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 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

POSITIVELY CHARGED NAF-1 DERIVED PEPTIDES AND USES THEREOFFIELD OF THE INVENTION

[0001] The disclosure is directed to peptides derived from human NAF-1 protein, analogs thereof and their use in treating various types of cancer.BACKGROUND OF THE INVENTION

[0002] Nutrient-deprivation autophagy factor-1 (NAF-1, CISD2) is a unique iron-sulfur (2Fe-2S) protein that belongs to the NEET protein family and is encoded by the CISD2 gene in humans. Recent studies implicated NAF-1, localized in the endoplasmic reticulum (ER), the mitochondrial outer membranes, and the ER-mitochondria-associated membranes (MAM). NAF-1 is involved in a diverse array of biological processes and diseases, such as the regulation of autophagy, apoptosis, ferroptosis, iron and reactive oxygen species (ROS) homeostasis, and cancer. Enhanced expression of NAF-1, is associated with different cancers, including breast, prostate, gastric, cervical, lung, liver, glioma, neuroblastoma, head and neck, 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, Y. Z. 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; E.H. Kim et al. Cancer Lett., 432 (2018), p.180-190, Karmi et al. Free Radicals in Biology and Medicine, 176, p. 92-104. (2021))

[0003] Reduction of NAF-1 expression in breast, or gastric cancer cells significantly inhibited cellular proliferation and tumorigenicity, whereas overexpression of NAF-1 in these cells significantly enhanced cellular proliferation. Darash-Yahana et al., (PNAS, 2016, 113(39), 10890- 10895) showed that NAF-1 overexpression in xenograft tumors results in a dramatic augmentation in tumor size and aggressiveness; conversely, inhibition of NAF-1 expression by shRNA dramatically reduced tumor size and aggressiveness.

[0004] Development of a therapy for cancer is a tedious task requiring a constant search for novel targets as well as the generation of novel therapeutic agents affecting these targets. There is a clear and constant need to develop both strategies and to provide novel therapeutic agents for treating cancer.SUMMARY OF THE INVENTION

[0005] The present invention is based in part on the surprising finding that adding positively charged amino acids to the C-terminus of the peptide first identified in US 11,208,443 significantly improved its biological cancer-destroying activity, in particular in vivo.

[0006] As described herein is a peptide comprising:(a) an amino acid sequence comprising FLGVLAL-X1-X2 (SEQ ID NOTO), wherein Xi represents a second amino acid sequence LGYLAVRPFLPKKKQQK (SEQ ID NOTO) or a fragment of this second amino acid sequence with sequential deletions at the C-terminus; wherein X2 represents 1-10 positively charged moieties; and wherein any one of the amino acids in the amino acid sequence can be a D-amino acid; or(b) an analog of (a); or(c) a retro-inverso sequence of either (a) or (b).

[0007] In embodiments, the positively charged moiety of the peptide is a positively charged amino acid, a lysine (K); or an arginine (R). Preferably, the positively charged moiety is poly-lysine. For example, X2 can be 3-10 positively charged moieties; 4-7 positively charged moieties; or 6 positively charged moieties.

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

[0009] In other embodiments, the peptide can be described as comprising the 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);(1) 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 exemplified 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) FLGVLALLGYLAVRPFLPKKKQQKX2-10 (SEQ ID NO:4);(e) FLGVLALLGyLAVRPfLPKkKQQK-X2-io (SEQ ID NO: 5); or(f) FLGVLALLGYLAVRPFLPKKKQQKKKKKK (SEQ ID NO:29).[Oi l] In other embodiments, the peptide analog as described herein comprises:(a) 1 to 10, 2 to 8, or 3 to 6 modifications of the amino acid sequence;(b) 1 to 6 modifications of claim of the amino acid sequence;(c) 1 to 3 modifications of claim of the amino acid sequence;(d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications of the amino acid sequence;(e) a polypeptide sequence 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 to any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31; or(f) the analog of (a)-(e) comprising conservative substitutions.

[0012] Additional embodiments include, but are not limited to, peptides 17 to 50 amino acids in length. Also included are retro-inverso sequence of the peptide. Additional embodiments include polynucleotides encoding the peptides described herein as well as conjugates comprising the peptides described herein along with a moiety.

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

[0014] Alternatively, the moiety is:(a) a radioactive isotope, preferably wherein the radioactive isotope is 1311, 68Ga, 177Lu, 153Sm,212Pb,166HO,n iIn, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90 and thorium-227;(b) a signaling moiety, such as a fluorescent compound, preferably such as fluorescein, cyanine dyes or Alexa Fluor®; or(c) a signaling moiety, such as a PET or SPECT label, such as18F, "mTc,68Ga,nC,13N, 15O,64Cu,86Y,55CO,123I,131I, "mT,89Zr,133Xe and201TI.

[0015] Also contemplated are pharmaceutical compositions comprising the peptide, including the peptide conjugates, as described herein. Uses of such pharmaceutical compositions are contemplated.

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

[0017] In other embodiments, the cancer that is treated as described herein is associated with an enhanced expression of NAF-1 protein.

[0018] In embodiments, the method of treatments further comprises administering an anti-cancer therapy, preferably wherein the anti-cancer therapy is an immune-modulator, an activated lymphocyte, a lymphocyte activating agent, a kinase inhibitor, a chemotherapeutic agent or an anti-cancer agent.

[0019] Additionally, a method of diagnosing cancer is also contemplated. In this embodiment, the peptide, the conjugate or the pharmaceutical composition can be used to detect cancer cells. In some embodiments, the peptide comprises a label that is used to detect the cancer.

[0020] In other aspects, provided is a peptide comprising:(a) the ammo acid sequence FLGVLALLGYLAVIU’FLPKKKQQK-X (SEQ ID NO:4), wherein X is a positively charged moiety;(b) an analog of (a);(c) a retro-inverso sequence of (a) or (b);(d) a fragment consisting of at least 17 consecutive amino acids of (a), (b) or (c),wherein the peptide consists of 17 to 50 amino acids.

[0021] By a preferred embodiment at least one, at least two, preferably at least three amino acids are replaced by D-amino acids (presented as small cap letters) so the peptide is:FLGVLALLGyLAVRPfLPKkKQQK-X (SEQ ID NO: 5)

[0022] The positively charged moiety X may be a non-amino acid-based moiety such as polyamines and others.

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

[0024] By a specific example, X is 6 Lys residues in tandem and the peptide has the following structure:FLGVLALLGYLAVRPFLPI<I<I<QQI<I<I<I<I<I<I< (SEQ ID NO:1)

[0025] By another example, 3 amino acids of the peptide above were replaced by D-amino acids (represented in small cap letters) so that the peptide has the following formula SEQ ID NO:2:FLGVLALLGyLAVRPILPKkKQQKKKKKKK (SEQ ID NO: 2) which is also referred to herein 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 specific embodiment, the peptide consists 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 analog of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 wherein said analog comprises from 1 to 6 amino acid substitutions, deletions or additions. According to some embodiments, the analog comprises 1-3 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, at least one amino acid substitution is into the corresponding D-amino acid beyond the already 3 amino acidssubstituted by its corresponding D analog of SEQ ID NO:2. According to some embodiments, the peptide comprises a sequence of all D-amino acids. According to a further embodiment, the peptide comprises the amino acid sequence flgvlallgylavrpflpkkkqqkkkkkkk (where the small case letters refer to D-amino acids) as set forth as SEQ ID NO:3. According to another embodiment, the peptide consists of SEQ ID NO:2. According to yet other embodiments, the peptide comprises a retro-inverso sequence of any one of SEQ ID NO: 1-3.

[0028] According to another embodiment, the peptide comprises an analog of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 wherein said analog comprises a substitution selected from the group consisting of N-methylation of the amide nitrogen, acetylation of the N-terminus and non-proteinogenic amino acids.

[0029] According to another embodiment, the peptide comprises an analog of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 wherein said analog comprises one or more peptidomimetics. In some embodiments, the peptidomimetic substitution is selected from the group consisting of reduced amides, azapeptides and peptoids.

[0030] According to a further embodiment, the peptide comprises a fragment of 17 to 28 consecutive amino acid residues of a sequence selected from SEQ ID NO: 1 or SEQ ID NO:2, analog of SEQ ID NO: 1 or SEQ ID NO:2, or a retro-inverso sequence of an analog 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, provided is a conjugate of a peptide as disclosed herein with another moiety. According to some embodiment, the conjugate comprises at least one polyethylene glycol (PEG) molecule and a peptide comprising an amino acid selected from any one 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 to 1,000 Da.

[0033] According to a specific embodiment, the conjugate comprises a peptide as described herein and a PEG moiety conjugated to at least one of its termini.

[0034] According to some aspects, provided is a pharmaceutical composition comprising at least one peptide or at least one conjugate as disclosed herein. According to some embodiments, the pharmaceutical composition comprises a peptide of 17-50 amino acids comprising:(a) the amino acid sequence FLGVLALLGYLAVRPFLPKKKQQK-X as set forth in SEQ ID NO: 4; or the amino acid sequence of FLGVLALLGyLAVRPfLPKk KQQKKKKKKK as set forth in SEQ ID NO: 2(b) an analog of (a);(c) a retro-inverso 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 a molecule connected to said peptide to form a conjugate.

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

[0036] According to another aspect, the pharmaceutical composition disclosed herein is for use in treating cancer. According to some embodiments, the cancer is associated with an enhanced expression of NAF-1 protein. According to another embodiment, the cancer is selected from breast, prostate, gastric, cervical, liver, pancreas, head, neck, leukemia, lymphoma, myeloma, ovarian, myeloma, glioblastoma, malignant melanoma, CML, AML, ALL, and laryngeal cancer. The pharmaceutical composition can also be used to treat cancer metastasis and not only the primary tumor.

[0037] According to a further aspect, provided are methods for treating cancer in a subject in need thereof comprising administering to said subject a peptide, a conjugate or a pharmaceutical composition as disclosed herein. According to one embodiment, the pharmaceutical composition comprises an effective amount of the peptide or of the conjugate as disclosed herein. The pharmaceutical composition may be administered using any suitable method. According to some embodiments, the pharmaceutical composition is administered parenterally, for example 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.BRIEF DESCRIPTION OF THE FIGURES

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

[0039] FIG. 1 shows the approximate location, spanning the membrane (mitochondrial / ER / MAM) and extending to the cytoplasm, of the native NAF-1 peptide SEQ ID NO:6. The NAF-1 peptide (described within the box) derives from the original sequence of NAF- 1 (upper line, from the amino acid 44 until the amino acid 67) and the modifications in the novel 3D-NAF-1-6K peptide (SEQ ID NO:2) sequence are highlighted in red (lower line).

