Bioactive peptides, compositions, methods of production, and uses of bioactive peptides as antitumor agents

Abstract

The present disclosure relates to bioactive peptides, compositions, methods of production, and uses of snake venom-based bioactive peptides as antitumor agents. The disclosed bioactive peptides are synthesized on the basis of the C-terminus of songtail toxin B and its derivatives. The disclosed peptides exhibit attenuated toxic effects and improved anti-tumor activity against tumor cells, particularly against triple negative breast cancer cells, compared to current standard care. Thus, the disclosed bioactive snake venom peptides derived from songtail toxins are used to modulate cell proliferation, apoptosis, necrosis and cycle progression of tumor cells, particularly triple negative breast cancer cells, providing suitable alternatives for separate or supplementary treatment of patients with invasive solid tumors.

Description

[0001] Statement Pursuant to 37 CFR 1.77(b)(5)

[0002] Attached to this application is a .XML file containing SEQ IDs 1 to 10 as described at the end of this application and in the claims, the material of which is incorporated herein by reference as if fully set forth in this application. Technical Field

[0003] The present invention provides bioactive peptides, compositions, production methods and uses of snake venom-based bioactive peptides as anti-tumor agents. Background Art

[0004] Data from the American Cancer Society support the expectation that in 2022, there will be 1.9 million new cases of cancer and 609,360 deaths from cancer in the United States.

[0005] Although the risk of dying from cancer has decreased over the past few years, there are still highly aggressive and life-ending cancer types that could benefit from new, alternative forms of treatment.

[0006] One of these deadly cancers is triple-negative breast cancer, which accounts for 10% to 15% of all breast cancers. This type of cancer differs from other known breast cancers because it grows and spreads faster, has a worse survival prognosis, and is affected by the limited availability of treatment options. In fact, the nomenclature used for this cancer type (triple-negative) refers to the negative results in three tests applied to detect the presence of estrogen or progesterone receptors or the HER2 protein in breast cancer cells.

[0007] Treatments available for triple-negative breast cancer include chemotherapy using conventional chemotherapeutic drugs such as anthracyclines (e.g., doxorubicin), taxanes, capecitabine, gemcitabine, eribulin, etc. Depending on certain tumor characteristics, surgery and radiation are also treatment options.

[0008] However, due to severe side effects, toxicity and low specificity, new alternatives for treating aggressive solid breast cancer tumors in patients are highly desired.

[0009] Certain diseases, including certain types of cancer, can be treated using specific proteins and peptides (protein fragments) that are biologically active in humans. It is well known that these biologically active proteins and peptides can be derived from other animals, such as terrestrial mammals, marine animals, amphibians, and animal toxins.

[0010] The first venomous animal toxin component to be purified and crystallized was Crotoxin (i.e., Crotalus toxin or CTX), the major toxin component from Crotalus durissus terrificus, also known as the South American rattlesnake.

[0011] This component consists of a heterodimeric complex of two proteins, the first being an alkaline, toxic phospholipase A2 (PLA2), also known as component B or CB; and the second protein being an acidic, non-toxic, non-enzyme component (crotapotin), also known as component A or CA, which acts as a chaperone in a synergistic manner, enhancing the toxicity of PLA2. There are different crotalus toxin isoforms, and there are individual differences in the toxin levels of this protein. The expression of different isoforms in the same venom gland can be used to maximize the activity of snake venom proteins. It has been shown that CA and CB isoforms can form at least 16 different CTX complexes. In addition, it has been shown that different CB isoforms show differences in the enzymatic and pharmacological activities of CTX. Therefore, according to the toxicity and enzymatic activity of CTX isoforms, they are divided into two categories (I and II). The CB b, CB c, and CB d isoforms complexed with CA are more toxic, have lower enzymatic activity, and dissociate more slowly from CA (class I isoform) than the CB a2 isoform (class II isoform).

[0012] It is known from the prior art that the crotalin venom protein contains different molecular regions responsible for specific pharmacological activities - neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity being the most common activities of this protein in vivo. Specifically, CB binds to receptors at the presynaptic membrane of several organisms, inhibits the release of acetylcholine and thus promotes neuromuscular blockade. However, it is important to emphasize that until recently, the exact location of the active site in the structure of the snake venom responsible for its toxic activity remained unclear, being attributed in different independent international studies to the N-terminus, the IBS (interface binding surface) or the C-terminus.

