KR20090059152A - Compositions and methods to prevent cancer with cupredoxins - Google Patents

Abstract

The present invention relates to compositions comprising peptides that may be variants, derivatives and structural equivalents of cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Specifically, these compositions may comprise azurin from Pseudomonas aeruginosa, and/or the 50-77 residue region of azurin (p28). The present invention further relates to compositions that may comprise cupredoxin(s), and/or variants, derivatives or structural equivalents of cupredoxins, that retain the ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. These compositions may be peptides or pharmaceutical compositions, among others. The compositions of the invention may be used to prevent the development of premalignant lesions in mammalian cells, tissues and animals, and thus prevent cancer.

Description

Compositions and Methods to Prevent Cancer with Cupredoxins

Cross Reference to Related Applications

This application claims 35 U.S.C. U.S., filed September 14, 2006, under §§ 119 and 120. Claims priority to provisional application 60 / 844,358, supra. Provisional application 60 / 844,358, filed U.S. Patent application No. Claim priority on 11 / 244,105, supra. Patent application No. 11 / 244,105, filed U.S. Provisional Application No. U.S. 60 / 616,782 and filed May 13, 2005. Provisional Application No. U.S., filed November 11, 2003, claims priority on 60 / 680,500. Patent application No. 10 / 720,603, filed part of the file, see U.S. Pat. Patent application No. 10 / 720,603, filed U.S. Provisional Application No. U.S., filed January 15, 2002, with priority on 60 / 414,550. Patent application No. 10 / 047,710, filed part of the file, see U.S. Pat. Patent application No. 10 / 047,710, filed U.S. Provisional Application No. Claim priority on 60 / 269,133. The entire contents of these prior applications are hereby incorporated by reference in their entirety.

Statement of Government Rights

The gist of this application was supported by Grant Number AI 16790-21, ES 04050-16, AI 45541, CA 09432 and N01-CM97567 from the National Institutes of Health (NIH), Bethesda, Maryland, U.S.A. The government may assert some rights in the invention.

Technical Field of the Invention

The present invention relates to compositions containing peptides that are structural equivalents of variants, derivatives and cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. The present invention also relates to the use of variants, derivatives and structural equivalents of cupredoxin and / or cupredoxin as chemopreventive agents in mammals for inhibiting the development of premalignant lesions and ultimately cancer. do.

Cancer chemoprevention is the use of natural, synthetic or biologic chemical agents to reverse, inhibit or prevent carcinogenic progression to invasive cancer. Recent clinical trials that prevent cancer in high-risk populations suggest that chemopreventive therapy is a realistic treatment for high-risk patients. Chemoprophylaxis is based on the concept of multifocal field carcinogenesis and multistep carcinogenesis. In field carcinogenesis, comprehensive carcinogen exposure throughout the tissue field results in diffuse epithelial injury and clonal proliferation of mutated cells within the tissue. cause. These genetic mutations throughout the field increase the likelihood of developing one or more premalignant or malignant lesions within the field. In multistage carcinogenesis, these genetic and phenotypic alterations accumulate in stages. Disabling one or more stages in multistage carcinogenesis can interfere with or prevent the development of cancer (Tsao et al. , CA Cancer J Clin 54: 150-180 (2004)).

Azurin and other cupredoxins exhibit specific cytotoxicity against cancer cells. Azurin induces apoptosis in J774 lung cancer cells (Yamada et al ., PNAS 99 (22): 14098-14103 (2002)). After invading J774 lung cancer cells, azurin concentrates in the cytosol and nuclear fraction and complexes with the tumor suppressor protein p53 to stabilize and enhance its intracellular levels ( Id ). Induction of azurin-mediated apoptosis is not limited to J774 cells. Azurin can also invade cancer cells, such as human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells (Yamada et al. , Infect Immun. 70: 7054-7062 (2002); Punj et al) , Oncogene. 23: 2367-2378 (2004)). In both cases, azurin enabled elevation of intracellular p53 levels, leading to enhanced Bax formation and apoptosis induction in these cells. Interestingly, intraperitoneal injection of azurin into nude mice carrying xenografted Mel-2 or MCF-7 human cancers induced statistically significant regression of these cancers ( Id ).

Potential chemistry for the development of hormone-induced structural differentiation of the mammary gland and DMBA-induced preneoplastic hyperplastic alveolar nodule-like lesions in the mammary gland Mouse mammary gland organ culture (MMOC) assays can be used to assess the inhibitory effect of the prophylactic agent. Mammary gland from young virgin animals can differentiate into fully grown glands if cultured for 6 days in the presence of insulin (I) + prolactin (P) + aldosterone (A). These glands are morphologically similar to those obtained from pregnant mice. Aldosterone can be replaced with estrogen (E) + progesterone (Pg). Inclusion of hydrocortisone (H) in the medium promotes functional differentiation of the mammary gland (Mehta and Banerjee, Acta Endocrinol. 80: 501 (1975); Mehta and Moon, Breast Cancer: Treatment and Prognosis 300 , 300 (Basil A Stoll ed., Blackwell Press 1986)). Thus, the hormone-induced structural and functional differentiation observed in this culture system mimics the response to hormones observed during the various physiological stages of the animal. Mice exhibit a clear precancerous stage before cancer formation in MMOC. These precancerous lesions in C3H mice are induced in BALB / c mice by the murine breast tumor virus, or by DMBA. Following exposure of the gland to 2 μg / ml DMBA between days 3 and 4 of the growth phase and subsequent degeneration of the gland for 2-3 weeks in a medium containing insulin alone, the degeneration of the mammary alveolar lesion (MAL) Causing formation (Hawthorne et al. , Pharmaceutical Biology 40: 70-74 (2002); Mehta et al ., Methods in Cell Science 19: 19-24 (1997)). Furthermore, transplantation into syngeneic hosts of epithelial cells, made from glands containing DMBA-induced breast lesions, has caused the development of mammary adenocarcinoma (Telang et al ., PNAS). 76: 5886-5890 (1979)). Pathologically, these tumors were similar to those observed in vivo when DMBA was administered to mice of the same line ( Id ).

DMBA-induced breast lesion formation in MMOC can be inhibited by various classes of chemopreventive agents such as retinoids. These agents include chemical prophylactic agents derived from natural products, such as brassinin and resveretrol, thiols, antioxidants, ornithine decarboxylase. ) Inhibitors such as OFMO and deguelin, inhibitors of prostaglandin synthesis, Ca modulators and the like (Jang et al. , Science 275: 218-220 (1997); Mehta, Eur. J. Cancer 36: 1275-1282 (2000); Metha et al ., J. Natl. Cancer Inst. 89: 212-219 (1997)). These studies clearly demonstrate that this organ culture system provides a unique model for determining the efficacy of compounds against breast carcinogenesis. These results are expected to correlate closely with the inhibition achieved by the in vivo administration of these compounds.