[0040] FIG. 2 shows analytical data for the 3D-NAF-1-6K peptide. Shown are the analytical HPLC and ESI-MS of the pure peptide. Molecular weight is 3496.5 and the visual appearance is a white powder. The analytical HPLC was run using a Cl 8 column, 20%-50% acetonitrile in TDW, 25 min gradient.

[0041] FIG. 3 shows that 3D-NAF-1-6K peptide has substantially higher cytotoxicity towards breast cancer cells than the prior art peptide disclosed in US 11,208,443 (SEQ ID No: 7, denoted here as the 3D-NAF-1 peptide), while remaining non-toxic towards healthy cells. Cytotoxicity of each peptide towards the malignant MDA-MB-231 cells (FIG. 3A) and control MCF-10A cells (FIG. 3B) is shown. Cells were plated in 96-well plates at a density of 15,000 per well before the experiment. 1 OpM of either peptide was added to cells and incubated for 1 - or 3-days. Cell viability was assessed using Presto-Blue after 1- and 3-days incubation. ***p < 0.001, ****p < 0.0001 by t-test.

[0042] FIG. 4 shows the cytotoxic effect of 3D-NAF-1-6K and the 3D-NAF-1-6R (FLGVLALLGyLAVRPfLPKkKQQKRRRRRR) (SEQ ID NO:31) peptides on MDA-MB-231 cells. Cells were seeded in a 96-well plate at a density of 1.2 x 104and allowed to culture for overnight. 3D-NAF-1-6K or 3D-NAF-1-6R peptides were incubated with cells for 3, 18 hours at different concentrations. The black bar indicates the cytotoxic effect of 3D-NAF-1-6K, while thegrey bar shows the cytotoxicity of 3D-NAF-1-6R. Cell viability was determined using the fluorescent redox probe, Presto-blue™. Cells are incubated with Presto-blue for 1 hour and Prestoblue fluorescence was measured at 37°C (XeX=560 nm, Xcni=590nm) by a plate reader (Tecan Safire). The results demonstrate that the 3D-NAF-1-6K is more toxic to malignant cells than the 3D-NAF-1-6R.

[0043] FIG. 5 shows the cytotoxic effect of the 3D-NAF-1 -6K peptide on the viability of different malignant cells: MDA-MB-231 (triple negative breast cancer cells), PC-3 (prostate cancer cells), HepG2 (liver cancer cells), and SKOV-3 (ovarian cancer cells), after 1 hour (FIG. 5 A) and 24 hours (FIG. 5B) incubation without peptide (control-blue bars) and at two 3D-NAF-1-6K peptide concentrations (lOpM (red bars) and 50pM (green bars)). Viability was determined by the Prestoblue method.

[0044] FIG. 6 shows two different batches of the 3D-NAF-1-6K peptide (in this figure, O.P. stands for “old” batch of 3D-NAF-1-6K peptide, and N.P. stands for “new” fresh batch of 3D- NAF-1-6K peptide). As shown in FIG. 6 both batches have similar / identical cytotoxic effects on MDA-MB-231 (a triple negative breast cancer cell line), HepG2 (a liver cancer cell line) and SKOV-3 (an ovarian cancer cell line). The O.P. and N.P. were incubated with the malignant cells for 1 hour (FIG. 6A) or 24 hours (FIG. 6B) at a concentration of 20 pM (red / purple bars) or 50 pM (green / blue bars).

[0045] FIG. 7 shows the toxicity of 3D-NAF-1-6K towards different cancer cell lines; Cell survival was measured using the Presto-blue assay after 3 hours incubation with the 3D-NAF-1- 6K peptide at the indicated concentrations. SKOV-3, ovarian cancer (black circle), U87MG, glioblastoma (black rectangle), A375, human malignant melanoma (black up-point triangle), MDA-MB-231, breast cancer (black down-point triangle), PANC-1, pancreas cancer (black diamond), MOLM-14, acute myeloid leukaemia (red circle), and K562, chronic myelogenous leukaemia (yellow rectangle), were treated or untreated with the peptide and assayed for viability and survival. Data are shown as mean ± SD from three independent experiments. ** P<0.01, *** P<0.001, **** p<0.0001 by t-test.

[0046] FIG. 8 shows the IC50 of 3D-NAF-1-6K toward different cancer cell lines. IC50 value for inducing cancer cell death by 3D-NAF-1-6K peptide. The IC50 values for each are indicated.

[0047] FIG. 9 shows the cytotoxic effect of the 3D-NAF-1-6K peptide on the viability of ovarian cancer SKOV-3 3D spheroids. The spheroids were incubated with 20pM or 50pM 3D-NAF-1-6K peptide for 0, 1, 20 and 40 hours. Cell death was determined by the IncuCyte® Red cytotoxicity reagent (Essen Bioscience Cat #4632) (a probe used to measure cell death). Upper panel contains the images of the spheroids and the lower panel shows the quantified cytotoxic effect of the tested peptide.

[0048] FIG. 10 shows the cytotoxic effect of the 3D-NAF-1-6K peptide on the viability of liver cancer HepG2 3D spheroids. The spheroids were incubated with 20pM and 50pM 3D-NAF-1-6K peptide for 0, 1, 20 or 40 hours. Cell death was determined by the IncuCyte® Red cytotoxicity reagent (Essen Bioscience Cat #4632) (a probe used to measure cell death). The upper panel contains the images of the spheroids and the lower panel shows the quantified cytotoxic effect of the peptide.

[0049] FIG. 11 shows the cytotoxic effect of the 3D-NAF- 1 -6K peptide on the viability of prostate cancer PC-3 3D spheroids. The spheroids were incubated with 20pM and 50pM of the 3D-NAF- 1-6K peptide for 0, 1, 20 or 40 hours. Cell death was determined by the IncuCyte® Red cytotoxicity reagent (Essen Bioscience Cat #4632) (a probe used to measure cell death). The upper panel contains the images of the spheroids, and the lower panel shows the quantified cytotoxic effect of the peptide.

[0050] FIG. 12 shows the permeation of 3D-NAF-1-6K into ovarian cancer cells and targeting of mitochondria. (FIG. 12A) Representative confocal fluorescence images of SKOV-3 ovarian cancer cells at different time points following treatment with 3D-NAF-1-6K peptide. (FIG. 12B and 12C) Quantitative analysis of 3D-NAF-1-6K and free Fluorescein permeation into the SKOV-3 cells (FIG. 12B) and mitochondrial damage (FIG. 12C) as accessed by mitochondria membrane potential loss. Data are shown as mean ± SD of 15 cells per field (10 fields in total) calculated for each time-point obtained from 3 independent experiments. ***P < 0.001 by t-test.

[0051] FIG. 13 shows that the 3D-NAF-1-6K peptide causes the fragmentation of mitochondria in SKOV-3 cells. FIG. 13A shows representative confocal fluorescence images of mitochondria in SKOV-3 ovarian cancer cells treated with different concentrations of the 3D-NAF-1-6K peptide peptide. FIG. 13B shows quantitative analysis of mitochondrial length at different concentrationsof the peptide. Data is shown as mean ± SD of 25 cells in 8 fields images calculated from 3 independent experiments. ***P < 0.001, ****p < 0.0001, by t-test.

[0052] FIG. 14 shows the measurements of mitochondrial length in SKOV-3 ovarian cancer cells after treatment with the 3D-NAF-1-6K peptide. FIG. 14A shows representative confocal fluorescence images of mitochondria in SKOV-3 cells treated / untreated with the 3D-NAF-1-6K peptide. FIG. 14B shows that mitochondrial length was translated into skeleton binary images in the control (without peptide) and 50pM of the peptide treatment. The mitochondrial length was measured in 25 cells from 8 field images and 156 mito / cell in the control and 58 mito / cell in 50pM peptide treatment were measured using the Image J program. Mitochondrial length in the magnified images was measured using the free hand tool in image J program.

[0053] FIG. 15 shows that the 3D-NAF- 1 -6K peptide inhibits the activity of thioredoxin reductase (tRXr) in MDA-MB-231 cancer cells. The upper panel (FIG.15A) shows that the green fluorescence of thioredoxin reductase was followed by epi-fluorescent microscope at 0, 0.5 hr, 1 hour, 3 hours and 6 hours after the addition of the 3D-NAF-1-6K peptide. The lower panel (FIG. 15B) quantifies the data presented in the different confocal images shown in FIG. 15A. In this experiment, 10 pM of TRFS-green was loaded for 6 h and 15 pM of 3D-NAF-1-6K peptide was added.

[0054] FIG. 16 schematically shows our hypothesis as to how the 3D-NAF-1-6K peptide disrupts the a-helices of the transmembrane domains of the NAF-1 homodimer and facilitates the disruption of the mitochondrial outer membrane.

[0055] FIG. 17 shows the impact of 3D-NAF-1 -6K on primary xenograft SKOV-3 ovarian cancer tumours. (FIG. 17A) Tumour volume (mm3) of three mice groups: control (saline; blue), intravenously (IV; orange), and subcutaneously (SC; grey) injected groups. Representative images are shown on right. (FIG. 17B) Changes in mice weight of the three groups over the treatment duration. 0.5 mg / Kg of 3D-NAF-1-6K peptide was used in each peptide treatment, injections were applied 6 times over the course of 3 weeks. All results are presented as mean ± SEM. Significance was determined using t-test; *P<0.1, **P<0.01.

[0056] FIG. 18 shows that immunohistochemistry of mice having SKOV-3 tumors that were then injected with the 3D-NAF-1-6K peptide and probed with an antibody to the F4 / 80 mouse macrophage-specific marker, indicating a high number of tumor-infiltrating macrophages.

[0057] FIG. 19 shows that the 3D-NAF-1-6K suppresses the metastatic growth of xenograft SKOV-3 ovarian cancer. FIG19A shows decrease in tumour nodules weight (grams) in lungs of mice injected IV with SKOV-3 cells and treated with 3D-NAF— 6K peptide at either 0.25 or 0.5 mg / Kg per mouse. Injections were performed twice a week intravenously (IV). Taxol 20 mg / Kg (once a week) was injected IV and used as positive control. FIG. 19B shows decrease in colon nodule metastasis (Nodules number) following treatment with Saline (control) or the 3D-NAF-1- 6K peptide. All results were presented as mean ± SEM. Significance was determined using t-test; **P<0.01, ***P <0.001, ****P <0.0001.

[0058] FIG. 20 shows the effect of 3D-NAF-1-6K peptides on the migration of SKOV-3 cells. FIG.20A shows time-lapse high-definition phase contrast images of the control untreated (upper panels) and treated with 3D-NAF-1-6K peptides (lOpM, middle panels, 20pM, bottom panels) at 0, 48, and 96 h after culture cover slide removal. The dotted lines define the area lacking cells. Scale bars, 100pm. FIG. 20B presents the quantification of the cell migration during 96 h by untreated (blue) and treated with 3D-NAF-1-6K peptides, 5pM (orange), lOpM (grey), 20pM (yellow). Results represent the mean of four measurements of cell migration, obtained in 3 independent experiments (n = 12). **P < 0.01, *** P < 0.001. The corresponding p-values obtained by t-test.

