Positively charged naf-1 derived peptides and uses thereof

Abstract

Disclosed are peptide sequences derived from human NAF-1 protein, wherein it is demonstrated that the addition of positively charged moieties, including positively charged amino acids, to the C-terminus improves cancer cell killing without increasing toxicity. Also disclosed are pharmaceutical compositions and methods of use of the peptides.

Description

Technical Field

[0001] This disclosure relates to peptides derived from human NAF-1 protein, analogues thereof, and their use in the treatment of various types of cancer. Background Technology

[0002] Nutritional deprivation autophagy factor-1 (NAF-1, CISD2) is a unique iron-sulfur (2Fe-2S) protein belonging to the NEET protein family and encoded by the CISD2 gene in humans. Recent studies have shown that NAF-1 is located in the endoplasmic reticulum (ER), the outer mitochondrial membrane, and the ER-mitochondrial-associated membrane (MAM). NAF-1 is involved in a wide variety of biological processes and diseases, such as regulating autophagy, apoptosis, ferroptosis, iron and reactive oxygen species (ROS) homeostasis, and cancer.Enhanced NAF-1 expression is associated with various cancers, including breast cancer, prostate cancer, gastric cancer, cervical cancer, lung cancer, liver cancer, glioma, neuroblastoma, head and neck cancer, and laryngeal cancer (Holt et al., 2015, Journal of Cell Science, 129, 155-165; Sohn, Y.-S. et al., 2013, Proc. Natl. Acad. Sci. USA, 110, 14676–14681; Ge, YZ et al., 2014, Gene, 551, 86-91; Wang, L. et al., 2015, Oncotarget 7, 3791-3805; Liu, L. et al., 2014, Med. Oncol., 31,183; Chen, B. et al., 2015, Int. J. Clin. Exp. Pathol. 8, 13725-13738; Yang, L. et al., 2016, Oncotarget, 7, 22720-22732; Karmi et al., Free Radical Biology and Medicine 176(2021): 92-104; Lin, Hung-Yu et al., "Translational Implications Present and Future", Cancers 13.18 (2021): 4576; A.-G. Sun et al., Mol. Med. Rep., 16 (2017), pp.7939-7948; A.-G. Sun et al., Mol. Med. Rep., 16 (2017), pp. 7939-7948; S.-M. Li et al., Sci. Rep., 7 (2017), p. 11893; Shao, Fangchun et al., Frontiers in Oncology (2021):3187; J. Li et al., Pathol. Oncol. Res., 26 (2020), pp. 1725-1733; EH Kim et al., CancerLett., 432 (2018), p.180-190; Karmi et al., Free Radicals in Biology and Medicine, 176, p. 92-104 (2021)).

[0003] Decreased NAF-1 expression in breast or gastric cancer cells significantly inhibited cell proliferation and tumorigenicity, while overexpression of NAF-1 in these cells significantly enhanced cell proliferation. Darash-Yahana et al. (PNAS, 2016, 113(39), 10890-10895) showed that overexpression of NAF-1 in xenograft tumors led to a sharp increase in tumor size and invasiveness; conversely, inhibition of NAF-1 expression by shRNA significantly reduced tumor size and invasiveness.

[0004] Developing cancer therapies is a tedious task, requiring the continuous search for new targets and the generation of novel therapeutics that affect these targets. There is a clear and ongoing need to develop strategies for treating cancer and to provide novel therapeutics for cancer treatment. Summary of the Invention

[0005] This invention is partly based on a surprising discovery that adding a positively charged amino acid to the C-terminus of a peptide first identified in US 11,208,443 significantly enhances its biological anticancer activity, particularly in vivo.

[0006] This article describes a peptide that comprises: (a) An amino acid sequence containing FLGVLAL-X1-X2 (SEQ ID NO: 10), Where X1 represents the second amino acid sequence LGYLAVRPFLPKKKQQK (SEQ ID NO: 30) or a fragment of the second amino acid sequence with a continuous deletion at the C-terminus; Where X2 represents 1-10 positively charged portions; and Any amino acid in the amino acid sequence may be a D-amino acid; or (b) analogues of (a); or (c) The reversed configuration sequence of (a) or (b).

[0007] In this embodiment, the positively charged portion of the peptide is a positively charged amino acid, lysine (K), or arginine (R). Preferably, the positively charged portion is polylysine. For example, X2 can be 3-10 positively charged portions, 4-7 positively charged portions, or 6 positively charged portions.

[0008] In other embodiments, at least one amino acid of the peptide is a D-amino acid. In other embodiments, at least 1, 2, 3, 4 to at most all of the amino acids in any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 are D-amino acids.

[0009] In other embodiments, the peptide may be described as comprising the following amino acid sequence: (a) FLGVLALLGYLAVRPFLPKKKQQK-X2 (SEQ ID NO: 11); (b) FLGVLALLGYLAVRPFLPKKKQQ-X2 (SEQ ID NO: 12); (c) FLGVLALLGYLAVRPFLPKKKQ-X2 (SEQ ID NO: 13); (d) FLGVLALLGYLAVRPFLPKKK-X2 (SEQ ID NO: 14); (e) FLGVLALLGYLAVRPFLPKK-X2 (SEQ ID NO: 15); (f) FLGVLALLGYLAVRPFLPK-X2 (SEQ ID NO: 16); (g) FLGVLALLGYLAVRPFLP-X2 (SEQ ID NO: 17); (h) FLGVLALLGYLAVRPFL-X2 (SEQ ID NO: 18); (i) FLGVLALLGYLAVRPF-X2 (SEQ ID NO: 19); (j) FLGVLALLGYLAVRP-X2 (SEQ ID NO: 20); (k) FLGVLALLGYLAVR-X2 (SEQ ID NO: 21); (l) FLGVLALLGYLAV-X2 (SEQ ID NO: 22); (m) FLGVLALLGYLA-X2 (SEQ ID NO: 23); (n) FLGVLALLGYL-X2 (SEQ ID NO: 24); (o) FLGVLALLGY-X2 (SEQ ID NO: 25); (p) FLGVLALLG-X2 (SEQ ID NO: 26); (q) FLGVLALL-X2 (SEQ ID NO: 29) or (r) FLGVLAL-X2 (SEQ ID NO: 28).

[0010] For example, specific examples of peptides include, but are not limited to: (a) FLGVLALLGYLAVRPFLPKKKQQKKKKKKK (SEQ ID NO: 1); (b) FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2); (c) flgvlallgylavrpflpkkkqqkkkkkkk (SEQ ID NO: 3); (d)FLGVLALLGYLAVRPFLPKKKQQKX 2-10 (SEQ ID NO: 4); (e)FLGVLALLGyLAVRPfLPKkKQQK-X 2-10 (SEQ ID NO: 5); or (f) FLGVLALLGYLAVRPFLPKKKQQKKKKKK (SEQ ID NO: 29).

[0011] In other embodiments, the peptide analogs described herein comprise: (a) Modifications of 1 to 10, 2 to 8, or 3 to 6 of the amino acid sequence; (b) Modifications of 1 to 6 of the amino acid sequence; (c) One to three modifications to the amino acid sequence; (d) Modifications of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acid sequence; (e) A polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31; or (f)(a)-(e) analogues containing conservative substitutions.

[0012] Other embodiments include, but are not limited to, peptides of 17 to 50 amino acids in length. They also include the reverse conformation sequence of said peptide. Other embodiments include polynucleotides encoding the peptides described herein, and conjugates comprising the peptides and portions thereof described herein.

[0013] Examples of such portions that can be coupled with the peptides described herein include, but are not limited to, PEG, wherein, for example, the molecular weight of the PEG is in the range of 350 to 10,000 Da; or wherein the PEG has 5 to 100 ethylene glycol monomers.

[0014] Alternatively, the part described is: (a) A radioactive isotope, preferably wherein the radioactive isotope is 131I, 68Ga, 177Lu, 153 Sm、 212 Pb, 166 Ho、 111 In, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90, and thorium-227; (b) The signal-generating component, such as a fluorescent compound, preferably such as fluorescein, anthocyanin dye, or Alexa Fluor. ® ;or (c) Signal generation components, such as PET or SPECT markers, for example 18 F, 99m Tc, 68 Ga、 11 C 13 N、 15 O、 64 Cu、 86 Y、 55 Co、 123 I, 131 I, 99m T, 89 Zr、 133 Xe and 201 TI.

[0015] Pharmaceutical compositions comprising the peptides described herein, including peptide conjugates, are also envisioned. Uses of such pharmaceutical compositions are envisioned.

[0016] For example, a method for treating a subject in need is envisioned, wherein the method comprises administering to the subject a therapeutically effective amount of the peptide, conjugate, or pharmaceutical composition described herein. Preferably, the method is for treating cancer. Examples of cancers include, but are not limited to, breast cancer, prostate cancer, gastric cancer, colon cancer, rectal cancer, cervical cancer, endometrial cancer, bile duct cancer, ovarian cancer, myeloma, glioma, glioblastoma, neuroblastoma, melanoma (including malignant melanoma), CML, AML, ALL, liver cancer, pancreatic cancer, head and neck cancer, lung cancer, bladder cancer, kidney cancer, non-Hodgkin's lymphoma, and laryngeal cancer.

[0017] In other embodiments, the cancer treated as described herein is accompanied by enhanced NAF-1 protein expression.

[0018] In an embodiment, the treatment method further includes the administration of anticancer therapy, preferably wherein the anticancer therapy is an immunomodulator, activated lymphocytes, lymphocyte activators, kinase inhibitors, chemotherapeutic agents, or anticancer agents.

[0019] Furthermore, a method for diagnosing cancer is envisioned. In this embodiment, the peptide, conjugate, or pharmaceutical composition can be used to detect cancer cells. In some embodiments, the peptide contains a marker for detecting the cancer.

[0020] In other respects, a peptide is provided comprising: (a) The amino acid sequence FLGVLALLGYLAVRPFLPKKKQQK-X (SEQ ID NO: 4), where X is the positively charged portion; (b) analogues of (a); (c) The reversed configuration sequence of (a) or (b); (d) A fragment consisting of at least 17 consecutive amino acids from (a), (b), or (c), The peptides described therein consist of 17 to 50 amino acids.

[0021] According to a preferred embodiment, at least one, at least two, preferably at least three amino acids are replaced by D-amino acids (presented as lowercase letters), thus the peptide is: FLGVLALLGyLAVRPfLPKkKQQK-X (SEQ ID NO: 5).

[0022] The positively charged portion X can be a portion not based on amino acids, such as polyamines.

[0023] According to a preferred embodiment, X is a portion composed of 3-10 positively charged amino acids, preferably 4-7 positively charged amino acids, and most preferably 6 positively charged amino acids. The amino acids may be the same or different.

[0024] In a specific example, X is six Lys residues in tandem, and the peptide has the following structure: FLGVLALLGYLAVRPFLPKKKQQKKKKKKK (SEQ ID NO: 1).

[0025] According to another example, three amino acids of the above peptide are replaced by D-amino acids (represented by lowercase letters), such that the peptide has the following formula: SEQ ID NO: 2: FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2), which is also referred to in this paper as 3D-NAF-1-6K.

[0026] According to some embodiments, the peptide comprises any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to a particular embodiment, the peptide is composed of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0027] According to another embodiment, the peptide comprises an analogue of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31, wherein the analogue comprises 1 to 6 amino acid substitutions, deletions, or additions. According to some embodiments, the analogue comprises 1 to 3 amino acid substitutions, deletions, or additions. According to some embodiments, at least one amino acid substitution is a conservative substitution. According to other embodiments, all amino acid substitutions are conservative substitutions. According to some embodiments, in addition to the 3 amino acids of SEQ ID NO: 2 that have been replaced by the corresponding D-analyte, at least one amino acid is replaced with the corresponding D-amino acid. According to some embodiments, the peptide comprises a sequence of all D-amino acids. According to another embodiment, the peptide comprises the amino acid sequence flgvlallgylavrpflpkkkqqkkkkkkk (where lowercase letters refer to D-amino acids) as described in SEQ ID NO: 3. According to another embodiment, the peptide consists of SEQ ID NO: 2. According to other embodiments, the peptide comprises the reversed, inverse sequence of any one of SEQ ID NO: 1-3.

[0028] According to another embodiment, the peptide comprises an analogue of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31, wherein the analogue comprises N-methylation of amide nitrogen, N-terminal acetylation, and substitution of non-protein amino acids.

[0029] According to another embodiment, the peptide comprises an analogue of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31, wherein the analogue comprises one or more peptide mimics. In some embodiments, the peptide mimic is replaced by a selection from reductamides, azapeptides, and peptide-like substances.

[0030] According to another embodiment, the peptide comprises a fragment of 17 to 28 consecutive amino acid residues selected from the following sequences: SEQ ID NO: 1 or SEQ ID NO: 2, analogs of SEQ ID NO: 1 or SEQ ID NO: 2, or reversed sequence of analogs of SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] According to some embodiments, the peptide consists of 17 to 28 amino acid residues.

[0032] According to another aspect, conjugates of the peptides disclosed herein with another component are provided. According to some embodiments, the conjugate comprises at least one polyethylene glycol (PEG) molecule and a peptide comprising an amino acid selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to one embodiment, the PEG molecule has a molecular weight between 350 and 1,000 Da.

