Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Sequence Listing submitted May 14, 2021 as a text file named “SequenceListing- 065715-000103WO00_ST25” created on May 14, 2021 and having a size of 82,684 bytes, is hereby incorporated by reference.
• Described herein are methods and compositions related to mitochondrial peptides, analogs and derivatives thereof for use as a therapy for age-related diseases, including cancer.
• Mitochondrial peptides represent a new class of molecules for treatment of human diseases. It is now well-established that mitochondria are key actors in generating energy and regulating cell death. Mitochondria communicate back to the cell via retrograde signals that are encoded in the nuclear genome, or are secondary products of mitochondrial metabolism. More recently, mitochondrial-derived peptides that are encoded by the mitochondrial genome have been identified as important actors in these regulatory processes. It is widely believed that mitochondrial-derived retrograde signal peptides will aid in the identification of genes and peptides with therapeutic and diagnostic to treat human diseases.
• compositions based on mitochondrial-derived peptides for treatment of age-related diseases, including cancer.
• FIG. 1 NOSH (/VD-One short open reading frames (sORF) in f/umans) is safe in mice.
• NOSH has a sequence of MRLFGLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• FIG. 3 NOSH alters mitochondrial function. Membrane potential was measured by JC-1 staining.
• FIG. 4. NOSH reduces viability of tumor cells. Viability was measured by LDH release into the media.
• FIG. 5A-5E depict that NOSH acts as a senolytic peptide, preferentially reducing viability of senescent cells.
• FIG 5 A and 5B are based on MTT assay.
• FIG. 5C is 10 pM NOSH treatment for 24hr in non-senescent (NS) and senescent cells.
• Alternative representations of FIG. 5 A and 5B are shown in FIG. 5D and 5E, respectively.
• NOSH induces an inflammatory response in senescent cells (primary dermal fibroblast (HDFa)).
• Doxorubicin induced the senescent cells.
• the non-senescent cells and senescent cells were treated with lOpM NOSH for 24hr. After 24hr, conditioned media were collected and measured for the cytokine levels by meso scale discovery (MSD) assay.
• MSD meso scale discovery
• FIG. NOSH analogues with increased efficacy.
• FIG. 8A and 8B depict that NOSH is effective in decreasing viability of a number of different cancer cell lines.
• FIG. 8 A depicts viability of J82 cells, OVCAR3 cells, SHSY5Y cells, HepG2 cell.
• FIG. 8B depicts viability of HCT116 cells and MCF7 cells (upper row: 10 pM for 24 hours; lower row: 100 pM).
• FIG. 9 depicts gene expression in M2 macrophages, demonstrating that NOSH converts M2 Macrophages to Ml Macrophages.
• THP-1 cells were treated in PMA (200ng/ml) for 24 hours. Subsequently, medium was changed and PMA was removed. Cells were washed with PBS for three times. Cells were then treated with LPS (lOOng/ml) or IL-4 (20ng/ml), with different doses of NOSH for 24 hours.
• FIG. 11A is a graph depicting efficacy of 214 NOSH analogues in DU145 cells compared to NOSH.
• FIG. 1 IB depicts the efficacy of NOSH analogs (from NOSH-1 to NOSH-78) in DU145 cells compared to NOSH, an enlarged version of relevant data in FIG. 11 A.
• FIG. l lC depicts the efficacy of NOSH analogs (from NOSH-78 to NOSH-153) in DU145 cells compared to NOSH, an enlarged version of relevant data in FIG. 11 A.
• FIG. 11D depicts the efficacy of NOSH analogs (from NOSH-153 to NOSH-214) in DU145 cells compared to NOSH, an enlarged version of relevant data in FIG. 11 A.
• FIG. 12. NOSH analogues efficacy in A549 Cells FIG. 13. NOSH structure. NOSH has a predicted double alpha-helix structure where the alpha-helices are predicted to be in amino acids 3-11 and amino acids 18-30. The hinge region between the helices seems to be of importance as mutations in this region significantly alter function.
