Antitumor active compounds, corresponding methods of preparation and corresponding uses

Abstract

The present invention relates to antitumor active compounds of natural extracts obtained from proteins collected from the serum of pythons, and the use of these compounds in the treatment of cancer cells.

Description

[Technical Field]

[0001] The present invention relates to an antitumor active compound, particularly an antitumor active compound of natural origin, a method for producing the same, and use thereof. [Background technology]

[0002] Chemotherapeutic drugs are classified according to their chemical structure and biological activity (e.g., Non-Patent Document 1). In many cases, antitumor drugs act on the cell cycle by inhibiting cell growth.

[0003] The class of chemotherapeutic agents also includes naturally occurring compounds or their derivatives (such as taxol or vincristine) that are capable of binding to tubulin and inhibiting mitosis.

[0004] The patent application WO 02 / 04999 describes proteins with apoptotic and anti-telomerase activity against cancer cells, which proteins are useful for the preparation of pharmaceutical preparations with selective apoptotic capacity against cancer cells and cancer stem cells. The protein known from this document is of natural origin, in particular obtained from the African rock python (Python sebae), and has a tetrameric structure made up of four subunits, the sequences of which are as follows: SEQ ID NO: 1 HKCEICHGFG DDCDGYEEEC PSPEDKCGKI LIDIALAPVS FRATHKNCFS SSICKLGRVD IHVWDGVYMR GRTNCCDNDQ CEDQPLPGLP LSLKNGLYCP GAFGI FTEDS TEHEVKCRGT ETMCLDLVGY REENYAGNIT YNIKGCVSSC PLLTLSERGH EGRKNDLKKV ECREALKPAS SD SEQ ID NO: 2 HKCEICHGFG DDCDGYQEEC PSPEDKCGKI LIDIALAPVS FRATHKNCFS SSICKLGRVD IHVWEGVYIR GRTNCCDNDQ CEDQPLPGLP LSLKNGLYCP GAFGI FTEYS TEHEVKCRGT ETMCLDLVGY REENYVGNIT YNIKGCVSSC PLVTLSERGH EGRKNDLKKV ECREALKYES SD SEQ ID NO: 3 HKCEICHGFG DDCDGYEEEC PSPEDQCGKI LIDIALAPLS FRATHKNCFS SSICKLGRVD IHVWDGVYMR GRTNCCDNDQ CEDEPLPGLP LSLKNGLYCP GAFGIFTEDS TEHEVKCRGT ETMCLDLVGY REENYAGNIT YNIKGCVSSC PLITLSERGH EGRKNDLKKV ECREALKPAS SD SEQ ID NO:4 HKCEICHGFG DDCDGYEEEC PSPEDQCGKI LIDIALAPLS FRATHKNCFS SSICKLGRVD IHVWEGVYIR GKTNCCDNDQ CEDEPLPGLP LSLKNGLYCP GAFGIFTEYS TEHEVKCRGT ETMCLDLVGY REENYVGNIT YNIKGCVSSC PLITLSERGH EGRKNDLKKV ECREALKYES SD [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 021339 [Non-patent literature]

[0006] [Non-Patent Document 1] "The Pharmacological Basis of Therapeutics" by A. Goodman and M. Gilman, Pergamon Press, New York, 2005 Summary of the Invention [Problem to be solved by the invention]

[0007] One drawback of known proteins is the fact that, despite their selective antitumor activity, their development into pharmaceuticals is problematic due to their oligomeric structure and high molecular weight, exceeding 20 kDa.

[0008] Another drawback of known proteins is that they are naturally derived and can only be obtained from natural sources, in this case from specific pythons on the African continent, making them difficult to obtain, which is problematic especially for industrial pharmaceutical production.

[0009] Yet another drawback of known proteins is that they are difficult to modify, which can be problematic when optimizing pharmaceuticals based on such proteins.

[0010] Therefore, it is necessary to arrive at an active antitumor compound that can overcome at least one of the drawbacks of the current technology.

[0011] To achieve this, it is necessary to solve the technical problem of providing active antitumor compounds suitable for pharmaceutical development.

[0012] In particular, one object of the present invention is to provide an active antitumor compound that has a lower molecular weight and a simpler structure than known proteins.

[0013] Another object of the present invention is to provide an antitumor active compound that is readily available.

