Polypeptide, nucleic acid, expression vector, pharmaceutical composition and application thereof

Abstract

The invention relates to a polypeptide, a nucleic acid, an expression vector, a pharmaceutical composition and application thereof. Described herein are isolated modulatory peptides of protein kinase C, chimeric peptides thereof, and variants thereof. The use of the peptides in compositions and methods for treating cell proliferation pathologies is also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a continuation-in-part of U.S. Patent Application No. 17 / 394,431, filed on August 5, 2021, which is a continuation-in-part of U.S. Patent Application No. 16 / 486,832, filed on August 18, 2019, which is a national phase of International Patent Application No. PCT / IL2018 / 050182, filed on February 19, 2018; International Patent Application No. PCT / IL2018 / 050182 in turn claims the benefit of U.S. Provisional Patent Application No. 62 / 460,810, filed on February 19, 2017. The contents of the foregoing patent applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to polypeptides, nucleic acids, expression vectors, pharmaceutical compositions and uses thereof, in particular to isolated regulatory peptide uORFs of protein kinase C, chimeric peptides thereof, and variants thereof. Also described are uses of the peptides in compositions and methods for treating cell proliferation pathologies. Background Art

[0004] Translational regulatory elements function in cells to rapidly alter the protein expression landscape in response to internal or external stimuli. Among these elements are upstream open reading frames (uORFs) located in the 5'-untranslated region of mRNAs. Bioinformatics studies have shown that uORFs are present in approximately 40% of human mRNAs. uORFs are often associated with reduced protein expression levels because they reduce the efficiency of translation initiation of downstream ORFs. Previously, we reported that the expression of the protein kinase C isoform PKCη is regulated by two uORFs and showed their potential to be translated into short peptides.

[0005] Protein kinases, including the various PKC isoforms, are recognized for their roles in regulating numerous biological processes, and aberrant PKC expression is thought to be a factor in a variety of pathologies, including those involving cell proliferation. Thus, there is a continuing need to regulate PKC expression. Summary of the invention

[0006] Described herein are synthetic peptides derived from uORFs of PKCη isoforms, and in particular polypeptides comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NOs: 5, 36, and 37.

[0007] Also described herein are combinations of synthetic peptides set forth herein as SEQ ID NOs: 5, 36, and 37 and chemotherapeutic agents for use in treating abnormal cell proliferation (eg, cancer), such as in methods of treatment and in the preparation of a medicament for treating cancer.

[0008] Additionally described herein are methods of inhibiting cell proliferation and / or cancer metastasis by administering to a subject in need thereof a therapeutically effective amount of a peptide described herein (SEQ ID NOs: 5, 36, and 37) and optionally administering to the subject a chemotherapeutic agent.

[0009] Also provided are methods of using the peptides and / or nucleic acids to treat diseases or disorders associated with abnormal cell proliferation.

[0010] The foregoing and other objects, features and advantages will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1C Schematic representation of the mRNA of human PKCη (PKCeta), its two uORFs and pseudosubstrate (PS) sequence, and their homology with other PKCs from other species. Figure 1A : The amino acid sequence uORF2 (SEQ ID NO: 1) and pseudosubstrate (PS) sequence (SEQ ID NO: 19) of PKCη are depicted in single letter code. Figure 1B The amino acid sequence of uORF2 shows high conservation. The amino acid sequence encoded by human uORF2 (SEQ ID NO: 1) was compared with the uORFs encoded by homologous PKCη in other mammalian species (SEQ ID NO: 20-24, respectively, rhesus monkey, elephant, dog and rat). Figure 1C : The amino acid sequence of the pseudosubstrate of PKCη (ηPS) (SEQ ID NO: 19) was compared with the PS sequences of all other human PKCs (SEQ ID NO: 25-31, PKCα, PKCβ, PKCγ, PKCδ, PKCε, PKCζ and PKCλ / ι, respectively) and with the sequence encoded by uORF2 (SEQ ID NO: 3). All sequences are represented by single letter codes; multiple sequence alignments were performed using the HHpred toolkit.

[0012] Figures 2A-2D Peptides derived from PKCη are shown and it is demonstrated that the uORF2-encoded peptide (uPEP2 1-26 ) Schematic representation of the ability of the peptides to inhibit PKC kinase activity. Figure 2A is a schematic representation of a peptide derived from PKCη and used in the Examples described herein. 1-26 Refers to the complete peptide encoded by uORF2; uPEP9-26 and uPEP 18-26 refers to a control peptide lacking the PS motif; ηPS refers to a peptide containing the internal PS domain of PKCη. Figure 2B The results of testing the effects of different peptides on the kinase activity of PKCη are shown. Myelin basic protein (p-MBP) was used as substrate and γ- 32 The kinase activity of PKCη was measured by P radioactive kinase assay. All values ​​were normalized to the control sample without peptide (Cont.). The data shown are the average of three independent experiments and are also Figure 2C Presented in gel form. Figure 2D : describes the use of uPEP at a concentration of 2 μM 1-26 like Figure 2B Kinase assays were performed as described in . All values ​​were normalized to control samples (without peptide). Data shown are the average of three independent experiments.

[0013] Figures 3A-3D The peptide encoded by uORF2 (uPEP2 1-26 ) ability to inhibit cell migration of breast cancer cells. Figure 3A are photographs taken from a scratch assay performed on MDA-MB-231 cells. Cells were seeded into 24-well plates and incubated with the indicated peptides for 4 hours in serum-free medium. After 4 hours, scratches were performed using a 200 μl pipette tip. Photos of selected areas of each scratch were taken at the indicated time points. Areas were measured by ImageJ software. Figure 3B-3D Is representative Figure 4A Figure 3 is a graph of the analysis of the scratch assay performed on the cell lines MDA-MB-231, MCF-7 and MCF10A, respectively, as described in. All values ​​are normalized to time 0 for each well and expressed as percentages.

[0014] Figures 4A-4D The peptide encoded by uORF2 (uPEP2 1-26 ) has the ability to reduce the viability of breast cancer and glioblastoma cells. Figure 4A :MCF-7 cells were seeded onto 48-well plates. After 24 hours, the cells were incubated with the indicated peptides for 24 hours. After incubation, the medium with the test peptides was gently aspirated from each well, and 25 μL of PrestoBlue reagent was added to each well of the 48-well plate and incubated at 37°C, 5% CO2 for 15 min as recommended. The absorbance was detected by an 'Infinite M200PRO' reader. Figures 4B-4D is with Figure 4A Results of the same experiments described but performed on the cell lines U251 MG (glioblastoma cell line), MDA-MB-231 and MCF10A, respectively.

[0015] Figure 5 The peptide encoded by uORF2 (uPEP2 1-26 ) and etoposide (etoposide) in combination to induce cell death. MCF-7 cells were inoculated in 96-well plates for 24 hours. The indicated peptides (5 μM) were added for 4 hours. Then, etoposide (50 μM) was added to the wells for 48 hours. The plates were analyzed using an XTT kit (Biological Industries, Israel) according to the manufacturer's instructions.

[0016] Figures 6A-6C The peptide encoded by uORF2 mutated at position 10 (uPEP 1-26[c / a] ) in reducing cell viability in various cell lines. Fig. 6A MCF-7 cells were seeded into 96-well plates and incubated with the indicated peptides for 24 h. After 24 h, the proliferation rate was monitored using an XTT kit (Biological Industries, Israel) according to the manufacturer's instructions. Figure 6B :like Fig. 6A The MDA-MB-231 cell line was tested as shown in . Figure 6C :like Fig. 6A HeLa cells as shown in the test

[0017] Figure 7A-7B showed that PKCη knockdown reduced tumor invasiveness and metastasis formation in the mammary 4T1 mouse model. Fig. 7A : 4T1 cells expressing sh-PKCη (SEQ ID NO: 33) or scrambled control were injected into the mammary fat pad of 8-week-old female BALB / c mice. Tumor diameter was measured with a vernier caliper every week. Figure 7B :8 weeks later, mice were sacrificed and the number of metastases formed in the lungs was counted using Bouin's solution. Error bars represent mean ± sem, and statistical analysis indicated statistical significance by two-tailed unpaired sample t-test.

[0018] Figures 8A-8B is a schematic diagram of the 5'UTR of the additional PKC containing the uORF and PS motif. Fig. 8A : Bioinformatics revealed the presence of uORFs in PKCη and PKCζ. The 5' cap structure is shown as a diamond, the 5' UTR is shown as a line, the CDS is shown as a black arrow, and the uORF is shown as a box. The uORF was found by ORF-finder software (NCBI). Figure 8B : Depicts the amino acid sequence of the uORF of PKCζ containing the PS motif (SEQ ID NO: 11).

[0019] Figures 9A-9E The mutant peptides uPEP2[R8A] (SEQ ID NO:36) and uPEP2[C10A] (SEQ ID NO:5) were shown to be more effective as kinase inhibitors compared to uPEP2 (SEQ ID NO:1): the novel PKC (PKCh[ Fig. 9A ]、PKCe[ Fig. 9B ]、PKCd[ Fig. 9C ] and PKCq[ Fig.9D ]) infected (HA-tagged) plasmid was transfected into HEK 293T cells. 48 hours after transfection, cells were lysed and immunoprecipitated using protein A / G beads and anti-HA antibodies. Immunoprecipitates were subjected to in vitro kinase assays with / without peptides uPEP2, uPEP2[R8A], uPEP2[C10A] (0 to 5 μM), γ-ATP and substrate MBP. According to the IC50 values ​​calculated and determined from the corresponding graphs for maximum activation (obtained in the absence of inhibitory peptides), both uPEP2[R8A] and uPEP2[C10A] peptides were better inhibitors of all novel PKCs compared to uPEP2. Fig.9E :The results shown in the table are the average of three separate experiments.