• administering refers to any route for delivering a pharmaceutical composition to a patient. Routes of delivery may include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art.
• Parenteral refers to a route of delivery that is generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
• the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
• Modulation or “modulates” or “modulating” as used herein refers to upregulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response or the two in combination or apart.
• “Pharmaceutically acceptable excipients” refer to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
• excipients include starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, pH buffering agents and combinations thereof.
• Promoter and/or “promoting” as used herein refer to an augmentation in a particular behavior of a cell or organism.
• Subject includes all animals, including mammals and other animals, including, but not limited to, companion animals, farm animals and zoo animals.
• the term “animal” can include any living multi-cellular vertebrate organisms, a category that includes, for example, a mammal, a bird, a simian, a dog, a cat, a horse, a cow, a rodent, and the like.
• the term “mammal” includes both human and non-human mammals.
• a subject, individual or patient refers to a human being.
• a “patient in need of’ or “subject in need” of treatment for a particular disease, disorder, or condition may be a subject suspected of having that disease, disorder, or condition, diagnosed as having that disease, disorder, or condition, already treated or being treated for that disease, disorder, or condition, not treated for that disease, disorder, or condition, or at risk of developing that disease, disorder, or condition.
• “Therapeutically effective amount” as used herein refers to the quantity of a specified composition, or active agent in the composition, sufficient to achieve a desired effect in a subject being treated.
• a therapeutically effective amount may vary depending upon a variety of factors, including but not limited to the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, desired clinical effect) and the route of administration.
• physiological condition of the subject including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, desired clinical effect
• One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation.
• Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disease or disorder (collectively “ailment”) even if the treatment is ultimately unsuccessful.
• Those in need of treatment may include those already with the ailment as well as those prone to have the ailment or those in whom the ailment is to be prevented.
• Senescence occurs when a cell is too damaged to continue dividing and therefore its growth is curtailed. Senescent cells may begin leaching out harmful proteins and other compounds that can damage cells around them, leading to inflammation and eventually cell death. Senolytics are a class of drugs which selectively induce death of senescent cells. In some embodiments, senoytics reverse damages done by senescent cells.
• “Retrograde signaling” refers to a process where a signal travels backwards from a target source to its original source.
• mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus.
• Mitochondria are thought to have transferred their genome to the host nucleus, leaving chromosomal “doppelgangers”, through the process of Nuclear Mitochondrial DNA-Transfer or nuclear insertions of mitochondrial origin (NUMTs).
• NUMTS come in various sizes from all parts of the mitochondrial DNA (mtDNA) with various degrees of homology with the original sequences. Entire mtDNA can be found in the nuclear genome, although in most cases with substantial sequence degeneration. Most NUMTs are small insertions of ⁇ 500 bp and only 12.85% are >1500 bp.
• the percentage identity is inversely correlated with size and the mean percentage between NUMTs and mtDNA is 79.2% with a range of 63.5% to 100% identity.
• Mitochondrial DNA (mtDNA) replication and transcription starts are regulated by nuclear-encoded proteins and is thought to be transcribed as a single polycistronic precursor that is processed into individual genes by excising the strategically positioned 22 tRNAs (tRNA punctuation model), giving rise to two rRNAs and 13 mRNA.
• the human mitochondrion has two promoters in the heavy strand (major and minor) in close proximity, and one in the light strand, thereby giving rise to three different single polycistronic transcripts.
• the heavy major promoter is responsible for 80% of all mitochondrial RNA (mtRNA) transcripts. Although the entire gene is thought to be transcribed as a single transcript, the abundance of individual rRNA, tRNA, and mRNA transcripts varies greatly, and the rRNAs are the most abundant. This processing structure indicates an unexplored class of posttranscriptional processing in the mitochondria.
• mitochondrially derived peptides also referred to mitochondrial -derived peptides or mitochondrial peptides
• the Inventors identified NOSH (ND-One sORF in Humans) with a potent biological activity against cancer cells while being safe for healthy cells.