MMOC may also be induced to form mammary ductal lesions (MDLs). MDL can be induced when estrogen and progesterone are included in the medium instead of aldosterone and hydrocortisone. Alveolar structures are very small in the presence of ovarian steroi, but intraductal lesions are observed in histopathological sections (Mehta et al. , J. Natl. Cancer Inst). 93: 1103-1106 (2001). Antiestrogens, such as tamoxifen, which selectively act on ovarian hormone dependent ER + breast cancer, inhibited MDL formation but did not inhibit MAL. Thus, in addition to traditional MAL induction protocols, such modified culture models can be used to evaluate the effects of chemopreventive agents on both MAL and MOL.

There is a need for chemopreventive agents that inhibit the development of premalignant lesions. Such chemopreventive agents should be able to prevent the first occurrence of premalignant lesions, induce cell death in the resulting premalignant lesions and / or prevent the development of premalignant lesions into malignant lesions. Such chemopreventive agents will be particularly useful for treating patients at higher risk of developing cancer due to the presence of high-risk features, the presence of premalignant lesions, or previous cancers or premalignant lesions.

Summary of the Invention

The present invention relates to compositions containing peptides that are structural equivalents of variants, derivatives and cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Specifically, these compositions contain 50-77 residue regions (P28) of azurin and / or azurin from Pseudomonas aeruginosa . The invention also relates to compositions containing variants of the cupredoxin and / or cupredoxin variants, derivatives or structural equivalents that retain the ability to inhibit the development of premalignant lesions in mammalian cells, tissues and animals. These compositions are especially isolated peptides or pharmaceutical compositions. The composition of the present invention can be used in a method of preventing the occurrence of cancer in a mammalian patient.

One aspect of the invention relates to an isolated peptide that is a variant, derivative or structural equivalent of cupredoxin and that can inhibit the development of premalignant lesions in mammalian tissue. These cupredoxins are azurin, pseudoazurin, platocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber. Cucumber basic protein, specifically, azurin. Cupredoxins are Pseudomonas aeruginosa , Alcaligenes faecalis , Ulva pertussis , Achromobacter xylosoxidan , Bordetella bronchiseptica Methylomonas sp. , Neisseria meningitidis , Neisseria gonorrhoeae , Pseudomonas fluorescens , Pseudomonas chlororaphis , Staphylococcus aureus, Xylella fastidiosa Vibrio parahaemolyticus , specifically Pseudomonas aeruginosa . In some embodiments, such isolated polypeptide is part of a sequence selected from SEQ ID NOs: 1, 3-19, or at least 80% amino acid sequence identity with the sequence selected from SEQ ID NOs: 1, 3-19 identity.

In some embodiments, the isolated peptide may be the truncation of cupredoxin. Such isolated peptides have at least about 10 residues and up to about 100 residues. Such isolated peptides include P. aeruginosa azurin residues 50-77, P. aeruginosa azurin residues 50-67, P. aeruginosa azurin residues 36-88, or SEQ ID NO: 20 Or, alternatively, consist of such a sequence.

Another aspect of the invention is a pharmaceutical composition containing at least one or two cupredoxins or isolated peptides of the invention in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are prepared for intravenous administration. In some embodiments, the cupredoxin in the pharmaceutical composition is Pseudomonas aeruginosa , Ulva pertussis , Alcaligenes faecalis , Achromobacter xylosoxidan , Bor Bordetella bronchiseptica , Methylomonas sp. , Neisseria meningitidis , Neisseria gonorrhoeae , Pseudomonas fluorescens , Pseudomonas chlorolapis chlororaphis Xylella fastidiosa , or Vibrio parahaemolyticus , specifically Pseudomonas aeruginosa . Cupredoxin is selected from SEQ ID NOs: 1, 3-19.

Another aspect of the invention is a method of treating a mammalian patient, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of the invention. The patient is a human and has a higher risk of developing cancer than the general population. In some embodiments, the cancer is melanoma, breast cancer, pancreatic cancer, glioblastoma, astrocytoma, lung cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, skin cancer or cervical cancer. In some embodiments, the patient has at least one high risk characteristic, has premalignant lesions, or has been treated for cancer or premalignant lesions.

 Pharmaceutical compositions are administered by intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection or oral, in particular intravenous injection. The pharmaceutical composition is co-administered with at least one other chemopreventive agent, in particular almost simultaneously with other chemopreventive agents.

Another aspect of the invention is a kit comprising the pharmaceutical composition of the invention in a vial. Such kits are designed for intravenous administration.

Another aspect of the invention is a method of investigating the development of cancer, the method comprising contacting a mammalian cell with a cupredoxin or peptide of the invention and measuring the development of premalignant and malignant cells. In some embodiments, these cells are human or breast cells. In some embodiments, these cells are induced to develop premalignant lesions.

Another aspect of the invention is an expression vector encoding a peptide of the invention.

These aspects, advantages and features of the present invention will become apparent from the following drawings and detailed description of specific embodiments.

Brief description of the sequence

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa .

SEQ ID NO: 2. P28, amino acid sequence of Pseudomonas aeruginosa azurin residues 50-77.

SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidium laminosum .

SEQ ID NO: 4. Amino acid sequence of Rusticyanine from Thiobacillus ferrooxidans .

SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobacter cycloclastes .

SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis .

SEQ ID NO: 7. Acromobacter xyloxicillus ssp. The amino acid sequence of from the very lean Denny pecan tree's (Achromobacter xylosoxidan ssp. Denitrificans) I .

SEQ ID NO: 8. Amino acid sequence of azurin from Bordetella bronchiseptica .

SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp .

SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidis Z2491 .

SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonas fluorescens .

SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonas chlororaphis .

SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa 9a5c .

SEQ ID NO: 14. Amino Acid Sequence of Stellacyanin from Cucumis sativus

SEQ ID NO: 15. Amino acid sequence of Auracianin A from Chloroflexus aurantiacus .

SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexus aurantiacus .

SEQ ID NO: 17. Amino acid sequence of cucumber basic protein from Cucumis sativus .

SEQ ID NO: 18. Neisseria gonorrhoeae Amino acid sequence of Laz from F62.

SEQ ID NO: 19. Amino acid sequence of azurin from Vibrio parahaemolyticus .

SEQ ID NO: 20. Amino acid sequence of amino acids 57 to 89 of Auracianin B of Chloroflexus aurantiacus .

SEQ ID NO: 21. Amino acid sequence of amino acids 51-77 of Pseudomonas syringae azurin.