[0014] Another object of the present invention is to provide active antitumor compounds that can be modified to allow the optimization of any drug candidate.

[0015] Another object of the present invention is to provide naturally occurring compounds with antitumor activity.

[0016] Applicant has conceived, tested and embodied the present invention to overcome the shortcomings of the current art and to obtain these and other objects and advantages. [Means for solving the problem]

[0017] The invention is set forth and characterized in the independent claims, while the dependent claims describe further features of the invention or variants of the main idea of ​​the invention.

[0018] In accordance with the above objectives, in order to solve the technical problems disclosed above in a new and original way and also to achieve significant advantages over the prior art, the antitumor active compound according to the present invention comprises a peptide isolated from one of the proteins of the current art, having the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0019] The peptide sequence is: SEQ ID NO:5 149 SCPLLTLSERGHEGRKNDLKKVECR 173 Or, SEQ ID NO: 6 149 SCPLVTLSERGHEGRKNDLKKVECR 173 or SEQ ID NO: 7 149 SCPLITLSERGHEGRKNDLKKVECR 173 It has.

[0020] In particular, the peptides correspond to region 149-173 of one of the above sequences, more specifically, SEQ ID NO: 5 corresponds to region 149-176 of SEQ ID NO: 1, SEQ ID NO: 6 corresponds to region 149-173 of SEQ ID NO: 2, and SEQ ID NO: 7 corresponds to region 149-173 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0021] Preferably, the peptide corresponds to the region 149 to 173 of the sequence of SEQ ID NO:1, ie has the sequence of SEQ ID NO:5.

[0022] In doing so, we achieve at least the advantage of having an active antitumor compound that, in addition to being of natural origin like an isolated protein, has a much simpler structure and a much lower molecular weight (approximately 2.9 kDa) compared to known proteins, making it more suitable for pharmaceutical development.

[0023] Furthermore, the compounds of the present invention can be obtained synthetically in the laboratory, thus overcoming the problem of limited supply sources. Because the compounds can be synthesized, they can obviously be easily modified and optimized with a view to pharmaceutical manufacturing.

[0024] According to another aspect of the present invention, a method for preparing an antitumor active compound provides for peptide synthesis, preferably in solid phase, which advantageously provides for the use of the compound Fmoc to protect the alpha amino groups of the amino acids of the peptide during synthesis.

[0025] According to another aspect of the present invention, the antitumor active compound disclosed above is provided for use in the treatment of cancer cells and cancer stem cells, advantageously, the cancer cells are human epidermoid carcinoma.

[0026] According to another aspect of the present invention, there is provided a use of the antitumor active compound disclosed above for preparing a pharmaceutical preparation having selective antitumor activity against cancer cells and cancer stem cells, advantageously, the cancer cells are human epidermoid carcinoma.

[0027] These and other aspects, features and advantages of the present invention will become apparent from the following description of embodiments, given by way of non-limiting example with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows a spectrum obtained by tandem mass spectrometry of the antitumor active compound according to the present invention. [Figure 2] FIG. 1 shows a chromatogram obtained by RP-HPLC of the antitumor active compound according to the present invention. [Figure 3] FIG. 1 shows a deconvoluted mass spectrum of an antitumor active compound according to the present invention. [Figure 4A] FIG. 1 shows the results of a cytotoxic activity test of the antitumor active compound according to the present invention, showing the survival rates of three cancer cell lines treated with and without the compound after one day. [Figure 4B] FIG. 1 shows the results of a cytotoxic activity test of the antitumor active compound according to the present invention, showing the survival rates after 6 days of three cancer cell lines treated with and without the compound. [Figure 5A] FIG. 1 shows the results of an apoptosis activity test of the antitumor active compound according to the present invention, showing apoptosis after one day in three cancer cell lines treated with and without the compound. [Figure 5B] FIG. 1 shows the results of an apoptosis activity test of the antitumor active compound according to the present invention, showing apoptosis after 6 days in three cancer cell lines treated with and without the compound. DETAILED DESCRIPTION OF THE INVENTION

[0029] It must be made clear that the phraseology and terminology used in the description of the invention, as well as the figures of the accompanying drawings, which are also related to the method of description, only serve to better illustrate and explain the invention, the scope of protection being defined by the claims, and their purpose is to provide a non-limiting example of the invention itself.