• NOSH ND- One sORF in Humans
• NOSH is a mitochondrially encoded open reading frame that leads to the production of a new polypeptide that we call NOSH, which has biological effects affecting mitochondria, cell growth, and cell death.
• This peptide is implicated in cancer, senescence, and aging.
• NOSH as well as synthetic NOSH analogues could be used in slower the progression of cancer, reducing tumor sizes, and treating other age-related diseases such as inflammatory diseases, obesity, and metabolic disorders.
• These peptides are key factors in retrograde mitochondrial signaling as well as mitochondrial gene expression.
• mitochondria have a modest sized circular genome of -16,570 bp, which ostensibly includes only 13 protein coding genes, which are all structural components of the electron transport chain system.
• This invention includes the composition of matter of a family of peptide analogues of NOSH. This invention includes methods of use for these peptides in these indications. The invention also includes antibodies and assays for the detection of the levels of the NOSH peptide in the circulation and tissues of humans.
• compositions comprising a mitochondrial -derived peptide or a synthetic or recombinant analog thereof.
• a composition comprises a mitochondrial-derived peptide that comprises, or consists of, a peptide with an amino acid sequence of SEQ ID NO:215, or set forth in SEQ ID NO:215.
• a composition comprises two or more mitochondrial-derived peptides selected from SEQ ID NOs: 1-215.
• a composition includes a peptide analog of SEQ ID NO:215, a peptide derivative of SEQ ID NO:215, or a combination thereof.
• the mitochondrial peptide is about 12-65, 14-40, 16-35, 18-32, 20 -32, or 25-32 amino acids in length. In a particular embodiment, the mitochondrial peptide is about 32 amino acids in length. In one embodiment, the mitochondrial peptide includes a synthetic amino acid. In one embodiment, the mitochondrial peptide possesses at least 25%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more percentage identity to MRLF GLLL A VRRS GRSL SLMLTLIRGL SKRLG (SEQ ID NO:215).
• the mitochondrial peptide possesses at least 87.5% identity to SEQ ID NO:215; or, the mitochondrial peptide is 28-36 amino acids in length or contains no more than four amino acids difference (including substitution, deletion, and/or insertion) compared to SEQ ID NO: 215. In some embodiments, the mitochondrial peptide possesses at least 93% identity to SEQ ID NO:215; or, the mitochondrial peptide is 30-34 amino acids in length or contains no more than two amino acids difference (including substitution, deletion, and/or insertion) compared to SEQ ID NO: 215.
• the mitochondrial peptide possesses at least 80% identity to SEQ ID NO:215; or, the mitochondrial peptide is 26-38 amino acids in length or contains no more than six amino acids difference (including substitution, deletion, and/or insertion) compared to SEQ ID NO: 215. In some embodiments, amino acids 3-11 and amino acids 18-30 are not replaced or deleted in SEQ ID NO:215.
• substitution of amino acids that disrupt the alpha-helixes are bad for activity; and therefore, in some aspects, the amino acid residues of SEQ ID NO:215 are not replaced with glycine, not replaced with proline, or not replaced with either glycine or proline; and in further aspects, the amino acid residues 3 through 11 and residues 18 through 30 of SEQ ID NO:215 are not replaced with glycine, not replaced with proline, or not replaced with either glycine or proline.
• replacement with alpha-helix enhancers improve activity of NOSH (SEQ ID NO:215); and therefore in some aspects, one or more residues of SEQ ID NO:215 are independently replaced with alanine, leucine, or both; and in further aspects, one or more residues 3 through 11 and residues 18 through 30 of SEQ ID NO:215 are independently replaced with alanine, leucine, or both.
• Mitochondrial-derived peptide of SEQ ID NO: 215 in some embodiments, can be considered as an analog of one or more peptides of SEQ ID NOs: 1-214.
• a composition comprising a peptide of any of SEQ ID NOs: 1-214 is provided, as well as its analogs or derivatives.