SEQ ID NO: 22. Amino acid sequence of amino acids 89-115 of Neisseria meningitidis Laz.

SEQ ID NO: 23. Amino acid sequence of amino acids 52-78 of Vibrio parahaemolyticus azurin.

SEQ ID NO: 24. Amino acid sequence of amino acids 51-77 of Bordetella bronchiseptica azurin.

Figure 1 shows photographs of all glands for the efficacy of p28 and azurin. 1A shows representative photographs of comparisons with DMBA treated gland with alveolar lesions and chemoprophylaxis in DMBA-treated lines. 1B-1G show representative photographs of the p28 effect on the development of alveolar lesions.

2 shows a graph demonstrating the efficacy of p28 on DMBA-induced breast alveolar lesions.

Figure 3 shows a representative section of breast duct lesions and pictures of the p28 effect.

4 shows a graph demonstrating the efficacy of p28 on DMBA-induced breast duct lesions.

Justice

As used herein, "cell" encompasses both the singular and the plural of the term unless specifically indicated as a "single cell."

In this specification, "polypeptide", "peptide" and "protein" are used as the same meaning, and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. The term also applies to naturally occurring amino acid polymers. "Polypeptides", "peptides" and "proteins" include glycosylation, lipid attachment, sulfation, and gamma-carboxylation and hydroxylation of glutamic acid residues. hydroxylation, ADP-ribosylation, including but not limited to variations. Polypeptides are not always perfectly linear. For example, polypeptides are branched as a result of ubiquitination and are generally post-translation, including natural processing events and phenomena caused by human manipulation that do not occur naturally. as a result of an event) (branched or unbranched). Cyclic polypeptides, branched polypeptides and branched cyclic polypeptides may be synthesized by non-translated natural processes and entirely by synthetic methods.

As used herein, "pharmacologically active" means the effect of a drug or other chemical on a biological system. The effects of chemicals are beneficial (therapeutic) or harmful (toxic). Pure chemicals or mixtures are of natural origin (plants, animals, or minerals) or are synthetic compounds.

As used herein, "premalignant" means before precancerous, or abnormal, cells are differentiated without control.

As used herein, "lesion" means a site of abnormal tissue.

As used herein, a "pathological condition" constitutes a damage of the normal state of a living animal or portion thereof; Disrupt or modify performance of physical functions; Various factors (such as malnutrition, industrial hazards, or climate), certain infectious pathogens (such as worms, parasites, bacteria or viruses), and genetic defects of organisms (such as genetic variants) genetic anomaly)), or a combination of these factors, covers anatomical and physiological deviations from normal.

As used herein, "disorder" encompasses anatomical and physiological deviations from normal, which constitute damage to the steady state of a living animal or portion thereof and disrupt or modify performance of body functions.

As used herein, “suffering” refers to the symptoms of a current pathological disorder and encompasses having a pathological disorder, recovering from, or recovering from a pathological disorder, even if there are no observable symptoms.

As used herein, "chemoprevention" is the use of drugs, vitamins, or other agents to reduce the risk of cancer or to delay the occurrence or recurrence of cancer.

As used herein, "treatment" encompasses preventing, decreasing, stopping or reversing the progression or severity of the disorder to be treated or the symptoms associated with the disorder. Thus, "treatment" includes appropriate medical, therapeutic and / or prophylactic administration. Treatment also includes preventing or reducing the occurrence of disorders such as cancer.

As used herein, "inhibition of cell growth" means retardation or interruption of cell division and / or cell expansion. The term also means inhibition of cell development or an increase in cell death.

A “therapeutically effective amount” is an amount effective to prevent, delay, stop or reverse the occurrence of a particular disorder in the individual being treated, or to partially or completely alleviate existing symptoms of such disorder. Determination of a therapeutically effective amount is within the ability of those skilled in the art.

As used herein, “substantially pure” used to modify a protein or other cell product of the invention is, for example, isolated from the growth medium or cell contents in a form that is substantially free or free of other proteins and / or other compounds. Direct the protein. "Substantially pure" means at least 75% factor, or at least "75% substantially pure" factor per dry weight of the separated fractions. More suitably, "substantially pure" refers to at least 85% of the compounds per building weight of the separated fractions, or at least "85% substantially pure" compounds. Most suitably, "substantially pure" refers to at least 95% of the compounds per building weight of the separated fractions, or at least "95% substantially pure" compounds. "Substantially pure" can also be used to modify the synthesized proteins or compounds of the present invention, where, by way of example, such synthetic proteins are separated from the reactants and by-products of the synthetic reaction.

As used herein, a “pharmaceutical grade” in connection with a peptide or compound of the present invention is substantially or essentially separated from components commonly associated in nature, including synthetic reactants and by-products, and as It indicates a peptide or compound that is substantially or essentially separated from the component that constrains its utilization. For example, a "pharmaceutical grade" peptide is isolated from any carcinogen. In some instances, “pharmaceutical grade” is modified by the intended method of administration to specify a peptide or compound that is substantially or essentially isolated from any material that makes the composition unsuitable for intravenous administration to a patient ( Eg, “intravenous pharmaceutical grade”). For example, "intravenous pharmaceutical grade" peptides are isolated from detergents such as SDS and anti-bacterial agents such as azide.

"Isolated", "purified" or "biologically pure" refers to a substance that is substantially or essentially free of components normally involved in its natural state. Thus, isolated peptides according to the present invention preferably do not comprise substances typically associated with these peptides in an in situ environment. An “isolated” region of a polypeptide refers to a region in which such region does not carry the entire sequence of the polypeptide from which it is derived. "Isolated" nucleic acids, proteins, or individual fragments thereof may be subjected to nucleotide sequencing, restriction digestion, site-directed mutagenesis, subcloning into expression vectors for nucleic acid fragments ( subcloning) and the acquisition of proteins or protein fragments in substantially pure amounts, and is substantially transferred from the in vivo environment by manipulation by those skilled in the art, including but not limited to these.

As used herein, “variant” in reference to a peptide, refers to an amino acid sequence variant in which an amino acid is substituted, deleted, or inserted as compared to a wild type polypeptide. The variant may be truncation of the wild type peptide. "Deletion" is the removal of one or more amino acids from within a wild-type polypeptide and "fragment" is the removal of one or more amino acids from one or both ends of a wild-type polypeptide. Thus, variant peptides are made by manipulation of genes encoding such polypeptides. Variants are made by changing the basic composition or properties without altering at least some of the pharmacological activity of the polypeptide. For example, an “variant” of azurin can be a mutated azurin that retains its ability to inhibit the development of premalignant mammalian cells. In some instances, variant peptides are synthesized with non-natural amino acids such as ε- (3,5-dinitrobenzoyl) -Lys residues (Ghadiri & Fernholz, J. Am. Chem. Soc., 112: 9633). -9635 (1990)). In some embodiments, a variant is substituted, deleted or inserted up to 20 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, the variant has no more than 15 amino acids substituted, deleted or inserted compared to the wild type peptide or a portion thereof. In some embodiments, the variant is substituted, deleted or inserted with up to 10 amino acids as compared to the wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 6 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 5 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 3 amino acids as compared to a wild type peptide or portion thereof.