[0013] The present invention is the first to link the synthetic C-terminus of Crotalin B and its derivatives with reduced toxic effects and enhanced anti-tumor activity against tumor cells, but specifically against triple-negative breast cancer cells.

[0014] The present study showed that other peptides, such as those located in the N-terminal region of Crotalin B, did not show promising effects.

[0015] Furthermore, in vitro and in vivo assays demonstrated that the identified peptides could affect cell proliferation, apoptosis, necrosis, and cycle progression in tumor cells, especially triple-negative breast cancer cells.

[0016] Therefore, the present invention describes biologically active snake venompeptides derived from rattlesnake toxins for use in regulating cell proliferation, apoptosis, necrosis and cycle progression of tumor cells, especially triple-negative breast cancer cells, providing a suitable alternative for monotherapy or complementary treatment of patients with aggressive solid tumors. Summary of the invention

[0017] The present invention describes non-toxic bioactive snake venom peptides for the treatment of aggressive solid tumors.

[0018] In another aspect, the present invention comprises a bioactive peptide derived from the C-terminus of the CB protein of Crotalus terrestris subspecies terrificus.

[0019] In one aspect, the invention comprises biologically active peptides in substantially pure form (>95%).

[0020] More specifically, the biologically active peptide is selected from the group comprising SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9 and / or SEQ ID 10.

[0021] The biologically active peptide may also be selected from the group having at least 70% similarity to the biologically active peptide SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9 and / or SEQ ID 10.

[0022] Additionally, the biologically active peptides can be incorporated into pharmaceutical compositions.

[0023] In one aspect, the isolated bioactive peptide or a composition comprising the bioactive peptide is used to modulate cell proliferation, apoptosis, necrosis and / or cell cycle progression in tumor cells.

[0024] In particular, the peptide is therapeutically effective for the treatment of aggressive solid tumors such as lung, prostate, eye cancer (ocular melanoma), colorectal, skin, brain, pancreatic, kidney or breast cancer. More specifically, it is used to treat patients with triple negative breast cancer.

[0025] In a different aspect, the present invention claims a method for producing a biologically active peptide selected from the group comprising SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9 and / or SEQ ID 10, wherein the biologically active peptide is obtained synthetically, the method comprising the following steps:

[0026] a) Microwave-assisted efficient synthesis and amino acid coupling;

[0027] b) purification of peptides using HPLC (high performance liquid chromatography);

[0028] c) Analyze the results by mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present disclosure will be more clearly understood by referring to the accompanying drawings listed below:

[0030] Figure 1 HPLC characterization of 3-NantC is shown, where the purity of the batch was observed to be greater than 95% (95.15%).

[0031] Figure 2 Shown is the MS / MS characterization of the 3-NAntC peptide (SEQ ID 1) sequence confirmed by m / z relationships.

[0032] Figure 3 The presence of abundant untreated triple-negative tumor cells is shown.

[0033] Figure 4 Shown are tumor cells after 24 hours of treatment with peptide 3-NantC (SEQ ID 1). Arrows indicate tumor cells that show fewer numbers and have vesicles when compared to untreated tumor cells.

[0034] Figure 5 The following results are shown: MTT assay for both MDAMB231 cells (triple negative) and MCF10A cells (breast benign cells) treated with peptides derived from the N-terminal region of Crotalin B (Peptide 1 and Peptide 2), where no antitumor effect or high toxicity was observed in tumor cells.

[0035] Figure 6 Results of an MTT assay are shown, showing decreased viability for the tumor cell lines, while the fibroblast (benign) cell line remained viable at 24, 48 and 72 hours of testing.

[0036] Figure 7Comparison of the antitumor effects of 3-NAntC and commercial drugs (doxorubicin and cisplatin) by MTT assay results is shown.

[0037] Figure 8 Results of BrdU labeling are shown, which show that proliferation of triple-negative tumor cells was reduced when treated with 3-NAntC, even at concentrations that did not show cytotoxicity (eg, 0.2 μg / mL).

[0038] Fig. 9 Cell cycle progression assay at 48 hours is shown, where 3-NAntC treatment results in a decrease in the G0 / G1 phase, while it causes a G2 / M arrest.

[0039] Fig.10 Shown is the release of lactate dehydrogenase from MDAMB231 cells after 24, 48 and 72 hours of treatment with 3-NAntC peptide. There were no significant differences in this biomarker, indicating that necrosis, necroptosis or pyroptosis were not involved as the primary cell death mechanism.