• a composition includes a peptide of SEQ ID NO: 1, and/or a peptide that possesses at least 25%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more percentage identity to (SEQ ID NO:l).
• a composition includes a peptide of SEQ ID NO:2, and/or a peptide that possesses at least 25%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more percentage identity to (SEQ ID NO:2).
• SEQ ID NO:2 One of ordinary skill in the art can establish percentage identity according to methods known in the art, including establishing a comparison window between a reference amino acid sequence and a second amino sequence, to establish the degree of percentage identity.
• the mitochondrial-derived peptide and/or its analogs possesses a post- translational modification or other type of modification such as an artificial modification.
• this includes for example, pegylation, fatty-acid conjugation lipidation, repeat polypeptide extension, the fragment crystallizable region of immunoglobulin G (IgG- Fc), camptothecin (CPT), human serum albumin (HAS), elastin-like polypeptide (ELP), transferrin, or albumin modification, among others.
• the peptide is 12-65 amino acids in length.
• the peptide at position 1 i.e., first N-terminal amino acid
• position 2 is (X2) and so on (X3, X4, X5, X6, etc.), wherein XI, X2, X3, X4, X5, X6, etc. is independently selected from a group consisting of a natural or synthetic amino acid.
• the mitochondrial peptide e.g., SEQ ID NO:215) possesses a post- translational modification or other type of modification such as an artificial modification.
• modifications could include formylation, phosphorylation, acetylation at corresponding XI, X2, X3, X4, X5, and/or X6, etc. positions in analogs thereof.
• the peptide possesses at least 25%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more percentage identity to MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• the peptide is 75%, 80%, 85%, 90%, 95% or more percentage identity to a portion of MRLFGLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), including for example, three or more, five or more, ten or more, fifteen or more, twenty or more, twenty five or more amino acids of MRLF GLLL AVRRSGRSL SLMLTLIRGL SKRLG (SEQ ID NO:215), wherein the portion begins at XI, X2, X3, X4, etc. This includes, any of the sequences in Table 1.
• the peptide includes one or more of the aforementioned portions of MRLF GLLL AVRRSGRSL SLMLTLIRGL SKRLG (SEQ ID NO:215), further including about 6-9 amino acids of XI 1 to X18 of MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• Various embodiments provide methods of treating a disease and/or condition using a mitochondrial-derived peptide and/or analogs, derivatives thereof including administering a quantity of the mitochondrial-derived peptide and/or analogs, derivative thereof, e.g., in a pharmaceutical composition with one or more pharmaceutically acceptable excipient, to a subject in need of treatment.
• the mitochondrial peptide is a MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), an analog or derivative thereof.
• the peptide is about 12-65 amino acids in length.
• the mitochondrial peptide is about 32 amino acids in length.
• the peptide administered possesses at least 80%, 87.5%, 90%, 93% sequence identity to SEQ ID NO:215.
• the peptide administered is selected from Table 1.
• the quantity of the mitochondrial peptide administered is a therapeutically effective amount of the mitochondrial peptide.
• the subject is a mammal. In one embodiment, the subject is a human.
• administration of NOSH or its analogs decreases mitochondrial function.
• administration of NOSH and/or its analogues decreases cancer cell viability.
• administration of NOSH or its analogs to healthy mice has no grossly toxic effects.
• administration of NOSH or its analogs induces autophagy.
• one or more NOSH and its analogs are administered as a senolytic drug, which selectively kills cells that are senescent.
• one or more of NOSH and its analogs are administered as an immunomodulator, increasing the immune response.
• the disease and/or condition suitable for treatment with the mitochondrial peptide or analogue composition described includes cancer.
• cancers are lung cancer, prostate cancer, colorectal cancer, stomach cancer, breast cancer, colorectal cancer, cervical cancer, melanoma, skin cancer, among others.
• disease and/or condition suitable for treatment with the mitochondrial peptide or analogue composition described includes cancer.