[0059] FIG. 21 shows internal specified organs isolated from mice injected IV with 3D-NAF-1- 6K peptide. All organs, e.g., liver, kidney, colon, brain lungs and others had normal colors.

[0060] FIG. 22-24 show that only slices of the shrunken breast MDA-MB-231 tumors had green fluorescence when analyzed and quantified in a confocal microscope, indicating uptake of the Fl- 3D-NAF-1-6K peptide (labeled in FIG. 22 as FL-3D-NAF-1-6K). FIG. 23 shows representative confocal images of tumor’s isolated cells from the mice treated with or without the 3D-NAF-1-6K peptides in initial stages of treatment. FIG. 24 shows tumor cells before and after treatment that were stained with RPA to identify mitochondria and H342 to identify the nucleus.

[0061] FIG. 25 shows that Cy5-3D-NAF-1-6K peptide injection into nude mice with tumors formed from MDA-MB-231 cells. FIG. 25A shows images from the mice which were injected intravenously with either saline control or 0.5mg / Kg of the Cy5-3D-NAF-1-6K at the following time points 0, 2, 12, 96, 140 and 170 hours. Mice were followed and imaged over time in order to track the peptide movement and accumulation. FIG.25B shows the fluorescence intensity overtime of the Cy5-3D-NAF-1-6K peptide in the tumor, liver and kidneys. FIG.25C shows the fluorescence accumulation in the organs of the mouse and the tumor following necropsy in order to investigate the peptide accumulation in the different organs and tumor.

[0062] FIG. 26 shows that the 3D-NAF-1-6K peptide is non-toxic. Mice were injected intraperitoneally with 3D-NAF-1-6K using a concentration of 0.5mg / Kg or 5mg / Kg, while the control group was injected with Saline. Mice were injected with either saline or the 3D-NAF-1- 6K peptide at a frequency of 3 times a week for 4 weeks. FIG. 26A. CBC results of male and female mice after treatment with the 3D-NAF-1-6K peptide. Abbreviations; WBC: white blood cells, RBC: Red Blood cells, HGB: Hemoglobin, Pit: Platelets, Lymph: Lymphocytes cells, Neut: Neutrophils cells. FIG. 26B. shows the change in body weight and change in organ weights over time, along with representative H&E-stained sections of kidney, liver, and spleen following the treatment of normal mice with either saline or with either 0.5mg / Kg 3D-NAF-1-6K or 5mg / Kg 3D-NAF-1-6K. (a) Male mice (upper) and female mice (lower) body weights over time.Concentrations used: 0.5mg / Kg 3D-NAF-1-6K (orange), 5mg / Kg 3D-NAF-1-6K (grey) and saline control (blue), (b) Bar graphs showing the weight of the different organs harvested from Male mice (Upper Panel) and Female mice (Lower Panel) mice, treated or untreaded with the 3D- NAF-1-6K peptide at two different concentrations, (c) representative H&E-stained sections of kidney, liver, and spleen, taken from the mouse organs shown in (b), showing similarity between the tissues treated or untreated.

[0063] FIG. 27 shows that increasing concentrations of BSA (bovine serum albumin) delayed the cytotoxicity activity of the 3D-NAF-1-6K peptide on the breast cancer cell line, MDA-MB-231. The concentrations of BSA were 0.125 mg / ml (purple bars), 0.25 mg / ml (green bars) and 0.5 mg / ml (red-orange bars). The peptide without BSA (red bars) served as a positive control and a negative control is shown with blue bars. Incubation of the cells without / with free / BSA present peptides were for 3, 6, 12, 24, and 72 hours. Cytotoxicity was determined by presto-blue method in cells imaging plate. These findings indicate that the peptide binds to Albumin and probably the latter assists its delivery in vivo.

[0064] FIG. 28: shows the effect of the 3D-NAF-1-6K peptide on A375 Melanoma cancer cells. FIG. 28A Shows that 3D-NAF-1 -6K peptide has high cytotoxicity towards melanoma cancer cells, in a concentration dependent manner. Cells were plated in 96 well plates at the density of 15,000per well before the experiment. 0, 5, 10, 25, and 50pM of the 3D-NAF-1-6K peptide was added to cells and incubated for 3 or 24 hours. Cell viability was assessed using Presto-Blue after 3- and 24-hours incubation. ****p < 0.0001 by t-test. FIG. 28B Mice carrying A375 cancer xenografts were distributed into 2 groups, a control group and the 3D-NAF-1-6K peptide treated group which received 0.75mg / Kg of the 3D-NAF-1-6K 3 peptide intravenously, three times a week for three weeks. Male group (upper panel) and female group (lower panel). Graphs representing the tumor growth volume (mm3), and the picture of a representative tumor after extracted from the mice flank, scale bar= 1.0 cm. FIG.28C Male group body weights (upper panel) and Female group body weights (lower panel). Graph represents the body weights of the mice (grams) during the course of the experiment. The fact that treatment with the peptide does not affect the body weights of the mice is indicative that the 3D-NAF-1-6K is not toxic.DETAILED DESCRIPTION OF THE INVENTION

[0065] Reference is made to US patent 11,208,443, by some of the current inventors which is hereby incorporated by reference in its entirety. This document teaches that peptides derived from a specific region of the NAF1 protein were found to selectively penetrate cancer cells and kill them. Specifically, as disclosed herein, the peptide first disclosed in US Patent 11,208,443 (FLGVLALLGYLAVRPFLPKKKQQK (SEQ ID NO:6) and a peptide derived from this sequence that includes three D-amino acids substitutions (FLGVLAELGyLAVRPfLPKkKQQK (SEQ ID NO:7)) were found to have substantially and unexpectedly improved cytotoxic activity when additional positively charged amino acids, preferably lysines, are added to the C-terminus. The sequences disclosed in US patent 11,208,443, including SEQ ID NO:6-7 are excluded (“disclaimed”) from the scope of the peptides as disclosed herein.

[0066] The term "peptide" as used herein refers to a short chain of amino acid monomers linked by peptide bonds, i.e., the covalent bond formed between carboxyl group of one amino acid and an amino group of another amino acid. According to some embodiments, the peptide can comprise up to 50 amino acids. According to some embodiments, the peptide consists of 12 to 45 amino acids. According to another embodiment, the peptide consists of 15 to 40 amino acids. According to some embodiments, the peptide comprises 17 to 35 amino acids. According to other embodiments, the peptide consists of 20 to 30 amino acids. According to another particularembodiment, the peptide consists of 17 to 29 amino acids. According to some embodiments, the 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 concerned, they are fragments (of 17-24 amino acids in length) of the prior peptide of US patent 11,208,443, FLGVLALLGYLAVRPFLPKKKQQK (SEQ ID NO: 6), while the positively charged moiety X, in particular that have 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 as long as in that fragment, the positively charged moiety, especially a stretch of 2- 10 positively charged amino acids, is not deleted and is present at the same length as of the parent peptide from which it is derived. According to other embodiments, the peptide comprises a fragment of an analog of SEQ ID NO:1 or SEQ ID NO:2. According to certain embodiments, the peptide comprises a fragment of a retro-inverse sequence of SEQ ID NO: 1 or SEQ ID NO:2 or analog 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 stretch of the positively charged amino acids at the C-terminal is maintained identical to that 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 such 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 SEQ ID NO: 1 or SEQ ID NO:2. According to some such embodiments, the fragment comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9) (attached to a stretch of positively charged amino acids). According to other embodiments, the fragment consists of 17 to 28 consecutive amino acids of an analog 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 of consecutive amino acids of the analog of SEQ ID NO: 1 or SEQ ID NO:2 as described herein.

[0070] Thus, starting with the fragment FLGVLAL (SEQ ID NO:9), the peptide disclosed herein comprises the following structure:FLGVLAL-X1-X22 (SEQ ID NO: 10) wherein Xi represents LGYLAVRPFLPKKKQQK (SEQ ID NO: 30) or a fragment of this sequence, sequentially deleted from the C-terminus; and wherein X2 represents 1-10 positively charged moieties.

[0071] Thus, 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) orFLGVLAL-X2(SEQ ID NO:28).

[0072] In these peptides (e.g., SEQ ID NO: 10-28), X2 is one to ten positively charged moieties. In some embodiments, the positively charged moiety is a positively charged amino acid. 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 acid is the same, and is either arginine or lysine, preferably lysine. As disclosed herein, the each of the positively charged amino acids in X2 can be either a D or L amino acid.

[0073] Exemplary representative peptide species encompassed by SEQ ID NO: 11 (for example) include, but are not limited to:FLGVLALLGYLAVRPFLPI<I<I<QQI<I<I<I<I<I<I< (SEQ ID NO:1)FLGVLALLGYLAVRPFLPI<I<I<QQI<I<I<I<I<I< (SEQ ID NO: 29)

[0074] As described in detail below, each of the peptides described herein, such as those represented by any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 can also include (a) substitutions (including conservative substitutions); (b) replacement of any one or more L amino acids with D amino acids at each amino position; (c) conjugation to other molecules, and / or (d) association with other molecules. Moreover, retro-inverso peptides of each of the peptides represented by any one of SEQ ID NO: 1 -5 or SEQ ID NO: 10-29 or 31 are also contemplated.

[0075] The term “analog” refers to an amino acid sequence in which at least one amino acid of the parent sequence is modified while retaining the functionality of the parent peptide. Examples of such modifications of 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 "unnatural" or "non-natural" amino acids.

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

[0078] The term “conservative substitution” as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and biological activity of thepeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:(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 may be affected with non-natural amino acids. The term “non-natural amino acids” refers to amino acids having structures different from those of natural amino acid species. In one embodiment, the non-natural amino acid is a D-amino acid. Another examples of non-natural amino acids include ornithine, 3 -substituted tyrosine, azidoalanine, azidohomoalanine, norleucine, norvaline, 4- aminotryptophan, 7-azatryptophan, 6-methyltryptophan, acetyllysine, s-Boc-lysine, s- methyllysine, 1 -naphthylalanine, 2-naphthylalanine, styrylalanine, diphenylalanine, thiazolylalanine, 2-pyridylalanine, 3 -pyridylalanine, 4-pyridylalanine, anthrylalanine, 2-amino-5- hexynoic acid, furylalanine, benzothienylalanine, thienylalanine, allylglycine, propargylglycine, phosphorylserine, phosphorylthreonine, and 2,3-diaminopropionic acid.

[0080] According to some embodiments, the analog comprises from 1 to 10, 2 to 8, or 3 to 6 modifications. According to one embodiment, the analog comprises from 1 to 6 modifications or 1 to 3 modifications. According to another embodiment, the analog comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. According to some embodiments, the modification is a substitution, such as a conservative substitution. According to one embodiment, the analog comprises 1 to 6 conservative substitutions.