[0020] Fig.10 Inhibition of the kinase activity of PKCη by [C10A] (SEQ ID NO: 5) and [R8A] (SEQ ID NO: 36) is shown. HEK293T cells were transfected with the corresponding HA-PKCη plasmids: 48 hours after transfection, cells were lysed and immunoprecipitated using protein A / G beads and anti-HA antibodies. Immunoprecipitates were subjected to in vitro kinase assays with / without peptides uPEP2, uPEP2[R8A], uPEP2[C10A] (0 to 10 μM), γ-ATP and substrate MBP. Results represent the mean of three independent experiments P values ​​calculated using one-way ANOVA. ***P≤0.001.

[0021] Figures 11A-11B Peptides with substitutions at positions 6, 8, 10, 12 show inhibition of PKCη kinase activity: [ Fig.11A]HEK293T cells were transfected with the corresponding HA-PKCη plasmids. 48 hours after transfection, the cells were lysed and immunoprecipitated using protein A / G beads and anti-HA antibodies. The immunoprecipitates were subjected to in vitro kinase assays with / without peptides (0 to 10 μM), γ-ATP and substrate MBP. The peptides used were uPEP2 (SEQ ID NO: 1), uPEP2[R8A] (SEQ ID NO: 36), uPEP2[C10A] (SEQ ID NO: 5), uPEP2[S12A] (SEQ ID NO: 38), uPEP2[A6S] (SEQ ID NO: 8) and uPEP2[A6T] (SEQ ID NO: 9). Among the peptides tested, alanine substitution at position 8 (uPEP2[R8A]) was the most potent PKCη kinase inhibitor. P values ​​were calculated using one-way ANOVA. ***P≤0.001. [ Fig. 11B ]represent( Fig.11A ) Autoradiographs and immunoblots of the data in ).

[0022] Figures 12A-12B Inhibition of cell viability by uPEP2[R8A] (SEQ ID NO: 36) and uPEP2[C10A] (SEQ ID NO: 5) was shown: The peptide [R8A] ( Fig. 12A ) and [C10A]( Fig. 12B ) were assayed for cell viability and IC50 values ​​were calculated. The data shown represent three independent experiments performed in the presence of the corresponding peptides (0-10 mM). In all analyzed cancer cell lines, the substituted peptides uPEP2[R8A] and uPEP2[C10A] were better inhibitors of cell viability than the unsubstituted peptide uPEP2.

[0023] Fig.13 : Inhibition of cell viability of leukemic MV-4-11 line by uPEP2-substituted peptides is shown: Peptides attenuated cell viability of MV-4-11 (AML-derived). Data shown are representative of three independent experiments performed in the presence / absence of uPEP2-substituted peptides (0 to 20 μM).

[0024] Figures 14A-14B The effect of alanine substitutions at positions 8 (SEQ ID NO: 36) and 10 (SEQ ID NO: 5) of uPEP2 and the mutation [R8A+C10A] on peptide stability in mouse serum is shown: ( Fig.14A). The stability of uPEP2 and its substituted forms in mouse serum (20%) was examined using Western blotting and antibodies against uPEP2. The introduction of alanine substitution at position 10 increased its stability compared to the unmutated peptide or the peptide with a mutation at position 8. The best serum stability was obtained with the peptide with two substitutions at positions [R8A+C10A]. ( Fig. 14B ) Time course analysis of peptides quantified and normalized from Western blots in the presence of mouse serum. The terminal half-life (t) estimated by fitting the experimental data to an exponential decay model is shown. 1 / 2 s) and corresponding 95% confidence intervals (CI). The percentage of intact peptide was calculated by western blot quantification and normalization in Image lab 6.1 (Bio-rad) software, expressed as a percentage of the amount at t0.

[0025] Fig.15 : The effect of uPEP2 peptide with [R8A] on the viability of cell lines derived from tumors of different tissue origins.Use different cell lines to carry out the cell viability assay of uPEP2 mutant peptide [R8A], and calculate the IC50 value of each line.The data shown represent three independent experiments carried out in the presence of the corresponding peptide (0-10mM).The lowest IC50 is described for AML (Kasumi) and triple negative breast cancer (MDA-MB-231), followed by osteosarcoma (U2OS), melanoma (SKMEL-2), AML (MV-4-11, HL-60), lymphoma (U937) and ALL (MOLT-4) tumor-derived cells.

[0026] Fig.16 : Effect of uPEP2 peptides with [C10A] on the viability of cell lines derived from tumors of different tissue origins. Cell viability assays of uPEP2 mutant peptide [C10A] were performed using different cell lines and IC50 values ​​were calculated for each line. The data shown represent three independent experiments performed in the presence of the corresponding peptide (0-10 mM). The lowest IC50 is described for cervical cancer (HeLa), osteosarcoma (U2OS), leukemia (HL-60), lymphoma (U937), melanoma (Hs940T), breast cancer (MDA-MB-231), choriocarcinoma (BeWo) and colorectal adenocarcinoma (C2BBe1) sources. DETAILED DESCRIPTION

[0027] A brief description of the sequence

[0028] The nucleic acid and / or amino acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. The sequence listing is submitted as an XML file named 3278_1_3003_seq of approximately 39 KB created on July 23, 2023, the entire contents of which are incorporated herein by reference.

[0029] SEQ ID NO: 1 is the peptide encoded by the PKCη derived uORF.

[0030] SEQ ID NO: 2 is a peptide encoded by a PKCη-derived uORF, with a CPP penetratin added to its C-terminus.

[0031] SEQ ID NO: 3 is a fragment of the PKCη derived uORF containing the pseudosubstrate.

[0032] SEQ ID NO:4 is a pseudosubstrate-containing fragment of a PKCη-derived uORF comprising an alanine substitution at Cys-10.

[0033] SEQ ID NO:5 is the full-length PKCη-derived uORF containing an alanine substitution at Cys-10.

[0034] SEQ ID NO:6 is a pseudosubstrate-containing fragment of a PKCη-derived uORF comprising a serine substitution at Ala-6.

[0035] SEQ ID NO:7 is a pseudosubstrate-containing fragment of a PKCη-derived uORF comprising a threonine substitution at Ala-6.

[0036] SEQ ID NO:8 is the full-length PKCη-derived uORF containing a serine substitution at Ala-6.

[0037] SEQ ID NO:9 is the full-length PKCη-derived uORF containing a threonine substitution at Ala-6.

[0038] SEQ ID NO: 10 is a CPP.

[0039] SEQ ID NO: 11 is PKCζuORF.

[0040] SEQ ID NO: 12 is the PKCζuORF without the two C-terminal amino acids.

[0041] SEQ ID NO: 13 is the CPP Arginine (R) repeat.

[0042] SEQ ID NOs: 14-18 are illustrative nuclear localization signals (NLS).

[0043] SEQ ID NO: 19 is a peptide sequence encoding a pseudosubstrate sequence of a novel PKCη isoform (ηPC).

[0044] SEQ ID NO:20 is the rhesus monkey encoded peptide sequence from uORF2 of PKCη.

[0045] SEQ ID NO:21 is the elephant-encoded peptide sequence from uORF2 of PKCη.

[0046] SEQ ID NO:22 is the dog-encoded peptide sequence from uORF2 of PKCη.

[0047] SEQ ID NO:23 is the mouse encoded peptide sequence from uORF2 of PKCη.

[0048] SEQ ID NO:24 is the rat encoded peptide sequence from uORF2 of PKCη.

[0049] SEQ ID NO: 25 is the conventional PKCα internal pseudosubstrate sequence.

[0050] SEQ ID NO: 26 is the conventional PKCβ internal pseudosubstrate sequence.

[0051] SEQ ID NO: 27 is the conventional PKCγ internal pseudosubstrate sequence.

[0052] SEQ ID NO: 28 is a novel PKCδ internal pseudosubstrate sequence.

[0053] SEQ ID NO: 29 is a novel PKCε internal pseudosubstrate sequence.

[0054] SEQ ID NO:30 is an atypical PKCζ internal pseudosubstrate sequence.

[0055] SEQ ID NO:31 is an atypical PKCλ / ι internal pseudosubstrate sequence.

[0056] SEQ ID NO: 32 is the summary sequence of the peptide encoded by the variant PKCη derived uORF.

[0057] SEQ ID NOs: 33-35 are shRNA sequences for targeting PKCη expression.

[0058] SEQ ID NO:36 is the full-length PKCη-derived uORF containing an alanine substitution at Arg-8.

[0059] SEQ ID NO:37 is the full-length PKCη-derived uORF comprising an alanine substitution at Arg-8 and an alanine substitution at Cys-10.

[0060] SEQ ID NO:38 is the full-length PKCη-derived uORF containing an alanine substitution at Ser-12.