• cancers are lung cancer, prostate cancer, colorectal cancer, stomach cancer, breast cancer, colorectal cancer, cervical cancer, melanoma, skin cancer, among others.
• the cancer for treatment with the mitochondrial peptide or analogue composition described includes prostate cancer.
• the cancer for treatment with the mitochondrial peptide or analogue composition described includes ovarian cancer.
• the cancer for treatment with the mitochondrial peptide or analogue composition described includes glioblastoma; or a malignant glioblastoma (e.g., grade IV).
• the cancer for treatment with the mitochondrial peptide or analogue composition described includes neuroblastoma. In some embodiments, the cancer for treatment with the mitochondrial peptide or analogue composition described includes bladder cancer. In some embodiments, the cancer for treatment with the mitochondrial peptide or analogue composition described includes liver cancer. In some embodiments, the cancer for treatment with the mitochondrial peptide or analogue composition described includes colon cancer. In some embodiments, the cancer for treatment with the mitochondrial peptide or analogue composition described includes breast cancer.
• a subject suitable for treatment with the mitochondrial peptide or analogue composition described has, suffers from, or has been diagnosed with one or more of ovarian cancer, glioblastoma, neuroblastoma, bladder cancer, liver cancer, colon cancer, and breast cancer.
• a subject suitable for treatment with the mitochondrial peptide or analogue composition described has, suffers from, or has been diagnosed with one or more of prostate cancer, glioblastoma, neuroblastoma, bladder cancer, liver cancer, and colon cancer.
• a subject suitable for treatment with the mitochondrial peptide or analogue composition described, or a subject in need of a treatment, a diagnosis, and/or an assay described is a human at an age above 40 years old, above 50 years old, above 55 years old, above 60 years old, above 65 years old, above 70 years old, above 75 years old, above 80 years old, or above 85 years old.
• he disease and/or condition suitable for treatment with the mitochondrial peptide or analogue composition described includes an inflammatory disease.
• the inflammatory disease is inflammatory bowel disease, pelvic inflammatory disease, Crohn’s disease, costochondritis, conjunctivitis, bursitis, contact dermatitis, sarcoidosis, bronchiolitis, seroma, or chronic simusitis.
• the inflammatory disease is in the nervous system, cardiovascular system, respiratory system, digestion system, accessory digestive organs, integumentary system, musculoskeletal system, urine system, reproductive system, endocrine system, or lymphatic system.
• disease and/or condition suitable for treatment with the mitochondrial peptide or analogue composition described includes age-related disease or condition.
• age-related diseases or conditions are neurodegenerative disorders.
• age-related diseases are atherosclerosis and cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, or Alzheimer’s disease.
• the subject does not express the peptide
• MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG SEQ ID NO:215.
• the subject expresses low amounts of
• MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215) relative to a healthy normal subject.
• the subject possesses a metabolic signature of low NOSH activity.
• the subject possesses a metabolic signature of high or abberrant NOSH activity.
• the subject is administered a dominant negative analog and/or derivative of NOSH.
• a dominant negative analog or derivative generally refers to an analog or derivative with a mutation/substitution/modification resulting in an adverse effect on the normal, wild-type molecule within the same cell. This usually occurs if the product can still interact with the same elements as the wild-type product, but block some aspect of its function.
• the method includes selecting a subject, detecting the presence, absence, or expression level of one or more biomarkers, and diagnosing the subject for a disease and/or condition, based on the presence, absence, or expression level of the one or more biomarkers.
• the biomarker includes a mitochondrial peptide.
• the biomarker includes
• MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG SEQ ID NO:215, or any of those peptides in SEQ ID NOs: 1-214.
• the subject may be diagnosed if expressing a low, high, or aberrant amount of MRLF GLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215) relative to a healthy normal subject.
• detection of the presence, absence, or expression level of the biomarker includes antibody detection of the one of or more biomarkers, including the use of, for example, a monoclonal antibody, polyclonal antibody, antisera, other immunogenic detection, and mass spectrometry detection methods.