[0081] According to some embodiments, the analog comprises the amino acid sequence FLGVLAL (SEQ ID NOV).

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

[0083] According to one embodiment, the analog comprises amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 or a modified SEQ ID NO:1 or SEQ ID NO:2 in which at least one Lys residue is substituted by another positively charged amino acid. According to one embodiment, at least one of the Lys residues is substituted by Arg.

[0084] According to some embodiments, the analog comprises at least one conservative 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, the analog comprises lOTyr, 16Phe, and 20Lys amino acids. To clarify, the number to the left of the amino acid name represents its sequential number in SEQ ID NO:1 (or the equivalent position in any one of SEQ ID NO:10-29 and 31). For example, lOTyr or 10Y refers to the tyrosine at the 10th position of SEQ ID NO: 1 (or the equivalent position in any one of SEQ ID NO: 10-29 and 31).

[0085] For example, representative examples of analogs of SEQ ID NO: 11, include, but are not limited to the following peptides (D-amino acids are represented by lowercase letters):FLGVLALLGyLAVRPfLPI<kI<QQI<I<I<I<I<I<I< (SEQ ID NO: 2) (also referred to herein 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 still 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 a further embodiment, at least 4 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 still another embodiment, the fragment consists of 17 to 28, 18 to 27, or 19 to 26 consecutive amino acids of sequence SEQ ID NO:3.

[0088] According to any one of the above embodiments, the analog comprises a parent sequence or a modified parent sequence in which at least one of the Phe or Tyr amino acids are substituted by an amino acid with a bulky hydrophobic residue. In one embodiment, IPhe and / or 16Phe is substituted with Tyr or Trp. According to another embodiment, lOTyr is substituted by Phe or Trp. According to a further embodiment, IPhe and / or 16Phe is substituted with Tyr or Trp, and lOTyr is substituted by Phe or Trp. As used herein, the number to the left of the amino acid name represents its sequential number in SEQ ID NO:1 (or the equivalent position in any one of SEQ ID NO:10-29 and 31). For example, lOTyr or 10Y refers to the tyrosine at the 10th position of SEQ ID NO: 1 (or the equivalent position in any one of SEQ ID NO: 10-29 and 31).

[0089] According to any one of the above embodiments, the analog comprises a parent sequence or a modified parent sequence in which at least one of the hydrophobic amino acid residues is substituted by Ala.

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

[0091] According to any one of the above embodiments, the modification may be a substitution 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 the D- configuration around the a-carbon as opposite to native L-amino acid having L-conformation. As used herein, the D-amino acid in the sequence is represented by a lower-case letter, whereas the L-amino acid by a capital letter. As such, the sequence of VyL represents a sequence in which Vai and Leu are native L-amino acids and Tyr is a D-amino acid.

[0092] According to any one of the above embodiments, the analog of any one of SEQ ID NO: 10- 29 and 31 comprises at least one D-amino acid. According to another embodiment, the analog comprises from 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 analog comprises 1, 2 or 3 D-amino acids. According to one embodiment, the analog 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 the equivalent position in any one of SEQ ID NO: 10-29 and 31) is a D-amino acid. According to a further embodiment, at least one of 10Y, 16F, and 20K of SEQ ID NO:1 (or the equivalent position in any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31) is a D-amino acid. According to yet another embodiment, the amino acids 10Y, 16F and 20K of SEQ ID NO: 1 (or the equivalent positions in any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31) are D-amino acids. According to one embodiment, the analog has the amino acid sequence: FLGVLALLGyLAVRPfLPI<kI<QQI<I<I<I<I<I<I< (SEQ ID NO:2). Thus, 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 4 D-amino acids. According to another embodiment, the analog comprises at least 5, at least 6 or at least 8 D-amino acids. According to one embodiment, all amino acids of the analog are D-amino acids. According to yet another embodiment, the analog comprises the amino acid sequence in which all amino acids of SEQ ID NO: 1 are substituted by corresponding D-amino acids. According to one embodiment, all residues of such a peptide are all D-amino acids.

[0094] According to some embodiments, the peptide comprises a retro-inverso 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 “retro-inverso peptide” and “retro-inverse peptide” are used herein interchangeably and refer to a reverse peptide composed of D-amino acids (retro-reversed; as described in Regenmortel and Muller: Current Opinion in Biotechnology 9, pp 377-382 ,1998).

[0096] As used herein the terms "comprise(s)," "include(s)," "having," "has," "contain(s)," and variants thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structure. The term “comprising” always includes the term “consisting” and when the term “comprising” appears in an embodiment of the disclosure, this same embodiment wherein the term “consisting” replaces the term “comprising” is always also an embodiment of the disclosure. Thus, in certain embodiments, the peptide consists of the amino acid sequence selected from any one of SEQ ID NO: 1-5 or any one of SEQ ID NO: 10-29 or 31 , an analog thereof, or a retro-inverso thereof.

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

[0098] According to some embodiments, provided is a peptide comprising an amino acid sequence having at least 70% identity to the sequences selected from SEQ ID NO: 1-5 or any one of 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 to the sequences selected from SEQ ID NO: 1-5 or any one of SEQ ID NO: 10-29 or 31.

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

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

[0101] The term “conjugate” as used herein refers to the association of a peptide with another moiety. According to some embodiments, the moiety is a non-peptidic moiety. According to some embodiment, the peptide is conjugated with polyethylene glycol (PEG), Poly(N-vinylpyrrolidone), polyglycerol, a permeability enhancing moiety and polysaccharides such as glycosyl. According to another embodiment, the peptide is conjugated with a marker. According to some embodiments, the marker is a fluorescent marker, such as fluorescein.

[0102] The term “permeability-enhancing moiety” refers to any moiety known in the art to facilitate actively or passively or enhance permeability of the compound through body barriers or into the cells. Non-limitative examples of permeability-enhancing moiety include: hydrophobic moieties such as fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides, nanoparticles and liposomes.

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

[0104] According to some embodiments, the PEG molecule has a molecular weight (MW) between about 300 daltons to about 100,000 daltons. According to other embodiments, the PEG molecule has a MW of from about 400 to about 10,000, about 1000 to about 8,000, about 2000 to about 6,000 and about 3,000 to about 5,000 dalton. According to other embodiments, PEG molecule have molecular weight selected from about 10,000 Da to about 20,000 Da, from about 20,000 Da to about 30,000 Da, from about 30,000 Da to about 40,000 Da, from about 40,000 Da to about 50,000 Da, from about 50,000 Da to about 60,000 Da, from about 60,000 Da to about 70,000 Da, and from about 70,000 Da to about 80,000 Da. Non-limiting examples of average molecular weights of the PEG moieties are about 350, 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 from 5 to 200 ethylene glycol monomers. According to other embodiments, PEG has from 6 to 150 ethylene glycol monomers. According to other embodiments, PEG has from 8 to 120, from 10 to 100, from 15 to 80 from 20 to 60 or from 30 to 50 ethylene glycol monomers.

[0106] According to some embodiments, the invention provides a conjugate of the peptide comprising or consisting of SEQ ID NO: 1 and PEG. According to some embodiments, the peptide is PEGylated at N-terminus. According to other embodiments, the peptide is PEGylated at C- Terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000Da, 350 to 8,000Da, 400 to 6,000Da, 1,000 to 4,000Da or 2,000Dato 3000Da. According to other embodiments, the PEG has from 5 to 20 or from 6 to 10 ethylene glycol monomers.

[0107] According to some embodiments, provided is a conjugate of the peptide comprising or consisting of any one 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 10,000Da, 350 to 8,000Da, 400 to 6,000Da, 1,000 to 4,000Da or2,000Da to 3,000Da. According to other embodiments, the PEG has from 5 to 20 or from 6 to 10 ethylene glycol monomers.

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

[0109] According to some embodiments, provided is a conjugate of any one of the peptides comprising or consisting of any one 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 N-Terminus. According to other embodiments, the peptide is PEGylated at C-Terminus. According to some embodiments, the PEG has a molecular weight selected from 300 to 10,000Da, 350 to 8,000Da, 400 to 6,000Da, 1,000 to 4,000Da or 2,000Da to 3,000Da. According to other embodiments, the PEG has from 5 to 20 or from 6 to 10 ethylene glycol monomers.

[0110] According to one embodiment, conjugates of a peptide consisting of any one of the amino acid sequences of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 bound through its N-terminus to PEG molecule having between 6 to 10 ethylene glycol monomers are provided. According to another embodiment, a conjugate of a peptide consisting of the amino acid sequence any one of SEQ ID NO: 1 -5 or SEQ ID NO: 10-29 or 31 bound through its C-terminus to PEG molecule having between 6 to 10 ethylene glycol monomers is provided. According to a further embodiment, a conjugate of a peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 bound through both its N-terminus and C-terminus to PEG molecule having between 6 to 10 ethylene glycol monomers is provided.

[0111] The conjugate may be more stable than the parent peptide. According to some embodiment, the half-life of a conjugate of a peptide of the present invention is at least 2 times longer than the parent peptide. According to some embodiments, the half-life of a conjugate of a peptide of the present invention is at least 3, 5, 8, or 10 times longer than the parent peptide.

[0112] According to another aspect, provided is a pharmaceutical composition comprising at least one peptide or at least one conjugate of the present invention. According to one embodiment, thepharmaceutical 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 consists of 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 an analog of 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 a further embodiment, the pharmaceutical composition comprises a peptide consisting of an analog of amino acid sequence of any one of SEQ ID NO: 1- 5 or any one of SEQ ID NO: 10-29 or 31. According to such embodiments, the analog is as defined in any one 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 conservative 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, 20K are D-amino acids (as shown in SEQ ID NO:1 or the equivalent positions in any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31). According to another embodiment, the analog has the amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. Thus, the pharmaceutical composition comprises at least one peptide comprising or consisting of amino acid sequence of any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31. According to a further embodiment, at least 4 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 the amino acid sequence SEQ ID NO:3. According to such embodiments, the pharmaceutical composition comprises at least one peptide comprising or consisting of the amino acid sequence SEQ ID NO:3.

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

[0115] According to a further embodiment, the pharmaceutical composition comprises at least one peptide comprising or consisting of 17 to 28 consecutive amino acids of the amino acid sequence selected from SEQ ID NO:1 or SEQ ID NO:2, an analog of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and a retro-inverse sequence of the analog of SEQ ID NO:1 or SEQID No:2 (as long as the stretch of positively charged amino acids at the C-terminal is the same as in the parent peptide). According to some such embodiments, the fragment comprises the amino acid sequence FLGVLAL (SEQ ID NO: 9).