[0061] I. Abbreviations

[0062] CPP cell penetrating peptide

[0063] PKC protein kinase C

[0064] PS pseudosubstrate

[0065] uORF upstream open reading frame

[0066] II. Terminology

[0067] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. The singular terms "a", "an" and "the" include plural indicators unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides and all molecular weight or molecular mass values ​​are approximate and are provided for description. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described as follows. The term "comprise" means "including / include". The abbreviation "eg (for example)" is derived from Latin exempli gratia and is used herein to indicate non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the term "for example". In the event of a conflict, this specification (including term explanation) shall prevail. In addition, all materials, methods and embodiments are illustrative and are not intended to be limiting.

[0068] Abnormal: Not normal. As used herein, abnormal cell proliferation or cell division refers to excessive or hyperproliferation, such as may occur in a subject diagnosed with cancer or a hyperproliferative disorder.

[0069] Administration: The composition is introduced into the subject through a selected route. The administration of the active compound or composition can be by any route known to those skilled in the art. Administration can be local or systemic. Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intraocular administration, or local ocular administration. In addition, local administration includes administration routes commonly used for systemic administration, such as by directing intravascular administration to the arterial supply of a specific organ. Therefore, in a specific embodiment, local administration includes intra-arterial administration and intravenous administration, as long as such administration targets the vascular system of a specific organ. Local administration also includes incorporating active compounds and agents into implantable devices or constructs, such as vascular stents or other cavities (reservoirs), and delivery devices such as biodegradable implants, which release active agents and compounds over extended time intervals to obtain sustained and / or local therapeutic effects.

[0070] Systemic administration includes any route of administration designed to distribute the active compound or composition extensively throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to, intra-arterial and intravenous administration. Systemic administration also includes, but is not limited to, topical administration, subcutaneous administration, intramuscular administration, or administration by inhalation, as long as such administration is directed to absorption and distribution throughout the body via the circulatory system.

[0071] Antagonist: A molecule or compound that tends to counteract the action of another molecule or compound, such as phosphatase activity, or in some cases blocks the ability of a given chemical to bind to its receptor or other interacting molecule, thereby preventing a biological response. Antagonists are not limited to a particular type of compound, and in various embodiments may include peptides, antibodies and fragments thereof, as well as other organic or inorganic compounds (e.g., peptide mimetics and small molecules). As used herein, "inhibitor" is synonymous with "antagonist."

[0072] Antisense inhibitor: refers to an oligomeric compound, which is at least partially complementary to the region of the target nucleic acid molecule to which it hybridizes. As used herein, an antisense inhibitor (also referred to as an "antisense compound") "specific to a target nucleic acid molecule is an inhibitor that specifically hybridizes with a target nucleic acid molecule and regulates the expression of the target nucleic acid molecule. As used herein, a "target" nucleic acid is a nucleic acid molecule to which an antisense compound is designed to specifically hybridize and regulate expression. Non-limiting examples of antisense compounds include primers, probes, antisense oligonucleotides, siRNA, miRNA, shRNA, and ribozymes. Therefore, these compounds can be introduced as single-stranded, double-stranded, circular, branched, or hairpin compounds, and can include structural elements, such as internal or terminal protrusions or loops. Double-stranded antisense compounds can be two chains that hybridize to form a double-stranded compound, or a single chain with enough self-complementarity to allow hybridization and form a complete or partial double-stranded compound.

[0073] Cancer: The product of neoplasia is a neoplasm (tumor or cancer), which is an abnormal growth of tissue due to excessive cell division. Tumors that do not metastasize are called "benign". Tumors that invade surrounding tissues and / or can metastasize are called "malignant". Neoplasia is an example of a proliferative disorder. "Cancer cells" are neoplastic, such as cells or cell lines isolated from a tumor.

[0074] Examples of hematological neoplasms include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic acute myeloid leukemia [AML] and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0075] Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer (such as small cell lung cancer and non-small cell lung cancer), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, neuroblastoma, and retinoblastoma).

[0076] Cell penetrating peptide (CPP): a short polypeptide, usually less than or equal to 40 amino acids in length, capable of translocation across the cell membrane and into the cytoplasm together with any molecule connected to the CPP. As used herein, CPP is synonymous with peptides and sequences known in the art as "protein transduction domain (PTD)", "Trojan protein" and "membrane translocation sequence (MTS)" and encompasses the latter. Typical CPPs are positively charged and are dominated by amino acids such as arginine and lysine in their sequences. Specific non-limiting examples of CPPs include polylysine or polyarginine peptides (e.g., but not limited to, between 3 and 20 amino acids), penetratin, TAT, SynB1, SynB3, PTD-4, PTD-5, FHV-Coat (35-49), BMV Gag (7-25), HTLV-II Rex (4-16), D-Tat, R9-Tat, Transportan, MAP, SBP, NLS, FBP, MPG, Pep-1, and Pep-2.

[0077] Nuclear localization signal (NLS): The terms "nuclear localization signal" and "NLS" sequences described herein are used interchangeably and refer to amino acid sequences that can induce the transport of molecules comprising or connected to such sequences into the nucleus. For example, NLS can guide the transport of proteins associated with it from the cytoplasm of the cell across the nuclear membrane barrier. NLS is intended to cover not only the nuclear localization sequence of a specific peptide, but also its derivatives that can guide the translocation of cytoplasmic polypeptides across the nuclear membrane barrier. When connected to the N-terminus, C-terminus, or both the N-terminus and the C-terminus of a polypeptide, NLS can guide the nuclear translocation of a polypeptide.

[0078] Chemotherapeutic agent: any chemical agent that has a therapeutic effect in treating a disease characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancers as well as diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent used to treat breast cancer, ovarian cancer or other tumors, such as an anti-tumor agent. In one embodiment, a chemotherapeutic agent is a radioactive compound. Those skilled in the art can easily identify the chemotherapeutic agent used (see, for example, Slapak and Kufe, Principles of Cancer Therapy). Non-limiting examples of chemotherapeutic agents include etoposide, cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone and vinorelbine. Combination chemotherapy is the administration of more than one agent to treat cancer, including a combination of a peptide described herein and one or more chemotherapeutic agents, such as chemotherapeutic agents that target tumor DNA, such as by inducing strand breaks or sequence errors.

[0079] Chemotherapeutic agents can be provided to subjects in various preparations and treatment regimens. In some embodiments, chemotherapeutic agents can be provided in the form of one or more preparations separated from other therapeutic agents such as peptides or nucleic acid agents as described herein. In other embodiments, chemotherapeutic agents are connected or conjugated to peptides and / or nucleic acids as described herein. Various methods for connecting molecules to peptides are known in the art. A non-limiting example used is to introduce non-conventional amino acids into peptides, followed by chemical reactions, and the molecule of interest is attached to the R group of the non-classical amino acids incorporated (see Kubyshkin et al, Biotechnology Journal, 3July 2017).

[0080] Chimera: A nucleic acid sequence, an amino acid sequence or a protein that includes nucleic acid sequences, amino acid sequences or proteins from two or more sources, such as amino acid sequences from two or more different species or two or more polypeptides from the same species. In general, chimeric sequences are the result of genetic engineering. A PKCηuORF2-derived peptide fused to at least one CPP described herein is an example of a polypeptide chimera.

[0081] Contact: To place in direct physical communication. Includes both solid and liquid forms. Contact can occur in vitro with isolated cells or in vivo by administration to a subject.

[0082] Effective amount of a composition: An amount of a composition (including an isolated peptide described herein) sufficient to achieve the desired effect in a treated subject. The effective amount of the composition can be administered in a single dose or multiple doses during the course of treatment, such as daily administration. However, the effective amount of the composition will depend on the composition used, the subject being treated, the severity and type of the ailment, and the mode of administration of the composition.

[0083] Encoding: A polynucleotide is considered to "encode" a polypeptide if the polynucleotide, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide or fragment thereof and / or the polypeptide or fragment thereof.

[0084] Expression control sequence: a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operably connected, such as the expression of the PKCηuORF2 derived polypeptide can be operably connected to an expression control sequence. When the expression control sequence controls and regulates the transcription of the nucleic acid sequence and the translation when appropriate, the expression control sequence is operably connected to the nucleic acid sequence. Therefore, the expression control sequence can include a start codon (ATG) in front of a suitable promoter, enhancer, transcription terminator, protein coding gene, an intronic splicing signal (maintaining the correct reading frame of the gene to allow correct translation of mRNA) and a stop codon. The term "control sequence" is intended to include at least components whose presence can affect expression, and can also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. The expression control sequence can include a promoter.

[0085] Functional fragments and variants of polypeptides: those fragments and variants of the PKCη / PKCζuORF2 derived polypeptides that include one or more functions of the parent polypeptide. It is recognized that a gene or cDNA encoding a polypeptide can undergo considerable mutations without substantially changing one or more functions of the polypeptide. First, it is well known that the genetic code is degenerate, so different codons encode the same amino acid. Secondly, even if amino acid substitutions are introduced, the mutations can be conservative and have no substantial effect on the basic function of the protein. Third, a portion of a polypeptide chain can be deleted without damaging or eliminating all of its functions. Fourth, insertions or additions can be made in the polypeptide chain, such as adding an epitope tag, without damaging or eliminating its function. Other modifications that can be made without substantially damaging one or more functions of the polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of uncommon amino acids. Such modifications include, for example, acetylation, carboxylation, myristoylation, phosphorylation, glycosylation, ubiquitination, labeling (e.g., with radionuclides) and various enzymatic modifications, as readily understood by those skilled in the art. Various methods for labeling polypeptides and markers for such purposes are well known in the art, and include radioisotopes such as32 P, ligands that bind to or are bound by labeled specific binding partners (eg, antibodies), fluorophores, chemiluminescent agents, enzymes, and anti-ligands. Functional fragments and variants may have different lengths.