[0033] According to one particular embodiment, the conjugate comprises the peptide described herein and a PEG moiety conjugated to at least one end thereof.

[0034] According to some aspects, a pharmaceutical composition is provided comprising at least one peptide or at least one conjugate disclosed herein. According to some embodiments, the pharmaceutical composition comprises a peptide of 17-50 amino acids, said peptide comprising: (a) The amino acid sequence FLGVLALLGYLAVRPFLPKKKQQK-X as described in SEQ ID NO: 4; or the amino acid sequence FLGVLALLGyLAVRPfLPKkKQQKKKKKKK as described in SEQ ID NO: 2; (b) analogues of (a); (c) The reversed configuration sequence of (a) or (b); or (d) A fragment consisting of at least 17 to 28 consecutive amino acids of (a), (b), or (c); and Optionally linked to the peptide to form a conjugate molecule.

[0035] According to some embodiments, the pharmaceutical composition comprises at least one peptide having an amino acid sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to some embodiments, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 coupled to at least one PEG molecule.

[0036] According to another aspect, the pharmaceutical compositions disclosed herein are used to treat cancer. According to some embodiments, the cancer is accompanied by enhanced expression of the NAF-1 protein. According to another embodiment, the cancer is selected from breast cancer, prostate cancer, gastric cancer, cervical cancer, liver cancer, pancreatic cancer, head cancer, neck cancer, leukemia, lymphoma, myeloma, ovarian cancer, glioblastoma, malignant melanoma, CML, AML, ALL, and laryngeal cancer. The pharmaceutical compositions can also be used to treat cancer metastases, not just primary tumors.

[0037] According to another aspect, a method for treating cancer in a subject in need is provided, the method comprising administering to the subject a peptide, conjugate, or pharmaceutical composition disclosed herein. According to one embodiment, the pharmaceutical composition comprises an effective amount of the peptide or conjugate disclosed herein. The pharmaceutical composition may be administered using any suitable method. According to some embodiments, the pharmaceutical composition is administered parenterally, such as intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally. According to some embodiments, the pharmaceutical composition is administered intratumorally. According to some specific embodiments, the pharmaceutical composition is administered intratumorally during surgery. Attached Figure Description

[0038] In the following figures, the peptide of SEQ ID NO: 2 is also referred to by the name "3D-NAF-1-6K".

[0039] Figure 1 The approximate location of the original NAF-1 peptide SEQ ID NO: 6 is shown, which spans the membrane (mitochondria / ER / MAM) and extends into the cytoplasm. The NAF-1 peptide (described within the box) is derived from the original NAF-1 sequence (top row, from amino acid 44 to amino acid 67), and modifications in the novel 3D-NAF-1-6K peptide (SEQ ID NO: 2) sequence are highlighted in red (bottom row).

[0040] Figure 2 Analytical data for the 3D-NAF-1-6K peptide are shown. Analytical HPLC and ESI-MS of the purified peptide are presented. The molecular weight is 3496.5, and the appearance is a white powder. Analytical HPLC was performed using a C18 column, 20%–50% acetonitrile in TDW solution, and a 25-minute gradient.

[0041] Figure 3 The 3D-NAF-1-6K peptide demonstrates significantly higher cytotoxicity against breast cancer cells compared to the prior art peptide disclosed in US 11,208,443 (SEQ ID NO: 7, named 3D-NAF-1 peptide herein), while remaining non-toxic to healthy cells. The effects of each peptide on malignant MDA-MB-231 cells (…) are shown. Figure 3 A) and control MCF-10A cells ( Figure 3 B) Cytotoxicity. Before the experiment, cells were seeded at a density of 15,000 cells / well in 96-well plates. 10 µM of any peptide was added to the cells, and the plates were incubated for 1 or 3 days. Cell viability was assessed using Presto-Blue after 1 and 3 days of incubation. Cells were then analyzed using a t-test. P< 0.001 , P < 0.0001 .

[0042] Figure 4 The cytotoxic effects of the peptides 3D-NAF-1-6K and 3D-NAF-1-6R (FLGVLALLGyLAVRPfLPKkKQQKRRRRRR) (SEQ ID NO: 31) on MDA-MB-231 cells are shown. Cells were cultured at 1.2 x 10⁻⁶ cells per cell line. 4 Cells were seeded at a density in 96-well plates and allowed to incubate overnight. 3D-NAF-1-6K or 3D-NAF-1-6R peptides were incubated with cells at different concentrations for 3 and 18 hours. Black bars represent the cytotoxic effect of 3D-NAF-1-6K, while gray bars indicate the cytotoxicity of 3D-NAF-1-6R. The fluorescent redox probe Presto-blue was used. TM Cell viability was determined. Cells were incubated with Presto-blue for 1 hour, and Presto-blue fluorescence (λ) was measured at 37°C using a Tecan Safire plate reader. ex =560 nm, λ em =590nm). The results showed that 3D-NAF-1-6K was more toxic to malignant cells than 3D-NAF-1-6R.

[0043] Figure 5 The results show the effects of incubation for 1 hour with no peptide (control, blue bar) and two concentrations of 3D-NAF-1-6K peptide (10 μM (red bar) and 50 μM (green bar)). Figure 5 A) and 24 hours ( Figure 5 Following B), the cytotoxic effects of 3D-NAF-1-6K peptide on the survival rates of different malignant cell types were investigated: MDA-MB-231 (triple-negative breast cancer cells), PC-3 (prostate cancer cells), HepG2 (liver cancer cells), and SKOV-3 (ovarian cancer cells). Survival rates were determined using the Presto-blue method.

[0044] Figure 6 Two different batches of 3D-NAF-1-6K peptides are shown (in this figure, OP represents the "old" batch of 3D-NAF-1-6K peptides, and NP represents the "new" fresh batch of 3D-NAF-1-6K peptides). Figure 6As shown, the two batches exhibited similar / identical cytotoxic effects against MDA-MB-231 (triple-negative breast cancer cell line), HepG2 (liver cancer cell line), and SKOV-3 (ovarian cancer cell line). OP and NP were incubated with malignant cells at concentrations of 20 μM (red / purple bars) or 50 μM (green / blue bars) for 1 hour. Figure 6 A) or 24 hours ( Figure 6 B).

[0045] Figure 7 The toxicity of 3D-NAF-1-6K to different cancer cell lines was demonstrated; cell viability was measured using the Presto-blue assay after incubation with a specified concentration of 3D-NAF-1-6K peptide for 3 hours. SKOV-3 (ovarian cancer (black circle)), U87MG (glioblastoma (black rectangle)), A375 (human malignant melanoma (black equilateral triangle)), MDA-MB-231 (breast cancer (black inverted triangle)), PANC-1 (pancreatic cancer (black rhombus)), MOLM-14 (acute myeloid leukemia (red circle)), and K562 (chronic myeloid leukemia (yellow rectangle)) were treated with or without the peptide, and viability was determined. Data are shown as mean ± SD of three independent experiments. Cell viability was assessed by t-test. P<0.01 , P<0.001 , P< 0.0001 .

[0046] Figure 8 The effects of 3D-NAF-1-6K on the IC50 of different cancer cell lines were demonstrated. 50 3D-NAF-1-6K peptide induces IC50 in cancer cell death. 50 The IC50 values ​​for each cell line are specified. 50 value.

[0047] Figure 9 The cytotoxic effect of the 3D-NAF-1-6K peptide on the survival of SKOV-3 ovarian cancer 3D globules is illustrated. The globules were incubated with 20 μM or 50 μM of the 3D-NAF-1-6K peptide for 0, 1, 20, and 40 hours. Cell death was measured using the IncuCyte® Red Cytotoxicity Reagent (Essen Bioscience Cat #4632) (a probe for measuring cell death). The top image contains the globules, and the bottom image shows the quantitative cytotoxic effect of the tested peptide.

[0048] Figure 10The cytotoxic effect of the 3D-NAF-1-6K peptide on the survival of HepG2 hepatocellular carcinoma 3D globules is illustrated. The globules were incubated with 20 μM and 50 μM of the 3D-NAF-1-6K peptide for 0, 1, 20, or 40 hours. Cell death was measured using the IncuCyte® Red cytotoxicity assay (Essen Bioscience Cat #4632) (a probe for measuring cell death). The top image contains the globules, and the bottom image shows the quantitative cytotoxic effect of the peptide.

[0049] Figure 11 The cytotoxic effect of the 3D-NAF-1-6K peptide on the survival of PC-3 3D globules from prostate cancer is illustrated. The globules were incubated with 20 μM and 50 μM of the 3D-NAF-1-6K peptide for 0, 1, 20, or 40 hours. Cell death was measured using the IncuCyte® Red cytotoxicity assay (Essen Bioscience Cat #4632) (a probe for measuring cell death). The top image contains the globules, and the bottom image shows the quantitative cytotoxic effect of the peptide.

[0050] Figure 12 This demonstrates that 3D-NAF-1-6K penetrates ovarian cancer cells and targets mitochondria. Figure 12 A) Representative confocal fluorescence images of SKOV-3 ovarian cancer cells at different time points after treatment with 3D-NAF-1-6K peptide. Figure 12 Quantitative analysis of B and 12C) 3D-NAF-1-6K and free luciferin infiltrating into SK-OV-3 cells ( Figure 12 B) and mitochondrial damage assessed by loss of mitochondrial membrane potential ( Figure 12 C). Data are presented as the mean ± SD of 15 cells per field of view (10 fields of view in total) at each time point, calculated from three independent experiments. The results were analyzed by a t-test. P < 0.001 .

[0051] Figure 13 The study demonstrated that the 3D-NAF-1-6K peptide leads to fragmentation of mitochondria in SKOV-3 cells. Figure 13 A shows representative confocal fluorescence images of mitochondria in SKOV-3 ovarian cancer cells treated with different concentrations of 3D-NAF-1-6K peptide. Figure 13 B shows the quantitative analysis of mitochondrial length at different peptide concentrations. Data are presented as mean ± SD of 25 cells from 8 fields of view calculated from 3 independent experiments. A t-test was performed. P < 0.001 , P < 0.0001 .

[0052] Figure 14 The measurement of mitochondrial length in SKOV-3 ovarian cancer cells after treatment with 3D-NAF-1-6K peptide is shown. Figure 14 A shows representative confocal fluorescence images of mitochondria in SKOV-3 cells treated / untreated with 3D-NAF-1-6K peptide. Figure 14 B shows the conversion of mitochondrial length into binary images of the backbone in both the control (peptide-free) and 50 µM peptide treatments. Mitochondrial length was measured in 25 cells from 8 fields of view using ImageJ, with 156 mitochondria / cells measured in the control and 58 mitochondria / cells in the 50 µM peptide treatment. Mitochondrial length in magnified images was measured using the full-capacity processing tools in ImageJ.

[0053] Figure 15 The 3D-NAF-1-6K peptide was shown to inhibit the activity of thioredoxin reductase (tRXr) in MDA-MB-231 cancer cells. (See the image above.) Figure 15 A) shows the green fluorescence of thioredoxin reductase tracked by epifluorescence microscopy at 0, 0.5, 1, 3, and 6 hours after the addition of 3D-NAF-1-6K peptide. (See figure below.) Figure 15 B) Quantified in Figure 15 Data presented in different confocal images shown in Figure A. In this experiment, 10 µM TRFS-green was loaded for 6 hours, and 15 µM 3D-NAF-1-6K peptide was added.

[0054] Figure 16 The illustration shows our hypothesis about how the 3D-NAF-1-6K peptide disrupts the α-helix of the transmembrane domain of the NAF-1 homodimer and promotes damage to the outer mitochondrial membrane.

[0055] Figure 17 The effect of 3D-NAF-1-6K on primary xenograft SKOV-3 ovarian cancer tumors was demonstrated. Figure 17 A) Tumor volume (mm) in the three mouse groups 3 : Control (saline; blue), intravenous (IV; orange), and subcutaneous (SC; gray) injection groups. Representative images are shown on the right. Figure 17 B) Changes in body weight of mice in the three groups during treatment. 0.5 mg / kg of 3D-NAF-1-6K peptide was administered six times over a 3-week period. All results are presented as mean ± SEM. Significance was determined using a t-test. P<0.1 , P<0.01 .

[0056] Figure 18 Immunohistochemistry of mice with SKOV-3 tumors, subsequently injected with 3D-NAF-1-6K peptide and detected with an antibody targeting macrophage-specific markers from F4 / 80 mice, indicated a large number of tumor-infiltrating macrophages.

[0057] Figure 19 The study demonstrated that 3D-NAF-1-6K inhibited the metastatic growth of xenograft SKOV-3 ovarian cancer. Figure 19 A shows a reduction in tumor nodule weight (g) in the lungs of mice treated with intravenous injection of SKOV-3 cells and 0.25 or 0.5 mg / kg of 3D-NAF-1-6K peptide per mouse. Injections were administered intravenously (IV) twice weekly. Paclitaxel 20 mg / kg was administered IV weekly as a positive control. Figure 19 B shows a reduction in colonic nodule metastasis (number of nodules) after treatment with saline (control) or 3D-NAF-1-6K peptide. All results are presented as mean ± SEM. Significance was determined using a t-test; P<0.01 , P< 0.001 , P<0.0001 .