[0030] To facilitate understanding, the same reference numbers have been used wherever possible to identify identical common elements in the figures, it being understood that elements and features of one embodiment may be conveniently combined or incorporated in other embodiments without further description.

[0031] Unless otherwise defined, all technical and scientific terms used herein and hereafter have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where methods and materials similar or equivalent to those described herein are used in the practice and testing of the present invention, the methods and materials are described below by way of example. In case of conflict, the present application, including definitions, shall control. The materials, methods, and examples are purely illustrative and should not be construed as limiting.

[0032] All measurements are made at 25°C (ambient temperature) and atmospheric pressure unless otherwise specified. All temperatures are in degrees Celsius unless otherwise specified.

[0033] Unless otherwise indicated, all percentages and ratios given should be understood to mean by weight (w / w) of the total composition.

[0034] Unless otherwise indicated, all percentage ranges given herein are given in terms of the total of the total composition, totaling 100%.

[0035] Unless otherwise indicated, all intervals reported herein, including those reporting intervals "between" two values, should be understood to include both extremes.

[0036] Unless otherwise indicated, the description of the present invention also includes intervals obtained by combining or superimposing two or more of the intervals described.

[0037] Unless otherwise indicated, the description of the invention also includes intervals that can be obtained from the combination of two or more precise values.

[0038] In describing the present invention, the following abbreviations have been used: SPPS, solid phase peptide synthesis; Fmoc, fluorenylmethyloxycarbonyl; NMP, N-methylpyrrolidone; DIEA, diisopropylethylamine; DMF, dimethylformamide; HOBt, hydroxybenzotriazole; Pbf, pentamethyldihydrobenzofuran-5-sulfonyl; TFA, trifluoroacetic acid; EDT, ethanedithiol; TIPS, triisopropylsilane; RP-HPLC, reversed-phase high performance liquid chromatography; ACN, acetonitrile; Da, Dalton; TRIS, tris(hydroxymethyl)aminomethane; β-ME, β-mercaptoethanol.

[0039] As a compound according to the present invention, a peptide having the following sequence in the sequence of SEQ ID NO: 1 of the corresponding known protein was identified. SEQ ID NO:5 149 SCPLLTLSERGHEGRKNDLKKVECR 173 This maintains the antitumor activity similar to that observed with the original protein. The peptide sequence corresponds to the sequence between positions 149 and 173 of SEQ ID NO: 1.

[0040] Since peptides are naturally occurring, even if they are obtained from natural sources, the method for their production provides peptide synthesis.

[0041] In particular, solid phase peptide synthesis (SPPS) was performed using a Protein Technologies (Tucson, AZ) PS3 peptide synthesizer, utilizing Fmoc chemistry for terminal deprotection during synthesis. The support used was a resin derivatized with the C-terminal amino acid of the peptide to be synthesized. The following reagents were used in the synthesis:

[0042] [Table 1]

[0043] The crude peptide was dried in a stream of nitrogen, then dissolved in H2O-TFA (0.1% v / v) and analyzed using RP-HPLC.

[0044] For RP-HPLC characterization, a Vydac 218TP54 C18 analytical column (4.6 × 250 mm, 5 μm, 300 Å) was used, equilibrated with 10% aqueous acetonitrile in the presence of 0.1% TFA. Elution was performed using a gradient of mobile phase B (acetonitrile + 0.078% TFA) from 10% to 30% in 20 min at a flow rate of 0.8 ml / min. The absorbance of the eluate was recorded at a wavelength of 226 nm.

[0045] The material eluting corresponding to the most intense chromatographic peak (approximately 32% acetonitrile) was lyophilized using a SpeedVac concentrator and chemically characterized by high-resolution mass spectrometry using a Xevo-G2S Q-TOF spectrometer (Waters, Milford, MO) equipped with an electrospray ionization source. The experimental mass obtained from peptide analysis is consistent with the theoretical monoisotopic molecular weight (2867.49 Da) of the amino acid sequence. The correct sequence of the peptide was also confirmed using tandem mass spectrometry (Figure 1).

[0046] After confirming the correct synthesis, the resulting peptide underwent oxidative regeneration to promote the formation of a disulfide bond between Cys150 and Cys172 present in the native protein sequence. The mixture used for the cysteine ​​oxidation reaction, which took 16 h and was monitored by RP-HPLC, consisted of the following: 100 mM TRIS·HCl pH 8.3, 250 μM β-ME, and a peptide concentration of 0.5 mg / mL.