As used herein, an "amino acid" is an amino acid moiety comprising any naturally-occurring or non-naturally occurring or synthetic amino acid residue, ie one, two, three or more carbon atoms, typically By any moiety comprising at least one carboxyl residue and at least one amino residue directly linked by one (α) carbon atom.

As used herein, “derivative” refers to a peptide derived from a target peptide. Derivation includes chemical modifications of the peptide such that the peptide still retains some of its essential activity. For example, an "derivative" of azurin can be, for example, a chemically modified azurin that maintains its ability to inhibit angiogenesis in mammalian cells. Desired chemical modifications include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, glycosylation of peptides. In addition, the derivative peptide may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as another peptide, drug molecule or other therapeutic or pharmaceutical agent, or detectable probe.

As used herein, “% amino acid sequence identity” is defined as the ratio of amino acid residues in a polypeptide that matches an amino acid residue in a candidate sequence when the two sequences are aligned. To determine the percent amino acid identity, the sequences are aligned and, if necessary, a gap is introduced to ensure the maximum percent sequence identity; Conservative substitutions are not considered as part of sequence identity. Amino acid sequence alignment procedures to determine percent identity are known to those skilled in the art. Publicly available computer software, such as BLAST, BLAST2, ALIGN or Megalign (DNASTAR) software, is used to align peptide sequences. In certain embodiments, Blastp (available from National Center for Biotechnology Information, Bethesda MD) is used in default parameters of long complexity filter, expect 10, word size 3, existence 11, extension 1.

When aligning the amino acid sequence, the amino acid sequence identity ratio of the constant amino acid sequence A to the constant amino acid sequence B (or the constant amino acid sequence A having or including the specific% amino acid sequence identity to the constant amino acid sequence B) (%) Can be calculated as follows:

% Amino acid sequence identity = X / Y * 100

Where X is the number of amino acid residues that are recorded in the same match of A and B by sequence alignment program or algorithm alignment,

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage amino acid sequence identity of A to B will not match the percentage amino acid sequence identity of B to A. If a longer sequence is compared to a shorter sequence, the shorter sequence will be the "B" sequence. For example, when comparing truncated polypeptides to corresponding wild-type polypeptides, the truncated peptide will be the "B" sequence.

General

The present invention provides compositions containing cupredoxin or variants, derivatives or structural equivalents of cupredoxin and methods for preventing the development of cancer in mammals. The present invention also provides variants, derivatives or structural equivalents of cupredoxin that maintain the ability to prevent the occurrence or recurrence of cancer in a mammal. Most specifically, the present invention relates to compositions and patients containing Pseudomonas aeruginosa azurin , or variants, derivatives or structural equivalents of azurin, in particular for treating patients at a higher risk of developing cancer than the general population. Its uses are presented. Finally, the present invention provides a method for investigating the occurrence of cancer in mammalian cells, tissues and animals, which method may be combined with cupredoxin, or variants, derivatives or structural equivalents thereof before and after inducing premalignant lesions. Contacting and observing the development of premalignant and / or malignant cells.

Previously, the redox protein, cupredoxin azurin, produced by P. aeruginosa , has been shown to selectively enter J774 lung cancer cells, not normal cells, and induce apoptosis (Zaborina et al. ., Microbiology 146: 2521-2530 (2000 )). Azurin also selectively enters and kills human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells (Yamada et al., PNAS 99: 14098-14103 (2002); Punj et al., Oncogene 23) . : 2367-2378 (2004). From P. aeruginosa , azurin preferentially enters into J774 murine reticulum cell sarcoma cells, complexes with and stabilizes the tumor suppressor protein p53, strengthens the intracellular concentration of p53, , Induces apoptosis (Yamada et al. , Infection and Immunity, 70: 7054-7062 (2002)). In a detailed study of the various domains of the azurin molecule, amino acids 50-77 (P28) (SEQ ID NO: 2) are protein transduction domains that are crucial for internalization and subsequent apoptotic activity. , PTD) (Yamada et al. , Cell. Microbial. 7: 1418-31, (2005)).

At present, azurin and peptides derived from azurin, such as p28, are known to have chemopreventive properties. Currently, azurin and p28 are known to prevent the formation of premalignant precancerous lesions in mouse mammary gland organ cultures. In the mouse mammary gland long-term culture model, azurin at 50 μg / ml was found to inhibit 67% of the formation of alveolar lesions. Similarly, at 25 μg / ml p28 was found to inhibit the formation of alveolar lesions by 67% (see Example 1). Furthermore, at 50 μg / ml, azurin was found to inhibit 79% of the formation of breast duct lesions, and at 25 μg / ml, p28 inhibited the formation of mammary duct lesions by 71% (see Example 1). In confocal microscopy and FAC, azurin and p28 were found to invade normal mammary epithelial cells (MM3MG) and breast cancer cells (4T1). P28 also penetrates human umbilical vein endothelial cells (HUVECs) in a temperature, time and concentration dependent manner and in a dose dependent manner, capillary formation of HUVECs plated on Matrigel ^VII ( capillary tube formation). For this reason, variants of azurin and azurin can be used in mammalian patients to inhibit the formation of premalignant precancerous lesions and the development of cancer, in particular breast cancer.

Due to the high structural similarity between cupredoxins, not only azurin but also other cupredoxins are likely to inhibit the formation of premalignant lesions in mammals. These cupredoxins can be found, for example, in bacteria or plants. Several cupredoxins are known to have pharmacokinetic activity similar to that of azurin from Pseudomonas aeruginosa . For example, Rusticyanine from Thiobacillus ferrooxidans can also invade macrophages to induce apoptosis (Yamada et al. , Cell Cycle 3: 1182-1187 (2004); Yamada et al ., Cell. Micro. 7: 1418-1431 (2005)). Pseudoazin from platocyanin and Achromobacter cycloclastes from Phormidium laminosum also exhibits cytotoxicity against macrophages (US Pat. Pub. No. 20060040269; 2 2006 Released on March 23). For this reason, it is contemplated that other cupredoxins may be used in the compositions and methods of the present invention. Furthermore, variants, derivatives and structural equivalents of cupredoxin that maintain the ability to inhibit cancer formation in mammals can also be used in the compositions and methods of the present invention. These variants and derivatives include, but are not limited to, protein modifications such as truncation of cupredoxin, conservative substitution and PEGylation of amino acids, and all-hydrocarbon stapling of α-helices.

