of the positive beads at the initial screening, which evidenced the greater affinity of these peptides for the CK2 phosphoacceptor site. The nanoESI-MS/MS spectra obtained from the nine positive beads were analyzed in parallel, by manual de novo sequencing and automated sequencing, using the Mascot program to search the BCP-1 databases. Table 1 shows the sequence of the nine peptides identified (SEQ ID NO: 1-SEQ ID NO: 9). From the analysis of the sequences assigned to each peptide, a complete correspondence of the assignment of the m/z signals between the manual identification and the automatic sequencing is obtained, which allowed the accurate identification of the peptide sequences in each case.
A 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).
To corroborate that the nine sequences identified by nanoESI-MS/MS interact with the phosphoacceptor site of the CK2 enzyme in the E7 protein, the peptides were synthesized directly on the Tentagel resin, using the Fmoc/tBu chemistry (Field G. B. et al., 1990, Int. J. Peptide Protein Res., 35(3):161-214). An enzymatic reaction was carried out on the peptide coupled to the resin, with the BIOT peptide at a concentration of 120 μg/mL The streptavidin-alkaline phosphatase conjugate was used as recognition molecule, and BCIP was used as 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 agitation. 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 on the E7 protein. The procedure was adjusted to reproduce the BCP-1 library screening assay in a 500 μL working volume for each reactor. The results showed that the nine peptides coupled to the resin (P1-P9) developed an intense blue color after enzymatic assay, which evidences 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 develop blue coloration.
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 confirmed by ESI-MS. For 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 seen in Table 2, all were obtained with more than 95% purity and ESI-MS analysis confirmed their identity.
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, where the E7 oncoprotein of HPV-16 expressed in Escherichia coli was used as substratum. This oncoprotein was obtained as a fusion protein to Glutathione S-Transferase. The assay was performed according to the procedure previously described. (Perea S. E., et al., 2004, Cancer Res., 64: 7127-7129). The results are shown in
In order to achieve intracellular action on endogenous CK2 substrates, the sequences identified as SEQ ID NO: 1-SEQ ID NO: 9 the intracellular penetration peptide Tat, corresponding to the Tat1 protein of HIV, was added at the N-terminus, (GRKKRRQRRRPPQ). A beta Alanine (BA) residue was introduced as a spacer between the peptide of interest and the Tat peptide. The polypeptides were coded as F11P13-F11P21 for synthesis, and correspond to the 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 carried out as shown in Example 2. As shown in Table 3, all of them were obtained with more than 95% purity and the ESI-MS analysis confirmed their identity.
The evaluation of the antiproliferative activity of polypeptides F11P13-F11P21 (SEQ ID NO: 12-SEQ ID NO: 20) was performed on the NCI-H125 cell line. The cell line exhibits 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 widely characterized in this line (Cirigliano S. M., et al., 2017, Cancer Cell International, 17, 42), therefore, it was selected as a positive control. Tat peptide was used as a negative control. The polypeptides were evaluated at the concentrations of 25, 50, 100 and 200 μM. The cell proliferation assay was performed by a previously described procedure (Perera Y., et al., 2012, J. Pept. Sci, 18 (4):215-23).
The Trp amino acid was excluded in the BCP-1 library synthesis 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 for the Trp in charge was performed (Table 4). This allowed us to study the contribution of Trp at each position of the cargo sequence.
Trp position-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), RP-HPLC purified and identity confirmed by ESI-MS. As can be seen in Table 4, which shows the amino acid sequences, all of them were obtained with more than 95% purity and the ESI-MS analysis confirmed the identity with the designed molecule.
To corroborate that the 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 coupled to the resin developed a blue color intense after enzyme assay, evidencing interaction with the phosphoacceptor site of the CK2 enzyme in the model E7 substrate. On the other hand, to demonstrate that the 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-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. Evaluation of the antiproliferative activity of synthetic polypeptides generated by positional scanning of Trp (SEQ ID NO: 21-SEQ ID NO: 44) was performed on the NCI-H125 cell line. The test was performed by the procedure previously described. (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. The polypeptides were evaluated at the concentrations of 12.5; 25; fifty; 100 and 200 μM.
The structural study performed on the peptide sequence X1X2X3X4X5X6X7X8, allowed to conclude that the change of D by W in position X7 is essential to enhance the biological activity (A14P46>>A14P33>A14P59), with the best response for the polypeptide A14P46. Additionally, it was concluded that the positions X4>X5>X1 also showed remarkable results regarding biological activity, which suggested the introduction of a second amino acid W in substitution of said positions.
WYRYKRDP
To corroborate that the biological activity of the polypeptide A14P46 (SEQ ID NO: 35) is only attributed to the charge position (RKRSRYWP), a similar peptide without the Tat and BA sequence was synthesized using the synthesis procedure described in this example. The peptide D14P165, identified as SEQ ID NO: 45, was obtained with 95% purity and the analysis by ESI-MS corroborated its MW of 1146.7 Da. The 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.
Taking into account that the polypeptide A14P46 showed biological activity results similar to CIGB-300 in the NCI-H125 cell line, the behavior of such polypeptide in the pancreatic adenocarcinoma tumor line (AsPC-1) was studied, compared to the CIGB-300. The cell proliferation assay was performed following the procedure previously described (Perera Y, et al., 2012, J. Pept. Sci, 18 (4):215-23). As shown in
It is shown that the polypeptide A14P46 (SEQ ID NO: 35) is comparable to CIGB-300 in its ability to inhibit cell proliferation in the NCI-H125 and AsPC-1 cell lines (
Based on the results obtained in Example 4, another Trp residue was introduced at positions X1, X4 and X5 of the A14P46 polypeptide, and its impact on biological activity was evaluated. 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 identity confirmed by ESI-MS. As shown in Table 6, the three peptides were obtained with more than 95% purity and ESI-MS analysis confirmed the identity of the molecule.