[0047] After the oxidation process was completed, the reaction mixture was acidified by adding TFA and analyzed by RP-HPLC using the same conditions as previously reported (Figure 2). The material eluted corresponding to the chromatographic peak formed after the oxidation reaction (approximately 28% acetonitrile) was lyophilized and analyzed using high-resolution mass spectrometry (Figure 3).

[0048] The experimental mass value obtained from the analysis of the oxidized peptide is consistent with the theoretical monoisotopic molecular mass (2865.49 Da) predicted for the amino acid sequence in which the two cysteines present are paired.

[0049] The peptides thus synthesized and characterized were then used to carry out tests of cytotoxic and apoptotic activity.

[0050] Cytotoxicity tests were performed using the KD31 human epidermoid carcinoma cell line (American Type Culture Collection) and non-tumor cell lines, normal dermal fibroblasts (NDFa) and immortalized human keratinocytes (HACAT), as controls.

[0051] The cells used were placed in culture dishes (Corning®) and treated with peptide (concentration 150 μM) for 1 and 6 days. In parallel, the same cell line without peptide treatment was prepared as a negative control.

[0052] Analysis was performed using a resazurin-based cytotoxicity test (Sigma Aldrich) and a trypan blue exclusion test. The results shown in Figures 4A and 4B are expressed as percentage means with standard error of the mean (SEM).

[0053] The apoptotic activity of the peptides was verified using the same cell lines, treatments, and negative controls. The percentage of apoptotic cells was determined using the BD™ Cell Viability Kit (BD Biosciences) according to the manufacturer's instructions. Results are expressed as mean percentages with standard error of the mean (SEM).

[0054] After one day of peptide treatment, a decrease in cell viability was observed in all three cell lines tested, with the KB31 line already showing a greater decrease than the other two, NDFa and HACAT. After six days of treatment, the viabilities of the NDFa and HACAT lines remained at roughly the same levels as those observed after one day (approximately 80% and 85%, respectively), whereas the viability of the KB31 line decreased from 77% after one day to approximately 45% after six days. The viability of untreated cells remained 100% in all three cell lines.

[0055] Similar results were obtained in apoptotic activity studies. After one day, less than 5% apoptosis was observed in all cell lines. After six days, apoptosis measured in non-tumorous NDFa and HACAT cells remained below 5%, while apoptosis detected in the KB31 tumor cell line treated with the peptide increased to over 50%. For the negative control, apoptosis remained below 5% in the non-tumor cell lines and increased to just over 10% in the KB31 tumor cell line.

[0056] These tests show that the peptides according to the invention have excellent selectivity for tumor cells, in particular human epidermal KB31 cells.

[0057] It is clear that modifications and / or additions of moieties to the antitumor active compounds can be made to the corresponding methods of manufacture and use described hereinabove without departing from the field and scope of the present invention as defined by the claims.

[0058] Although the present invention has been described with reference to some specific examples, it is also clear that a person skilled in the art can create other equivalent forms of the antitumor active compounds, corresponding methods of preparation and corresponding uses, which have the characteristics described in the claims and therefore all fall within the field of protection defined by the claims.

[0059] In the following claims, references in parentheses are for ease of reading only and should not be considered as limitations on the field of protection defined by the claims.

Claims

1. SEQ ID NO: 5 149 SCPLLTLSERGHEGRKNDLKKVECR 173 SEQ ID NO: 6 149 SCPLVTLSERGHEGRKNDLKKVECR 173 and SEQ ID NO:7 149 SCPLITLSERGHEGRKNDLKKVECR 173 1. A peptide-type antitumor active compound having a sequence selected from the group consisting of:

2. The compound of claim 1 having the sequence of SEQ ID NO:

5.

3. 3. A method for the preparation of an antitumor active compound according to claim 1 or 2, comprising peptide synthesis.

4. 4. The method of claim 3, wherein the peptide synthesis is carried out on a solid phase.

5. 5. The method according to claim 3 or claim 4, wherein the compound Fmoc is used for amino acid protection during synthesis.

6. 3. A peptide-type antitumor active compound according to claim 1 or claim 2 for use in the treatment of cancer cells and cancer stem cells.

7. The peptide-type antitumor active compound for use according to claim 6, wherein the cancer cells are human epidermoid carcinoma.

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