[0086] Heterologous: A type of sequence that is not normally (i.e., in a wild-type sequence) adjacent to a second sequence. In one embodiment, the sequence is from a different genetic source than the second sequence, such as a virus or organism. Chimeric polypeptides, such as those described herein, are typically composed of heterologous sequences.

[0087] Hyperproliferative disease: A disease or condition characterized by uncontrolled proliferation of cells. Hyperproliferative diseases include, but are not limited to, malignant and non-malignant tumors and psoriasis, and can be treated by agents that inhibit cell proliferation, such as the PKCηuORF2-derived peptides described herein.

[0088] Isolated: A biological component (such as a nucleic acid, protein or organelle) that has been substantially separated or purified from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles) in the cells of an organism in which the component naturally occurs. Isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids and proteins, such as the PKCηuORF2 derivative peptides described herein.

[0089] Neoplasia, malignancy, cancer and tumor: A neoplasm is an abnormal growth of tissue or cells due to excessive cell division (cell proliferation). Neoplastic growth can give rise to a tumor. The number of tumors in an individual is the "tumor burden", which can be measured as the number, volume or weight of tumors. Tumors that do not metastasize are called "benign". Tumors that invade (or migrate to) surrounding tissues and / or can metastasize are called "malignant". Malignant tumors are also called "cancers".

[0090] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0091] Pharmaceutically acceptable carrier: pharmaceutically acceptable carriers that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by Lloyd V.Allen, Jt. (Ed.), 22nd edition (2012) describes compositions and formulations suitable for drug delivery of compounds disclosed herein. In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, dextrose aqueous solutions, glycerol, etc. as vehicles. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also include a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0092] Agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or cell. Incubation includes exposing the target to the agent for a sufficient time for the agent to interact with the cell. Contacting includes incubating the agent in solid or liquid form with the cell.

[0093] Protein Kinase C (PKC): is a family of protein kinase enzymes that are thought to play a key role in cell signal transduction cascades and are associated with mediating immune responses, cell growth and regulating cell division, as well as learning and memory. The PKC family is divided into three subfamilies: conventional, novel, and atypical. Each subfamily can be further divided into multiple isoforms. Specific examples of conventional isoforms include α, β, and γ; novel isoforms include δ, ε, and η; atypical isoforms include ζ and λ / ι.

[0094] Polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. The terms polypeptide, protein or peptide as used herein encompass any amino acid sequence and include modified sequences such as glycoproteins. The term polypeptide is specifically intended to encompass naturally occurring proteins as well as recombinantly or synthetically produced proteins.

[0095] The term polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope. The phrase "functional fragment or variant of a polypeptide" refers to all polypeptide fragments that retain an activity or a measurable portion of the activity of the polypeptide from which the fragment is derived.

[0096] Providing conservative amino acid substitution tables of functionally similar amino acids is well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions of each other:

[0097] 1) Alanine (A), serine (S), threonine (T);

[0098] 2) Aspartic acid (D), glutamic acid (E);

[0099] 3) Asparagine (N), glutamine (Q);

[0100] 4) Arginine (R), Lysine (K);

[0101] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0102] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0103] Preventing or treating a disease: Preventing a disease means inhibiting the full development of a disease, such as inhibiting the development of a myocardial infarction in a person with coronary artery disease or inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment means therapeutic intervention to ameliorate the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0104] Purified: The term purified does not require absolute purity; rather, it is a relative term. Thus, for example, a purified protein preparation is one in which the referenced protein is purer than the protein is in its native environment within a cell.

[0105] Radiation therapy (radiotherapy): The treatment of disease (e.g., cancer or other hyperproliferative diseases or conditions) by exposing a subject or its tissues to radioactive substances. Radiation therapy is the medical use of ionizing radiation to control malignant cells as part of cancer treatment. Radiation therapy can be used for curative or adjuvant cancer treatment. It is used as a palliative treatment when cure is not possible, with the goal of local disease control or symptom relief.

[0106] Recombinant: A nucleic acid having a sequence that does not occur naturally or having a sequence that is produced by the artificial combination of two originally isolated sequence fragments. This artificial combination can be achieved by chemical synthesis or more commonly by artificial manipulation of isolated fragments of nucleic acids, such as by genetic engineering techniques. The expression vectors expressing PKCηuORF2-derived peptides described herein are exemplary recombinant nucleic acids.

[0107] Sequence identity: The similarity between two nucleic acid sequences or two amino acid sequences is expressed as the similarity between the sequences, also called sequence identity. Sequence identity is usually measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Sequence alignment methods for comparison are well known in the art, such as the NCBI Basic Local Alignment Search Tool (BLAST), available from a number of sources, used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. It can be accessed on the NCBI website, along with instructions on how to use the program to determine sequence identity.

[0108] Subject: Living multicellular organisms, including vertebrate organisms, a category that includes humans and non-human mammals.

[0109] Therapeutically effective amount: The amount of a compound sufficient to achieve the desired effect in a subject being treated. The effective amount of a compound can be administered in a single dose or multiple doses during the course of treatment, such as daily administration. However, the effective amount will depend on the compound used, the subject being treated, the severity and type of the ailment, and the mode of administration of the compound. For example, a therapeutically effective amount of an active ingredient can be measured as the concentration (moles per liter or moles-M) of the active ingredient (such as a small molecule, peptide, protein, or antibody) that produces the effect in blood (in vivo) or a buffer (in vitro).

[0110] Vector: A nucleic acid molecule introduced into a host cell to produce a transfected host cell. A recombinant DNA vector is a vector with recombinant DNA. A vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selective marker genes and other genetic elements known in the art. A viral vector is a recombinant DNA vector with at least some nucleic acid sequences derived from one or more viruses.

[0111] III. Overview of Several Implementation Methods

[0112] Provided herein are synthetic peptides having point mutations derived from uORFs of PKCη isoforms, particularly polypeptides comprising an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NOs: 5, 36 and 37.

[0113] The polypeptide of SEQ ID NO: 5 is further described herein. The polypeptide of SEQ ID NO: 36 is additionally described herein. The polypeptide of SEQ ID NO: 37 is additionally described herein.

[0114] In a specific embodiment, the polypeptide comprises a myristoyl group.

[0115] In some embodiments, the polypeptide includes at least one cell penetrating peptide (CPP), such as a penetrating peptide CPP sequence.

[0116] Additionally, nucleic acids encoding the provided polypeptides are described herein.In a specific embodiment, the nucleic acid encoding the polypeptide is part of an expression vector and is operably linked to the expression vector.

[0117] Further described herein are pharmaceutical compositions comprising the polypeptide or nucleic acid and a pharmaceutically acceptable carrier or excipient.

[0118] In some embodiments, the polypeptide or nucleic acid is used to treat a disease or condition associated with abnormal cell proliferation, such as a disease or condition that causes benign abnormal cell growth or a benign tumor. In other examples, the disease or condition is cancer. In a specific embodiment, such use includes the preparation of a medicament for treating a disease or condition associated with abnormal cell proliferation.

[0119] In specific embodiments, the cancer is breast cancer, leukemia, glioblastoma, osteosarcoma, melanoma, lymphoma, lung cancer, neuroblastoma, choriocarcinoma, colorectal adenocarcinoma, colon cancer, prostate cancer, and cervical cancer.

[0120] In some embodiments, the polypeptide or nucleic acid is administered together with a chemotherapeutic agent, surgery, or radiation therapy (which may itself be administered in a separate formulation or conjugated to the polypeptide or nucleic acid).

[0121] Also described are methods for treating a disease or condition associated with abnormal cell proliferation, comprising administering a therapeutically effective amount of the peptide to a subject in need thereof, thereby treating the disease or condition. In some embodiments, the peptide is provided to the subject as a nucleic acid capable of expressing the peptide. In specific embodiments, the disease or condition results in benign abnormal cell growth or benign tumors. In other embodiments, the disease is cancer.

[0122] The provided treatment methods also include methods of inhibiting cell proliferation or cancer metastasis in a subject, which is achieved by administering a therapeutically effective amount of the peptide and a chemotherapeutic agent (alone or conjugated to the peptide) to a subject in need; thereby inhibiting cell proliferation or cancer metastasis. Specific embodiments of such methods utilize nucleic acids capable of expressing isolated peptides.

[0123] IV. Protein Kinase C Peptide Inhibitors

[0124] The following discovery is provided herein: an upstream open reading frame (uORF) identified in the 5'-untranslated region (5'-UTR) of protein kinase C (PKC) isoform η encodes a 26 amino acid peptide that can inhibit PKC kinase activity, cell migration and cell proliferation, particularly in cancer cells. Within the peptide, a 17 amino acid subpeptide with homology to a PKC pseudosubstrate was identified. Collectively, these peptides and those described thereby are referred to as PKCηuORF-derived peptides.

[0125] Thus, described herein are isolated peptides of at least 17 amino acids, comprising an amino acid sequence having at least 70% identity to the sequence set forth herein in SEQ ID NO: 3. The isolated peptides that can be produced synthetically include functional variants and sequence variants of at least 17 amino acids having at least 70-99% identity to SEQ ID NO: 3, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 3.