[0040] Fig.11 Comparison of MTT assays of MDAMB231 cells treated with 3-NAntC alone or together with the autophagy inhibitor 3-methyladenine (3-MA) ​​is shown. Since no difference was observed, the 3-NAntC peptide does not induce cell death primarily through an autophagic mechanism.

[0041] Fig.12 Shown are the results of flow cytometry labeled with Annexin V-FITC and Propidium Iodide (PI), which indicated that 3-NAntC induced cell death by apoptosis and very little necrosis.

[0042] Fig.13 Cell viability of MDAMB231 cells in the presence of various 3-NAntC forms (SEQ 2 to 10) is shown, indicating that the linear form as well as the monomeric form have interesting anti-tumor effects.

[0043] Fig.14 It shows the cell viability of MDAMB231 cells when SEQ ID 4 is combined with SEQ ID 5. Although the cell viability is reduced, 3-NAntC still has a good anti-tumor effect.

[0044] Fig.15 The 3-NAntC peptide was shown to be highly tolerated in zebrafish.

[0045] Fig.16Antitumor and anti-metastatic effects of high doses of 3-NantC against a xenograft model of MDAMB231 in zebrafish are shown.

[0046] Fig.17 It was shown that the in vivo anti-metastatic effect of 3-NAntC still existed even at low doses of 3-NAntC. DETAILED DESCRIPTION

[0047] The term "biologically active peptide" comprises the C-terminal portion of the rattlesnake venom protein obtained from Crotalus terrestris subspecies and its derivatives. In a specific manner, the biologically active peptide shows a high purity (purity greater than 70%, preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95%). The biologically active peptide of the present invention is active against tumor cells, more specifically, against triple-negative breast cancer cells. It comprises one or more of SEQ ID 1 (also referred to herein as "3-Nantc") and its derivatives (SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10). In a more preferred embodiment, the biologically active peptide has at least 70%, 80%, 90% or 100% identity with SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10.

[0048] The term "modulating tumor cells" includes modulating (increasing, reducing or maintaining) at least one of the following parameters in tumor cells when using bioactive peptides: cell proliferation, apoptosis, necrosis and cycle progression when using bioactive peptides. Once it is expected that the bioactive peptides are active against the growth or survival of tumor cells at the same time, but have no or little effect on benign cells, then the regulation of tumor cells also depends on the regulation of benign cells. In a preferred manner, the use of bioactive peptides to regulate tumor cells reduces the viability of cancer cells while not reducing the viability of benign skin cells. In addition, the bioactive peptides reduce the proliferation rate of tumor cells while not reducing the proliferation of benign cells. The bioactive peptides preferably make it significantly difficult for tumor cells to enter the G0 / G1 phase, while causing stagnation in both the S phase and the G2 / M phase. In addition, the bioactive peptides do not induce tumor cell death through autophagy. The bioactive peptides induce apoptosis of tumor cells. In addition, when compared with the use of drugs known to treat tumor cells (such as doxorubicin) to induce necrosis, the rate of induction of undesirable necrosis by the bioactive peptides is very low.

[0049] The term "therapeutically effective" includes the use of bioactive peptides as a single treatment or in combination with other treatments or as a supplement to other treatments for regulating one or more aspects of tumor cells while retaining benign cells in cancer patients, preferably solid tumor cancer patients, more preferably breast cancer patients, and even more preferably triple-negative breast cancer patients. In addition, the bioactive peptides can be administered in vivo or in vitro. The bioactive peptides of the present invention can be effectively used in combination with a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier as a carrier, diluent and / or excipient.

[0050] Protein synthesis was performed by microwave-assisted high efficiency solid phase synthesis (HE-SPPS) and amino acid coupling using the equipment Liberty Blue HT12 (CEM Corporation, USA).

[0051] Peptide purification was performed using HPLC (high performance liquid chromatography). A gradient of two eluents was used: deionized water acidified with trifluoroacetic acid-TFA (0.1%) and UV / HPLC grade acetonitrile in TFA (ACN / 0.08% TFA, phase B). For detection, a UV detector with a λmax of 220 nm (amide absorption range of 215 nm to 220 nm) was used. Purification was performed at room temperature on a semi-preparative scale (5 mg crude sample and a flow rate of 1 mL / min). For HPLC, a Varian model Pro Star 210 chromatograph was used, which had a detector in the ultraviolet region: model Pro Star 330. The columns used were: 4.6×250 mm, Boston Green 0DS-AQ and the rings were 5 μL, 250 μL, respectively. The results were analyzed by mass spectrometry (matrix-assisted laser desorption / ionization) in an ESI-Q-ToF Micromass Micro mass spectrometry instrument (Micromass, Manchester, UK) and MALDI-ToF / ToFMS (Applied Biosystems, USA). The data obtained were analyzed by the MassLynx 4.0 program.