• the biomarker includes a single nucleotide polymorphism (SNP).
• SNP single nucleotide polymorphism
• the present invention further provides a method of enhancing efficacy of a treatment disease and/or condition using a mitochondrial peptide, including the steps of selecting a subject in need of treatment, and administering a quantity of the mitochondrial peptide to a subject receiving treatment a disease and/or condition, wherein the mitochondrial peptide enhancing the efficacy of the disease and/or condition, thereby enhancing efficacy of the treatment.
• the mitochondrial peptide is administered simultaneously with a composition capable of treating a cancer.
• the mitochondrial peptide is administered sequentially, before or after administration, of a composition capable of treating a disease and/or condition.
• the subject is a human.
• the mitochondrial peptides and analog compositions of the invention can be co-administered with other therapeutic agents for the treatment of cancer, including for example cancers such as lung cancer, prostate cancer, colorectal cancer, stomach cancer, breast cancer, colorectal cancer, cervical cancer, melanoma, skin cancer, among others.
• Co-administration can be simultaneous, e.g., in a single pharmaceutical composition or separate compositions.
• the compositions of the invention can also be administered separately from the other therapeutic agent(s), e.g., on an independent dosing schedule.
• the present invention further provides a pharmaceutical composition.
• the pharmaceutical composition includes a mitochondrial peptide and a pharmaceutically acceptable carrier.
• the mitochondrial peptide is MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• the peptide is 75%, 80%, 85% or more percentage identity to a portion of MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), including for example, three or more, five or more, ten or more, fifteen or more, twenty or more, twenty-five or more amino acids of MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), wherein the portion begins at XI, X2, X3, X4, etc. This includes, any of the sequences in Table 1.
• the peptide includes one or more of the aforementioned portions of MRLFGLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), further including about 6-9 amino acids of XI 1 to XI 8 of MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• the bioactive mitochondrial peptide is as small as about 6-9 amino-acids, about 9-15 amino acids, about 15-20 amino acid, about 20-25 amino acids, about 25-35 amino acids, about 30-40 amino acids, as well as some that are about 12-65 amino acids in length.
• the mitochondrial peptide is about 32 amino acids in length.
• the mitochondrial peptide in the pharmaceutical composition includes a therapeutically effective amount of the mitochondrial peptide.
• pharmaceutical composition includes one or more mitochondrial peptides and a pharmaceutically acceptable carrier. In some embodiments, an amount of the mitochondrial - derived peptide of 0.1-20 mM is administered or added to a sample containing tumor cells.
• a pharmaceutical composition includes a mitochondrial-derived peptide at 1-10 mg, 10-50 mg, 50-100 mg, 100-300 mg, 300-500 mg, 500 mg-1 g, 1 g-5 g, or 5 g-10 g.
• the amount of a mitochondrial-derived peptide is administered at a dose 0.1-1 mg/kg, 1-10 mg/kg, 10-50 mg/kg, 50-100 mg/kg, 100-200 mg/kg, 200-300 mg/kg, 300-400 mg/kg, 400-500 mg/kg, 500-600 mg/kg, 600-700 mg/kg, 700-800 mg/kg, 800-900 mg/kg, 0.9-1 g/kg, 1-5 g/kg, 5-10 g/kg to a subject.
• the present invention further provides a method of manufacturing a mitochondrial peptide.
• the method of manufacturing includes the steps of providing one or more polynucleotides encoding a mitochondrial peptide, expressing the one or more polynucleotides in a host cell, and extracting the mitochondrial peptide from the host cell.
• the method of manufacturing includes the steps of expressing the one or more polynucleotides in a host cell, and extracting the mitochondrial peptide from the host cell.
• the one or more polynucleotides are a sequence encoding MRLFGLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), or a mitochondrial peptide possessing at least 25%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more percentage identity to
• MRLF GLLL AVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215).