[0126] Specific embodiments of the peptides include peptide sequences of at least 26 amino acids in length having the amino acid sequence of the peptide produced by PKCηuORF2 and set forth herein as SEQ ID NO: 1. Specific embodiments of the peptides include peptides having at least 70-99% identity to SEQ ID NO: 1, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 1.

[0127] Specific embodiments of the peptides include peptide sequences of at least 26 amino acids in length, which although similar to uPEP 1-26 (SEQ ID NO: 1), but including at least one alanine substitution, particularly at Cys-10. Specific embodiments of the peptide include peptides having at least 70-99% identity to SEQ ID NO: 5, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 5.

[0128] Specific embodiments of the peptides include peptide sequences of at least 26 amino acids in length, which although similar to uPEP 1-26 (SEQ ID NO: 1), but including at least one alanine substitution, particularly at Arg-8. Specific embodiments of the peptide include peptides having at least 70-99% identity to SEQ ID NO: 36, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 36.

[0129] Another embodiment of the peptide comprises a peptide sequence of at least 26 amino acids in length, which although similar to uPEP 1-26 (SEQ ID NO: 1), but including at least two alanine substitutions, particularly at Cys-10 and Arg-8. Specific embodiments of the peptide include peptides having at least 70-99% identity to SEQ ID NO: 37, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 37.

[0130] Also described herein is the sequence of the peptide encoded by the PKCη derived uORF set forth herein as SEQ ID NO:32 (MASRGALRRXLSPGLPRLLHLSRGLA); wherein X is C (SEQ ID NO: 1) or A (SEQ ID NO: 5).

[0131] Further described herein are isolated polypeptides having at least 70%-99% identity to a peptide derived from PKCζuORF and set forth herein as SEQ ID NO:11, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO:11.

[0132] Specific embodiments of the peptides include peptide sequences of at least 26 amino acids in length, which although similar to uPEP 1-26 (SEQ ID NO: 1), but including at least one alanine substitution, particularly at Ser- 12. Specific embodiments of the peptide include peptides having at least 70-99% identity to SEQ ID NO: 38, including peptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to SEQ ID NO: 38.

[0133] Specific non-limiting embodiments of the isolated synthetic polypeptides include polypeptides having sequences as shown herein as SEQ ID Nos 2, 4-9, 12 and 36-38.

[0134] In a specific embodiment, the polypeptide that can be produced synthetically or produced by an expression vector and purified from cell culture by standard methods is fused with at least one cell-penetrating peptide (CPP), which can promote the separated polypeptide (such as PKCηuORF derived peptide) to enter the cell interior from the external environment. As described herein, there are many examples of CPP, and its fusion with PKCηuORF derived peptide can be used to produce chimeric PKCη (or PKCζ) uORF derived peptides, such as described herein. PKCηuORF derived peptide chimeras can include one, two, three or more CPPs from the same or multiple sources, which can be fused to PKCηuORF derived peptides at the C-terminus and / or N-terminus of the polypeptide. Specific examples of CPPs for the chimeric peptides include penetrating peptide CPPs (included herein as SEQ ID NO: 10) and arginine repeat CPPs (included herein as SEQ ID NO: 13), and they include poly-arginine sequences of 3-20 arginine residues. In some embodiments, the at least one CPP is directly fused to the other CPPs in the PKCηuORF derived peptide and / or polypeptide chimera. In other embodiments, the at least one CPP is fused to the other CPPs in the PKCηuORF derived peptide and / or polypeptide chimera via a peptide linker, which may be one or more amino acids connecting one or more CPPs and / or PKCηuORF derived peptides.

[0135] In some embodiments, the CPP comprises repeats of arginine as shown in SEQ ID NO: 13 (RRRX), wherein R is arginine and X is an integer ranging from 0 to 17. In some embodiments, X is an integer ranging from 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 6 to 17, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, or 6 to 8. Each possibility represents a separate embodiment of the present invention.

[0136] In some embodiments, the peptide of the present invention further comprises a nuclear localization signal (NLS) fused to the carboxyl or amino terminus, wherein the NLS sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 (KKKRR), SEQ ID NO: 15 (PKKKRRV), SEQ ID NO: 16 (KRRMKWKK), SEQ ID NO: 17 (KKKRK), and SEQ ID NO: 18 (KKKRK).

[0137] The polypeptide can be produced by any method known in the art. In a specific embodiment, the polypeptide is chemically synthesized. In other embodiments, the polypeptide is produced and purified by a suitable prokaryotic, fungal, plant or animal cell host into which a suitable polypeptide expression vector has been introduced. Protein separation and purification methods are also standard (e.g., affinity chromatography, size exclusion chromatography, etc.).

[0138] As described above, variants, fragments and analogs of the polypeptides are included in the present disclosure. Such polypeptides include polypeptides having about 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with the PKCη or PKCζu ORF derived peptides and chimeras. Other exemplary variants include peptides that differ from the peptides described herein by only one, two or three amino acids. In a specific embodiment, variations from those sequences explicitly described herein can be conservative substitutions that would not significantly change the shape or charge of the polypeptide as would be expected by a person skilled in the art.

[0139] The polypeptides also include those having 100% sequence identity to those shown, but differing from the native sequence in post-translational or post-synthetic modifications. For example, the synthetic polypeptides may be acetylated at the N- or C-terminus of the polypeptide. Other examples include conjugation of a palmitic acid group at either end of the peptide or other modifications common in the field of polypeptide synthesis, such as myristoylation (see also, e.g., sigmaaldrich.com / technical-documents / articles / biology / peptide-modifications-n-terminal-internal-and-c-terminal.html).

[0140] In a specific embodiment, the PKCη or PKCζu ORF-derived peptides, peptides and chimeric peptides are provided as discrete biomolecules. In other embodiments, the polypeptide is a domain of a larger polypeptide, such as an independently folded structural domain or an environmentally accessible functional domain.

[0141] Also provided herein are nucleic acids encoding the PKCη or PKCζu ORF-derived polypeptides and chimeric polypeptides, including variations due to codon degeneracy and specific nucleic acid sequences optimized for codon bias in bacteria, animals and plant cells.

[0142] In a specific embodiment, the nucleic acid sequence is included in a DNA clone and / or expression plasmid of standard in the art. It should be understood that any standard expression plasmid can be used to express one or more of the polypeptides and chimeric polypeptide encoding nucleic acids, as discussed herein. Such a plasmid will contain a replication origin, a selection sequence (such as but not limited to an antibiotic resistance gene) and an expression control sequence operably connected to a PKCη or PKCζuORF derived peptide or chimeric peptide encoding nucleic acid at a minimum. In a specific embodiment, the expression plasmid includes a post-translational sequence (e.g., a signal sequence for instructing polypeptide processing and output) encoded in frame with a PKCη or PKCζuORF derived polypeptide or chimeric polypeptide encoding nucleic acid.

[0143] Specific non-limiting examples of bacterial expression plasmids include IPTG inducible plasmids, arabinose inducible plasmids, etc. Other non-limiting examples of expression induction include light induction, temperature induction, nutrient induction and self-induction and mammalian specific DNA expression plasmids. Custom expression plasmids can be purchased from suppliers such as New England Biolabs (Ipswich, MA) and DNA 2.0 (Menlo Park, CA). In a specific embodiment, PKCη or PKCζu ORF derived polypeptide or chimeric polypeptide expression plasmids can be designed to respond to local cell microenvironment (such as the local environment of a specific cancer type) and carry out specific local induction.

[0144] V. Compositions for treating diseases associated with abnormal cell proliferation

[0145] In addition, the present invention provides compositions comprising the isolated PKCη or PKCζu ORF-derived polypeptides, chimeric polypeptides and encoding nucleic acids for use in treating or preventing diseases or disorders characterized by abnormal cell proliferation. Exemplary uses include preparing a medicament for the treatment or prevention, and a method of treating by administering a therapeutically effective amount of the composition to a subject in need thereof.

[0146] In addition to peptides (synthetic or produced in vivo by transfected nucleic acids) agents for reducing PKCη expression and activity, compositions and methods for treating diseases and / or disorders associated with abnormal cell proliferation are also described herein, which utilize RNA targeting methods, such as using siRNA and shRNA agents. RNA therapeutic agents (including those shown in SEQ ID NO 33-35 herein) can be provided by any standard method in the art of delivering nucleic acid agents to therapeutic targets. Non-limiting examples include lipid-based transfection agents, solid polymer delivery systems, and other systemic and local delivery methods and preparations described herein.

[0147] Diseases and conditions characterized by abnormal cell proliferation are most often associated with benign and cancerous tumors. In the specific example where the disease or condition is cancer, the peptides and methods of use thereof allow for the treatment of cancers particularly associated with abnormal or dysregulated PKC (particularly PKCη or PKCζ) function. Such cancers include breast cancer, lung cancer, colon cancer, glioma, head and neck cancer, ovarian cancer, gastric cancer, and pancreatic cancer.

[0148] In some examples, the PKCη or PKCζu ORF-derived polypeptides, chimeric polypeptides and encoding nucleic acids, RNA targeting agents (RNA interference (RNAi) agents) are used in methods for treating specific characteristic symptoms of cell proliferative diseases. In specific examples, the peptide or RNAi agent can be administered to a subject to inhibit cell proliferation, thereby reducing and / or preventing cancer cell or tumor growth. As described herein, the PKCηu ORF-derived peptide or RNAi agent can inhibit cell migration, which is required for tumor metastasis in a subject. Therefore, in addition to inhibiting proliferation, the peptide can also be used in compositions and methods for inhibiting or reducing cancer metastasis, thereby reducing or even eliminating the spread of cancer beyond its tumor of origin.