[0052] The present invention is illustrated below by reference to the following examples. However, it will be appreciated by those skilled in the art that the specific methods and results discussed are merely illustrative of the present invention, because numerous variations, modifications, applications and extensions may be made to these embodiments and principles without departing from the spirit and scope of the present invention.

[0053] Example

[0054] Characterization of the peptide (3-NantC) using MS / MS and HPLC-UV

[0055] To obtain peptide purity, HPLC was performed under these conditions:

[0056] Column: 4.6×250mm, Boston Green 0DS-AQ

[0057] Solvent A: 0.1% trifluoroacetic acid in 100% water

[0058] Solvent B: 0.1% trifluoroacetic acid in 100% acetonitrile

[0059]

[0060] Flow rate: 1.0mL / min

[0061] Wavelength: 220nm

[0062] Volume: 10 μL

[0063] To confirm the sequence, MS / MS was performed after HPLC under these conditions:

[0064] Probe: ESI Probe bias: 4.5kV

[0065] Nebulizer gas flow: 1.5L / min

[0066] Detector: 1.5kV

[0067] CDL: -20.0V CDL temperature: 250℃

[0068] Block temperature: 200°C

[0069] T. Flow rate: 0.2mL / min

[0070] B. Concentration: 50% H2O / 50% ACN

[0071] HPLC analysis of 3-NAntC peptide (SEQ ID 1) Figure 1 ) and MS / MS( Figure 2 ) characterization showed a purity of 95.15% and a molecular mass of 1645.95, confirming the sequence of [H]-MFYPDSRCRGPSET-[OH] (SEQ ID 1), also referred to herein as 3-NAntc.

[0072] In vitro assay methods and results

[0073] Compared with untreated ( Figure 3 ), when treated with 3-NAntC for 24 h, triple-negative breast tumor cells (MDAMB231) were present in fewer numbers and had a greater number of vesicles, such as Figure 4 as shown in .

[0074] Cell viability

[0075] For the MTT method, cells were plated in 96-well sterile plates with lids for cell culture at a density of 2 × 10 for treatments lasting 24 h. 4 cells / well, and for treatments lasting 48 h or longer, the density was 1 × 10 4 cells / well and make up the volume to 100 μL with culture medium. Incubate the plate in an incubator at 37°C and 5% CO2 for 8 to 16 hours. The next day, prepare the treatment in culture medium, apply 200 μL / well at the desired concentration, and place the plate in an incubator at 37°C and 5% CO2 for the duration of the treatment. After the treatment period, add 20 μL / well of MTT solution at a concentration of 5 mg / mL in PBS (1×) and incubate the dye at 37°C and 5% CO2 for 2.5 hours. After incubation with MTT, add 200 μL of dimethyl sulfoxide solution (DMSO): isopropanol (3:1) to dissolve the formaldehyde. Salt and stir at room temperature for at least 15 minutes. After complete dissolution of the salt, read the absorbance at 570 nm in a spectrophotometer using an endpoint protocol.

[0076] The cell viability was calculated using the following formula:

[0077] Cell viability % = (OD cell + test substance) - (OD base) x 100 (OD cell + medium) - OD base

[0078] OD: Optical density captured by a spectrophotometer

[0079] NOTE: Basal OD is the OD present in the blank wells.

[0080] To determine whether it was the region of crotoxin B that showed promising antitumor activity (comparison of dose and time for the MDAMB231 cell line) and safety (comparison between the effects of MDAMB231 and MCF10A), analysis of variance (ANOVA) and Bonferroni post hoc test were applied in GraphPad Prism software version 6.0 with a significance level of 5%.

[0081] MTT showed that both peptides [H]-HLLQFNKMIKFETRKNAIPF-[OH] (peptide A) and [H)-AIPFYAFY-(OH] (peptide B) derived from the N-terminal region of Crotalin B showed no promising antitumor activity, as they were unable to reduce the viability of tumor cells or maintain the viability of benign cells. The first peptide (A) did not reduce the viability of tumor cells, and the second peptide (B), although it did reduce the viability of MDAMB231 at 24 hours (about 82% of viable cells), also reduced the viability of MCF10A benign cells at 48 hours (about 85% of viable cells) ( Figure 5 ).