[0116] According to a further embodiment, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence 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 consisting of an amino acid sequence SEQ ID NO:1. According to some embodiments, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence SEQ ID NO:2. According to another embodiment, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence SEQ ID NO:3. According to another embodiment, the pharmaceutical composition comprises at least one fragment of said peptide consisting of 17-28 amino acids of the amino acid sequence selected from SEQ ID NO: 1, 2 or 3. According to a further embodiment, the pharmaceutical composition comprises at least one peptide consisting of an amino acid sequence 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 said peptides. According to one embodiment, the conjugate is PEG conjugate. According to certain embodiments, the pharmaceutical composition comprises at least one conjugate of a peptide comprising an amino acid sequence selected from any one 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 consisting of an amino acid sequence selected from any one 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 N-terminus. According to other embodiments, the peptide is PEGylated at 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 6,000Da, 1,000 to 4,000Da or 2,000Da to 3,000Da. 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 the peptide comprising any one of the amino acid sequences of SEQ IDNO: 1 -5 or SEQ ID NO: 10-29 or 31 and PEG having from 5 to 15 ethylene glycol monomers, such as 8 monomers, bound to the C-terminus or to the N-terminus of the peptide.

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

[0120] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein refer to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical 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 providing supplemental, additional, or enhanced therapeutic functions.

[0121] According to any one of the above embodiments, the peptide may be administered by any known route of administration. The term “administering” or “administration of’ a substance, a compound, an agent or a pharmaceutical composition to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound, an agent or a composition can be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally. A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. In other embodiments, routes of administration include, but are not limited to: local, oral, sublingual, buccal, mucosal, nasal, intravenous, subcutaneous, enteral, intra-arterial, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intra-articular, intraosseous infusion, intracardiac, intravitreal, parenteral, vaginal, intracavernous, intravesicle,rectal, topical, transdermal, inhalation, perivascular, or any combination thereof. In some embodiments, the administering can be in the form of a cream, dressing or spray.

[0122] In one embodiment, the pharmaceutical composition comprising the peptide or the conjugate of the present invention is administered via a systemic administration. For example, the pharmaceutical composition comprising the peptide or the conjugated 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 composition according the present invention may be prepared in any known method. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration. In one particular embodiment, the pharmaceutical composition is formulated as a solid dosage form selected from tablets, capsules, powder or granules. In another embodiment, the pharmaceutical composition is formulated as a liquid or semi-liquid dosage form selected from an elixir, tincture, suspension, syrup, emulsion or gel.

[0124] Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binders; and lubricating agents. The tablets are optionally coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide an extended release of the drug over a longer period.

[0125] According to one aspect, the pharmaceutical composition, according to any one of the above embodiments, is for use in treating 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 NOs: 1-5, a fragment thereof consisting of 17-23 amino acids, or an analog of said peptide or said fragment. Additionally, the pharmaceutical compositioncomprises at least one peptide consisting of the amino acid sequence of SEQ ID NO: 10-29 and 31 or an analog thereof. According to other embodiments, the pharmaceutical composition for use comprises a conjugate of such peptides, e.g. PEG conjugate.

[0126] The term neoplasm means an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. A "cancer" or "tumor" is a neoplasm that has undergone characteristic anaplasia with loss of differentiation, increase rate of growth, invasion of surrounding tissue, and is capable of metastasis. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and / or can metastasize is referred to as "malignant." Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived.

[0127] The term "treating” as used herein refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or parameters associated with cancer, delay or slowing of that impairment, amelioration, palliation or stabilization of that impairment, and other beneficial results. In connection with cancer the treatment refers to reduction of tumor load, malignancy, or metastasis or slowing down the growth of the rate of tumor growth, reduction of the rate tumor grade increase , and reduction in the rate of tumor metastasis spread

[0128] According to some embodiments, the cancer is associated with enhanced or increased expression of 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 one of the above embodiments, the cancer is selected from breast, prostate, gastric, colon, rectal, cervical, endometrial, liver, bile duct, pancreas, head, neck, ovarian, myeloma, glioblastoma, malignant melanoma, CML, AML, ALL, lung, bladder, kidney, non Hodgkins lymphoma, and laryngeal cancer.

[0130] According to other embodiments, the composition is for use in treating cancer selected from mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, myeloma, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarian carcinomas, cervical carcinomas, lung cancer,prostate cancer, kidney cell carcinomas and / or metastases in any organ including metastasis in liver, lymph nodes, bone or lungs.

[0131] According to other embodiments, the composition is for use in treating cancer selected from the following hematological tumors including leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, nonHodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0132] According to other embodiments, the composition is for use in treating cancer selected from the following solid tumors, such as sarcomas and carcinomas, that may be treated according to the methods provided herein include fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

[0133] The pharmaceutical composition comprising the peptide described herein may be administered in combination with at least one other anti-cancer therapy. The peptide can be administered before, during or after the other anti-cancer therapy. The peptide can be administered by the same route of administration or a different route than that of the other anti-cancer therapy.

[0134] Examples of such anti-cancer therapies include, but are not limited to, immune-modulators, activated lymphocytes, lymphocyte activating agent, kinase inhibitor, chemotherapeutic agent or any other anti-cancer agent.

[0135] For example, one example of an anti-cancer therapy includes, but is not limited to, a protein synthesis inhibitor. Examples of a protein synthesis inhibitor include, but are not limited to dactinomycin, plicamycin, omacetaxine, ranpirnase, brefeldin A, and KRN-5500.

[0136] Another example of an anti-cancer therapy includes, but is not limited to, a proteasome inhibitor. Examples of a proteasome inhibitor include, but are not limited to, bortezomib, carfilzomib, and ixazomib.

[0137] Another example of an anti-cancer therapy includes, but is not limited to, a Flt3 inhibitor. Examples of a Flt3 inhibitor include, but are not limited to midostaurin, sorafenib, sunitinib, ponatinib, quizartinib, gilteritinib, and crenolab.

[0138] Another example of an anti-cancer therapy includes, but is not limited to, an IDH inhibitor. Examples of an IDH inhibitor include, but are not limited to ivosidenib, olutasidenib, enasidenib 3BP, di chloroacetate, AGI-5198, and AGI-6780.

[0139] Another example of an anti-cancer therapy includes, but is not limited to, an inhibitor of one or more members of the BCL-2 family. Examples of members of the BCL-2 family, 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 an anti-cancer therapy includes, but is not limited to, an anthracy cline. Examples of anthracycline include, but are not limited to daunorubicin, doxorubicin epirubicin, idarubicin, mitoxantrone, CPX-351, and valrubicin.

[0141] Another example of an anti-cancer therapy includes, but is not limited to, a taxane. Examples of taxane include, but are not limited to paclitaxel, docetaxel, cabazitaxel and Abraxane®.

[0142] Another example of an anti-cancer therapy includes, but is not limited to, a platinum complex. Examples of a platinum complex include, but are not limited to cisplatin, carboplatin and oxaliplatin.

[0143] Another example of an anti-cancer therapy includes, but is not limited to, a vinca alkaloid. Examples of a vinca alkaloid, include, but are not limited to vinblastine, vinorelbine, vincristine and vindesine.

[0144] Another example of an anti-cancer therapy includes, but is not limited to, a ROCK inhibitor. Examples of a ROCK inhibitor, include, but are not limited to ripasudi and netarsudil.

[0145] Another example of an anti-cancer therapy includes, but is not limited to, an antitumor alkylphospholipid. Examples of an antitumor alkylphospholipid include, but are not limited to, edelfosine, ilmofosine, miltefosine, perifosine, erucylphosphocholine and erufosine.

[0146] Another example of an anti-cancer therapy includes, but is not limited to, a Ezrin inhibitor. Another example of an anti-cancer therapy includes, but is not limited to, temozolomide.

[0147] In some other embodiments, the anti-cancer therapy includes, but is not limited to metformin, tamoxifen, 3 -bromopyruvate, 2-deoxy-d-glucose, rotenone, CCCP, IACS-010759, TAXI 65, mubritinib, arsenic trioxide, antimycin A, 5 -fluorouracil, buthionine sulfoximine, auranofin, chlorambucil, alpha-tocopheryl succinate, PX12, clodronate, disulfiram, gracillin, glasdegib, elesclomol, tigecycline, mitotane oxymatrine, capsaicin, casticin, myricetin, forchlorfenuron, piperlongumine, oleanolic acid, celastrol, plumbagin, valproic acid, venetoclax setanaxib, brusatol, trigonneline, and resveratrol.

[0148] In some embodiments, the anti-cancer therapy is an immunotherapy.

[0149] In some embodiments, the immunotherapy is a cellular immunotherapy. In some embodiments, the cellular immunotherapy is selected from the group consisting of 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 the group consisting of CTLA-4 and PD- 1 inhibitors.

[0151] In some embodiments the anti-cancer therapy is a cancer vaccine. In some embodiments the cancer vaccine is a neoantigen vaccine.

[0152] Any of these anti-cancer therapies may result in synergistic activity with the peptide when administered as part of a combination therapy. As used herein, the terms “synergistic,” “synergy,” or “synergism” refers to a combination of two or more medicaments that is more effective intreating a cancer than would be predicted by the additive effect of the two when used separately as single agents. In some aspects, the synergistic combination can kill tumor cells that are resistant to either medicament alone. In some aspects, synergy refers to a more than additive effect of the two or more medicaments at a particular dose or at a suboptimal dose of one or more of the medicaments. Lowering the dose of one or more of the medicaments in a synergistic combination can result in greater efficacy with lower toxicity.

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

[0154] According to some embodiments, the peptide described herein can be conjugated to a radioactive isotope. Examples of radioactive isotopes include, but are not limited to131I,68Ga,177LU,153Sm,212Pb,166HO,n iIn, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90 and thorium-227. 7. Besides its use in treating cancer as described herein, peptides conjugated to a radioactive isotope can also be used as a diagnostic.Formulations and Excipients, Carriers and Diluents

[0155] As described herein, a peptide can be formulated into a “pharmaceutical preparation” or “pharmaceutical composition.” In some cases, a composition herein can comprise a pharmaceutical composition. In some cases, a pharmaceutical composition can be in unit dose form. In some cases, a formulation containing at least one of the peptides described herein, can take the form of liquid, solid, semi-solid or lyophilized powder forms, such as, for example, solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

[0156] In some embodiments, the pharmaceutical composition can include a conventional pharmaceutical carrier or excipient and can additionally include other medicinal agents, carriers, adjuvants, additives and the like. In some cases, the composition can be about 0.1% to about 85%, about 0.5% to about 75% by weight of a compound or compounds of the disclosure, with the remainder consisting essentially of suitable pharmaceutical excipients.