Compositions of the Invention

The present invention provides peptides which are variants, derivatives or structural equivalents of cupredoxin that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Furthermore, the present invention provides peptides that are variants, derivatives or structural equivalents of cupredoxin that inhibit the development of cancer in mammalian cells, tissues and animals. In some embodiments, the peptides are isolated. In some embodiments, the peptide is substantially pure or pharmaceutical grade. In other embodiments, the peptide is present in a composition comprising or consisting essentially of such a peptide. In another specific embodiment, the peptide is non-antigenic and does not elicit an immune response in mammals, more preferably in humans. In some embodiments, the peptide is cupredoxin up to full length and retains some of the pharmacological activity of cupredoxin. Specifically, in some embodiments, the peptide maintains the ability to inhibit the development of premalignant lesions in mouse mammary organ culture.

The present invention also provides a pharmaceutical composition containing at least one peptide which is a cupredoxin, or a variant, derivative or structural equivalent of cupredoxin. In certain embodiments, the pharmaceutical composition is designed to be suitable for a particular mode of administration, including but not limited to oral, intraperitoneal, intravenous. These compositions are hydrated in water or dried for later hydration (eg by lyophilization). These compositions may be dissolved in a solvent other than water, such as an alcohol.

Due to the high structural homology between cupredoxin, cupredoxin is considered to have the same chemopreventive properties as azurin and P28. In some embodiments, cupredoxin includes, but is not limited to, azurin, pseudoazurine, plastocyanin, rusticyanin, auracyanin, stelacyanin, cucumber based protein, Laz. In certain embodiments, the azurin is Pseudomonas aeruginosa ; Alcaligenes faecalis ; Acromobacter xylopsis ssp. Denitripicans ( Achromobacter cycloclastes ssp denitrificans ) I; Bordetella bronchiseptica ; Methylomonas sp . ; Meningitis ( Nisseria meningitidis ); Neisseria gonorrhoeae ; Pseudomonas fluorescens ; Pseudomonas chlororaphis ; Staphylococcus aureus ( Xylella fastidiosa ); It is derived from Ulva pertussis , or Vibrio parahaemolyticus . In a more particular embodiment, the azurin is derived from Pseudomonas aeruginosa . In another specific embodiment, the cupredoxin comprises an amino acid sequence of SEQ ID NOs: 1, 3-19.

The present invention provides peptides in which amino acid sequences are substituted, deleted or inserted amino acid sequences compared to wild type cupredoxin. Variants of the invention may be truncated of wild type cupredoxin. In some embodiments, peptides of the invention comprise a region of cupredoxin, which is a full length or less wild type polypeptide. In some embodiments, a peptide of the invention comprises at least 10 residues, at least 15 residues, or at least 20 residues of truncated cupredoxin. In some embodiments, a peptide of the invention comprises up to 100 residues, up to 50 residues, up to 40 residues, up to 30 residues or up to 20 residues of truncated cupredoxin. In some embodiments, the cupredoxin is the peptide, more specifically, at least 70% amino acid sequence identity, at least 80% amino acid sequence identity, at least 90% amino acid sequence in SEQ ID NOs: 1, 3-19 as the peptide of the invention Identity, at least 95% amino acid sequence identity or at least 99% amino acid sequence identity.

In certain embodiments, the variant of cupredoxin comprises P. aeruginosa azurin residues 50-77 (P28, SEQ ID NO: 2), azurin residues 50-67, or azurin residues 36-88 . In another embodiment, the variant of cupredoxin consists of P. aeruginosa azurin residues 50-77, azurin residues 50-67, or azurin residues 36-88. In another specific embodiment, the variant of cupredoxin consists of an equivalent residue of cupredoxin other than azurin. In addition, other cupredoxin variants may be designed that have similar pharmacological activity to azurin residues 50-77, or azurin residues 36-88. To this end, the target cupredoxin amino acid sequence is a target residue, target that is found on P. aeruginosa azurin sequence, P. aeruginosa azurin amino acid sequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR). The equivalent residues are found on the cupredoxin sequence, and thus, equivalent variants are designed.

In one embodiment of the invention, the cupredoxin variant comprises at least amino acids 57 to 89 (SEQ ID NO: 20) of auracyanin B of Chloroflexus aurantiacus . In another embodiment of the invention, the cupredoxin variant comprises at least amino acids 51-77 (SEQ ID NO: 21) of Pseudomonas syringae azurin. In another embodiment of the present invention, the cupredoxin variant comprises at least amino acids 89-115 (SEQ ID NO: 22) of Neisseria meningitidis Laz. In another embodiment of the invention, the cupredoxin variant comprises at least amino acids 52-78 (SEQ ID NO: 23) of Vibrio parahaemolyticus azurin. In another embodiment of the invention, the cupredoxin variant comprises at least amino acids 51-77 (SEQ ID NO: 24) of Bordetella bronchiseptica azurin.

Variants also include peptides made from synthetic amino acids that do not occur naturally. For example, non-naturally occurring amino acids are incorporated into variant peptides to extend or optimize the half-life of the composition in the bloodstream. Such variants include D, L-peptides (diastereomers) (eg, Futaki et al ., J. Biol. Chem. 276 (8): 5836-40 (2001); Papo et al. , Cancer Res. 64 (16): 5779-86 (2004); Miller et al, Biochem. Pharmacol. 36 (1): 169-76, (1987)), peptides with specific amino acids (eg, Lee et al ., J. Pept. Res. 63 (2): 69-84 (2004)), olefin-bearing non-natural amino acids followed by hydrocarbon stapling (eg, Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)), including but not limited to peptides comprising ε- (3,5-dinitrobenzoyl) -Lys residues Do not.

In another embodiment, the peptides of the invention are derivatives of cupredoxin. Derivatives of cupredoxin are chemical modifications of the peptide such that the peptide still retains some of its essential activity. For example, an "derivative" of azurin may be a chemically modified azurin that maintains its ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. Desired chemical modifications include, but are not limited to, hydrocarbon stapling, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, glycosylation of peptides. It doesn't work. In addition, the derivative peptide may be a fusion of a cupredoxin, or variant, derivative or structural equivalent thereof to a chemical compound, such as another peptide, pharmaceutical molecule or other therapeutic or pharmaceutical agent, or detectable probe. Derivatives of interest include, for example, circularized peptides (eg, Monk et al. , BioDrugs 19 (4): 261-78, (2005); DeFreest et al ., J. Pept. Res. 63 (5): 409- 19 (2004)), followed by N- and C-terminal modifications (eg, Labrie et al ., Clin. Invest. Med. 13 (5): 275-8, (1990)), followed by hydrocarbon stapling Integration of an olefin-bearing non-natural amino acid (eg, Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)) These methods include chemical modifications that can extend or optimize the half-life of the peptides and compositions of the invention in the bloodstream by a number of methods well known to those skilled in the art, including but not limited to these.