[0058] Figure 20 The effect of 3D-NAF-1-6K peptide on SKOV-3 cell migration was shown. Figure 20 A shows the untreated control (top) and the control treated with 3D-NAF-1-6K peptide (10 μM, middle); 20 High-resolution time-lapse phase-contrast images of the M (see below) treated group at 0, 48, and 96 h after removal of the culture coverslip. Dashed lines define areas lacking cells. Scale bar, 100 μm. Figure 20 B shows the untreated (blue) and treated with 3D-NAF-1-6K peptide (5 M (orange), 10 M (grey), 20 Quantification of cell migration within 96 h in the M (yellow) treated group. Results are expressed as the mean of four measurements of cell migration obtained in three independent experiments (n = 12). P < 0.01， P < 0.001 The corresponding p-values ​​were obtained through a t-test.

[0059] Figure 21The internal organs designated from mice injected IV with 3D-NAF-1-6K peptide are shown. All organs, such as the liver, kidneys, colon, brain, lungs, and other organs, have normal coloration.

[0060] Figure 22-24 The results showed that only sections of shrunken breast MDA-MB-231 tumors exhibited green fluorescence when analyzed and quantified under confocal microscopy, indicating the presence of the Fl-3D-NAF-1-6K peptide (in...). Figure 22 Intake of (labeled as Fl-3D-NAF-1-6K). Figure 23 Representative confocal images of tumor-isolated cells from mice treated with or without 3D-NAF-1-6K peptide at the initial stage of treatment are shown. Figure 24 Tumor cells before and after treatment are shown, with RPA staining used to identify mitochondria and H342 staining used to identify nuclei.

[0061] Figure 25 The study demonstrated the injection of Cy5-3D-NAF-1-6K peptide into nude mice with tumors formed by MDA-MB-231 cells. Figure 25 Image A shows images of mice that were intravenously injected with saline control or 0.5 mg / Kg Cy5-3D-NAF-1-6K at the following time points: 0, 2, 12, 96, 140, and 170 hours. Mice were tracked and imaged over time to monitor peptide movement and accumulation. Figure 25 B shows the fluorescence intensity of Cy5-3D-NAF-1-6K peptide over time in tumors, liver, and kidney. Figure 25 C shows the fluorescent accumulation in mouse organs and tumors after autopsy, in order to study peptide accumulation in different organs and tumors.

[0062] Figure 26 shows that the 3D-NAF-1-6K peptide is non-toxic. Mice were injected intraperitoneally with 0.5 mg / kg or 5 mg / kg of 3D-NAF-1-6K, while the control group was injected with saline. Mice were injected with saline or 3D-NAF-1-6K peptide three times a week for 4 weeks. Figure 26A CBC results in male and female mice after treatment with 3D-NAF-1-6K peptide. Abbreviations: WBC: white blood cells, RBC: red blood cells, HGB: hemoglobin, Plt: platelets, Lymph: lymphocytes, Neutral: neutrophils. Figure 26BThe changes in body weight and organ weight over time after normal mice were treated with saline or 0.5 mg / Kg 3D-NAF-1-6K or 5 mg / Kg 3D-NAF-1-6K, and representative H&E stained sections of the kidney, liver, and spleen are shown. (a) Changes in body weight over time in male mice (top) and female mice (bottom). Concentrations used: 0.5 mg / Kg 3D-NAF-1-6K (orange), 5 mg / Kg 3D-NAF-1-6K (grey), and saline control (blue). (b) Bar graph showing the weight of different organs harvested from male mice (top) and female mice (bottom) treated or untreated with two different concentrations of 3D-NAF-1-6K peptide. (c) Representative H&E stained sections of the kidney, liver, and spleen obtained from the mouse organs shown in (b), showing the similarity between treated and untreated tissues.

[0063] Figure 27 Increasing BSA (bovine serum albumin) concentrations delayed the cytotoxic activity of the 3D-NAF-1-6K peptide against the breast cancer cell line MDA-MB-231. BSA concentrations were shown as 0.125 mg / ml (purple bar), 0.25 mg / ml (green bar), and 0.5 mg / ml (red-orange bar). The peptide without BSA (red bar) served as a positive control, and the negative control is shown as a blue bar. Cells were incubated for 3, 6, 12, 24, and 72 hours in the presence of the peptide (without / in the presence of free / BSA). Cytotoxicity was measured on a cell imaging plate using the presto-blue method. These findings suggest that the peptide binds to albumin, and that the latter may facilitate its delivery in vivo.

[0064] Figure 28 The effect of 3D-NAF-1-6K peptide on A375 melanoma cancer cells was demonstrated. Figure 28 A study showed that 3D-NAF-1-6K peptide exhibited high cytotoxicity against melanoma cancer cells in a concentration-dependent manner. Prior to the experiment, cells were seeded at a density of 15,000 cells / well in 96-well plates. Cells were then treated with 0, 5, 10, 25, and 50 µM of 3D-NAF-1-6K peptide and incubated for 3 or 24 hours. Cell viability was assessed using Presto-Blue after 3 and 24 hours of incubation. Cell viability was determined by t-test. P < 0.0001 . Figure 28 B: Mice carrying A375 cancer xenografts were randomly divided into two groups: a control group and a 3D-NAF-1-6K peptide treatment group. The latter received intravenous administration of 0.75 mg / kg of 3D-NAF-1-6K peptide three times a week for three weeks. Male group (top image) and female group (bottom image). The graphs represent tumor growth volume (mm). 3(and images of representative tumors extracted from the flanks of mice, scale bar = 1.0 cm). Figure 28 C: Body weight of males (top) and females (bottom). The charts represent the body weight (grams) of mice during the experiment. The fact that treatment with the peptide did not affect the body weight of mice indicates that 3D-NAF-1-6K is non-toxic. Detailed Implementation

[0065] Reference is made to U.S. Patent 11,208,443, to some of the inventors of this application, the entire patent of which is incorporated herein by reference. That document teaches that peptides derived from specific regions of the NAF1 protein selectively penetrate and kill cancer cells. Specifically, as disclosed herein, it has been found that the peptide first disclosed in U.S. Patent 11,208,443 (FLGVLALLGYLAVRPFLPKKKQQK (SEQ ID NO: 6)) and a peptide derived from that sequence comprising three D-amino acid substitutions (FLGVLALLGyLAVRPfLPKkKQQK (SEQ ID NO: 7)) exhibit significantly and unexpectedly enhanced cytotoxic activity when an additional positively charged amino acid (preferably lysine) is added to the C-terminus. The sequences disclosed in U.S. Patent 11,208,443 (including SEQ ID NOs: 6-7) are excluded from the scope of the peptides disclosed herein (“waived”).

[0066] As used herein, the term "peptide" refers to a short chain of amino acid monomers linked by peptide bonds (i.e., covalent bonds formed between the carboxyl group of one amino acid and the amino group of another amino acid). According to some embodiments, a peptide may contain up to 50 amino acids. According to some embodiments, a peptide consists of 12 to 45 amino acids. According to another embodiment, a peptide consists of 15 to 40 amino acids. According to some embodiments, a peptide contains 17 to 35 amino acids. According to other embodiments, a peptide consists of 20 to 30 amino acids. According to another particular embodiment, a peptide consists of 17 to 29 amino acids. According to some embodiments, a peptide consists of 17 to 28, 18 to 27, 19 to 22, or 20 to 21 amino acids.

[0067] It should be noted that, when fragments are involved, they are fragments of the prior art peptide FLGVLALLGYLAVRPFLPKKKQQK (SEQ ID NO: 6) of US Patent 11,208,443 (17-24 amino acids in length), while the positively charged portion X (part X having 3-10 amino acids, preferably 6 amino acids, most preferably 6 consecutive lysines) is not fragmented (i.e. its sequence is not shortened).

[0068] According to some embodiments, the peptide comprises a fragment of SEQ ID NO: 1 or SEQ ID NO: 2, provided that the positively charged portion (particularly a segment of 2-10 positively charged amino acids) is not missing and is present at the same length as the parent peptide from which it is derived. According to other embodiments, the peptide comprises a fragment of an analogue of SEQ ID NO: 1 or SEQ ID NO: 2. According to some embodiments, the peptide comprises a fragment of the reversed, inversely configured sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or an analogue thereof. According to some embodiments, the peptide comprises a peptide fragment consisting of 10 to 28 consecutive amino acids of the parent sequence (provided that the segment of positively charged amino acids at the C-terminus is consistent with the segment appearing in the parent peptide). In one embodiment, the fragment consists of 11 to 27, 12 to 26, 13 to 21, 14 to 20, 15 to 19, 16 to 18, or 17 such consecutive amino acids. According to another embodiment, the fragment consists of 17 to 23, 18 to 22, or 19 to 21 consecutive amino acids.

[0069] According to some embodiments, the fragment consists of 10 to 28 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the fragment consists of 11 to 23, 12 to 22, 13 to 21, 14 to 20, 15 to 19, 16 to 18, or 17 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. According to another embodiment, the fragment consists of 17 to 23, 18 to 22, or 19 to 21 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. According to some such embodiments, the fragment contains the amino acid sequence FLGVLAL (SEQ ID NO: 9) (attached to a segment of a positively charged amino acid). According to other embodiments, the fragment consists of 17 to 28 consecutive amino acids of an analogue of SEQ ID NO: 1 or SEQ ID NO: 2. According to another embodiment, the fragment consists of 18 to 27 or 19 to 26 consecutive amino acids of an analogue of SEQ ID NO: 1 or SEQ ID NO: 2 as described herein.

[0070] Therefore, starting with the fragment FLGVLAL (SEQ ID NO: 9), the peptide disclosed herein comprises the following structure: FLGVLAL-X1-X 22 (SEQ ID NO: 10) Where X1 represents LGYLAVRPFLPKKKQQK (SEQ ID NO: 30) or a C-terminal sequential deletion of that sequence; and Where X2 represents 1 to 10 positively charged moieties.

[0071] Thus, the peptides represented by FLGVLAL-X1-X2 (SEQ ID NO: 10) are as follows: FLGVLALLGYLAVRPFLPKKKQQK-X2 (SEQ ID NO: 11); FLGVLALLGYLAVRPFLPKKKQQ-X2 (SEQ ID NO: 12); FLGVLALLGYLAVRPFLPKKKQ-X2 (SEQ ID NO: 13); FLGVLALLGYLAVRPFLPKKK-X2 (SEQ ID NO: 14); FLGVLALLGYLAVRPFLPKK-X2 (SEQ ID NO: 15); FLGVLALLGYLAVRPFLPK-X2 (SEQ ID NO: 16); FLGVLALLGYLAVRPFLP-X2 (SEQ ID NO: 17); FLGVLALLGYLAVRPFL-X2 (SEQ ID NO: 18); FLGVLALLGYLAVRPF-X2 (SEQ ID NO: 19); FLGVLALLGYLAVRP-X2 (SEQ ID NO: 20); FLGVLALLGYLAVR-X2 (SEQ ID NO: 21); FLGVLALLGYLAV-X2 (SEQ ID NO: 22); FLGVLALLGYLA-X2 (SEQ ID NO: 23); FLGVLALLGYL-X2 (SEQ ID NO: 24); FLGVLALLGY-X2 (SEQ ID NO: 25); FLGVLALLG-X2 (SEQ ID NO: 26); FLGVLALL-X2 (SEQ ID NO: 29) or FLGVLAL-X2 (SEQ ID NO: 28).

[0072] In these peptides (e.g., SEQ ID NO: 10-28), X2 consists of 1 to 10 positively charged portions. In some embodiments, the positively charged portions are positively charged amino acids. Examples of such positively charged amino acids include, but are not limited to, arginine and / or lysine, preferably lysine. In other embodiments, the positively charged amino acids are the same and are either arginine or lysine, preferably lysine. As disclosed herein, each positively charged amino acid in X2 can be a D or L amino acid.

[0073] Exemplary representative peptides covered by SEQ ID NO: 11 (for example) include, but are not limited to: FLGVLALLGYLAVRPFLPKKKQQKKKKKKK (SEQ ID NO: 1), FLGVLALLGYLAVRPFLPKKKQQKKKKKK (SEQ ID NO: 29).

[0074] As described in detail below, each peptide described herein, such as the peptide represented by any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31, may also include: (a) substitution (including conserved substitution); (b) substitution of any one or more L-amino acids at each amino acid position with D-amino acids; (c) coupling with other molecules; and / or (d) binding with other molecules. Furthermore, reverse-conformation peptides of each peptide represented by any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 are also contemplated.

[0075] The term "analyte" refers to an amino acid sequence in which at least one amino acid of the parent sequence is modified while retaining the function of the parent peptide. Examples of such modifications to the amino acid sequence are substitutions, rearrangements, deletions, additions, and / or chemical modifications in the amino acid sequence of the parent peptide.

[0076] The term "amino acid" includes both "natural" and "non-natural" amino acids.

[0077] According to one embodiment, the analogue includes at least one modification selected from substitution, deletion, or addition. According to some embodiments, the modification is a substitution. According to one embodiment, the substitution is a conservative substitution.