[0082] To determine the efficacy (comparison of doses and time against the MDAMB231 cell line) and safety (comparison between the effects against MDAMB231 and HDFa cells) of the peptide named 3-NAntC, SEQ ID 1 - MFYPDSRCRGPSET (C-terminal region of Crotalin B), analysis of variance (ANOVA) with Bonferroni post hoc test was applied at a significance level of 5% in GraphPad Prism software version 6.0.

[0083] MTT showed that 3-NAntC significantly reduced the viability of triple-negative cancer cell line (MDAMB231) within 24, 48 and 72 hours without reducing the viability of benign skin cell-fibroblasts (HDFa) ( Figure 6 )——(the viable cells of tumors were reduced to 70.98%, 59.68% and 17.60%, respectively). The cytotoxicity ratio averaged six tumor cells to one benign cell (6:1). Data are shown as mean ± SEM of at least three independent determinations in triplicate.

[0084] MTT also showed that 3-NAntC surpassed the antitumor effect of cisplatin as early as 48 h of treatment, and the biological effect at 72 h was similar to that recorded by doxorubicin at 48 h ( Figure 7 ). Data are shown as mean ± SEM of at least three independent determinations performed in triplicate.

[0085] Cell proliferation

[0086] The bromodeoxyuridine (BrdU) quantification method was performed according to the standard operating protocols No. 10 and 12 of the Laboratory of Clinical Cytology of the Faculty of Pharmaceutical Sciences of Ribeirao Preto (FCFRP-USP). In this method, for BrdU quantification, cells were plated in 96-well sterile plates with lids for cell culture at a density of 2×10 for a treatment lasting 24 hours. 4 cells / well, and for treatments of 48 h or longer, the density was 1 × 10 4 Cells / well, the volume is made up to 100 μL with culture medium. The plate is incubated in an incubator at 37 ° C and 5% CO2 for 8 to 16 hours. The next day, the treatment is prepared in the culture medium, 200 μL / well is applied at the desired concentration, and the plate is placed in an incubator at 37 ° C and 5% CO2 for the duration of the treatment. After the treatment period, 20 μL / well of BrdU labeling is added and incubated for 2.5 hours at 37 ° C and 5% CO2. After incubation with BrdU labeling, the plate is stored at 2 ° C to 4 ° C for up to 1 week. After storage, the antibody anti-BrdU-POD is used by ELISA method, followed by washing and using substrate solution to show the reaction. The substrate is developed for 15 minutes, and the reaction is stopped with HCl6M solution and read at 495nm in a spectrophotometer at the end point.

[0087] The cell proliferation was calculated using the following formula:

[0088] Cell proliferation % = (OD cell + test substance) - (OD base) × 100 (OD cell + medium) - OD base

[0089] OD: Optical density captured by a spectrophotometer

[0090] NOTE: Basal OD is the OD present in the blank wells.

[0091] To determine the effect of the peptide in proliferation (MDAMB231 and HDFa cell lines), analysis of variance (ANOVA) with Dunnet and Bonferroni post hoc test was applied at 5% significance in GraphPad Prism software version 6.0.

[0092] Thus, BrdU labeling showed that 3-NAntC reduced the proliferation rate of MDAMB231 cells (minimum reduction of 33% at 24 hours for 0.2 μg / mL concentration). In addition, the data showed that at the maximum exposure time, the peptide did not alter the proliferation of the HDFa benign cell line ( Figure 8 ).

[0093] Effects on cell cycle progression of tumor cells

[0094] For the cell cycle progression method, plate the cells in 6-well sterile plates with lids for cell culture at a density of 2.5 × 10 for treatments lasting 24 h or longer. 5 Cells / well were added and the volume was made up with 1000 μL culture medium. The plate was incubated in an incubator at 37°C and 5% CO2 for 8 to 16 hours. The next day, the treatment was prepared in the culture medium, 2000 μL / well was applied at the desired concentration, and the plate was placed in an incubator at 37°C and 5% CO2 for the duration of the treatment. After the treatment period, the cells were collected in a 15mL conical tube and washed with 1000 μL PBS (1×). The cells were resuspended in 500 μL / tube of PBS (1×) and 4.5mL ethanol 70°. Then, the tube was sealed and stored at 2°C to 4°C for up to 2 months. The cells were washed with PBS (1×) and resuspended with 135 μL PBS (1×). 100 μg / mL of RNase A solution was added and incubated at room temperature for 30 minutes. After incubation, cells were labeled with 2 μL of 100 μg / mL propidium iodide and read on a flow cytometer for size and DNA content.