In another embodiment, the peptide is a structural equivalent of cupredoxin. Examples of studies that determine significant structural homology between cupredoxin and other proteins include Toth et al ., Developmental Cell 1: 82-92 (2001). Specifically, significant structural homology between cupredoxin and structural equivalents is determined by using the VAST algorithm (Gibrat et al., Curr Opin Struct Biol 6: 377-385 (1996); Madej et al., Proteins 23: 356). -3690 (1995). In certain embodiments, the VAST p value from the structural comparison of cupredoxin to structural equivalent is about 10 ⁻³ or less, about 10 ⁻⁵ or less, or about 10 ⁻⁷ or less. In another embodiment, significant structural homology between cupredoxin and structural equivalents is determined by using the DALI algorithm (Holm & Sander, J. Mol. Biol. 233: 123-138 (1993)). In certain embodiments, the DALIZ score for pairwise structural comparison is at least 3.5, at least 7.0, or at least 10.0.

The peptide of the composition of the present invention may be one or more of the variants, derivatives and / or structural equivalents of cupredoxin. For example, these peptides are truncated of PEGylated azurin, so both variants and derivatives are possible. In one embodiment, the peptides of the present invention possess an olefin-bearing tether and are free of full-hydrocarbon “staples” by ruthenium catalyzed olefin metathesis. Synthesized from the following α, α-disubstituted non-natural amino acids (Scharmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walensky et al ., Science 305: 1466-1470 (2004)). In addition, peptides that are structural equivalents of azurin are fused to other peptides, thus yielding peptides that are both structural equivalents and derivatives. These examples merely illustrate the invention and do not limit it. Variants, derivatives or structural equivalents of cupredoxin may or may not bind to copper.

In some embodiments, the cupredoxin, or variant, derivative or structural equivalent thereof has some of the pharmacological activity of P. aeruginosa azurin , specifically P28. In certain embodiments, cupredoxin and variants, derivatives or structural equivalents of cupredoxin inhibit or prevent the development of premalignant lesions in mammalian cells, tissues or animals, including but not limited to mammary cells. In the present invention, mammalian premalignant lesions, specifically, melanoma, breast cancer, pancreatic cancer, glioblastoma, astrocytoma, lung cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, skin cancer, or uterus Cupredoxins and variants, derivatives or structural equivalents of cupredoxin that have the ability to inhibit the growth of cervical cancer cells are presented. Inhibition of cancer cell development is a reduction in the incidence or rate of increase of statistically significant premalignant lesions compared to control treatment.

This chemoprophylactic activity is known because cupredoxin can inhibit the development of premalignant lesions and ultimately cancer in mammalian cells, tissues or animals, specifically breast cells, more specifically mouse mammary cells. It is possible to design variants, derivatives or structural equivalents of cupredoxin. Such variants, derivatives or structural equivalents make, for example, a "library" of various variants, derivatives and structural equivalents of cupredoxin, and then described in the art, for example as described in Example 1. Typical methods can be used to investigate chemopreventive activity in each of these, specifically, chemopreventive activity in mouse mammary gland organ cultures. The resulting variants, derivatives and structural equivalents of cupredoxin having chemopreventive activity can be used in the methods of the invention in place of, or in addition to, azurin or p28.

In some specific embodiments, the variants, derivatives or structural equivalents of cupredoxin are selected from 7,12-dimethylbenz (a) in a mouse mammary gland organ culture (MMOC) to an unstated degree from the untreated control. ) Inhibits the development of anthracene (7,12-dimethylbenz (a) anthracene (DMBA) induced premalignant lesions. Peptides are described in Example 1, or Mehta et al. . This activity can be investigated by using the MMOC model system as described in J Natl Cancer Inst 93: 1103-1106 (2001) and Mehta et al ., Meth Cell Sci 19: 19-24 (1997). Other methods of determining if cancer development is inhibited are well known in the art.

In some specific embodiments, variants, derivatives or structural equivalents of cupredoxin inhibit the development of mammary alveolar lesions (MAL) in a model of MMOC to an unstated degree from the untreated control. In some specific embodiments, variants, derivatives or structural equivalents of cupredoxin inhibit the development of mammary ductal lesions (MDLs) in the MMOC model to un statistically different degrees from untreated controls. Peptides can be examined for these activities by using a MMOC model system induced to form premalignant lesions by DMBA, as described in Example 1. Assessment of the incidence of premalignant lesions in an MMOC model system can be determined by morphometic analysis, or histopathological analysis, as described in Example 1.

Cupredoxin

These small blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or whose function is unknown. Copper ions are bound only by the protein matrix. Twisted triangular two-dimensional alignment, unique to two histidines and one cysteine around copper, results in a very unique electronic property and intense blue color of the metal sites. Many cupredoxins have been crystallographically characterized at medium to high resolution.

Cupredoxin generally has low sequence homology but high structural homology (Gough & Clothia, Structure 12: 917-925 (2004); De Rienzo et al., Protein Science 9 : 1439-1454 (2000)). For example, the amino acid sequence of azurin is 31% identical to the amino acid sequence of auracyin B, 16.3% of the amino acid sequence of Rusticyanin, 20.3% of the amino acid sequence of Plastocyanin, and 17.3% of the amino acid sequence of Pseudoazurin. (See Table 1). However, the structural similarity of these proteins is more pronounced. The VAST p value for the structural comparison of ^Azurin vs. ^auracyanin B is 10 ^-7.4 , the VAST p value for the structural comparison of ^Azurin vs. ^Rusticinine is 10 ^-5 , and the structure of the azurin vs. plastocyanin The VAST p value for the comparison is 10 ^−5.6 and the VAST p value for the structural comparison of ^azurin versus ^pseudoazurin is 10 ^−4.1 .

All cupredoxins share an eight-stranded Greek key beta-barrel or beta-sandwich fold and have a highly conserved site architecture (De Rienzo et al., Protein Science 9: 1439-1454 (2000)). Significant hydrophobic patches due to the presence of many long-chain aliphatic residues, such as methionine and leucine, are found in azurin, amicyanin, cyanobacterial plastocyanin, Cucumber-based proteins and, to a lesser extent, are present around the copper site in pseudoazurin and eukaryotic plastocyanin ( Id. ). Hydrophobic patches, although lesser, are also found within the stelacyanine and rustiocyanin copper sites, but differ in character ( Id. ).