[0078] As used herein, the term "conservative substitution" refers to the replacement of one amino acid residue with another without altering the overall conformation and biological activity of the peptide, including but not limited to the replacement of one amino acid with an amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, shape, hydrophobicity, aromaticity, etc.). Amino acids with similar properties are well known in the art. For example, according to a table known in the art, the following six groups each contain amino acids that are conservedly substituted for each other: (1) Alanine (A), serine (S), threonine (T); (2) Aspartic acid (D), glutamic acid (E); (3) Asparagine (N), glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), leucine (L), methionine (M), valine (V); and (6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0079] According to some embodiments, the substitution is not limited to natural amino acids and can be implemented using non-natural amino acids. The term "non-natural amino acid" refers to an amino acid having a structure different from that of a natural amino acid. In one embodiment, the non-natural amino acid is a D-amino acid. Another example of non-natural amino acids includes ornithine, 3-substituted tyrosine, azidoalanine, azidohomalanine, leucine, valine, 4-aminotryptophan, 7-azatryptophan, 6-methyltryptophan, acetyllysine, ε-Boc-lysine, ε-methyllysine, 1-naphthylalanine, 2-naphthylalanine, styrylalanine, diphenylalanine, thiazolylalanine, 2-pyridylalanine, 3-pyridylalanine, 4-pyridylalanine, anthraquinonealanine, 2-amino-5-hexanoic acid, furanylalanine, benzothiophenealanine, thiophenealanine, allylglycine, propargylglycine, phosphoserine, phosphothreonine, and 2,3-diaminopropionic acid.

[0080] According to some embodiments, the analogues comprise 1 to 10, 2 to 8, or 3 to 6 modifications. According to one embodiment, the analogues comprise 1 to 6 modifications or 1 to 3 modifications. According to another embodiment, the analogues comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. According to some embodiments, the modifications are substitutions, such as conservative substitutions. According to one embodiment, the analogues comprise 1 to 6 conservative substitutions.

[0081] According to some embodiments, the analogue comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9).

[0082] According to one embodiment, the analog has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% amino acid sequence identity with a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the analog has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0083] According to one embodiment, the analogue comprises the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 or a modified SEQ ID NO:1 or SEQ ID NO:2, wherein at least one Lys residue is replaced by another positively charged amino acid. According to one embodiment, at least one Lys residue is replaced by Arg.

[0084] According to some embodiments, the analogue comprises at least one conserved substitution and / or comprises at least one D-amino acid. According to another embodiment, the analogue comprises 1 to 3 D-amino acids. According to one embodiment, the analogue comprises 10Tyr, 16Phe, and 20Lys amino acids. For clarity, the numbers to the left of the amino acid names indicate their sequence number in SEQ ID NO: 1 (or their equivalent position in any of SEQ ID NO: 10-29 and 31). For example, 10Tyr or 10Y refers to the tyrosine residue at position 10 of SEQ ID NO: 1 (or its equivalent position in any of SEQ ID NO: 10-29 and 31).

[0085] For example, representative examples of analogues of SEQ ID NO: 11 include, but are not limited to, the following peptides (D-amino acids are represented by lowercase letters): FLGVLALLGyLAVRPfLPKkKQQKKKKKKKK (SEQ ID NO: 2) (also referred to in this document as NAF-1-3D-6K), flgvlallgylavrpflpkkkqqkkkkkkk (SEQ ID NO: 3).

[0086] According to another embodiment, the analog has the amino acid sequence SEQ ID NO: 2. According to another embodiment, the fragment consists of 17 to 28, 18 to 27, or 19 to 26 consecutive amino acids of sequence SEQ ID NO: 2. According to another embodiment, at least four amino acids of the analog are D-amino acids. According to one embodiment, all amino acids of the analog are D-amino acids.

[0087] According to one embodiment, the analog has the amino acid sequence SEQ ID NO: 3. According to another embodiment, the fragment consists of 17 to 28, 18 to 27, or 19 to 26 consecutive amino acids of the sequence SEQ ID NO: 3.

[0088] According to any of the above embodiments, the analogue comprises a parent sequence or a modified parent sequence, wherein at least one of the Phe or Tyr amino acids is replaced by an amino acid having a large hydrophobic residue. In one embodiment, 1Phe and / or 16Phe is replaced by Tyr or Trp. According to another embodiment, 10Tyr is replaced by Phe or Trp. According to another embodiment, 1Phe or 16Phes is replaced by Tyr or Trp, and 10Tyr is replaced by Phe or Trp. When used herein, the number to the left of the amino acid name indicates its sequence number in SEQ ID NO: 1 (or its equivalent position in any of SEQ ID NO: 10-29 and 31). For example, 10Tyr or 10Y refers to the tyrosine residue at position 10 of SEQ ID NO: 1 (or its equivalent position in any of SEQ ID NO: 10-29 and 31).

[0089] According to any of the above embodiments, the analogue comprises a parent sequence or a modified parent sequence, wherein at least one hydrophobic amino acid residue is replaced by Ala.

[0090] According to any of the above embodiments, the analogue comprises a parent sequence or a modified parent sequence, wherein 14Arg in SEQ ID NO: 1 (or the equivalent position in any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31) is replaced by Ala.

[0091] According to any of the above embodiments, the modification may be replaced by a non-natural amino acid as defined above. According to one embodiment, the non-natural amino acid is a D-amino acid. The term "D-amino acid" refers to an amino acid having a D-configuration around the α-carbon, as opposed to a natural L-amino acid having an L-configuration. When used herein, D-amino acids in a sequence are represented by lowercase letters, while L-amino acids are represented by uppercase letters. Thus, the sequence VyL represents a sequence where Val and Leu are natural L-amino acids and Tyr is a D-amino acid.

[0092] According to any of the above embodiments, the analogue of any one of SEQ ID NO: 10-29 and 31 comprises at least one D-amino acid. According to another embodiment, the analogue comprises 1 to 24, 2 to 23, 3 to 22, 4 to 21, 5 to 20, 6 to 19, 7 to 18, 8 to 17, 9 to 16, 10 to 15, 11 to 14, or 12 to 13 D-amino acids. According to one embodiment, the analogue comprises 1, 2, or 3 D-amino acids. According to one embodiment, the analogue comprises 4, 5, or 6 D-amino acids. According to some embodiments, at least one of the amino acids at positions 10, 16, and 20 of SEQ ID NO: 1 (or equivalent positions in any one of SEQ ID NO: 10-29 and 31) is a D-amino acid. According to another embodiment, at least one of 10Y, 16F, and 20K (or equivalent positions in any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31) in SEQ ID NO: 1 is a D-amino acid. According to another embodiment, amino acids 10Y, 16F, and 20K (or equivalent positions in any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31) in SEQ ID NO: 1 are D-amino acids. According to one embodiment, the analog has the amino acid sequence: FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2). Therefore, in one embodiment, the peptide comprises the amino acid sequence FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2).

[0093] According to some embodiments, the analog comprises at least four D-amino acids. According to another embodiment, the analog comprises at least five, at least six, or at least eight D-amino acids. According to one embodiment, all amino acids in the analog are D-amino acids. According to another embodiment, the analog comprises an amino acid sequence in which all amino acids of SEQ ID NO: 1 are replaced by corresponding D-amino acids. According to one embodiment, all residues of this peptide are D-amino acids.

[0094] According to some embodiments, the peptide comprises the reversed sequence of the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0095] The terms “reverse transconfiguration peptide” and “reverse reverse peptide” are used interchangeably in this document and refer to a reverse peptide composed of D-amino acids (reverse transconfiguration; as described in Regenmortel and Muller, Current Opinion in Biotechnology 9, pp 377-382, 1998).

[0096] The terms “comprising,” “including,” “having,” “containing,” and variations thereof, as used herein, are intended as open-ended transitional phrases, terms, or words that do not exclude the possibility of other behaviors or structures. The term “comprising” always includes the term “consisting of,” and when the term “comprising” appears in one embodiment of this disclosure, the same embodiment in which the term “consisting of” replaces the term “comprising” is always an embodiment of this disclosure. Therefore, in some embodiments, the peptide consists of an amino acid sequence selected from any of SEQ ID NO: 1-5 or any of SEQ ID NO: 10-29 or 31, its analogues, or their reversed or inverted sequences.

[0097] In one embodiment, the peptide consists of the sequence SEQ ID NO: 1.

[0098] According to some embodiments, a peptide is provided comprising an amino acid sequence having at least 70% identity with a sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to one embodiment, the peptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0099] According to some embodiments, a peptide is provided that comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. According to another embodiment, a fragment of such a peptide comprising 17 to 28 amino acids, or an analogue of said peptide or said fragment, is provided.

[0100] According to another aspect, the present invention provides a peptide conjugate as described above. According to another embodiment, the conjugate is a peptide conjugate comprising an analogue of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to another embodiment, the conjugate is a peptide composed of an analogue of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to one embodiment, the analogue comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9).

[0101] As used herein, the term "conjugate" refers to the binding of a peptide to another moiety. According to some embodiments, the moiety is a non-peptide portion. According to some embodiments, the peptide is conjugated with polyethylene glycol (PEG), poly(N-vinylpyrrolidone), polyglycerol, a permeability-enhancing moiety, and a polysaccharide (e.g., a glycosyl group). According to another embodiment, the peptide is conjugated with a label. According to some embodiments, the label is a fluorescent label, such as fluorescein.

[0102] The term "permeability-enhancing moiety" refers to any moiety known in the art that actively or passively promotes or enhances the permeability of a compound across body barriers or into cells. Non-limiting examples of permeability-enhancing moieties include: hydrophobic moieties, such as fatty acids, steroids, and bulky aromatic or aliphatic compounds; and moieties that may have cell membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transport peptides, nanoparticles, and liposomes.

[0103] According to some embodiments, the conjugate comprises the peptide and PEG molecule described herein. According to some embodiments, the PEGylation is carried out via the C-terminus of the peptide. According to other embodiments, the PEGylation is carried out via the N-terminus of the peptide. According to a further embodiment, the PEGylation is carried out via the amino acid side chain of the peptide.

[0104] According to some embodiments, the PEG molecule has a molecular weight (MW) between about 300 Daltons and about 100,000 Daltons. According to other embodiments, the PEG molecule has a MW of about 400 to about 10,000, about 1,000 to about 8,000, about 2,000 to about 6,000, and about 3,000 to about 5,000 Daltons. According to other embodiments, the PEG molecule has a molecular weight selected from about 10,000 Da to about 20,000 Da, about 20,000 Da to about 30,000 Da, about 30,000 Da to about 40,000 Da, about 40,000 Da to about 50,000 Da, about 50,000 Da to about 60,000 Da, about 60,000 Da to about 70,000 Da, and about 70,000 Da to about 80,000 Da. Non-limiting examples of the average molecular weight of the PEG portion are about 350 Da, about 400 Da, about 600 Da, about 1,000 Da, about 2,000 Da, about 6,000 Da, about 8,000 Da, about 10,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da, about 50,000 Da, about 60,000 Da, about 70,000 Da, and about 80,000 Da.

[0105] According to some embodiments, the PEG has 5 to 200 ethylene glycol monomers. According to other embodiments, the PEG has 6 to 150 ethylene glycol monomers. According to other embodiments, the PEG has 8 to 120, 10 to 100, 15 to 80, 20 to 60, or 30 to 50 ethylene glycol monomers.

[0106] According to some embodiments, the present invention provides a conjugate comprising a peptide comprising or consisting of SEQ ID NO: 1 and PEG. According to some embodiments, the peptide is PEGylated at the N-terminus. According to other embodiments, the peptide is PEGylated at the C-terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000 Da, 350 to 8,000 Da, 400 to 6,000 Da, 1,000 to 4,000 Da, or 2,000 Da to 3,000 Da. According to other embodiments, the PEG has 5 to 20 or 6 to 10 ethylene glycol monomers.

[0107] According to some embodiments, a conjugate comprising a peptide and PEG comprising any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31, or thereof. According to some embodiments, the peptide is PEGylated at the N-terminus. According to other embodiments, the peptide is PEGylated at the C-terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000 Da, 350 to 8,000 Da, 400 to 6,000 Da, 1,000 to 4,000 Da, or 2,000 Da to 3,000 Da. According to other embodiments, the PEG has 5 to 20 or 6 to 10 ethylene glycol monomers.

[0108] According to some embodiments, a conjugate comprising a peptide consisting of SEQ ID NO: 3 or thereto and PEG is provided. According to some embodiments, the peptide is PEGylated at the N-terminus. According to other embodiments, the peptide is PEGylated at the C-terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000 Da, 350 to 8,000 Da, 400 to 6,000 Da, 1,000 to 4,000 Da, or 2,000 Da to 3,000 Da. According to other embodiments, the PEG has 5 to 20 or 6 to 10 ethylene glycol monomers.

[0109] According to some embodiments, a conjugate comprising any peptide and PEG, comprising any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 or thereof. According to some embodiments, the peptide is PEGylated at the N-terminus. According to other embodiments, the peptide is PEGylated at the C-terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000 Da, 350 to 8,000 Da, 400 to 6,000 Da, 1,000 to 4,000 Da, or 2,000 Da to 3,000 Da. According to other embodiments, the PEG has 5 to 20 or 6 to 10 ethylene glycol monomers.