[0095] To determine the role of the peptide in cell cycle progression (MDAMB231 cell line), analysis of variance (ANOVA) and Dunnet's post hoc test were applied at a significance level of 5% in GraphPad Prism software version 6.0.

[0096] At 48 hours, 3-NAntC caused a decrease in cells in G0 / G1 (8.72% decrease at the highest dose) while increasing the percentage of G2 / M (8.08% increase at the highest dose). Thus, the peptide significantly hindered the cells from entering the G0 / G1 phase while causing an arrest in the G2 / M phase (see Fig. 9 ). Data are shown as mean ± SEM of at least three independent determinations.

[0097] Cell death (apoptosis / necrosis, necroptosis, pyroptosis / autophagy)

[0098] Lactate dehydrogenase (LDH) is a cytosolic enzyme present in most mammalian cells. When the plasma membrane is damaged, LDH is released into the extracellular matrix and can be quantified as an important indicator of cell death caused by necrosis, necroptosis and pyroptosis, because, for example, in cell death caused by apoptosis, LDH extravasation does not occur due to the formation of apoptotic bodies and phagocytosis of the material by leukocytes.

[0099] For the LDH release protocol, 20 μL of supernatant per well was transferred to the EIA / RIA plate after the MTT protocol. Then, 20 μL / well of CyQUANT TM The reaction mixture of the LDH cytotoxicity assay kit and the reactants were incubated at room temperature in the dark for 30 minutes. Then, 60 μL / well of the stop solution from the kit was added and the plate was read in a spectrophotometer at both 490 nm and 650 nm.

[0100] The calculation of LDH release was performed as follows:

[0101] 1. Subtract the absorbance measured at 650 nm from the absorbance measured at 490 nm.

[0102] 2. Apply the formula:

[0103] % LDH release = (OD cell + test substance) - (OD base) x 100 (OD cell + Triton) - OD base

[0104] OD: Optical density captured by spectrophotometer Note: Basal OD is the OD present in blank wells (reaction mixture and stop solution only).

[0105] 3-Methyladenine (3-MA) ​​is an inhibitor of autophagy (blocks activation of this mechanism of action). For molecules that trigger autophagy as a mechanism of cell death, when this pathway is blocked, the molecule loses its effect.

[0106] The MTT method was performed as described previously herein and the effect of 3-NAntC in the presence or absence of 3-methyladenine (3-MA) ​​was compared.

[0107] Annexin V is an effective marker of apoptosis because it binds to phosphatidylserine of the cell membrane. This binding occurs only when phosphatidylserine is externalized in the membrane, which is one of the first signals of apoptotic death. Meanwhile, propidium iodide enters the cell only after membrane rupture and can indicate the final steps of cell death. Dual labeling with these markers allows differentiation of apoptotic and necrotic death by the following characteristics:

[0108] Annexin V - / PI - :Living Cells

[0109] Annexin V + / PI - :Early apoptotic cells

[0110] Annexin V + / PI + : Late apoptotic cells

[0111] Annexin V - / PI + : Necrotic cells

[0112] For the apoptosis / necrosis protocol, cells were plated in 6-well sterile plates with lids for cell culture at a density of 2.5 × 10 for treatments lasting 24 h or longer. 5 Cells / well, make up the volume with 1000 μL culture medium. Plates are incubated in an incubator at 37 ° C and 5% CO2 for 8 to 16 hours. The next day, treatments are prepared in culture medium, 2000 μL / well is applied with the desired concentration, and the plate is placed in an incubator at 37 ° C and 5% CO2 for the treatment of duration. After the treatment period, cells are collected in 15 mL conical tubes and washed with 200 μL of binding buffer (1×) from FITC Annexin V Apoptosis Detection Kit I. Cells are resuspended in 100 μL / tube binding buffer (1×), and 1 μL Annexin V-FITC is added to each tube except the blank tube. Cells are then incubated in ice and darkness for 20 minutes. Then, 50 μL / tube binding buffer is added, and cells are labeled with 2 μL 100 μg / mL of propidium iodide (PI), and size and fluorescence content (FITC and PI) are read on a flow cytometer.

[0113] To determine the role of the peptide in LDH release, autophagy induction and apoptosis / necrosis characteristics of the MDAMB231 cell line, analysis of variance (ANOVA) with Dunnet and Bonferroni post hoc test was applied at a significance level of 5% in GraphPad Prism software version 6.0.