Sequence and structure alignment of azurin (1JZG) from P. aeruginosa against other proteins using the VAST algorithm PDB Sort Length ^One aa % Identity P-value ² Score ³ RMSD ⁴ Explanation 1AOZ A 2 82 18.3 10e-7 12.2 1.9 Ascorbic acid oxidase 1QHQ_A 113 31 10e-7.4 12.1 1.9 Auracianin B 1V54 B 1 79 20.3 10e-6.0 11.2 2.1 Cytochrome c oxidase 1GY2 A 92 16.3 10e-5.0 11.1 1.8 Rusticyanine 3MSP A 74 8.1 10e-6.7 10.9 2.5 Motility Main Sperm Protein ⁵ 1IUZ 74 20.3 10e-5.6 10.3 2.3 Plastocyanin 1KGY E 90 5.6 10e-4.6 10.1 3.4 ephrinB2 1PMY 75 17.3 10e-4.1 9.8 2.3 Pseudoazurine

¹ Ordered length: The number of equivalent pairs of C-alpha atoms superimposed between two structures, ie the number of residues used to calculate 3D overlap.

² P-VAL: The VAST p value is a measure of the significance of the comparison, expressed as probability. For example, if the p value is 0.001, the odds of seeing a match of this quality by chance is 1000 to 1. The p value from VAST is adjusted for the effect of multiple comparisons using the assumption that there are 500 independent and unrelated types of domains in the MMDB database. The p value thus obtained corresponds to the p value for the pairwise comparison of each domain pair, divided by 500.

³ score: VAST structure-similarity score. This number relates to the number of nested secondary structural elements and the nature of this overlap. Higher VAST scores correlate with higher similarities.

⁴ RMSD: root mean square overlapping residue (in Angstrom units). This number is calculated as the square root of the mean square distance between equivalent C-alpha atoms after the best overlap of the two structures. Note that the RMSD numbers are proportional to the degree of structural alignment, and this size should be taken into account when using RMSD as a descriptor of overall structural similarity.

⁵ C. elegans major sperm protein (Kuwabara, Genes and Development, 17: 155-161 (2003)) that has been demonstrated to be an ephrin antagonist in oocyte maturation.

Azurin

Azurin is a copper-bearing protein consisting of 128 amino acid residues belonging to the cupredoxin population involved in electron transfer in certain bacteria. Azurin includes those derived from P. aeruginosa (PA) (SEQ ID NO: 1), Alcaligenes xylosoxidans and Alcaligenes denitrifican ( Murphy et al., J. Mol. Biol. 315: 859-71 (2002)). Although amino sequence identity varies between 60-90% between these azurins, these proteins exhibit strong structural homology. All azurins possess characteristic β-sandwiches containing Greek key motifs, and a single copper atom is always located in the same region of the protein. In addition, azurin retains essentially neutral hydrophobic patches surrounding the copper site (Murphy et al.).

Plastocyanin

Plastocyanin is a soluble protein of cyanobacteria, algae and plants that has one copper molecule per molecule and is bluish in oxidized form. They are produced in chloroplasts, where they function as electron carriers. In 1978, after the determination of non-Poplar Place Toshi structure, birds (ttemok end (Scenedesmus), Parlay in (Enteromorpha), Chlamydomonas (Chlamydomonas)) and the structure of the plastic Toshi, not plants (kidney beans (French bean)) crystallographic Method, or NMR method, this poplar structure was carefully distinguished at 1.33 Å resolution. SEQ ID NO: 3 shows the amino acid sequence of plastocyanin from Phormidium laminosum , a thermophilic cyanobacteria. Other plastocyanines of interest are derived from Ulva pertussia .

Despite the sequence divergence (eg, 62% sequence identity between Chlamydomonas and poplar proteins) between the plastocyanins of algae and vascular plants, these three-dimensional structures are preserved (Eg, a 0.76 μs rms deviation in C alpha position between Chlamydomonas and poplar protein). Structural features include warped tetrahedral copper binding sites, prominent negative patches, and flat hydrophobic surfaces at one end of an eight-strand antiparallel beta-barrel. . Copper sites are optimized for electron transfer function, and negative and hydrophobic patches are proposed to be involved in the recognition of physiological response partners. Chemical modification, cross-linking and site-specific mutagenesis experiments confirmed the importance of negative and hydrophobic patches in binding interactions with cytochrome f and identified two functionally important electron transfer pathway models involving plastocyanin. The first putative electron transfer pathway is relatively short (approximately 4 μs) and involves solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other pathway is longer (approximately 12-15 μs) and almost conserved in the negative patch. With residue Tyr-83 (Redinbo et al., J. Bioenerg. Biomembr. 26 (1): 49-66 (1994)).

Rusticyanine

Loose tea is when non-Tio bacilli (Thiobacillus) (currently, ah Sidi Tio bacilli (Acidithiobacillus)) obtained from the bronze-chain polypeptide is a single-hold. The oxidized form of the highly stable highly acidic cupredoxin rustiyanin (SEQ ID NO: 4) from Thiobacillus ferrooxidans was determined by multiwavelength anomalous diffraction. And are finely differentiated at 1.9 Å resolution. Rusticyanine consists of a core beta-sandwich fold consisting of 6- and 7-stranded β-sheets. Similar to other cupredoxins, copper ions are coordination by clusters of four conserved residues (His85, Cys138, His143, Met148) aligned within the twisted coordination site (Walter et al., J. Mol). Biol. 263: 730-51 (1996)).

Pseudoazurine

Pseudoazin is a bronze-bearing single-chain polypeptide population. The amino acid sequence of pseudoazurine obtained from Achromobacter cycloclastes is listed in SEQ ID NO: 5. X-ray structure analysis of pseudoazurine shows that pseudoazurin has a similar structure to azurin although it has low sequence homology with azurin. Two major differences exist between the overall structure of pseudoazurin and azurin. Compared to azurin, pseudoazurine has a carboxy terminal extension consisting of two alpha-helices. In the mid-peptide region, azurin has an extended loop compared to pseudoazurin, which forms a flap with a short α-helix. The only major differences at the copper atom sites are the conformation of the MET side chain and the Met-S copper bond length, which is much shorter in pseudoazurin than in azurin.