[0110] According to one embodiment, a conjugate is provided in which a peptide consisting of any of the amino acid sequences of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 binds to a PEG molecule having 6 to 10 ethylene glycol monomers via its N-terminus. According to another embodiment, a conjugate is provided in which a peptide consisting of any of the amino acid sequences of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 binds to a PEG molecule having 6 to 10 ethylene glycol monomers via its C-terminus. According to yet another embodiment, a conjugate is provided in which a peptide consisting of any of the amino acid sequences of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 binds to both its N-terminus and C-terminus to a PEG molecule having 6 to 10 ethylene glycol monomers.

[0111] The conjugate can be more stable than the parent peptide. According to some embodiments, the half-life of the peptide conjugate of the present invention is at least twice that of the parent peptide. According to some embodiments, the half-life of the peptide conjugate of the present invention is at least 3, 5, 8, or 10 times that of the parent peptide.

[0112] According to another aspect, a pharmaceutical composition is provided comprising at least one peptide or at least one conjugate of the present invention. According to one embodiment, the pharmaceutical composition comprises at least one peptide comprising the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to another embodiment, the peptide comprises the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0113] According to another embodiment, the pharmaceutical composition comprises at least one peptide analog, the peptide comprising the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to another embodiment, the pharmaceutical composition comprises a peptide composed of an analog of the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to such embodiments, the analog is as defined in any of the above embodiments. According to some embodiments, the analog comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9). According to some embodiments, the analog comprises at least one conserved substitution and / or comprises at least one D-amino acid. According to another embodiment, the analog comprises 1 to 3 D-amino acids. According to one embodiment, 10Y, 16F, and 20K are D-amino acids (as shown in SEQ ID NO: 1, or equivalent positions in SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31). According to another embodiment, the analog has an amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. Therefore, the pharmaceutical composition comprises at least one peptide, the peptide comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to another embodiment, at least four amino acids of the analog are D-amino acids. According to one embodiment, all amino acids of the analog are D-amino acids. According to one embodiment, the analog has an amino acid sequence SEQ ID NO: 3. According to such embodiments, the pharmaceutical composition comprises at least one peptide, the peptide comprising or consisting of an amino acid sequence SEQ ID NO: 3.

[0114] According to some embodiments, the pharmaceutical composition comprises at least one peptide, the peptide comprising, or consisting of, the reversed sequence of SEQ ID NO: 1 or an analogue thereof. In one embodiment, the peptide comprises the amino acid sequence SEQ ID NO: 2.

[0115] According to another embodiment, the pharmaceutical composition comprises at least one peptide comprising, or consisting of, 17 to 28 consecutive amino acids of an amino acid sequence selected from analogs of SEQ ID NO: 1 or SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and the reversed transconformation sequence of analogs of SEQ ID NO: 1 or SEQ ID NO: 2 (provided that the segment of the positively charged amino acid at the C-terminus is identical to that in the parent peptide). According to some such embodiments, the fragment comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9).

[0116] According to another embodiment, the pharmaceutical composition comprises at least one peptide, the peptide consisting of an amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. For example, according to some embodiments, the pharmaceutical composition comprises at least one peptide, the peptide consisting of an amino acid sequence of SEQ ID NO: 1. According to some embodiments, the pharmaceutical composition comprises at least one peptide, the peptide consisting of an amino acid sequence of SEQ ID NO: 2. According to another embodiment, the pharmaceutical composition comprises at least one peptide, the peptide consisting of an amino acid sequence of SEQ ID NO: 3. According to another embodiment, the pharmaceutical composition comprises at least one fragment of the peptide, which consists of 17-28 amino acids selected from the amino acid sequence of SEQ ID NO: 1, 2 or 3. According to another embodiment, the pharmaceutical composition comprises at least one peptide, which consists of an amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31.

[0117] According to some embodiments, the pharmaceutical composition comprises at least one conjugate of the peptide. According to one embodiment, the conjugate is a PEG conjugate. According to some embodiments, the pharmaceutical composition comprises at least one conjugate of a peptide having an amino acid sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 and PEG. According to other embodiments, the pharmaceutical composition comprises at least one conjugate of a peptide having an amino acid sequence selected from any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 and PEG. According to some embodiments, the peptide is PEGylated at the N-terminus. According to other embodiments, the peptide is PEGylated at the C-terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000 Da, 350 to 8,000 Da, 400 to 6,000 Da, 1,000 to 4,000 Da, or 2,000 Da to 3,000 Da. According to some embodiments, the PEG has a molecular weight selected from 300 to 1,000 Da.

[0118] According to some embodiments, the pharmaceutical composition comprises at least one conjugate comprising a peptide containing any of the amino acid sequences of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 and a PEG having 5 to 15 ethylene glycol monomers (e.g., 8 monomers) attached to the C-terminus or N-terminus of the peptide.

[0119] The terms “pharmaceutical composition” and “pharmaceuticalally acceptable composition” are used interchangeably herein and refer to a composition comprising a peptide or conjugate thereof disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0120] As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic agents and absorption delay agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active agents to provide complementary, additional, or enhanced therapeutic functions.

[0121] According to any of the above embodiments, the peptide can be administered via any known route of administration. The term "administer" a substance, compound, agent, or pharmaceutical composition to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound, agent, or composition can be administered enterically or parenterally. Enterally means administration via the gastrointestinal tract, including oral, sublingual, or rectal administration. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal administration. The compound or agent may also be suitably introduced via a refillable or biodegradable polymer device or other device (e.g., patches and pumps) or a formulation providing an extended, slow, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods. In other embodiments, routes of administration include, but are not limited to: local, oral, sublingual, buccal, mucosal, nasal, intravenous, subcutaneous, intraenteral, intraarterial, intramuscular, intraperitoneal, epidural, intrathecal, intraventricular, intra-articular, intraosseous, intracardiac, intravitreal, parenteral, vaginal, cavernous sinus, intravesical, rectal, local, transdermal, inhalation, perivascular, or any combination thereof. In some embodiments, administration may be in the form of a cream, dressing, or spray.

[0122] In one embodiment, the pharmaceutical composition comprising the peptide or conjugate of the present invention is administered systemically. For example, the pharmaceutical composition comprising the peptide or conjugate of the present invention is administered orally, intravenously, or transdermally. Alternatively, the composition is administered intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, or intranasally.

[0123] The pharmaceutical compositions according to the invention can be prepared by any known method. Specifically, the pharmaceutical compositions can be formulated using methods known in the art to provide a rapid, continuous, or delayed release of the active ingredient upon administration. In one particular embodiment, the pharmaceutical composition is formulated into a solid dosage form selected from tablets, capsules, powders, or granules. In another embodiment, the pharmaceutical composition is formulated into a liquid or semi-liquid dosage form selected from elixirs, tinctures, suspensions, syrups, emulsions, or gels.

[0124] Pharmaceutical compositions intended for oral use may be prepared according to any method of preparing pharmaceutical compositions known in the art, and may also contain one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically palatable formulation. Tablets contain an active agent mixed with non-toxic, pharmaceutically acceptable excipients suitable for tablet manufacturing. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binders; and lubricants. The tablets may optionally be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a prolonged release of the drug over a longer period.

[0125] According to one aspect, the pharmaceutical composition according to any of the above embodiments is used to treat cancer. According to some more specific embodiments, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence selected from SEQ ID NO: 1-5, a fragment consisting of 17-23 amino acids thereof, or an analogue of said peptide or said fragment. Furthermore, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence of SEQ ID NO: 10-29 and 31, or an analogue thereof. According to other embodiments, the pharmaceutical composition used comprises a conjugate of such peptides, such as a PEG conjugate.

[0126] The term vegetation refers to the abnormal growth of tissue or cells caused by excessive cell division. Vegetative growth can lead to tumors. "Cancer" or "tumor" refers to a vegetation that has undergone characteristic dysplasia with loss of differentiation, increased growth rate, invasion of surrounding tissues, and the ability to metastasize. The amount of tumor in an individual is called the "tumor burden," which can be measured as the number, volume, or weight of tumors. Non-metastatic tumors are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant." Metastatic cancer is cancer located in one or more sites in the body other than the site of origin of the primary (original) cancer from which the metastatic cancer described herein originates.

[0127] As used herein, the term "treatment" refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, reducing or improving one or more symptoms or parameters associated with cancer, delaying or slowing the damage, improving, mitigating or stabilizing the damage, and other beneficial outcomes. In the context of cancer, treatment refers to reducing tumor burden, malignancy or metastasis, or slowing the rate of tumor growth, lowering tumor grade, or reducing the rate of tumor metastasis and spread.

[0128] According to some implementations, the cancer is accompanied by enhanced or elevated expression of the NAF-1 protein. The terms "NAF-1" and "CISD2" refer to a protein, wherein the human protein has gene ID: 493856.

[0129] According to any of the above embodiments, the cancer is selected from breast cancer, prostate cancer, gastric cancer, colon cancer, rectal cancer, cervical cancer, endometrial cancer, liver cancer, bile duct cancer, pancreatic cancer, head cancer, neck cancer, ovarian cancer, myeloma, glioblastoma, malignant melanoma, CML, AML, ALL, lung cancer, bladder cancer, kidney cancer, non-Hodgkin's lymphoma, and laryngeal cancer.

[0130] According to other embodiments, the composition is used to treat cancer selected from breast cancer, melanoma, skin growths, lymphoma, leukemia, myeloma, gastrointestinal tumors (including colon cancer, stomach cancer, pancreatic cancer, small bowel cancer), ovarian cancer, cervical cancer, lung cancer, prostate cancer, renal cell carcinoma and / or metastases in any organ (including metastases in the liver, lymph nodes, bone or lungs).

[0131] According to other embodiments, the composition is used to treat cancers selected from the following hematologic malignancies: leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythrocytic leukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplastic syndrome.

[0132] According to other embodiments, the composition is used to treat cancers selected from: solid tumors, such as sarcomas and carcinomas that can be treated according to the methods provided herein, including fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphomas, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma. Cancer, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, myeloma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0133] Pharmaceutical compositions comprising the peptides described herein may be administered in combination with at least one other anticancer therapy. The peptide may be administered before, during, or after the other anticancer therapy. The peptide may be administered via the same or different routes of administration as the other anticancer therapy.

[0134] Examples of such anticancer therapies include, but are not limited to, immunomodulators, activated lymphocytes, lymphocyte activators, kinase inhibitors, chemotherapy agents, or any other anticancer agents.

[0135] For example, one example of anticancer therapy includes, but is not limited to, protein synthesis inhibitors. Examples of protein synthesis inhibitors include, but are not limited to, styracin, procainamide, homoharringtonine, ranpirnase, brefidobacterium A, and KRN-5500.

[0136] Another example of anticancer therapy includes, but is not limited to, proteasome inhibitors. Examples of proteasome inhibitors include, but are not limited to, bortezomib, carfilzomib, and ixazomib.

[0137] Another example of anticancer therapy includes, but is not limited to, Flt3 inhibitors. Examples of Flt3 inhibitors include, but are not limited to, midotutolin, sorafenib, sunitinib, panatinib, quezatinib, giretinib, and crenolab.

[0138] Another example of anticancer therapy includes, but is not limited to, IDH inhibitors. Examples of IDH inhibitors include, but are not limited to, evanib, olutanib, enxidipine 3BP, dichloroacetate, AGI-5198, and AGI-6780.

[0139] Another example of anticancer therapy includes, but is not limited to, inhibitors of one or more members of the BCL-2 family. Examples of BCL-2 family members include, but are not limited to, BCL-2, BCL-XL, BCL-W, MCL-1, BFL-1 / A1, BAX, BAK, BOK, BAD, BID, BIK, BIM, BMF, HRK, NOXA, and PUMA. Examples of BCL-2 inhibitors include, but are not limited to, navitoclax, venetoclax, ABT-737, ECPU-0001, TW-37, GX15-070, and BM-1197.

[0140] Another example of anticancer therapy includes, but is not limited to, anthracyclines. Examples of anthracyclines include, but are not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, CPX-351, and pentorubicin.

[0141] Another example of anticancer therapy includes, but is not limited to, taxanes. Examples of taxanes include, but are not limited to, paclitaxel, docetaxel, cabazitaxel, and abraxane. .

[0142] Another example of anticancer therapy includes, but is not limited to, platinum complexes. Examples of platinum complexes include, but are not limited to, cisplatin, carboplatin, and oxaliplatin.

[0143] Another example of anticancer therapy includes, but is not limited to, vinca alkaloids. Examples of vinca alkaloids include, but are not limited to, vinorelbine, vincristine, and vindesine.

[0144] Another example of anticancer therapy includes, but is not limited to, ROCK inhibitors. Examples of ROCK inhibitors include, but are not limited to, ripasudi and netarsudil.

[0145] Another example of anticancer therapy includes, but is not limited to, antitumor alkyl phospholipids. Examples of antitumor alkyl phospholipids include, but are not limited to, edifoxine, imifocin, perifoxine, berberine, and erufosine.

[0146] Another example of anticancer therapy includes, but is not limited to, Ezrin inhibitors. Another example of anticancer therapy includes, but is not limited to, temozolomide.