[0149] In addition to methods for inhibiting cell proliferation and metastasis, the PKCηuORF derived polypeptides, chimeric polypeptides and encoding nucleic acids and RNAi agents can also be used in methods for treating cerebral infarction, rheumatoid arthritis, Alzheimer's disease and pain. Similarly, the PKCζuORF derived polypeptides, chimeric polypeptides and encoding nucleic acids can be used in methods for treating memory and learning disorders, chronic obstructive pulmonary disease and type 2 diabetes.

[0150] In a specific embodiment, the therapeutic composition described herein can be provided in any pharmaceutically acceptable composition. In such an embodiment, one or more PKCη or PKCζu ORF derived polypeptides or chimeric polypeptide expression nucleic acids and RNAi agents are provided in the form of a pharmaceutical preparation having a therapeutically effective dose of each therapeutic agent as described herein, and including standard pharmaceutically acceptable salts, excipients, fillers, etc.

[0151] A variety of delivery systems are known and can be used for the administration of polypeptides and nucleic acids as therapeutic agents. Such systems include, for example, being encapsulated in liposomes, microparticles, microcapsules, recombinant cells that can express one or more therapeutic molecules, and therapeutic nucleic acids are constructed as a part of retrovirus or other vectors, etc. Methods of introduction include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural and oral routes. Therapeutic agents can be formulated as administration by any convenient route, including, for example, infusion or bolus injection, external use, absorption by epithelium or mucocutaneous membranes (e.g., oral mucosa, rectum and intestinal mucosa, etc.), eyes, nose and transdermal administration, and can be administered together with other bioactive agents. Pulmonary administration (e.g., by inhaler or nebulizer) can also be adopted, for example, using a preparation comprising an aerosolizer.

[0152] In specific embodiments, it may be desirable to administer the drug therapy by injection, catheter, suppository, or implant (e.g., implants formed of porous, non-porous, or gel-like materials, including membranes, such as silicone rubber membranes or fibers), etc. In another embodiment, the therapeutic agent is delivered in a vesicle, particularly a liposome.

[0153] In specific embodiments, the polypeptides and nucleic acids can be formulated for immediate release, whereby they are immediately available to the surrounding environment, thereby providing an effective amount of one or more active agents when administered to a subject, and until the administered dose is metabolized by the subject.

[0154] In another embodiment, the polypeptide and nucleic acid can be formulated in a sustained release formulation or system. In such a preparation, a therapeutic agent is provided for a longer duration, such as 1, 2, 3, 4 or more days, including 1-72 hours, 24-48 hours, 16-36 hours, 12-24 hours, and any time length therebetween. In a specific embodiment, a sustained release formulation is available immediately after administration, and an effective dose of the therapeutic composition is provided, and is available with an effective dose over an extended period of time. In other embodiments, a sustained release formulation is not immediately available in the subject, but only becomes available after the formulation is metabolized or degraded to release one or more active compounds into the surrounding environment, thereby providing one or more active compounds of a therapeutically effective amount. Illustrative non-limiting examples of sustained release formulations include hydrogels and nanoparticles (e.g., Raza et al., Pharmaceutics 10, 2018; and Rivzi et al., Saud Pharm. J. 26, 2018).

[0155] In one embodiment, a pump may be used. In another embodiment, the sustained release formulation comprises polymeric materials commonly used in the art, such as in implants, gels, capsules, and the like.

[0156] In a specific embodiment, the polypeptide and nucleic acid are prepared using methods well known to those skilled in the art. For example, in some embodiments, the compound is prepared together with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to a pharmaceutically acceptable compound approved by a federal or state government regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias worldwide for use in animals, and more particularly for humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent. Such a drug carrier can be a sterile liquid, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions, plasma media, dextrose aqueous solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. The medium can also include conventional pharmaceutical auxiliary materials, such as, for example, pharmaceutically acceptable salts for regulating osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methylcellulose, detergents, buffers, preservatives, etc.

[0157] The example of pharmaceutical excipients includes starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also include a small amount of wetting agent or emulsifier, or pH buffer. The composition can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, all of which are in the immediate and sustained release formulations understood in the art. The therapeutic agent can be formulated as a suppository together with traditional adhesives and carriers (such as triglycerides). Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0158] Therapeutic formulations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier to provide for proper administration to the patient. The formulation should suit the mode of administration.

[0159] The ingredients of the formulation can be provided separately or mixed together in a unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, or as lyophilized powders or water-free concentrates in a hermetically sealed container, such as an ampoule or a sachet, indicating the amount of active agent. Therefore, the present invention also encompasses a kit comprising the PKCη or PKCζu ORF derivative polypeptide or chimeric polypeptide or encoding nucleic acid.

[0160] In a specific embodiment of the pharmaceutical composition and its use method, the PKCη or PKCζu ORF derived polypeptide or chimeric polypeptide or encoding nucleic acid is administered to a subject as a polypeptide. In other embodiments, it is administered to a subject via an expression vector comprising the nucleic acid. It should be understood that in such embodiments, the expression of the polypeptide can be constitutive or inducible, as is well known in the art. In some embodiments, an inducible expression system can allow specific targeting of regions, such as a local tumor environment containing an induction signal.

[0161] In some embodiments, the PKCη or PKCζuORF derived polypeptide or chimeric polypeptide, or its encoding nucleic acid is administered to a subject in combination with other agents for treating the disease or condition being treated. For example, in a method for treating breast cancer, the polypeptide or nucleic acid can be combined with trastuzumab (Herceptin) therapy. In other embodiments of cancer treatment, the peptide or nucleic acid can be combined with a DNA damaging chemotherapeutic agent. Non-limiting examples of such agents include cisplatin, doxorubicin, 5-fluorouracil, etoposide and gemcitabine (see also Cheung-Ong et al., Chem and Biol. 20, 2013). In other examples of cancer treatment, the composition can be combined with surgery and / or radiotherapy. When provided as part of a treatment regimen or treatment method combined with other therapies, the PKCη or PKCζuORF derived polypeptide or chimeric polypeptide or encoding nucleic acid can be administered to a subject in sequence (before or after) or simultaneously with the composition. Where applicable, in specific embodiments, the combination of active ingredients may be administered to a subject in a single or multiple formulations and via a single or multiple routes of administration.

[0162] In a specific embodiment, the chemotherapeutic agent is provided together with the peptide and nucleic acid agent as a separate molecule or formulation. In other embodiments in which the chemotherapeutic agent is administered "together" with the peptide or nucleic acid therapeutic agent, the chemotherapeutic agent is conjugated to the peptide or nucleic acid. Conjugation methods are known in the art. In a specific non-limiting example, the chemotherapeutic component and the peptide (or nucleic acid) component can be connected by a bioreleasable bond, such as by a selectively cleavable joint or amino acid sequence. For example, a peptide joint including a cleavage site for an enzyme is contemplated. An exemplary form of such a peptide joint is one that is cut by urokinase, plasmin, thrombin, factor IXa, factor Xa, or metalloproteinases such as collagenase, gelatinase, or stromelysin. In addition, any other connection / coupling agent and / or mechanism known to those skilled in the art can be used to combine the therapeutic agents described herein, such as, for example, antibody-antigen interactions, avidin biotin bonds, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphate bonds, phosphoramide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies, and antibody fragments or combinations thereof. In a specific embodiment, an unconventional amino acid is incorporated into the peptide sequence or at a terminus of the peptide sequence. Modification and reaction of the R group of such unconventional amino acids using methods such as those described can be used to conjugate chemotherapeutic agents to the peptide.

[0163] The amount of each therapeutic agent used in the composition and method and effectively will depend on the nature of the disease or disease to be treated and the stage of the illness or disease. The therapeutically effective amount can be determined by standard clinical techniques. The precise dosage adopted in the therapeutic composition will also depend on the route of administration used by the composition, and should be determined according to the judgment of the health care practitioner and the situation of each patient. The specific dosage level and the frequency of administration used for any particular subject can vary, and will depend on multiple factors, including the activity of the specific compound, the metabolic stability and the duration of action of the compound, age, body weight, general health, sex, diet, mode of administration and time, excretion rate, drug combination and the severity of the condition of the host being treated.

[0164] The therapeutic compounds and compositions of the present disclosure can be formulated or administered at approximately the same dose throughout the treatment period, in an ascending dose regimen, or in a loading dose regimen (e.g., wherein the loading dose is about two to five times the maintenance dose). In some embodiments, the dose varies during the course of treatment depending on the condition of the subject being treated, the severity of the disease or condition, the apparent response to treatment, and / or other factors determined by one of ordinary skill in the art. In some embodiments, long-term treatment with a drug is contemplated.

[0165] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0166] Example

[0167] Example 1: Method

[0168] Peptides

[0169] The peptides were synthetically produced by GL Biochem. (China).