WKRSRYWP
The evaluation of the antiproliferative activity of synthetic polypeptides A15P35, A15P36 and A15P37 (SEQ ID NO: 46-SEQ ID NO: 48) was performed on the NCI-H125, AsPC-1, PanC-1 (pancreatic carcinoma) and OCI-AML3 (acute myeloid leukemia-3) cell lines. The test was carried out according to the procedure previously described (Perera Y, et al., 2012, J. Pept. Sci, 18 (4):215-23).
Table 7 summarizes the IC50 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 IC50 values of the polypeptide A15P35 are similar to those obtained for its precursor A14P46 and for CIGB-300, in the four lines studied, which shows that the introduction of Trp in position X1 of the sequence is irrelevant. In contrast, the IC50 values obtained for polypeptides A15P36 and A15P37 were lower than those obtained for polypeptide A14P46 and peptide CIGB-300, especially in pancreatic cancer cell lines (AsPC-1, PanC-1). and acute myeloid leukemia (OCI-AML3).
To corroborate that the biological activity of the A15P36 polypeptides (SEQ ID NO: 47) and A15P37 (SEQ ID NO: 48) it is only attributed to the charge RKRWRYWP and RKRSWYWP, both peptides were synthesized without the Tat or BA sequence, using the synthesis procedure described in this example. The peptides D15P105 (SEQ ID NO: 49) and D15P106 (SEQ ID NO: 50) they were obtained with more than 95% purity, and the ESI-MS analysis corroborated 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 in protein HPV-16 E7.
The results of this example show that the introduction of a second Trp in positions X4 and X5 of the polypeptide sequence A14P46 (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 linear sequences of smaller size, chemically more stable, since they do not have sensitive residues such as methionine and cysteine. In addition, they are more attractive from the productive point of view.
In order to protect polypeptides A15P36 (SEQ ID NO: 47) and A15P37 (SEQ ID NO: 48) from the action of endopeptidases in biological fluids, non-natural amino acids were introduced in those sites of the sequence that were more prone to degradation by trypsin and chymotrypsin. The sites most sensitive 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 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. Polypeptides E17P01, E17P02 and E17P03 (SEQ ID NO: 51-SEQ ID NO: 53) are derivatives of A15P36 and 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 shown in Table 8, all were obtained with more than 95% purity and ESI-MS analysis confirmed the identity of the molecule.
To corroborate 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. The six resin-coupled polypeptides developed a deep blue color after enzyme assay, evidencing interaction with the phosphoacceptor site of the CK2 enzyme on the model E7 substrate. On the other hand, to demonstrate that the modification does not affect the effect of the peptides on the phosphorylation by the enzyme CK2 in the E7 protein, the procedure described in Example 2 was followed. The 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 E7 protein of HPV-16, phosphorylation inhibition values between 83 and 90% were obtained.
Antiproliferative assays were performed on the NCI-H125, Hep2C (laryngeal carcinoma) cell lines, and on two bladder cancer lines (MGH-U3 and MGH-U4). The test was carried out according to the procedure previously described (Perera Y., et al, 2012, Pept. Sci, 18 (4):215-23).
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, in comparison with their precursors and the control peptide CIGB-300.
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 those obtained for the peptide CIGB-300, which shows that they are more powerful in terms of their antiproliferative effect. Surprisingly, in the case of the bladder cancer lines (MGH-U3 and MGH-U4), the IC50 values are one order of magnitude lower than the CIGB-300, which is very attractive, as it is a little explored niche, unresolved and with a high impact on health. Modifications to the A15P36 and A15P37 polypeptides were introduced to increase stability to proteases. Surprisingly, however, the ability to inhibit CK2 phosphorylation was also enhanced.
To corroborate that the biological activity of the peptides identified as SEQ ID NO: 51-SEQ ID NO: 56 is only attributed to their charges, peptides without the Tat and BA sequence were synthesized, using the synthesis procedure described in this same example. Peptides D17P81-D17P86 (SEQ ID NO: 57-SEQ ID NO: 62) were obtained with more than 95% purity and ESI-MS analysis confirmed the identity of the molecules obtained with the designed ones (Table 10).
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-16 E7 protein.
In summary, the polypeptides identified as SEQ ID NO: 51-SEQ ID NO: 56 are more potent than the peptide CIGB-300, in terms of their ability to inhibit cell proliferation, in particular their inhibitory activity in lines of bladder cancer (MGH-U3 and MGH-U4). In addition, these peptides are linear, smaller in size, and chemically more stable, which brings undoubted advantages from the production point of view.
For these tests, athymic female BalbC mice, between 6 and 8 weeks old, were used. MGH-U3 cells, from bladder cell carcinoma, were used. For tumor implantation in this model, cells were suspended 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. Administration of E17P01 polypeptide (identified as SEQ ID NO: 51) or CIGB-300 was started 5 days after the tumor cells were inoculated, and continued for 5 consecutive days, intraperitoneally. The polypeptide and peptide were dissolved in PBS. The administered dose was 20 μg, which corresponds to 1 mg/kg of weight. At different times, the tumor volume (in mm3) was measured, as a parameter to evaluate the antitumor effect of the polypeptide. As seen in
A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on Feb. 16, 2024, is entitled “976-123_PCTUS_ST26”, and is 110,993 bytes in size.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-0058 | Jul 2021 | CU | national |
This application is the U.S. National Phase of International Patent Application Number PCT/CU2022/050007 filed 7 Jul. 2022, which claims priority from CU 2021-0058 filed 9 Jul. 2021, each of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CU2022/050007 | 7/7/2022 | WO |