[0147] In some other embodiments, the anticancer therapy includes, but is not limited to, metformin, tamoxifen, 3-bromopyruvate, 2-deoxy-d-glucose, rotenone, CCCP, IACS-010759, TAX165, lilotinib, arsenic trioxide, antimycin A, 5-fluorouracil, sulfonylurea, auronoxine, chlorambucil, α-tocopherol succinate, PX12, clophosphonate, disulfiram, diosgenin, glaggib, irismo, tigecycline, mitotane, oxymatrine, capsaicin, vitexin, myricetin, chlorpyrifos, long pepperamide, oleanolic acid, triptolide, valproic acid, venetoclax, setanaxib, crotonol, trigonelline, and resveratrol.

[0148] In some implementations, the anticancer therapy is immunotherapy.

[0149] In some embodiments, the immunotherapy is cellular immunotherapy. In some embodiments, the cellular immunotherapy is selected from CAR-T, CAR-M, CAR-NK, NKT, TILS, and NK cells.

[0150] In some embodiments, the immunotherapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from CTLA-4 and PD-1 inhibitors.

[0151] In some embodiments, the anticancer therapy is a cancer vaccine. In some embodiments, the cancer vaccine is a neoantigen vaccine.

[0152] When administered as part of a combination therapy, any of these anticancer therapies can result in synergistic activity with the peptide. As used herein, the terms "synergistic," "synergistic," or "synergistic effect" refer to a combination of two or more drugs that is more effective in treating cancer than predicted by the additive effect of the two drugs when used alone as single agents. In some respects, the synergistic combination can kill tumor cells resistant to either drug alone. In other respects, synergy refers to a greater than additive effect of two or more drugs at a specific dose or a suboptimal dose of one or more drugs. Reducing the dose of one or more drugs in a synergistic combination can lead to higher efficacy and lower toxicity.

[0153] According to some implementations, the peptide sequence is fused into an albumin-binding domain.

[0154] According to some implementation methods, the peptides described herein can be coupled to radioisotopes. Examples of radioisotopes include, but are not limited to, those mentioned above. 131 I, 68 Ga、 177 Lu、 153 Sm、 212 Pb, 166 Ho、 111 In, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90, and thorium-227. Besides their use in cancer treatment as described herein, peptides coupled with radioactive isotopes can also be used as diagnostic agents.

[0155] Formulations and excipients, carriers and diluents

[0156] As described herein, peptides can be formulated into “pharmaceutical formulations” or “pharmaceutical compositions.” In some cases, compositions described herein may include pharmaceutical compositions. In some cases, pharmaceutical compositions may be in unit dose form. In some cases, formulations containing at least one peptide described herein may be in liquid, solid, semi-solid, or lyophilized powder form, such as solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches, etc., preferably in unit dose form suitable for easy administration with precise dosage.

[0157] In some embodiments, the pharmaceutical composition may include conventional pharmaceutical carriers or excipients, and may additionally include other pharmaceutical agents, carriers, adjuvants, additives, etc. In some cases, the composition may have about 0.1% to about 85%, or about 0.5% to about 75% by weight of one or more compounds of this disclosure, with the remainder consisting substantially of suitable pharmaceutical excipients.

[0158] In some embodiments, the peptides described herein may be packaged and / or resuspended in one or more of the following excipients: gum arabic, acesulfame potassium, glacial acetic acid, acetone, tributyl acetyl citrate, triethyl acetyl citrate, adipic acid, agar, albumin, alcohol, alginic acid, aliphatic polyester, arabinose, allantoin, almond oil, α-hydroxy acid, α-tocopherol, aluminum hydroxide adjuvant, aluminum monostearate, alumina, aluminum phosphate adjuvant, ammonia solution, ammonium alginate, ammonium chloride, argan oil, ascorbic acid, ascorbate glucoside, ascorbate palmitate, aspartame, attapulgite, azelaic acid, chamomile, psoralen, β-glucan, β-hydroxy acid, bentonite, benzalkonium chloride, benzyl chloride, benzoic acid, benzyl alcohol, benzyl benzoate. Methyl ester, boric acid, bromonitol, butylated glycol, butylated hydroxyanisole, butylated hydroxytoluene, butanediol, butyl p-hydroxybenzoate, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium hydroxide, calcium lactate, anhydrous dicalcium hydrogen phosphate, dicalcium hydrogen phosphate dihydrate, tricalcium phosphate, calcium silicate, calcium stearate, calcium sulfate, rapeseed oil, decanediol, decanoic acid triglyceride, carbomer, carbon dioxide, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, carrageenan, castor oil, hydrogenated castor oil, microcrystalline cellulose, microcrystalline cellulose and sodium carboxymethyl cellulose, powdered cellulose, silicified microcrystalline cellulose, cellulose acetate, cellulose acetate phthalate, ceramide, ceresin, cetearyl alcohol, cetrimonium bromide, cetearyl alcohol, cetyl alcohol, cetylpyridine chloride, Chitosan, chlorhexidine, chlorobutanol, chlorocresol, dichlorofluoroethane (HCFC), chlorofluorocarbons (CFC), dichloroxylenol, cholesterol, citrate monohydrate, coconut oil, collagen, colloidal silica, coloring agents, copper peptides, copovidone, corn oil, corn starch and pregelatinized starch, cottonseed oil, cresol, croscarmellose sodium, croscarmellose, cyclodextrin, cyclomethylsiloxane, denatonium benzoate, desitin, glucose binding agent, dextrin, glucose, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HCC), polydimethylsiloxane, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethyl acetamide, disodium ethylenediaminetetraacetate, sodium docusate, ethylenediaminetetraacetate Aminotetraacetic acid, isoascorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, ethyl vanillin, ethyl cellulose, ethylene glycol stearate, ethylene-vinyl acetate, ethyl p-hydroxybenzoate, fatty acids, ferulic acid, fructose, fumaric acid, gelatin, liquid glucose, glycerol, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitate, glycine, glycogen, glycolic acid, ethylene glycol stearate, guar gum, lithium saponite, heptafluoropropane (HFC), hexocide, hydrocarbons (HC), hyaluronic acid, hydrochloric acid, hydrocortisone, hydrophobic colloidal silica, mesoporous silica, hydroquinone, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl β-cyclodextrin, hydroxypropyl celluloseLow-substituted hydroxypropyl cellulose, hydroxypropyl starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, imidazolidinyl urea, inulin, iron oxide, isomaltulitol, isoparaffin, isopropanol, isopropyl myristate, isopropyl palmitate, jojoba oil, kaolin, kojic acid, lactic acid, lactitol, anhydrous lactose, inhaled lactose, lactose monohydrate, lactose monohydrate and corn starch, lactose monohydrate and microcrystalline cellulose, lactose monohydrate and povidone, lactose monohydrate and powdered cellulose, spray-dried lactose, lanolin, hydrated lanolin, lanolin alcohol, lauric acid, lecithin, leucine, linoleic acid, polyethylene glycol 15-hydroxystearate, magnesium aluminum silicate, magnesium carbonate, magnesium oxide, magnesium silicate, stearic acid Magnesium, magnesium trisilicate, maleic acid, malic acid, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium-chain triglycerides, meglumine, menthol, methionine, methylcellulose, methylparaben, mineral oil, light mineral oil, mineral oil and lanolin alcohol, monoethanolamine, monosodium glutamate, monothioglycerol, myristic acid, myristol, neohesperidin dihydrochalcone, neotame, nicotinamide, nitrogen, nitrous oxide, octyldodecyl alcohol, oleic acid, oleyl alcohol, olive oil, palmitic acid, paraffin, peanut oil, pectin, PEG-8 stearate, pentiformic acid, petrolatum, petrolatum and lanolin alcohol, phenol, phenoxyethanol, phenylethanol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phospholipids, phosphoric acid, phytic acid, phytosphoethanol Potassium polacolin, poloxamer, polycarboflavone, polydextrose, polyl-lactic acid, polyethylene glycol, polyethylene oxide, polymethacrylate, poly(methyl vinyl ether / maleic anhydride), polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, polyoxyglycerol, polyparaben, polysorbate 60, polysorbate 80, polyvinyl acetate, polyvinyl alcohol, potassium alginate, potassium alum, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium metabisulfite, potassium sorbate, povidone, propionic acid, propyl gallate, propylene carbonate, propylene glycol, propylene glycol alginate, propylparaben, sodium propylparaben, pyrrolidine Ketones, raffinose, retinoic acid, retinol and retinoic acid derivatives, saccharin, sodium saccharin, safflower oil, salicylic acid, soapstone, sesame oil, shellac, simethicone, sodium acetate, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium borate, sodium carbonate, sodium chloride, sodium citrate dihydrate, sodium cyclohexanoate, sodium formaldehyde sulfoxylate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium propionate, sodium starch glycolate, sodium stearyl fumarate, sodium ascorbate phosphate, sodium deoxycholate, sodium hydroxide, sodium lauroyl lactylate, sodium lauryl sulfate, sodium palmitate, sorbitan stearate, sodium sulfite (E221), spironolactone, sodium sulfite, sodium thiosulfate, sorbic acid.Sorbitol esters (sorbitol fatty acid esters), sorbitol monostearate, sorbitol, soybean oil, sphingomyelin, starch, pregelatinized starch, sterilizable corn starch, stearic acid, stearyl alcohol, squalene, sucralose, sucrose, sucrose octaacetate, compressible sugar, confectionery sugar, sugar balls, sulfobutyl ether β-cyclodextrin, sulfur dioxide, sulfuric acid, sunflower oil, suppository base—stearic acid, tagatose, talc, tartaric acid, tetrafluoroethane (HFC), kiwifruit protein, sulfur Mercury, thymol, titanium dioxide, tragacanth gum, trehalose, retinoic acid, triacetin, tributyl citrate, tricaprylic acid, triethanolamine, triethyl citrate, triethanolamine, trioleic acid, undecenoic acid, vanillin, hydrogenated vegetable oil, vitamins, vitamin E, polyethylene glycol succinate, water, anionic emulsified wax, carnauba wax, cetyl ester wax, microcrystalline wax, nonionic emulsified wax, white wax, yellow wax, xanthan gum, xylitol, zein, zinc acetate and / or zinc stearate.

[0159] In some cases, compositions, such as pharmaceutical compositions, may contain a carrier or diluent. In some cases, the carrier or diluent may include water, alcohol, a salt solution (e.g., saline), or mixtures thereof. In some cases, the carrier may include carbohydrates, buffers, salts, pH adjusters, or any combination thereof. In some cases, the compositions herein may contain buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tension modifiers, flavoring agents, coloring agents, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, or any combination thereof.

[0160] In some embodiments, the liquid composition can be prepared by dissolving or dispersing one or more peptides described herein (about 0.5% by weight to about 20% by weight or more) and optional pharmaceutical adjuvants in a carrier such as an aqueous saline solution, an aqueous glucose solution, glycerol, or ethanol to form a solution or suspension. In some cases, for use in oral liquid formulations, the composition can be prepared as a solution, suspension, emulsion, or syrup, supplied in liquid form or a dry form suitable for hydration in water or physiological saline.

[0161] In some embodiments, the peptides described herein can be formulated as injectable compositions for parenteral administration (e.g., intravenous, intramuscular, intracapsular, or intrathecal). The peptides for use in injectable compositions can be in a suitable intravenous solution, such as sterile saline solution. The compositions can also be formulated as suspensions in aqueous emulsions.

[0162] In some embodiments, the peptides described herein can be delivered in film patches or microneedle patches.

[0163] Furthermore, the peptides described herein can be formulated with penetration enhancers. When used herein, "penetration enhancer" refers to an excipient that promotes the delivery of peptides into tissues. Examples of penetration enhancers include, but are not limited to, terpenes, terpenoids, sulfoxides, laurocapram, pyrrolidone, 1-phenylpiperazine, sodium deoxycholate, fatty acids, fatty alcohols, urea, calcium chelating agents (including, but not limited to, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediamine-N,N'-disuccinic acid (EDDS)); glyceryl oleate, boric acid; hyaluronic acid, crown ethers, surfactants, saponins, benzalkonium chloride, parabens, and transcutol. ® P, Labrasol ® Sodium decanoate, acylcarnitine, geranium pigment, citric acid, sodium sapolixalate, bile acids, nanoparticles, cell-penetrating peptides, gelucires, Azone™ (1-dodecylazacycloheptan-2-one), borneol, trioleic acid glycerides, Miglyol, oleic acid, sesame oil, picrocoba oil, Capryol PGMC, sweet almond oil, lemon oil, clove oil, eucalyptus oil, diacylglycerol esters, polyoxyethylene ethers, eucalyptol, geraniol, or combinations thereof.

[0164] As needed, the peptides described herein may be administered once daily, twice daily, three times daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, seven times weekly, once every two weeks, once every three weeks, once monthly, once quarterly, once every six months, once a year, and / or for life.

[0165] This article also describes the use of the peptide in cancer diagnosis. For example, the peptide can be coupled to a signaling moiety that provides a signal detectable by imaging techniques. According to this embodiment, the accumulation of the coupled peptide in tumor tissue is detected by visual or imaging techniques.

[0166] For example, the signal component can be a fluorescent compound that can be detected by fluorescence detection (whether visually or using a fluorescence imaging device). Examples of fluorescent dyes that can be used include, but are not limited to, fluorescein, anthocyanin dyes, or AlexaFluor®.