[0157] In some embodiments, the peptide described herein can be packaged and / or resuspended in one or more of the following excipients: acacia, acesulfame potassium, acetic acid-glacial,acetone, acetyltributyl citrate, acetyltriethyl citrate, adipic acid, agar, albumin, alcohol, alginic acid, aliphatic polyesters, alitame, allantoin, almond oil, alpha hydroxy acids, alpha tocopherol, aluminum hydroxide adjuvant, aluminum monostearate, aluminum oxide, aluminum phosphate adjuvant, ammonia solution, ammonium alginate, ammonium chloride, argan oil, ascorbic acid, ascorbyl glucoside, ascorbyl palmitate, aspartame, attapulgite, azelaic acid, azulene, bakuchiol, beta glucan, beta-hydroxy-acids, bentonite, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, boric acid, bronopol, butylated glycol, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, butylparaben, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium hydroxide, calcium lactate, calcium phosphate-dibasic anhydrous, calcium phosphate-dibasic dihydrate, calcium phosphate- tribasic, calcium silicate, calcium stearate, calcium sulfate, canola oil, capric glycol, capric triglyceride, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carrageenan, castor oil, castor oil-hydrogenated, cellulose-microcrystalline, cellulose- microcrystalline and carboxymethylcellulose sodium, cellulose-powdered, cellulose-silicified microcrystalline, cellulose acetate, cellulose acetate phthalate, ceramides, ceresin, cetostearyl alcohol, cetrimide, cetearyl alcohol, cetyl alcohol, cetylpyridinium chloride, chitosan, chlorhexidine, chlorobutanol, chlorocresol, chlorodifluoroethane (hcfc), chlorofluorocarbons (cfc), chloroxylenol, cholesterol, citric acid monohydrate, coconut oil, collagen, colloidal silicon dioxide, coloring agents, copper peptide, copovidone, corn oil, corn starch and pregelatinized starch, cottonseed oil, cresol, croscarmellose sodium, crospovidone, cyclodextrins, cyclomethicone, denatonium benzoate, desitin, dextrates, dextrin, dextrose, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (hfc), dimethicone, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, disodium edetate, docusate sodium, edetic acid, erythorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, ethyl vanillin, ethylcellulose, ethylene glycol stearates, ethylene vinyl acetate, ethylparaben, fatty acids, ferulic acid, fructose, fumaric acid, gelatin, glucose — liquid, glycerin, glycerol, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, glycine, glycofurol, glycolic acid, glycol stearate, guar gum, hectorite, heptafluoropropane (hfc), hexetidine, hydrocarbons (he), hyaluronic acid, hydrochloric acid, hydrocortisone, hydrophobic colloidal silica, mesoporous silica, hydroquinone, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl betadex, hydroxypropyl cellulose, hydroxypropyl cellulose-low-substituted, hydroxypropyl starch, hypromellose, hypromellose acetate succinate, hypromellose phthalate, imidurea, inulin, iron oxides, isomalt, isoparaffin, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, jojoba oil, kaolin, kojic acid, lactic acid, lactitol, lactose-anhydrous, lactose-inhalation, lactose-monohydrate, lactose-monohydrate and corn starch, lactosemonohydrate and microcrystalline cellulose, lactose-monohydrate and povidone, lactose- monohydrate and powdered cellulose, lactose-spray-dried, lanolin, lanolin-hydrous, lanolin alcohols, lauric acid, lecithin, leucine, linoleic acid, macrogol 15 hydroxystearate, magnesium aluminum silicate, magnesium carbonate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, maleic acid, malic acid, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium-chain triglycerides, meglumine, menthol, methionine, methylcellulose, methylparaben, mineral oil, mineral oil-light, mineral oil and lanolin alcohols, monoethanolamine, monosodium glutamate, monothioglycerol, myristic acid, myristyl alcohol, neohesperidin dihydrochalcone, neotame, niacinamide, nitrogen, nitrous oxide, octyldodecanol, oleic acid, oleyl alcohol, olive oil, palmitic acid, paraffin, peanut oil, pectin, PEG-8 stearate, pentetic acid, petrolatum, petrolatum and lanolin alcohols, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phospholipids, phosphoric acid, phytic acid, phytosphingosine, polacrilin potassium, poloxamer, polycarbophil, polydextrose, poly (dl-lactic acid), polyethylene glycol, polyethylene oxide, polymethacrylates, poly(methyl vinylether / maleic anhydride), polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, polyparaben, polysorbate 60, polysorbate 80, polyvinyl acetate phthalate, 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, propylparaben sodium, pyrrolidone, raffinose, Retin- A, retinol and retinoic acid derivatives, saccharin, saccharin sodium, safflower oil, salicylic acid, saponite, sesame oil, shellac, simethicone, sodium acetate, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium borate, sodium carbonate, sodium chloride, sodium citrate dihydrate, sodium cyclamate, sodium formaldehyde sulfoxylate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate — dibasic, sodium phosphate — monobasic, sodium propionate, sodium starchglycolate, sodium stearyl fumarate, sodium ascorbyl phosphate, sodum deoxycholate, sodium hydroxide, sodium lauroyl lactylate, sodium lauryl sulfate, sodium palmitate, sorbitan stearate, sodium sulfite (E221), sprino lactone, sodium sulfite, sodium thiosulfate, sorbic acid, sorbitan esters (sorbitan fatty acid esters), sorbitan monostearate, sorbitol, soybean oil, sphingomyelins, starch, starch-pregelatinized, starch — sterilizable maize, stearic acid, stearyl alcohol, squalene, sucralose, sucrose, sucrose octaacetate, sugar-compressible, sugar-confecti oner’s, sugar spheres, sulfobutylether b-cyclodextrin, sulfur dioxide, sulfuric acid, sunflower oil, suppository bases — hard fat, tagatose, talc, tartaric acid, tetrafluoroethane (hfc), thaumatin, thimerosal, thymol, titanium dioxide, tragacanth, trehalose, tretinoin, triacetin, tributyl citrate, tricaprylin, triethanolamine, triethyl citrate, triethanolamine, triolein, undecylenic acid, vanillin, vegetable oil- hydrogenated, vitamins, vitamin e polyethylene glycol succinate, water, wax-anionic emulsifying, wax-carnauba, wax-cetyl esters, wax-microcrystalline, wax-nonionic emulsifying, wax-white, wax-yellow, xanthan gum, xylitol, zein, zinc acetate, and / or zinc stearate.

[0158] In some cases, a composition such as a pharmaceutical composition can comprise a carrier or a diluent. In some instances, a carrier or diluent can comprise a water, an alcohol, a salt solution (e.g., saline), or a mixture thereof. In some instances, a carrier can comprise a carbohydrate, a buffer, a salt, a pH adjuster, or any combination thereof. In some cases, a composition herein can comprise a buffering agent, a polymer, an antioxidant, a preservative, a chelating agent, a viscomodulator, a tonicifier, a flavorant, a colorant, an odorant, an opacifier, a suspending agent, a binder, a filler, a plasticizer, a lubricant, or any combination thereof.

[0159] In some embodiments, a liquid composition can be prepared by dissolving or dispersing the peptide described herein in one or more of the above formulations (about 0.5% to about 20% by weight or more), and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension. In some cases, for use in an oral liquid preparation, the composition can be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

[0160] In some embodiments, the peptide described herein can be formulated as an injectable composition for parenteral administration (e.g., intravenous, intramuscular, intravesicular or intrathecal). The peptide for injectable composition can be in a suitable intravenous solution, such31as sterile physiological salt solution. The composition can also be formulated as a suspension in an aqueous emulsion.

[0161] In some embodiments, the peptides described herein can be delivered in a thin film patch or microneedle patch.

[0162] Additionally, the peptides described herein can be formulated with a penetration enhancer. As used herein, “penetration enhancer” refers to excipients that promote the transit of a peptide into a tissue. Examples of penetration enhancers include, but are not limited to, terpenes, terpenoids, sulfoxides, laurocapram, pyrrolidones, 1 -phenylpiperazine, sodium deoxy cholate, fatty acids, fatty alcohols, urea, calcium chelators (including, but not limited to, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl)-N,N,N’,N’- tetraacetic acid (EGTA), l,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, paraben, Transcutol® P, Labrasol®, sodium caprate, acyl carnitine, pelargonidin, citric acid, salcaprozate sodium, bile acids, nanoparticles, cell penetrating peptides, gelucires, Azone™ (l-dodecylazacycloheptan-2-one), borneol, Triolein, Miglyol, oleic acid, sesame oil, Copaiba oil, Capryol PGMC, sweet almond oil, lemon oil, clove oil, eucalyptus oil, diacyl glyceride, poly-oxyethylene ether, cineole, geraniol or a combination thereof.

[0163] Administering the peptide as described herein can be as needed, once per day, twice per day, three times per day, once per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per week, once every two weeks, once every three weeks, once per month, quarterly, semi-annually, yearly, and / or for life.

[0164] Also described herein is the use of the peptide in diagnosing cancer. For example, the peptide can be conjugated with a signaling moiety that gives a signal that can be detected by an imaging technique. According to this embodiment, the accumulation of the conjugated peptide in tumor tissue is detected visually or through an imaging technique.

[0165] For example, the signaling moiety is a fluorescent compound that can be detected through fluorescence detection, either visually or using a fluorescent imaging device. Examples of fluorescent dyes that can be used include, but are not limited to fluorescein, cyanine dyes or Alexa Fluor®.

[0166] Other examples of signaling moieties include a PET or SPECT label that can be detected through a PET or SPECT scan. Examples of such labels include, but are not limited to18F, "mTc,68Ga,nC,13N,15O,64CU,86Y,55CO,123I,131I, "mT,89Zr,133Xe and201TI.

[0167] Also disclosed herein are kits comprising: at least one peptide described herein, a pharmaceutically appropriate excipient, carrier, diluent, or any combination thereof, and a sterile storage container.

[0168] Also disclosed herein are kits comprising: a lyophilized peptide of the present invention; a pharmaceutically appropriate: excipient, carrier, diluent, or any combination thereof; separate sterile storage containers for each; and instructions on how to reconstitute the lyophilized peptide with the pharmaceutically appropriate: excipient, carrier, diluent, or any combination thereof.

[0169] According to another aspect, provided is a method of treating cancer in a subject in need thereof comprising administering the peptide, the conjugate or the pharmaceutical composition as described herein to said subject. According to one embodiment, the pharmaceutical composition comprises an effective amount of the peptides or the conjugates as described herein. According to some specific embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of the peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31 or conjugates thereof. According to other specific embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of the peptide consisting of an amino acid sequence selected SEQ ID NO: 1, 2 or 3 or conjugates thereof. According to some embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of the peptide comprising or consisting of an amino acid sequence selected SEQ ID NO: 1, 2 or 3, a fragment thereof consisting of 17-28 amino acids, or an analog of said peptide or said fragment. According to one embodiment, the pharmaceutical composition comprises the conjugates or fusion proteins of the present invention. According to some embodiments, the method comprises administering the pharmaceutical composition of the present invention in combination with another anti-cancer therapy, as described above.