[0114] 3-NAntC did not show any significant increase in LDH release from MDAMB231 cells ( Fig.10 ), indicating no significant relationship with cell death caused by necrosis, necroptosis, and pyroptosis. Data are shown as mean ± SEM of at least three independent determinations in triplicate.

[0115] The activity of 3-NAntC in the presence and absence of autophagy inhibitors remained unchanged. Therefore, 3-NAntC does not induce death through autophagy ( Fig.11 ). Data are shown as mean ± SEM of at least three independent determinations in triplicate. This highlights the huge difference between 3-NAntC and Crotoxin B, as natural molecules are known to promote autophagy (Yanet.al.Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells.Acta Pharmacol Sin, 2007).

[0116] Compared with doxorubicin, 3-NAntC mainly induced apoptosis and very little necrosis in tumor cells. At 24 and 48 hours, 0.8 μg / mL of 3-NAntC significantly increased the apoptosis rate while maintaining the percentage of necrotic cells (about 20% in each case). At 72 hours, 3-NantC increased the apoptosis rate at all tested doses (increased by 28.73% at the highest dose) ( Fig.12 ). Data are shown as mean ± SEM of at least three independent determinations.

[0117] Derivatized peptides – in vitro comparative potency

[0118] SEQ ID 2 to 10 were compared with SEQ ID 1 by MTT assay (as previously described in the in vitro MTT method) to determine the best potency in reducing the viability of MDAMB231 cells. Thus, the results showed that SEQ ID 1 promoted the highest anti-tumor effect, followed by SEQ 4 and SEQ 8 ( Fig.13 ). It was observed that the combination of SEQ ID 4 and SEQ ID 5 also had a promising anti-tumor effect ( Fig.14 ). In addition, SEQ ID 10 shows that 3-NAntC monomer also has interesting anti-tumor effects, similar to 3-NAntC at the highest concentration.

[0119] In vivo methods and results

[0120] Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were raised in E3 embryo medium 0.2mM 1-phenyl-2-thiourea (also known as PTU) at 28°C for 48 hours. Unfertilized eggs or larvae that looked unhealthy or showed any obvious developmental defects were excluded before the start of treatment. MDA-MB-231 cells were labeled with Dil red fluorescent dye. Approximately 700 Dil-labeled cancer cells were subcutaneously implanted into the perivitelline space of 2-day-old larvae ± 75, 100 or 125mg / mL of 3-NantC peptide. Larvae in which tumor cells were accidentally injected into the circulation or tumors were mistakenly implanted into the yolk rather than the perivitelline space were excluded from this study. The selected tumor-bearing embryos were divided into experimental groups (20 embryos / group) and photos of primary tumors were taken immediately after injection. According to the study plan, 2.5μg / mL doxorubicin treatment was added to the embryo water. Tumor-bearing embryos were incubated in E3 / PTU medium at 36°C for 72 hours. After incubation, photographs of primary tumors and CVP were taken using a red fluorescent filter. Larvae that died or were otherwise lost during the study were not included in the final analysis.

[0121] Images obtained immediately after implantation (day 0) and after 72 hours of incubation (day 3) were analyzed using in-house developed software. Tumor growth regression was calculated and normalized relative to the negative control group. The number of metastatic cells was manually counted. A one-way ANOVA was performed, followed by a two-tailed Student t test. Clear outliers were detected using the ROUT method, with Q=1%. GraphPad Prism v9.0.2 was used to obtain / analyze charts and example images and experimental data.

[0122] The 3-NAntC peptide was well tolerated by zebrafish embryos, even at doses higher than the IC of MDAMB231 in vitro assays at 24 h. 50 50,000-fold higher, yet still showed high embryo survival (see Fig.15 Data are shown as mean ± SEM (20 embryos / group). Therefore, and given the very high dose levels, the LD could not be determined. 50 (Dose that kills 50% of the in vivo model).

[0123] 3-NantC peptide had an anticancer effect on MDAMB231 breast cancer cells at all concentrations tested in vivo; this effect was significantly more effective than doxorubicin treatment alone (75% reduction in tumor cells against approximately 35% of doxorubicin) (see Fig.16 ).

[0124] At concentrations of 75 mg / mL and 125 mg / mL, 3-NantC peptide exhibited potent in vivo anti-metastatic activity against the originally highly metastatic MDAMB231 breast cancer cells (metastatic cells were reduced by approximately 60%) (see Fig.16 ).