[0167] Other examples of production signal components include PET or SPECT markers that can be detected by PET or SPECT scans. Examples of such markers include, but are not limited to, those shown below. 18 F, 99m Tc, 68 Ga、 11 C 13 N、15 O、 64 Cu、 86 Y、 55 Co、 123 I, 131 I, 99m T, 89 Zr、 133 Xe and 201 TI.

[0168] This article also discloses a kit comprising: at least one peptide described herein, a pharmaceutically suitable excipient, a carrier, a diluent or any combination thereof, and a sterile storage container.

[0169] This document also discloses a kit comprising: the lyophilized peptide of the present invention; a pharmaceutically suitable excipient, carrier, diluent or any combination thereof; a separate sterile storage container for each; and instructions on how to reconstitute the lyophilized peptide with the pharmaceutically suitable excipient, carrier, diluent or any combination thereof.

[0170] According to another aspect, a method of treating cancer in a subject in need is provided, the method comprising administering to the subject a peptide, conjugate, or pharmaceutical composition described herein. According to one embodiment, the pharmaceutical composition comprises an effective amount of the peptide or conjugate as described herein. According to some specific embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of a peptide or conjugate thereof comprising an amino acid sequence selected from any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to other specific embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of a peptide or conjugate thereof comprising an amino acid sequence selected from SEQ ID NO: 1, 2, or 3. According to some embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of a peptide comprising an amino acid sequence selected from SEQ ID NO: 1, 2, or 3, or a peptide comprising 17-28 amino acids, or an analogue of the peptide or the fragment thereof. According to one embodiment, the pharmaceutical composition comprises the conjugate or fusion protein of the present invention. According to some embodiments, the method comprises administering the pharmaceutical composition of the present invention in combination with another anticancer therapy as described above.

[0171] As used herein, the term “about” when referring to a measurable value (e.g., quantity, duration of time, etc.) means to cover a variation of + / -10% or + / -5%, + / -1% or even + / -0.1% from the specified value.

[0172] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided as illustrative purposes and are not intended to limit the invention.

[0173] Example

[0174] Example 1. 3D-NAF-1-6K peptide selectively penetrates into breast cancer cells.

[0175] A peptide with the amino acid sequence FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2, also referred to herein as NAF-1-3D-6K peptide) is derived from a peptide corresponding to amino acids 44-67 of human NAF-1, which is part of the transmembrane domain and adjacent cytoplasmic region of human NAF-1. Figure 1 This region of the protein does not participate in the coordination of the [2Fe-2S] cluster of NAF-1. Six Lys (KKKKKK) were added to the original sequence (Seq ID No. 6), and three amino acids were converted to D-amino acids (appearing in lowercase in SEQ ID NO: 2) to produce the substance shown in SEQ ID NO: 2. The addition of 6K increased the cytotoxicity of the 3D-NAF-1-6K peptide by approximately three times compared to the original 3D-NAF-1 peptide. Figure 3 This increased efficiency is not simply achieved by adding a positive charge, because... Figure 4 This indicates that 3D-NAF-1-6K is more cytotoxic to breast epithelial cancer cells (MDA-MB-231) at 3 and 18 hours compared to 3D-NAF-1-6R.

[0176] The ability of a peptide having the sequence SEQ ID NO: 2 to penetrate into malignant cells and its effect on cell viability were tested as described below. A green fluorescent (Fl) / Cy-5 label was conjugated to the peptide having SEQ ID NO: 2 (i.e., the 3D-NAF-1-6K peptide). Figure 12 It was confirmed that the FL-3D-NAF1-6K peptide infiltrated into ovarian cancer (SKOV-3) cells. Figure 13 and Figure 14 Its effects on mitochondrial division / decomposition have been confirmed.

[0177] Example 2. Efficacy of peptide SEQ ID NO: 2 in killing different cancer cells

[0178] To validate cell-based results, in 2D tissue cultures ( Figure 5 , 6 and 7) and "tumor-like" 3D cancer cell spheroids ( Figure 9-11The efficacy of the peptide with SEQ ID NO: 2 was studied in both studies. In addition to the ability of the 3D-NAF-1-6K peptide to kill MDA-MB-231 breast cancer cells (…), Figure 3 In addition, the efficacy of 3D-NAF-1-6K peptide in killing prostate cancer (PC-3), ovarian cancer (SK-OV-3), and liver cancer (HepG2) cells was also measured.

[0179] For 2D cytotoxicity studies, cancer cells (MDA_MB-231 / HepG2 / SKOV-3 / PC-3) were cultured at 2 × 10⁻⁶ cells per well. 3 The cells were seeded in quadruplicates in 96-well ULA plates (Corning 7007) at a density of 1,000 cells, and cultured for 1 hour and 1 day after the addition of 3D-NAF-1-6K peptide. Figure 5 , 6 (and 7). 3D spheroids were treated with a peptide having SEQ ID NO: 2, and apoptosis in the spheroids was determined using the IncuCyte® Red cytotoxicity assay (Essen Bioscience Cat #4632) cell death probe. The spheroids were imaged at 10x magnification in an IncuCyte Zoom live-cell analysis system (Essen Bioscience, Ann Arbor, MI) at 37°C and 5% CO2. Images of the spheroid cells were recorded every two hours, and the resulting red color reflecting apoptosis was analyzed using IncuCyte software (Sartorius AG, Göttingen, Germany).

[0180] Figure 9 The results showed that after incubating the peptide with 3D spheroids of ovarian cancer SKOV-3 cells for 20 hours ( Figure 9 (See figure below). Most tumor-like cells were killed. Survival rate was determined by fluorescence obtained using the IncuCyte® Red cytotoxic reagent (Essen Bioscience Cat #4632) cell death probe. Similar effects of the 3D-NAF-1-6K peptide on Hep-G2 globular cells of liver cancer and PC-3 globular cells of prostate cancer were shown, respectively. Figure 10 and Figure 11 middle.

[0181] Example 3. The 3D-NAF-1-6K peptide penetrates only the plasma membrane (PM) of cancer cells and disrupts the intracellular organelles mitochondria and ER.

[0182] NAF-1-3D-6K peptide targets and damages intracellular organelles mitochondria and endoplasmic reticulum (ER). Figure 33D-NAF-1-6K was shown to be non-toxic to normal breast epithelial cells (MCF10A) while killing breast cancer cells (MDA-MB-231).

[0183] Of particular importance is the validation that the 3D-NAF-1-6K peptide specifically targets cancer cells. Figure 3 The results showed that the 3D-NAF-1-3D-6K peptide did not affect normal MCF-10A mammary epithelial cells, but it had a significant cytotoxic effect on MDA-MB-231 breast cancer cells.

[0184] Figure 5 , 6 Studies 7 also demonstrated the cytotoxic effects of 3D-NAF-1-6K peptide against other malignant cells, such as prostate cancer (PC-3 cells), ovarian cancer (SKOV-3), liver cancer (HepG2), melanoma (A375), glioblastoma (U87MG), two types of leukemia: acute myeloid leukemia (MOLM-14 cells) and chronic myeloid leukemia (K562 cells), and pancreatic cancer cells (PANC-1 cells). Although 3D-NAF-1-6K peptide showed efficacy against solid tumor cells in IC50... 50 The efficacy of 3D-NAF-1-6K was highest against leukemia K562 cells in the range of 7.8-15 μM (1.9 μM). Figure 7 .

[0185] Example 4. The 3D-NAF-1-6K peptide is highly effective in animal model systems of ovarian cancer and melanoma, where it specifically targets xenograft tumors.

[0186] In an in vivo animal model system, SKOV-3 xenograft tumors were developed in 5-6 week old mice after subcutaneous injection of 2.5 million ovarian cancer cells. Figure 17 The effect of injection of the 3D-NAF-1-6K peptide with SEQ ID NO: 2 at specified time on the size of SKOV-3 ovarian cancer tumors was demonstrated. The peptide (0.5 mg / kg) was injected intravenously (IV) or subcutaneously (SC), and the effects were compared with those in control mice (negative controls) injected with the carrier. Although the 3D-NAF-1-6K peptide had a significant inhibitory effect on tumor growth, it had no toxic effects on mice, as indicated by their weight gain.

[0187] Figure 21-24 The 3D-NAF-1-6K peptide was shown to specifically arrive at and accumulate in the tumor. Figure 21 This demonstrates the intravenous injection of fluorescently labeled Fl-3D-NAF-1-6K peptide (in... Figure 22All internal organs isolated from mice (referred to as "Fl-3D-NAF-1 peptide") were tested, confirming that the liver, kidneys, colon, brain, lungs, and other organs maintained their characteristic normal color. Figure 22-24 The results showed that only sections of shrunken breast MDA-MB-231 tumors exhibited green fluorescence when analyzed and quantified using confocal microscopy. Figure 24 In the image, representative confocal images of tumor cells from mice treated with or without 3D-NAF-1-6K peptide are shown. The mitochondria of the tumor cells are stained with RPA, and the nuclei are stained with H342. Figure 25 The kinetics of Cy5-3D-NAF-1-6K peptide after injection into nude mice with tumors formed from MDA-MB-231 breast cancer cells were demonstrated. Following injection, the Cy5-3D-NAF-1-6K peptide reached the liver and kidneys, from which it was cleared, but it accumulated and remained within the tumor.

[0188] exist Figure 28 In this study, mice carrying A375 cancer xenografts were divided into two groups: a control group and a 3D-NAF-1-6K peptide treatment group. The latter received intravenous administration of 0.75 mg / kg of 3D-NAF-1-6K peptide three times a week for three weeks. Figure 28 As shown in B, although 3D-NAF-1-6K peptide significantly inhibited the growth of A375 tumors, it had no toxic effects on mice, as evidenced by the increase in mouse body weight during the experiment. Figure 28 As indicated by C).

[0189] Example 5. The 3D-NAF-1-6K peptide is highly effective in animal model systems of primary and metastatic ovarian cancer.

[0190] Figure 17 The effects of injection of NAF-1-3D-6K peptide on the size and tumor development of ovarian cancer SKOV-3 cells were demonstrated. The peptide (50 μM = 0.5 mg / kg) was administered intravenously (IV) via the tail vein or subcutaneously (SC) in the neck of mice. Control mice were injected with saline. Immunohistochemistry of tumors injected with 3D-NAF-1-6K peptide and detected with an antibody targeting the macrophage-specific mouse cell surface glycoprotein F4 / 80 marker indicated a large number of tumor-infiltrating macrophages (…). Figure 18 ).

[0191] Figure 19The study showed that 3D-NAF-1-6K peptide (SEQ ID NO: 2) reduced the incidence of lung metastasis in SKOV-3 ovarian cancer cells. Nude mice were IV-seeded with 1 million SKOV-3 cells on day 0. NAF-1-3D-6K peptide was administered via SC injection twice weekly at a dose of 0.25 mg / kg or 0.5 mg / kg. Administration of NAF-1-3D-6K peptide began 7 days after the appearance of lung metastasis in satellite animals. Figure 19 Paclitaxel (positive control) was also indicated: IV, twice a week, starting from day 7, p=0.00094, paclitaxel - 20 mg / kg; P=0.000998 3D-NAF-1-6K 0.5mg / kg.

[0192] In addition to reducing lung metastases, the peptide 3D-NAF-1-6K also reduces metastatic lesions in the colon of mice. Figure 19 B. Figure 20 The study demonstrated that the 3D-NAF-1-6K peptide (SEQ ID NO: 2) inhibits the migration of SKOV-3 cells. Cell migration is one of the prerequisites for metastasis formation, and the fact that the 3D-NAF-1-6K peptide reduces cancer cell migration may contribute to the reduction of lung / colon metastasis formation.

[0193] Example 6. 3D-NAF-1-6K peptide promotes mitochondrial division and mitochondrial morphology disruption in SKOV-3 cells.

[0194] Figure 12-14 This indicates that 3D-NAF-1-6K (SEQ ID NO: 2) induces significant damage to mitochondria after infiltrating SKOV-3 cells. The 3D-NAF-1-6K peptide induces this division / fragmentation. Our hypothesis is that the peptide disrupts the dimer structure of its derived protein, NAF-1 (CISD2). Figure 1 This process induces the destruction of the outer mitochondrial membrane and the endoplasmic reticulum membrane.

[0195] Example 7. Binding of 3D-NAF-1-6K peptide to albumin

[0196] Unintentionally constrained by any assumptions, the binding of the peptide to serum albumin can prolong its half-life and / or form a delivery system that enhances the availability of the peptide to tumors. Figure 27 ).

[0197] Figure 27The results showed that increasing the concentration of BSA (bovine serum albumin) delayed the cytotoxic activity of the NAF-1-3D-6K peptide against mammary MDA-MB-231 cells. BSA concentrations were defined as 0.125 mg / ml (purple bar), 0.25 mg / ml (green bar), and 0.5 mg / ml – ~10% serum albumin concentration (red-orange bar). The peptide without BSA (red bar) served as a positive control, and the negative control was represented by the blue bar. Cells were incubated for 3, 6, 12, 24, and 72 hours in the presence of the peptide (with or without free BSA). Cytotoxicity was measured on a cell imaging plate using the presto-blue method. Although these results were obtained in vitro, they may suggest that albumin can act as a carrier to facilitate the translocation of the peptide in the bloodstream in vivo.