[0170] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -10%, or -+7-5%, +7-1%, or even + / -0.1% from the specified value.

[0171] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.EXAMPLESExample 1. 3D-NAF-1-6K peptide selectively penetrates into cancer breast cells

[0172] The peptide having the amino acid sequence FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID NO: 2, also referred to herein as NAF- 1-3D-6K peptide), is derived from the peptide corresponding to amino acids 44-67 of human NAF- 1, which is part of the transmembrane domain and the adjacent cytoplasmic region of human NAF- 1 (FIG. 1) This region of the protein is not involved 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 the D-amino acids (appear in the SEQ ID NO: 2 in small letters) to produce the species shown as SEQ ID NO:2. The addition of the 6K increased the cytotoxicity of the 3D-NAF-1-6K peptide in comparison to the original 3D-NAF-1 peptide by -three fold (FIG. 3). This higher efficacy does NOT result solely by the addition of the positive charge as FIG 4 indicates that 3D-NAF-1-6K is more cytotoxic to breast epithelial cancer cells (MDA-MB-231) after 3 and 18 hours when compared to 3D-NAF-1-6R

[0173] The ability of the peptide having the sequence SEQ ID NO:2 to permeate into the malignant cells and its effect on cells’ viability was tested as follows. Green fluorescein (Fl) / Cy-5 labels were conjugated to the peptide having SEQ ID NO:2 (i.e., 3D-NAF-1-6K peptide). FIG. 12 demonstrates the permeation of the FL-3D-NAF-1 -6K peptide into ovarian cancer (SKOV-3) cells. FIG. 13 and FIG. 14 demonstrate its effects on the fission / break down of their mitochondria.Example 2. Efficacy of the peptide SEQ ID NO:2 in killing of different cancer cells

[0174] In order to validate the cell-based results, the efficacy of peptide having SEQ ID NO:2 the peptide was studied both in 2D tissue cultures (FIG. 5, 6 and 7) and in “a tumor like” 3D cancer cell spheroids (FIG. 9-11). In addition to the 3D-NAF-1-6K peptide’s ability to kill MDA-MB- 231 breast cancer cells (FIG. 3), the efficacy of the 3D-NAF-1-6K peptide to kill prostate cancer (PC-3), ovarian cancer (SK-OV-3) and liver cancer (HepG2) cells was determined.

[0175] For 2D cytotoxicity studies, the cancer cells (MDA_MB-231 / HepG2 / SKOV-3 / PC-3) were plated in quadruplicate at 2 x 103cells per well in a 96 well ULA plate (Corning 7007) and the cells were further cultured for 1 hour and 1 day after adding the 3D-NAF-1-6K peptide (FIG. 5, 6 & 7). The 3D-spheroids were treated with the peptides having SEQ ID NO:2 and the apoptosis of spheroids was assayed using The IncuCyte® Red cytotoxicity reagent (Essen Bioscience Cat #4632) probes cell death. Spheroids were imaged at x 10 magnification in an IncuCyte Zoom Livecell analysis system (Essen Bioscience, Ann Arbor, MI) at 37 °C with 5% CO2. Spheroid cell images were recorded every two hours and the resulting red colors, reflecting cell apoptosis, were analyzed with the IncuCyte software Sartorius AG, Gottingen, Germany)

[0176] FIG. 9 shows that after a 20 hours incubation of the peptide (FIG. 9, lower panel) with the 3D spheroids of the ovarian cancer SKOV-3 cells, most tumor-like cells were killed. Viability was determined by the fluorescence obtained the IncuCyte® Red cytotoxicity reagent (Essen Bioscience Cat #4632) probes for cell death.. Similar effects of the 3D-NAF-1-6K peptide on liver cancer Hep-G2 spheroids and on prostate cancer PC-3 spheroids are shown in FIG. 10 and FIG. 11, respectively.Example 3. The 3D-NAF-1-6K peptide permeates the plasma membrane (PM) of ONLY cancer cells and disrupts the inner cellular organelles mitochondria and ER.

[0177] NAF-1-3D-6K peptide is targeted to and damages the intra cellular organelles Mitochondria and Endoplasmic reticulum (ER). FIG. 3 shows that 3D-NAF-1-6K is non-toxic to normal breast epithelial cells (MCF10A) while it kills the breast cancer cells (MDA-MB-231) cells.

[0178] Of special importance is the validation that the 3D-NAF-1-6K peptide specifically targets cancer cells. FIG. 3 shows that the 3D-NAF-1-3D-6K peptide does not affect the MCF-10A normal breast epithelial cells versus its dramatic cytotoxic effect on MDA-MB-231 breast cancer cells.

[0179] FIG. 5, 6 & 7 shows the killing efficacy of the 3D-NAF-1-6K peptide on other malignant cells as well, e.g., Prostate cancer (PC-3 cells), Ovarian cancer (SKOV-3) Liver cancer (HepG2), Melanoma (A375), Glioblastoma (U87MG), two types of Leukemias: Acute Myeloid Leukemia (MOLM-14 cells) and Chronic Myelogenous Leukemia (K562 cells) as well as Pancreatic cancer cells (PANC-1 cells). While the IC50 of the 3D-NAF-1-6K peptide towards solid tumors’ cells isin the ranges of 7.8-15pM, the highest efficacy of the 3D-NAF-1-6K is towards the Leukemia K562 cells (1.9pM) - FIG. 7.Example 4. The 3D-NAF-1-6K peptide is efficient in animal model systems of Ovarian and Melanoma Cancers - where it specifically targets the xenograft tumors.

[0180] In an in vivo animal model system SKOV-3 xenograft tumors were developed in 5-6 weeks old mice following injection of 2.5 million ovarian cancer cells to the mice subcutaneously. FIG. 17 shows the effect of the injection of the 3D-NAF-1-6K peptide having SEQ ID NO:2 on the size of Ovarian cancer SKOV-3 tumors at specified times. The peptide (at 0.5mg / KG) was injected intravenous (IV), or subcutaneously (SC) and the effect was compared to control mice, injected with the vehicle (negative control) Despite the significant inhibition that the 3D-NAF-1-6K peptide had on the tumor growth, the 3D-NAF-1-6K peptide had no toxic effect on the mice as indicative by their weight gain.

[0181] FIG. 21-24 show that the 3D-NAF-1-6K peptide specifically reaches and accumulates in the tumor. FIG. 21 shows all internal organs isolated from mice injected IV with fluorescently labeled F1-3D-NAF-1-6K peptide (referred to in FIG. 22 as “F1-3D-NAF-1 Peptide”,”), demonstrating that the liver, kidney, colon, brain, lungs, and others organs maintained their characteristics normal colors. FIG. 22-24 show that only slices of the shrunken breast MDA-MB- 231 tumors had green fluorescence when analyzed and quantified in a confocal microscope. In FIG. 24, the representative confocal images of tumor cells from the mice treated with or without the 3D-NAF-1-6K peptides, the tumor cells were stained with RPA for mitochondria and H342 for the nucleus. FIG. 25 shows the dynamics of Cy5-3D-NAF-1-6K peptide following its injection into nude mice with tumor formed from MDA-MB-231 breast cancer cells. Following the injection, the Cy5-3D-NAF-1-6K peptide reaches the liver and kidney from where it is cleared, yet the Cy5-3D-NAF-1-6K peptide accumulates and is retained in the tumor.

[0182] In FIG. 28, mice carrying A375 cancer xenografts were distributed into 2 groups, a control group and the 3D-NAF-1-6K peptide treated group which received 0.75mg / Kg of the 3D-NAF-1- 6K 3 peptide intravenously, three times a week for three weeks. As shown in FIG28B, despite the significant inhibition that the 3D-NAF-1-6K peptide had on the A375 tumor growth as shown in, the 3D-NAF-1-6K peptide had no toxic effect on the mice as indicative by their weight gain (FIG28C) during the course of the experiment.Example 5. The 3D-NAF-1-6K Peptide is efficient in an animal model systems of ovarian cancer primary tumor and metastasis.

[0183] FIG. 17 shows the effect of the injection of the NAF-1-3D-6K peptide on the size and tumor development of ovarian cancer SKOV-3 cells. The peptide (at 50pM = 0.5 mg / kg) ) was injected intravenously -(IV) via the tail vein, or subcutaneously (SC) at the neck of the mice. The control mice were injected with saline. The immunohistochemistry of tumors injected with the 3D-NAF-1-6K peptides and probed with an antibody to a macrophage specific mouse cell surface glycoprotein F4 / 80 marker which indicated high amounts of tumor infiltrating macrophages (FIG. 18).

[0184] FIG. 19 shows that the 3D-NAF-1-6K peptide (SEQ ID NO:2) reduces the development of lung metastasis of SKOV-3 ovarian cancer cells. IV inoculation of 1 million SKOV-3 cells, IV, to Nude mice, was performed on day 0. The NAF-1-3D-6K peptide was administered by SC injection, twice weekly at doses of 0.25mg / kg or 0.5mg / kg. Dosing of the NAF-1-3D-6K peptide was initiated 7 days following the appearance of lung metastases in satellite animals, FIG. 19 also indicate that Taxol (positive control) dosing: IV from day 7, twice weekly p= 0.000942 Taxol - 20mg / kg; P=0.000998 3D-NAF-1-6K 0.5mg / kg.

[0185] In addition to the 3D-NAF-1-6K reduction of lung metastasis, the peptide also reduces metastatic lesions in the colon of mice. FIG. 19B. FIG. 20 shows that the 3D-NAF-1-6K peptide (SEQ ID NO:2) inhibits 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 the migration of cancer cells, probably contributes to the reduction in lung / colon metastasis formation.Example 6. The 3D-NAF-1-6K peptide facilitates mitochondrial fission and the disruption of mitochondrial morphology in SKOV-3 cells.

[0186] FIG. 12-14 indicate that following the permeation of the 3D-NAF-1-6K (SEQ ID NO:2) into the SKOV-3 cells, it induces a significant damage to the cells’ mitochondria. The fission / fragmentation is induced by the 3D-NAF-1-6K peptide. Our hypothesis is that the peptide disrupts the dimer structure of NAF-1 (CISD2), the protein from which the peptide is derived (FIG. 1), and this induces the process in which the outer mitochondrial and endoplasmic reticulum membranes are destroyed.Example 7. The 3D-NAF-1-6K Peptide binds to albumin

[0187] Without meaning to be bound by any hypothesis, the binding of peptide to serum albumin could extend its half-life and / or form a delivery system increasing the availability of the peptide to the tumor (FIG. 27).