[0170] Immunoprecipitation and in vitro kinase assays

[0171] Immunoprecipitation was performed by using pre-absorbed mAb on protein A / G-agarose beads at room temperature for 1 hour. Excess Ab was removed by washing 3 times in cold phosphate buffered saline (PBS), and the Ab-coated beads were incubated with cell lysates at 4°C overnight. Immune complexes were precipitated by centrifugation, and then fully washed with RIPA buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 5mM EGTA, 1% NP-40, 45mM β-mercaptoethanol and 50mM NaF) without SDS. Immunoprecipitate samples were aliquoted into Eppendorf tubes for kinase assay. Myelin basic protein 5 μg (MBP, Sigma #M1891) was added as substrate and specified peptide (2 or 10 μM). Prepare the kinase reaction mixture: MgCl2 5mM, HEPES1M, CaCl2 300μM, TRIS-HCl pH 7.4 20mM, PMA 1μM, phosphatidylserine 40μg / ml, 'cold' ATP and γ-ATP 5mM (PerkinElmer). All preparations were performed in ice at 0-4°C. An Eppendorf tube containing 20μl beads mixed with 100μl kinase assay mixture was incubated at 30°C for 30min. The reaction was terminated with 25μl sample buffer *5, denatured at 95°C for 5min, and then centrifuged. All samples were run on SDS-PAGE 10% and transferred to nitrocellulose membranes (Sigma-Aldrich, USA). Kinase activity was assessed with exposure reagents. After detection of the phosphorylated substrate (p-MBP), the membrane was exposed to anti-HA (Bio-legends#901513) as a loading control.

[0172] Wound healing assay

[0173] MCF-7 and MDA-MB-231 cells are seeded in 24-well plates and grown to complete confluence. Subsequently, low serum medium (2% for MCF-7 and 0.1% for MDA-MB-231) containing peptides is added to the wells for 4 hours. Then, a scratch is made in the middle of the well with a sterile 200 μl pipette tip (Eppendorf). After washing once with PBS, return to each processing. Use IX70 Olympus Optical optical microscope (Tokyo, Japan) to take pictures of the wells with 4 × total magnification. Take 3 images of each well every 24 hours.

[0174] Cell viability assay

[0175] Presto Blue assay

[0176] MCF-7 and MDA-MB-231 cells were cultured at 2×10 4 Cells / well were seeded in a 48-well plate. After 24 h, serum-free medium containing peptides was added to the wells for 4 h. Then, serum-containing medium was added and the plates were incubated in an incubator (5% CO2 humidified atmosphere, 37°C) for 24 h. After incubation, the medium with the test peptide was gently aspirated from each well and 25 μL PrestoBlue was added. TM The reagent (Thermo #A13261) was added to each well of a 48-well plate and incubated for 15 min at 37°C, 5% CO2 as recommended. The absorbance was recorded at 570 nm and detected by an 'Infinite M200 PRO' reader.

[0177] XTT assay

[0178] Cells were seeded on 96-well plates at a cell density of 20,000 cells / well and grown for 48 hours. Cells were treated for 48 hours as indicated. XTT (#20-300-1000, Biological Industries) reaction solution (containing XTT and activation solution) was prepared and added (50 μl / well) to the cell culture medium for 2-4 hours at 37°C. The plate was immediately read at 450-500nm in an ELISA reader. Background reading was performed at 630nm, and the results were calculated as the difference between the values.

[0179] Mouse cell transplantation procedure

[0180] Mice were anesthetized with 10 ml / mg ketamine and 1.17 ml / mg xylazine in saline. The mice were anesthetized by intraperitoneal (IP) injection after the inoculation area was disinfected with 70% ethanol solution. 4T1 control and shPKCη knockdown cells (transfected with SEQ ID NO 33-35 or scrambled control (obtained from ATCC)) were injected subcutaneously a few millimeters from the nipple using a 27-gauge needle (120,000 cells in 50 μl per mouse).

[0181] Animal Analysis During the Study: Mice were tested for tumors weekly and primary tumors were examined with calipers. After 10 weeks, mice were euthanized, metastases in the lungs, liver, and spleen were examined, and samples were prepared for immunostaining.

[0182] Stability of peptides in serum

[0183] The peptide solution (2 mM) in H2O (lo-bind tube) was mixed with 20% mouse serum (#M5905, Sigma Aldrich, USA) and incubated at 37°C. At the specified time points, 20 μl of sample was taken from each tube, and the reaction was immediately terminated by adding Tricine sample buffer (#161-0739; Bio-Rad) with (β-mercaptoethanol), followed by incubation at 95°C for 5 min. The samples were run on 16.5% tris-tricine gels and immunoblotted using a rabbit polyclonal antibody raised against the last (C-terminal) 17 amino acids of the predicted uPEP2 sequence. The percentage of intact peptide was calculated by protein blot quantification and normalization in Image lab 6.1 (Bio-rad) software, expressed as a percentage of the amount at t0. The data were fitted to a single-phase exponential decay model using GraphPad Prism version 9 to estimate t 1 / 2 .

[0184] Example 2: Identification and Characterization of PKCηu ORF-Derived Kinase Inhibitors

[0185] Previously, it was reported that the translation of the protein kinase C isoform PKCη is regulated in part by two uORFs. Sequence alignment of human and mammalian PKCη uORF2 sequences surprisingly demonstrated sequence conservation within species ( Figure 1A and 1B ). The high degree of conservation supports its potential functional role. In addition, uORF2 was found to contain a sequence motif similar to the PS motif of PKC, which inhibits their kinase activity, so the similarity between various PKC PS sequences and uORF2 supports the presence of the PS motif in uORF2. ( Figure 1C ).

[0186] To test the possibility that the translated uORF2 peptide could modulate the kinase activity of PKC, full-length human PKCηuORF2 peptides (also described herein as MA-26 and / or uPEP2 1-26 , SEQ ID NO: 1). Peptide uPEP lacking the PS motif 9-26 and uPEP 18-26 Used as control ( Figure 2A ).like Figure 2B and 2C As can be seen in the figure, compared with the control peptide uPEP 9-26 Compared with ηPS, uPEP 1-26 is the strongest inhibitor. uPEP2 was tested as described above 1-26 Effects on the kinase activity of the PKC subfamily. Figure 2D As shown in , uPEP2 strongly inhibits the kinase activity of PKCs from a novel PKC subfamily.

[0187] The in vitro effects of PKCηuORF2 on cancer cell migration were tested in vivo by a standard wound healing (scratch) assay as described above. 1-26 、uPEP 9-26 、uPEP 18-26 The cells were treated with uPEP and ηPS and then streaked on confluent breast cancer cell lines MCF7 (breast cancer), MDA-MB-231 (breast cancer), and MCF10A (untransformed breast cells). 1-26 Inhibits the migration of breast cancer cells MCF7 and MDA-MB-231, but not non-transformed MCF10A ( Figures 3A to 3D ).

[0188] Similarly, the effects of PKCηuORF2 on the proliferation of cell lines MCF7, MDA-MB-231, U251 MG, and MCF10A were tested in vitro. 1-26 、uPEP 9-26 、uPEP 18-26 and ηPS, we observed that uPEP increased compared with untransformed MCF10A cells. 1-26 The greatest inhibition of cell proliferation in transformed cell lines ( Figures 4A to 4D Further sequence comparison of the 5' untranslated regions of several PKC variants revealed the presence of uORFs and sequences similar to PKCη pseudosubstrates ( Fig. 8A ). The uORF of PKCζ containing the PS motif was successfully sequenced, such as Figure 8B As shown in (SEQ ID No.11).

[0189] These results suggest that in PKCηuORF2 uPEP 1-26A short sequence similar to the PKC pseudosubstrate (PS) sequence was identified in uPEP. The PKC PS is part of the backbone of all PKC isoforms and functions as an internal inhibitor of PKC kinase activity. Our results suggest that uPEP 1-26 The peptide is a potent inhibitor of PKC kinase activity. We also showed that it inhibits cell proliferation and migration of breast cancer cells and cell proliferation of glioblastoma cells. In addition, we found a PS motif in the uORF of another PKC isoform, PKCζ. Our study shows for the first time the presence of a functional signature motif in uORF. Our study introduces uORFs as new players in protein network regulation, adding a new layer to the complexity of protein control and signaling cascades that has not been explored before.

[0190] Example 3: PKCη knockdown inhibits tumor formation

[0191] Example 2 demonstrates that inhibition of PKCη activity has an inhibitory effect on the proliferation and migration of breast cancer cells. To test the in vivo effect of PKCη on tumor formation, the establishment and growth of breast cancer cells with and without PKCη (PKCη knockdown 4T1 cells) were compared. Comparison of tumor volume over time showed that PKCeta knockdown cells had a significant inhibitory effect on tumor size ( Fig. 7A PKCη knockdown also inhibited the formation of tumor metastasis ( Figure 7B ). The results shown herein are a knockdown effect of the expression of SEQ ID NO:33, but similar results were observed using the shRNA represented by SEQ ID NO:34 and SEQ ID NO:35 listed herein. In summary, these results indicate that inhibition of PKCη, such as with MA-26 or an active subpeptide derived therefrom, can inhibit tumor growth and metastasis.