[0125] This anti-metastatic effect was observed even at very low concentrations of 3-NAntC (about 40% reduction in metastatic cells) (see Fig.17 ). Data are shown as mean ± SEM normalized to the control group (20 embryos / group).

[0126] Mechanism of action

[0127] Western blot protein expression showed that 3-NantC peptide inhibited the activation of pro-survival signals (e.g., NFKB) when compared to negative controls, while causing greater activation of caspases (i.e., caspase 9). In addition, proteomic analysis showed that 3-NantC peptide, in addition to consistently disabling the protein elongation machinery, also reduced Rho-GTPase expression (e.g., Rho-A and Rho-C), which strongly suggests activation of apoptosis (data not shown).

[0128] SEQ ID List

[0129] SEQ ID 1

[0130]

[0131] SEQ ID 2

[0132]

[0133] (Linear)

[0134] SEQ ID 3

[0135]

[0136] (Cyclic: disulfide bridges on the first and last C; the middle C is hindered)

[0137] SEQ ID 4

[0138]

[0139] SEQ ID

[0140]

[0141] SEQ ID 6

[0142]

[0143] (Linear)

[0144] SEQ ID 7

[0145]

[0146] (Cyclic: disulfide bridges on the first and last C)

[0147] SEQ ID 8

[0148]

[0149] (Linear)

[0150] SEQ ID 9

[0151]

[0152] (Cyclic: disulfide bridges on the first and last C)

[0153] SEQ ID 10

[0154]

Claims

1. A biologically active synthetic peptide derived from the C-terminal portion of Crotalus B snake venom toxin with anti-tumor activity against aggressive solid tumors.

2. The biologically active synthetic peptide according to claim 1, wherein the solid tumor is selected from the group consisting of lung, prostate, eye cancer (ocular melanoma), colorectal, skin, brain, pancreas, kidney, breast or any combination thereof.

3. The biologically active synthetic peptide according to claim 1 or 2, wherein the solid tumor is triple-negative breast cancer.

4. The biologically active synthetic peptide according to any one of claims 1 to 3, wherein the anti-tumor activity is selected from the group consisting of regulating cell proliferation, reducing metastasis, apoptosis, necrosis, cycle progression and any combination thereof.

5. The biologically active synthetic peptide according to any one of claims 1 to 4, wherein the anti-tumor activity comprises making it difficult for the solid tumor cells to enter the G0 / G1 phase while causing arrest in the G2 / M phase.

6. The biologically active synthetic peptide according to any one of claims 1 to 5, wherein the biologically active peptide is selected from SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10 and any combination thereof.

7. The biologically active synthetic peptide according to any one of claims 1 to 6, wherein the purity of the biologically active peptide is 70% to greater than 95%.

8. The bioactive peptide according to any one of claims 1 to 7, wherein the bioactive protein has at least 70% similarity to a bioactive peptide selected from SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, and any combination thereof.

9. The biologically active peptide according to any one of claims 1 to 8, wherein the biologically active protein comprises SEQ ID 1.

10. The bioactive peptide according to any one of claims 1 to 9, wherein the anti-tumor activity of the bioactive protein does not comprise promoting autophagy.

11. A pharmaceutical composition comprising the bioactive peptide according to any one of claims 1 to 10 and at least one pharmaceutically acceptable carrier as a carrier, diluent and / or excipient.

12. The pharmaceutical composition according to claim 11, which is administered in vivo or in vitro.

13. A method for preparing a bioactive peptide, wherein the bioactive peptide is selected from SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10 or any combination thereof, the method comprising the following steps: a) using microwave-assisted efficient solid phase synthesis to couple amino acids to obtain the bioactive peptide; b) purifying the obtained bioactive peptides using HPLC (high performance liquid chromatography); as well as c) analyzing the obtained bioactive peptides by mass spectrometry to confirm the composition of the obtained bioactive peptides.

14. The method of claim 13, wherein a gradient of two eluents is used in the purification step: (a) deionized water acidified with trifluoroacetic acid-TFA and UV / HPLC grade acetonitrile in TFA.

15. A method for treating a solid tumor selected from the group consisting of lung, prostate, eye cancer (ocular melanoma), colorectal, skin, brain, pancreas, kidney, breast, or any combination thereof, the method comprising administering to a patient a therapeutically effective amount of a biologically active peptide selected from the group consisting of SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, or any combination thereof, in a pharmaceutically acceptable carrier.

16. The method for treating solid tumors according to claim 15, wherein the bioactive peptide consists of SEQ ID 1.

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