[0198] Example 8. NAF-1-3D-6K peptide also exhibits activity in controlling the redox state of cancer cells.

[0199] 3D-NAF-1-6K peptide (SEQ ID NO: 2) inhibits the activity of thioredoxin reductase. Figure 15 Thioredoxin reductase is the reductase of thioredoxin, a major essential redox regulator in cells that ensures the maintenance of thiols (-SH) in proteins undergoing nonfunctional (SS) transformations under oxidative stress. Thioredoxin reductase is the enzyme that ensures the reduction of thioredoxin when it contributes its hydrogen. Thioredoxin is an essential protein that ensures normal redox status in cells. Thioredoxin reductase is upregulated in cancer cells as a defense against oxidative stress. The ability of the 3D-NAF-1-6K peptide (SEQ ID NO: 2) to inhibit thioredoxin reductase activity may be an important aspect of its anticancer activity.

[0200] Example 9: 3D-NAF-1-6K peptide exhibits enhanced cytotoxicity against cancer cells.

[0201] We tested whether adding an extra lysine residue to the C-terminus of 3D-NAF-1 (SEQ ID NO: 7) would improve solubility and / or cytotoxic activity. The modified peptide (with 6K) was tested using healthy and epithelial breast cancer cells. Figure 3 The cytotoxic effect of the peptide known as 3D-NAF-1-6K (SEQ ID NO: 2) compared to the original 3D-NAF-1 peptide (SEQ ID NO: 7) Figure 3Both peptides reduced cancer cell survival at 1 and 3 days compared to unaffected control cells. However, 3D-NAF-1-6K (SEQ ID NO: 2) showed unexpectedly and significantly higher cytotoxicity against cancer cells compared to 3D-NAF-1 (SEQ ID NO: 7).

[0202] In this experiment, cells were seeded at a density of 15,000 cells / well in 96-well plates before the experiment. 10 µM of 3D-NAF-1 (SEQ ID NO: 7) or 3D-NAF-1-6K (SEQ ID NO: 2) peptide was added to the cells, and they were incubated for 1 or 3 days. Cell viability was assessed using Presto-Blue after 1 and 3 days of incubation. Cell viability was determined by t-test. P < 0.001 , P < 0.0001 .

[0203] Example 10: Broad-spectrum anticancer activity of NAF-1-3D-6K peptide

[0204] We investigated the effects of 3D-NAF-1-6K on several cancers, including acute myeloid leukemia (MOLM-14), chronic myeloid leukemia (K562), pancreatic cancer cells (PANC-1), glioblastoma cells (U87MG), and human malignant melanoma (A375). Figure 8 As shown, at low concentrations below 5 μM, the peptide reduced the survival rate of all cancer cells, with leukemia cancer cells being the most sensitive to the cytotoxic effect. At higher concentrations of 25 μM, the peptide significantly reduced the survival rate of all tested cancer cell lines. Combined with our findings in breast and ovarian cancer, these findings suggest that the 3D-NAF-1-6K peptide could be used to combat a broad range of cancers.

[0205] Cell viability was measured using the Presto-Blue assay after incubation with the specified peptide concentration for 3 hours. SKOV-3 (ovarian cancer (black circle)), U87MG (glioblastoma (black rectangle)), A375 (human malignant melanoma (black equilateral triangle)), MDA-MB-231 (breast cancer (black inverted triangle)), PANC-1 (pancreatic cancer (black rhombus)), MOLM-14 (acute myeloid leukemia (red circle)), and K562 (chronic myeloid leukemia (yellow rectangle)) were treated with or without the stated peptide, and viability was determined. Data are shown as the mean ± SD of three independent experiments. Results were analyzed by t-test. P<0.01 , P<0.001 , P<0.0001 .

[0206] Example 11: 3D-NAF-1-6K peptide promotes mitochondrial division / fragmentation

[0207] By labeling peptide 3D-NAF-1-6K (SEQ ID NO: 2) with fluorescent labeling (in... Figure 12 The peptide, referred to as Fl-3D-NAF-1-6K, was labeled with Rhodamine 800 in mitochondria. The entry of this peptide into SK-OV-3 cancer cells and its effects on mitochondria were investigated. The cells were then imaged using confocal microscopy. Following the addition of the peptide to SK-OV-3 ovarian cancer cells, Fl-3D-NAF-1-6K fluorescence was primarily observed near the PM of SK-OV-3 cells. This was observed through high levels of Fl-3D-NAF-1-6K fluorescence at the PM of cancer cells in the early stages and within SK-OV-3 cancer cells at 4 and 6 hours of incubation. Figure 12 AB). These findings indicate that fluorescein-labeled 3D-NAF-1-6K (SEQ ID NO: 2) infiltrates SK-OV-3 cancer cells. Following infiltration of the peptide into the cancer cells, staining of mitochondria with Rhodamine 800 in the treated cells was reduced, suggesting that the peptide has a negative impact on mitochondrial function / integrity. Figure 12 C). This effect was also observed using Cy5, another fluorescent probe coupled to 3D-NAF-1-6K.

[0208] The effects of the 3D-NAF-1-6K (SEQ ID NO: 2) peptide on the mitochondria of SK-OV-3 cells were further investigated by tracking the mitochondrial length of SK-OV-3 cells at different concentrations of peptide. Figure 12-14 The peptide exhibits a dose-dependent effect on mitochondria, leading to a reduction in mitochondrial length. This result indicates that the 3D-NAF-1-6K (SEQ ID NO: 2) peptide targets the mitochondria of SKOV-3 cells. Figure 12-14 This leads to mitochondrial damage.

[0209] Figure 13 The measurement of mitochondrial length in SKOV-3 ovarian cancer cells after treatment with the 3D-NAF-1-6K (SEQ ID NO: 2) peptide is shown. Figure 13 A shows representative confocal fluorescence images of mitochondria in SKOV-3 cells treated with or untreated with the 3D-NAF-1-6K (SEQ ID NO: 2) peptide. Figure 13B shows the conversion of mitochondrial length into binary images of the backbone in both the control (peptide-free) and 50 µM peptide treatments. Mitochondrial length was measured in 25 cells from 8 fields of view using ImageJ, with 156 mitochondria / cells measured in the control and 58 mitochondria / cells in the 50 µM peptide treatment. Mitochondrial length in magnified images was measured using the full-capacity processing tools in ImageJ.

[0210] Figure 28 The efficacy of 3D-NAF-1-6K on the growth rate of A375 melanoma tumors in both female and male mice was demonstrated. Figure 28 A). The fact that the treated mice continued to gain weight ( Figure 28 B) further supports the findings shown in Figure 26, which indicate that the 3D-NAF-1-6K peptide is non-toxic based on CBC results, mouse organ weight, and H&E histochemical analysis of the organs.

[0211] All publications, published patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference.

Claims

1. A peptide comprising: (a) An amino acid sequence containing FLGVLAL-X1-X2 (SEQ ID NO: 10), Where X1 represents the second amino acid sequence LGYLAVRPFLPKKKQQK (SEQ ID NO: 30) or a fragment of the second amino acid sequence with a continuous deletion at the C-terminus; Where X2 represents 1-10 positively charged portions; and Any amino acid in the amino acid sequence may be a D-amino acid; or (b) analogues of (a); or (c) The reversed configuration sequence of (a) or (b).

2. The peptide according to claim 1, wherein the positively charged portion is: (a) Positively charged amino acids; (b) Lysine (K); or (c) Arginine (R).

3. The peptide according to any one of the preceding claims, wherein X2 is (a) 3-10 positively charged portions; (b) 4-7 positively charged portions; or (c) Six positively charged parts.

4. The peptide according to any one of the preceding claims, wherein at least one amino acid of the peptide is a D-amino acid.

5. The peptide according to claim 4, wherein at least 1, 2, 3, 4 to at most all of the amino acids in any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 are D-amino acids.

6. The peptide according to any one of the preceding claims, wherein the peptide comprises the following amino acid sequence: (a) FLGVLALLGYLAVRPFLPKKKQQK-X2 (SEQ ID NO: 11); (b) FLGVLALLGYLAVRPFLPKKKQQ-X2 (SEQ ID NO: 12); (c) FLGVLALLGYLAVRPFLPKKKQ-X2 (SEQ ID NO: 13); (d) FLGVLALLGYLAVRPFLPKKK-X2 (SEQ ID NO: 14); (e) FLGVLALLGYLAVRPFLPKK-X2 (SEQ ID NO: 15); (f) FLGVLALLGYLAVRPFLPK-X2 (SEQ ID NO: 16); (g) FLGVLALLGYLAVRPFLP-X2 (SEQ ID NO: 17); (h) FLGVLALLGYLAVRPFL-X2 (SEQ ID NO: 18); (i) FLGVLALLGYLAVRPF-X2 (SEQ ID NO: 19); (j) FLGVLALLGYLAVRP-X2 (SEQ ID NO: 20); (k) FLGVLALLGYLAVR-X2 (SEQ ID NO: 21); (l) FLGVLALLGYLAV-X2 (SEQ ID NO: 22); (m) FLGVLALLGYLA-X2 (SEQ ID NO: 23); (n) FLGVLALLGYL-X2 (SEQ ID NO: 24); (o) FLGVLALLGY-X2 (SEQ ID NO: 25); (p) FLGVLALLG-X2 (SEQ ID NO: 26); (q) FLGVLALL-X2 (SEQ ID NO: 29) or (r) FLGVLAL-X2 (SEQ ID NO: 28).

7. The peptide according to any one of the preceding claims, wherein the peptide is: (a) FLGVLALLGYLAVRPFLPKKKQQKKKKKKK (SEQ ID NO: 1); (b) FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2); (c) flgvlallgylavrpflpkkkqqkkkkkkk (SEQ ID NO: 3); (d)FLGVLALLGYLAVRPFLPKKKQQKX 2-10 (SEQ ID NO: 4) (e) FLGVLALLGyLAVRPfLPKkKQQK-X 2-10 (SEQ ID NO: 5); or (f) FLGVLALLGYLAVRPFLPKKKQQKKKKKK (SEQ ID NO: 29).

8. The peptide according to any one of the preceding claims, wherein the analog comprises: (a) Modifications of 1 to 10, 2 to 8, or 3 to 6 of the amino acid sequence; (b) Modifications of 1 to 6 of the amino acid sequence; (c) One to three modifications to the amino acid sequence; (d) Modifications of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acid sequence; (e) A polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31; or (f)(a)-(e) analogues containing conservative substitutions.

9. The peptide according to any one of the preceding claims, wherein the peptide has a length of 17 to 50 amino acids.

10. The peptide according to any one of the preceding claims, wherein the peptide is a reverse-configuration sequence.

11. A polynucleotide encoding a peptide according to any one of claims 1-10.

12. A conjugate comprising the peptide and portion according to any one of claims 1-10.

13. The coupling according to claim 12, wherein the portion is: (a) PEG; (b) PEG of (a), wherein the molecular weight of the PEG is in the range of 350 to 10,000 Da; or (c) (a)-(b) PEG, wherein the PEG has 5 to 100 ethylene glycol monomers.

14. The coupling according to any one of claims 12-13, wherein the portion is: (a) A radioactive isotope, preferably wherein the radioactive isotope is 131I, 68Ga, 177Lu, 153 Sm、 212 Pb, 166 Ho、 111 In, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90, and thorium-227; (b) The signal-generating component, such as a fluorescent compound, preferably such as fluorescein, anthocyanin dye, or Alexa Fluor. ® ;or (c) Signal generation components, such as PET or SPECT markers, for example 18 F, 99m Tc, 68 Ga、 11 C 13 N、 15 O、 64 Cu、 86 Y、 55 Co、 123 I, 131 I, 99m T, 89 Zr、 133 Xe and 201 TI.

15. A pharmaceutical composition comprising a peptide according to any one of claims 1-10 or a conjugate according to any one of claims 12-14.

16. A method of treating a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of the peptide according to any one of claims 1-10, the conjugate according to any one of claims 12-14, or the pharmaceutical composition according to claim 15.

17. The method of claim 16, wherein the method is used to treat cancer.

18. The method of claim 17, wherein the cancer is selected from breast cancer, prostate cancer, gastric cancer, colon cancer, rectal cancer, cervical cancer, endometrial cancer, bile duct cancer, ovarian cancer, myeloma, glioma, glioblastoma, neuroblastoma, melanoma (including malignant melanoma), CML, AML, ALL, liver cancer, pancreatic cancer, head and neck cancer, lung cancer, bladder cancer, kidney cancer, non-Hodgkin's lymphoma, and laryngeal cancer.

19. The method of claim 17 or 18, wherein the cancer is accompanied by enhanced NAF-1 protein expression.

20. The method according to any one of claims 16-19, wherein the method further comprises administering an anticancer therapy, preferably wherein the anticancer therapy is an immunomodulator, activated lymphocytes, lymphocyte activators, kinase inhibitors, chemotherapeutic agents, or anticancer agents.

21. A method for diagnosing cancer, wherein the method comprises detecting cancer cells using a peptide according to any one of claims 1-10, a conjugate according to any one of claims 12-14, or a pharmaceutical composition according to claim 15.

22. The method of claim 21, wherein the peptide of any one of claims 1-10, the conjugate of any one of claims 12-14, or the pharmaceutical composition of claim 15 is labeled.

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