[0192] Example 4: uPEP 1-26 Synergistic effects of chemotherapeutic agents in inducing cell death

[0193] The breast cancer cell line MCF-7 was treated with the indicated peptides ( Figure 5 ) were treated with or without the DNA-targeted chemotherapeutic agent etoposide. 1-26 When cells were treated under conditions that resulted in only 10-30% cell death, the presence of both drugs increased cell death to approximately 95-100%, demonstrating a synergistic effect. Treatment with etoposide alone induced some cell death, however, when etoposide was combined with uPEP 1-26 When administered together, a synergistic effect was observed, resulting in almost 100% apoptosis (see Figure 5, arrows). These results strongly suggest the potential for successful targeted dual cancer therapy.

[0194] Example 5: PKCηuORF2 peptide mutants effectively reduce cell viability

[0195] uPEP2 synthesized PKCηuORF2 peptide (SEQ ID NO: 1) 1-26[10C / A] A mutation comprising an alanine substitution at Cys-10, referred to herein as SEQ ID No 5. The cell line MCF-7 ( Fig. 6A )、MDA-MB-231( Figure 6B ) and HeLa( Figure 6C ) with 5 μM and 10 μM doses of uPEP2 18-26 、uPEP2 1-26 、uPEP2 1-26[10C / A] 、uPEP2 1-17 and ηPS were treated to determine the effect on cell proliferation. As shown in the figure, compared with other peptides, mutant uPEP 1-26[10C / A] Effectively reduced cell viability.

[0196] Example 6: PKCηuORF2 peptide mutants

[0197] Additional mutants of the PKCηuORF2 peptide (SEQ ID NO: 1) were synthesized and assayed for activity in kinase, cell proliferation and cell migration assays as described in Examples 1 and 2. Variants used in the assays included sequence variants set forth herein as SEQ ID NO: 2, 8 and 9. Myristoylated PKCηuORF2 peptides were also assayed.

[0198] Also tested were a 17 amino acid subpeptide fragment of SEQ ID NO: 1, which is limited to a putative PKC pseudosubstrate (described herein as SEQ ID NO: 3), and variants thereof (described herein as SEQ ID NO: 4, 6, and 7). The effects of C- or N-terminal addition of a penetrating peptide CPP (SEQ ID NO: 10) or myristoylation were also tested.

[0199] The peptides were also tested for their in vivo effects on cell proliferation, tumor growth and metastasis.

[0200] Example 7: PKCζuORF peptide

[0201] The sequence alignment discussed in Example 2 describes a putative uORF peptide in PKCζ, also listed herein as SEQ ID NO: 11. This peptide and variants thereof (including the peptide listed as SEQ ID NO: 12) were tested for kinase activity as described above, as well as for the effects of the peptide on cell proliferation and migration in vitro, and for its effects on tumor formation, memory and learning in vivo.

[0202] Example 8: Effects of mutated uPEP2 peptides on kinase activity

[0203] As shown in the above examples, uPEP2 (SEQ ID NO: 1) is believed to be a specific novel PKC inhibitor for certain novel subfamily members (PKCδ, PKCε, PKCη and PKCθ), but not an inhibitor of classical or atypical PKCs. 32 Kinase activity assays were performed using P-gATP and myelin basic protein (MBP) as substrates (described in Example 1). It can be seen that both peptide [C10A] mutation (SEQ ID NO: 5) and peptide [R8A] mutation (SEQ ID NO: 36) are better kinase inhibitors for the new PKC isoforms (PKCδ, PKCε, PKCη and PKCθ) than the original uPEP2 (SEQ ID NO: 1), as indicated by the peptide concentration that results in 50% inhibition of the maximal MBP phosphorylation for a specific PKC isoform ( Figures 9A-9E The inhibition of PKCη kinase activity by peptides [C10A] and [R8A] was also shown in Fig.10 , indicating that in this experiment, [R8A] inhibited about 95% of the activity at a concentration of 2.5 μM, while [R8A] almost completely inhibited it.

[0204] Subsequently, all generated mutant peptides were tested as inhibitors of PKCη kinase activity ( Fig.11A and Fig. 11B The results showed that among all the mutants tested, uPEP2[R8A] (SEQ ID NO: 36) most effectively inhibited the kinase activity of the novel PKCη ( Fig.10 and Figures 11A-11B ).

[0205] Notably, uPEP2 with a serine substitution for alanine at position 6 [A6S] (SEQ ID NO: 8) or a threonine substitution for alanine at position 6 [A6T] (SEQ ID NO: 9) was not a better inhibitor than uPEP2 (SEQ ID NO: 1) or [C10A] (SEQ ID NO: 5). uPEP2 with alanine substitution for serine at position 12 [S12A] (SEQ ID NO: 38) was ineffective as a PKCη inhibitor ( Figures 11A-11B ).

[0206] It can be seen that the mutant [R8A] (SEQ ID NO: 36) is the best inhibitor of the novel PKC family member.

[0207] These results suggest potential therapeutic applications for treating cancer.

[0208] Example 9: Effects of mutated uPEP2 peptides on cell survival

[0209] The uPEP2 derivatives uPEP2[R8A] (SEQ ID NO:36) and uPEP2[C10A] (SEQ ID NO:5) were considered to have the greatest therapeutic potential. Cell viability experiments were performed using wild-type uPEP2 and its derivatives (using XTT analysis as described in Example 1). It can be seen that in the various cancer cells analyzed, the two derivatives uPEP2[R8A] and uPEP2[C10A] reduced cancer cell viability to a greater extent than WT uPEP2 ( Figures 12A-12B The half maximal inhibitory concentration (IC50) for cell survival of specific uPEP2 mutants compared to WT uPEP2 is shown. Fig. 12A )uPEP2[R8A] depicts low IC50 values ​​for Kasumi-1, a myeloblast-derived cell line (acute myeloblastic leukemia), followed by triple-negative breast cancer MDA-MB-231. Fig. 12B )uPEP2[C10A] was most effective in HL60 (a lymphoblastoid-derived leukemia cell line). From these experiments, it was confirmed that the [R8A] and [C10A] mutant peptides were more effective in reducing the viability of different cancer cells compared to uPEP2, with [R8A] showing the lowest IC50 value ( Fig. 12A ).

[0210] Furthermore, the experiments demonstrated herein showed that the uPEP2 mutants were most effective in reducing cell viability in breast cancer and leukemia cells. Furthermore, the effects of the mutant peptides on a human AML leukemia line were also shown ( Fig.13 ). Mutant [R8A] was the most effective in reducing viability compared to [C10A]. Notably, peptides with a lauryl modification instead of a myristoyl modification at the N-terminus of the peptide were less effective in reducing viability, suggesting that the lauryl group is less effective in cell penetration than the peptide with a myristoyl group at the N-terminus.

[0211] In addition to the above, further cell survival experiments in response to treatment with the [R8A] or [C10A] mutant peptides were performed on various cell lines (using XTT as described above). The mutant peptides were tested on cell lines derived from various cancers, including breast cancer, leukemia, cervical cancer, glioblastoma, osteosarcoma, cutaneous melanoma, lymphoma, non-small cell lung cancer, neuroblastoma, choriocarcinoma, colorectal adenocarcinoma, colon cancer, and prostate cancer. The IC50 values ​​obtained from each of these experiments were as follows: Fig.15 [R8A] and Fig.16 [C10A] is shown.

[0212] A peptide with two mutations [R8A+C10A] (SEQ ID NO: 37) was synthesized. It can be seen that when compared with each substitution alone ( Figures 14A-14B ) compared with the combined mutant peptides, which were more stable in serum. 1 / 2 In these cells, the effect of [R8A+C10A] on MV-4-11 cell survival was comparable to that of C10A ( Fig.13 ).

[0213] These results strongly suggest the potential for successful cancer therapy utilizing individual mutants [R8A] or [C10A] and / or combined mutants [R8A+C10A].

Claims

1. A polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 5, 36 or 37.

2. The polypeptide according to claim 1, wherein The amino acid sequence is SEQ ID NO:

5.

3. The polypeptide according to claim 1, wherein The amino acid sequence is SEQ ID NO:

36.

4. The polypeptide according to claim 1, wherein The amino acid sequence is SEQ ID NO:

37.

5. The polypeptide of claim 1, wherein the amino acid sequence further comprises a myristoyl group.

6. The polypeptide of claim 1, further comprising at least one cell-penetrating peptide.

7. The polypeptide according to claim 6, wherein The cell-penetrating peptide is a penetrating peptide.

8. A nucleic acid comprising a nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid according to claim 8.

10. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.

11. Use of the polypeptide according to any one of claims 1 to 7 in the preparation of a medicament for treating a disease or disorder associated with abnormal cell proliferation.

12. The use according to claim 11, wherein The polypeptide is provided to the subject as a nucleic acid capable of expressing the polypeptide.

13. The use according to claim 11, wherein The disease or condition results in benign abnormal cell growth or a benign tumor.

14. The use according to claim 11, wherein The disease is cancer.

15. The use according to claim 14, wherein The cancer is selected from the group consisting of breast cancer, leukemia, glioblastoma, osteosarcoma, melanoma, lymphoma, lung cancer, neuroblastoma, choriocarcinoma, colorectal adenocarcinoma, colon cancer, prostate cancer and cervical cancer.

16. The use according to claim 14, wherein The cancer is additionally treated with a chemotherapeutic agent.

17. Use of a polypeptide according to any one of claims 1 to 7 and optionally a chemotherapeutic agent in the preparation of a medicament for inhibiting cell proliferation and / or cancer metastasis in a subject.

18. The use according to claim 17, wherein The polypeptide is provided to the subject as a nucleic acid capable of expressing the isolated polypeptide.

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