• the peptide is 75%, 80%, 85%, 90%, 95% or more percentage identity to a portion of MRLFGLLLAVRRSGRSLSLMLTLIRGLSKRLG (SEQ ID NO:215), including for example, three or more, five or more, ten or more, fifteen or more, twenty or more, twenty five or more amino acids of MRLF GLLL AVRRSGRSL SLMLTLIRGL SKRLG (SEQ ID NO:215), wherein the portion begins at XI, X2, X3, X4, etc.
• the method of manufacturing includes the steps of peptide synthesis using liquid-phase synthesis or solid-phase synthesis.
• the solid- phase synthesis is Fmoc or BOC synthesis.
• a family of new peptide analogues were prepared for use as a therapy for age-related diseases, including cancer. While looking for biologically active small, mitochondrially derived peptides, the Inventors found NOSH (ND-One sORF in Humans) that had potent biological activity.
• PC-3 is a human prostate cancer cell line.
• OV90 is an ovarian cancer cells.
• U251 cell line was derived from a malignant glioblastoma tumor.
• NOSH acts as a senolytic peptide, preferentially reducing viability of senescent cells.
• Figure 5A and Figure 5B Treatment with 10 pM NDDP4 for 24hr in non-senescent (NS) and senescent cells showed a higher caspase-3/7 activation in senescent cells than in non-senescent cells. Activation of caspase-3 is an essential event during apoptosis.
• Figure 5C Treatment with 10 pM NDDP4 for 24hr in non-senescent (NS) and senescent cells showed a higher caspase-3/7 activation in senescent cells than in non-senescent cells. Activation of caspase-3 is an essential event during apoptosis.
• Figure 5C Treatment with 10 pM NDDP4 for 24hr in non-senescent (NS) and senescent cells showed a higher caspase-3/7 activation in senescent cells than in non-senescent cells.
• NOSH induced an inflammatory response in senescent cells, for example in doxorubin induced senescent primary dermal fibroblast (HDFa).
• 10 pM NOSH were incubated for 24hr in both non senescent cells and senescent cells. After 24hr, conditioned media collected and measured the cytokine levels by meso scale discovery (MSD) assay.
• MSD meso scale discovery
• FIGS 8 A and 8B J82 cells are human bladder cancer cells; OVCAR3 is a cell line modeling ovarian carcinoma; SHSY5Y is a thrice-subcloned cell line derived from the SK-N-SH neuroblastoma cell line; HepG2 is a human liver cancer cell line; HCT116 is HCT116 is a human colon cancer cell line; and MCF7 is a breast cancer cell line isolated from a 69-year-old woman. Moreover, NOSH converted M2 Macrophages to Ml Macrophages, measured by gene expression in M2 macrophages.
• THP-1 cells Monocytic cells derived from a acute monocytic leukemia patient, i.e., THP-1 cells, were treated with phorbol 12-myristate 13- acetate (PMA) (200 ng/ml) for 24 hours. PMA at this concentration induces differentiation of THP-1 monocytes into macrophages. Medium was changed and PMA removed. Cells were washed with PBS for three times. Cells were then treated with lipopolysaccharide (LPS) (lOOng/ml) or IL-4 (20ng/ml), with different doses of NOSH for 24 hours.
• LPS lipopolysaccharide
• IL-4 20ng/ml
• NOSH Biochemical Structure Figure 13 depicts NOSH structure.
• NOSH has a predicted double alpha-helix structure where the alpha-helices are predicted to be in AA 3-11 and AA 18-30.
• the hinge region between the helices seem to be of importance as mutations in this region significantly alter function.
• Figure 14 depicts the plasma levels of inflammatory markers induced in mice following LPS (10 mg/kg) administration with or without NOSH (10 mg/kg). Compared to LPS-treated mice, mice administered with NOSH following/concurrently with LPS had a statistically significantly lowered level of inflammatory markers. Table 1 - NOSH Analogs
• Various embodiments of the invention can specifically include or exclude any of these variations or elements.
• the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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