[0188] FIG. 27 shows that increasing concentrations of BSA (bovine serum albumin) delays the cytotoxicity activity of the NAF-1-3D-6K peptide on breast MDA-MB-231 cells. The concentrations of BSA were 0.125 mg / ml (purple bars); 0.25 mg / ml (green bars) and 0.5 mg / ml - -10% of the albumin serum concentration (red-orange bars). The peptide without BSA (red bars) served as positive control and the negative control (blue bars). Incubation of the cells without / with free / BSA present peptides were for 3, 6, 12, 24 and 72 hours. Cytotoxicity was determined by presto-blue method in cells imaging plate. These results, although obtained in vitro, may indicate that, in vivo, albumin may serve as a carrier to assist the transfer of the peptide in the blood.Example 8. The NAF-1-3D-6K Peptide is also active in controlling the cancer cells’ redox state

[0189] The 3D-NAF-1-6K peptide (SEQ ID NO:2) inhibits the activity of thioredoxin reductase (FIG. 15) Thioredoxin reductase is the reducing enzyme of thioredoxin - the master essential redox regulator in cells which secures the maintenance of thiols (-SH) in proteins that undergo, in oxidative stress, (S-S) unfunctional transition. When thioredoxin donated its hydrogens, thioredoxin reductase is the enzyme that secures the thioredoxin reduction. Thioredoxin is an essential protein that secures the normal redox state of 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 the activity of thioredoxin reductase, could be an important aspect of its anticancer activityExample 9: The 3D-NAF-1-6K Peptide has enhanced cytotoxicity towards cancer cells

[0190] We tested whether additional lysines added to the C-terminus of 3D-NAF-1(SEQ ID NO:7) would improve the solubility and / or cytotoxic activity. The modified peptide (with 6K) (referred to in FIG. 3 as 3D-NAF-1-6K (SEQ ID NO: 2)) was tested for its cytotoxic effect vis-a-vis the original 3D-NAF-1 peptide (SEQ ID NO:7) using healthy and epithelial breast cancer cells (FIG. 3). Both peptides decreased viability of cancer cells after 1 and 3 days in comparison to the control cells that were not affected. However, compared to 3D-NAF-1 (SEQ ID NO:7), 3D-NAF-1-6K(SEQ ID N0:2)) showed unexpectedly and significantly higher cytotoxic ability towards the cancer cells.

[0191] In this experiment, cells were plated in 96 well plates at the density of 15,000 per well before the experiment. lOpM of the 3D-NAF-1 (SEQ ID NO:7) or 3D-NAF-1-6K (SEQ ID NO:2) peptides were added to cells and incubated for 1 or 3 days. Cell viability was assessed using Presto- Blue after 1- and 3 -days incubation. ***p < 0.001, ****p < 0.0001 by t-test.Example 10: Broad-Spectrum Anti-Cancer Activity of the NAF-1-3D-6K Peptide

[0192] We studied the effect of the 3D-NAF-1-6K on several cancers including acute myeloid leukaemia (MOLM-14), chronic myelogenous leukaemia (K562), pancreatic cancer cells (PANC- 1), brain glioblastoma cancer cells (U87MG), and human malignant melanoma (A375). As shown in FIG. 8, at a low concentration than 5pM, the peptide decreased the viability of all cancer cells, with leukaemia cancer cells being most sensitive to the cytotoxic effect. At the higher concentrations of 25 pM, the peptide dramatically decreased the viability of all tested cancer cells lines. Taken together with our findings with breast and ovarian cancers, these findings suggest that the 3D-NAF-1-6K peptide could be used against a wide spectrum of cancers.

[0193] Cell survival was measured using presto-blue assay after 3 hours incubation with the peptide at the indicated concentrations. SKOV-3, ovarian cancer (black circle), U87MG, glioblastoma (black rectangle), A375, human malignant melanoma (black up-point triangle), MDA-MB-231, breast cancer (black down-point triangle), PANC-1, pancreas cancer (black diamond), MOLM-14, acute myeloid leukaemia (red circle), and K562, chronic myelogenous leukaemia (yellow rectangle) were treated or untreated with the peptide and assayed for viability and survival. Data are shown as mean ± SD from three independent experiments. ** P<0.01, *** P<0.001, **** p<0.0001 by t-test.Example 11: The 3D-NAF-1-6K Peptide Facilitates Mitochondrial Fission / Fragmentation

[0194] The entry of the peptide into the SK-OV-3 cancer cells and its effects on mitochondria were investigated by labelling the peptide 3D-NAF-1-6K (SEQ ID NO:2) with fluorescein (referred to in FIG. 12 as F1-3D-NAF-1-6K), and labelling mitochondria with Rhodamine 800. The cells were then imaged with confocal microscopy. Upon addition of the peptide to the ovarian cancer, SK-OV-3 cells, F1-3D-NAF— 6K fluorescence was primarily found near the SK-OV-3cells’ PM. This was observed by high F1-3D-NAF-1-6K fluorescence at the PM of cancer cells at an early stage, and inside SK-OV-3 cancer cells at 4 and 6 hours of incubation (FIG. 12A-B.) These findings indicated that F1-3D-NAF-1-6K fluorescein labelled 3D-NAF-1-6K (SEQ ID NO:2) permeates SK-OV-3 cancer cells. Following the permeation of the peptide into the cancer cells, the staining of mitochondria with Rhodamine 800 in treated cells decreased indicating that the peptide negatively impacted mitochondrial function / integrity (FIG. 12C). This effect was also observed using another fluorescence probe, Cy5 conjugated with 3D-NAF-1-6K.

[0195] The effect of the 3D-NAF-1-6K (SEQ ID NO:2) peptide on the mitochondria of SK-OV-3 cells was further investigated by following the length of mitochondria in SK-OV-3 cells at different concentrations of the peptide (FIG. 12-14). The peptide showed a dose-dependent effect on mitochondria, causing a decrease in mitochondrial length. This result indicates that the 3D- NAF-1-6K (SEQ ID NO:2) peptide targets the mitochondria of SKOV-3 cells (FIG. 12-14), causing mitochondrial destruction.

[0196] FIG. 13 shows the measurement of mitochondrial length in SKOV-3 ovarian cancer cells after treatment with the 3D-NAF-1-6K (SEQ ID NO:2) peptide. FIG. 13A shows representative confocal fluorescence images of mitochondria in SKOV-3 cells treated or untreated with the 3D- NAF-1-6K (SEQ ID NO:2) peptide. FIG. 13B shows that mitochondrial length was translated into skeleton binary images in the control (without peptide) and 50pM of the peptide treatment. The mitochondrial length was measured in 25 cells from 8 field images and 156 mitochondria / cell in the control and 58 mitochondria / cell in 50pM peptide treatment were measured using the Image J program. Mitochondrial length in the magnified images was measured using the free hand tool in image J program.

[0197] FIG. 28 shows the efficacy of the 3D-NAF-1-6K on the A375 Melanoma tumor growth rate of both female and male mice (FIG. 28A). The fact that the treated mice continued to gain weight (FIG. 28B) further support the findings shown in FIG. 26 that indicate that the 3D-NAF- 1-6K peptide is non-toxic based on CBC results, mice organs’ weights and the organs’ H&E histochemical analysis.

[0198] All publications, issued patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS1. A peptide comprising(a) an amino acid sequence comprising FLGVLAL-X1-X2 (SEQ ID NO: 10), wherein Xi represents a second amino acid sequence LGYLAVRPFLPKKKQQK (SEQ ID NO: 30) or a fragment of this second amino acid sequence with sequential deletions at the C-terminus; wherein X2 represents 1-10 positively charged moieties; and wherein any one of the amino acids in the amino acid sequence can be a D-amino acid; or(b) an analog of (a); or(c) a retro-inverso sequence of either (a) or (b).

2. The peptide of claim 1, wherein the positively charged moiety is:(a) a positively charged amino acid;(b) lysine (K); or(c) arginine (R).

3. The peptide of any one of the previous claims, wherein X2 is(a) 3-10 positively charged moieties;(b) 4-7 positively charged moieties; or(c) 6 positively charged moieties.

4. The peptide of any one of the previous claims, wherein at least one of the amino acids of the peptide is a D-amino acid.

5. The peptide of claim 4, wherein at least 1, 2, 3, 4, up to all 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 of any one of the previous claims, wherein said peptide comprises the 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 of any one of the previous claims, wherein said peptide is:(a) FLGVLALLGYLAVRPFLPKKKQQKKKKKKK (SEQ ID NO:1);(b) FLGVLALLGyLAVRPfLPKkKQQKKKKKKK (SEQ ID N0:2);(c) flgvlallgylavrpflpkkkqqkkkkkkk (SEQ ID NO: 3);(d) FLGVLALLGYLAVRPFLPKKKQQKX2-10 (SEQ ID NO:4);(e) FLGVLALLGyLAVRPfLPKkKQQK-X2-io (SEQ ID NO: 5); or(f) FLGVLALLGYLAVRPFLPKKKQQKKKKKK (SEQ ID NO:29).

8. The peptide of any one of the previous claims, wherein said analog comprises:(a) 1 to 10, 2 to 8, or 3 to 6 modifications of the amino acid sequence;(b) 1 to 6 modifications of claim of the amino acid sequence;(c) 1 to 3 modifications of claim of the amino acid sequence;(d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications of the amino acid sequence;(e) a polypeptide sequence 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 to any one of SEQ ID NO: 1-5 or SEQ ID NO: 10-29 or 31; or(f) the analog of (a)-(e) comprising conservative substitutions.

9. The peptide of any one of the previous claims, wherein said peptide is 17 to 50 amino acids in length.

10. The peptide of any one of the previous claims, wherein said peptide is the retro-inverso sequence.

11. A polynucleotide encoding the peptide of any one of claims 1-10.

12. A conjugate comprising the peptide of any one of claims 1-10 and a moiety.

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

14. The conjugate of either claim 12-13, wherein the moiety is:(a) a radioactive isotope, preferably wherein the radioactive isotope is 1311, 68Ga, 177Lu, 153Sm,212Pb,166HO,n iIn, astatine-211, bismuth-213, actinium-225, radium-223, yttrium-90 and thorium-227;(b) a signaling moiety, such as a fluorescent compound, preferably such as fluorescein, cyanine dyes or Alexa Fluor®; or(c) a signaling moiety, such as a PET or SPECT label, such as18F, "mTc,68Ga,nC,13N, 15O,64Cu,86Y,55CO,123I,131I, "mT,89Zr,133Xe and201TI.

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

16. A method of treating a subject in need thereof, wherein said method comprises administering to a subject a therapeutically effective amount of 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.

17. The method of claim 16, wherein the method is for the use of treating cancer.

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

19. The method of claim 17 or 18, wherein the cancer is associated with an enhanced expression ofNAF-1 protein.

20. The method of any one of claims 16-19, wherein the method further comprises administering an anti-cancer therapy, preferably wherein the anti-cancer therapy is an immune-modulator, an activated lymphocyte, a lymphocyte activating agent, a kinase inhibitor, a chemotherapeutic agent or an anti-cancer agent.

21. A method of diagnosing a cancer, wherein said method comprises use of 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 to detect cancer cells.

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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