Phytocyanin

Proteins identified as phytocyanin include cucumber base protein, stelacyanin, mavicyanin, umecyanin, cucumber peeling cupredoxin, and pea pod. Putative bronze protein, including but not limited to bronze protein from Arabidopsis thaliana . With the exception of cucumber base proteins and pi-pod proteins, the axial methionine ligands commonly found in bronze sites are replaced with glutamine.

Auracianin

Three small bronze proteins, named Auracyanin A, Auracyanin B-1 and Auracyanin B-2, are used for thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus ). The two B forms are glycoproteins and have nearly identical properties to each other, but differ from the A forms. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent monomer molecular masses were found to be 14 (A), 18 (B-2) and 22 (B-1) kDa.

The amino acid sequence of auracyanin A was found to be a 139 residue polypeptide (Van Dreissche et al, Protein Science 8: 947-957 (1999)). His58, Cys123, His128 and Met132 are located at a certain distance in the manner expected in the case where they are evolutionarily conserved metal ligands such as the known small copper proteins plastocyanin and azurin. Secondary structure prediction also suggests that auracyanin has a beta-barrel structure that is generally similar to the beta-barrel structure of plastocyanin from azurin and poplar leaves from Pseudomonas aeruginosa . However, auracianin appears to have both sequence characteristics of both small copper protein sequence classes. The overall similarity of the azurin with the consensus sequence is largely identical to the similarity with the consensus sequence of plastocyanin (ie, 30.5%). The N-terminal sequence regions 1-18 of auracyanin are markedly rich in glycine and hydroxy amino acids ( Id ). See the typical amino acid sequence SEQ ID NO: 15 (NCBI Protein Data Bank Accession No. AAM12874) for chain A of auracianin from Chloroflexus aurantiacus .

The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of auracyanin B from Chloroflexus aurantiacus was investigated (Bond et al., J. Mol. Biol. 306: 47-67 (2001)). With the exception of the additional N-terminal strands, the molecule is very similar to the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands is located on strand 4 of the polypeptide and the other three are located along the large loop between strands 7 and 8. Cu site geometry is discussed with reference to amino acid spacing between the three latter ligands. The crystallographically characterized Cu-binding domains of auracyanin B are probably surrounding the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other electron-transfer proteins that are membrane-bound. It is bound to the periplasmic part. The amino acid sequence of form B is described by McManus et al., J Biol Chem. 267: 6531-6540 (1992). See the typical amino acid sequence SEQ ID NO: 16 (NCBI Protein Data Bank Accession No. 1QHQA) for chain B of auracianin from Chloroflexus aurantiacus .

Stellacyanine

Stellacyanin is a subclass of phytocyanin, a ubiquitous group of plant cupredoxins. Typical sequences of stellacyanine herein are listed in SEQ ID NO: 14. Umecyanin (Koch et al., J. Am. Chem. Soc . 127: 158-166 (2005)) and cucumber stelacyanin (Hart et al., Protein) Science 5: 2175-2183 (1996)) is also known. The protein has a total fold similar to other phytocyanins. The ephrinB2 protein ectodomain tertiary structure has significant similarity to stelacyanin (Toth et al., Developmental Cell 1: 83-92 (2001)). A typical amino acid sequence of Stellacyanin is found in SEQ ID NO: 14 (National Center for Biotechnology Information Protein DataBank Accession No. 1JER).

Cucumber Basic Protein

Typical amino acid sequences from cucumber based proteins are listed herein in SEQ ID NO: 17. The crystal structure of cucumber-based protein (CBP), a type 1 bronze protein, was carefully differentiated at 1.8 에서 resolution. The molecule is similar to other bronze proteins in that it has a Greek key beta-barrel structure, but the barrel is open on one side and is "beta-sandwich" or "beta-taco." (Guss et al., J. Mol. Biol. 262: 686-705 (1996)). This ephrinB2 protein ectodomain tertiary structure has a high similarity to the cucumber basal protein (rms deviation 1.5 kPa for 50 α carbon) (Toth et al., Developmental Cell 1: 83-92 (2001)).

Cu atoms have a bond length of Cu-N (His39) = 1.93 kPa, Cu-S (Cys79) = 2.16 kPa, Cu-N (His84) = 1.95 kPa, Cu-S (Met89) = 2.61 kPa, normal bronze NNSS ' It has co-ordination. Disulfide linkages, (Cys52) -SS- (Cys85), seem to play an important role in stabilizing this molecular structure. Polypeptide folding is typical of the ragweed allergen Ra3, a sub-family of bronze proteins (phytocyanins) and a non-metalloprotein with high sequence homology to CBP. Proteins currently identified as phytocyanin are presumed bronze proteins from CBP, Stellacyanin, Maviscyanin, Umecyanin, Cucumber Husk Cupredoxin, Piper and Bronze Protein from Arabidopsis thaliana . With the exception of CBP and P-Pad proteins, the axial methionine ligands commonly observed at bronze sites are substituted with glutamine. Typical sequences for cucumber basal proteins are found in SEQ ID NO: 17 (NCBI Protein Data Bank Accession No. 2CBP).

How to use

The present invention provides a method of preventing de novo malignancy in a patient, the method comprising administering to the patient at least one polypeptide that is cupredoxin, or a variant, derivative or structural equivalent of cupredoxin, as described above. It includes a step. Chemoprophylaxis is based on the assumption that disruption of the processes involved in carcinogenesis will prevent the development of cancer. The azurin peptide p28, truncated with Pseudomonas aeruginosa azurin, inhibits the initial formation of premalignant lesions, or kills or inhibits the growth of current premalignant lesions. It is known to inhibit development. For this reason, as described above, it is contemplated that cupredoxin, or a variant, derivative or structural equivalent of cupredoxin, having the ability to inhibit the development of premalignant lesions may be used for chemopreventive therapy in patients. In some embodiments, such patients are at a higher risk of developing cancer than the general population. Cancers that can be prevented by treatment with the compositions of the present invention include, but are not limited to, melanoma, breast cancer, pancreatic cancer, glioblastoma, astrocytoma, lung cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, skin cancer, cervical cancer It doesn't work. In some embodiments, the patient is a human. In other embodiments, the patient is not human.

In addition, the present invention includes a method of investigating the occurrence of cancer, wherein the method comprises contacting a mammalian cell with a composition containing cupredoxin, or a variant, derivative or structural equivalent thereof, before or after induction into a carcinogenic factor. Observing the development of these cells. In some embodiments, the cells are mouse mammary gland cells, and in other embodiments, these cells are other cells that turn malignant in a mammal.

Patients with a higher risk of developing cancer than the general population are patients with high-risk features, patients with premalignant lesions, and patients with early cancer or with clear treatment of premalignant tumors (Tsao et al. ., CA Cancer J Clin 54: 150-180 (2004)). High risk characteristics are the patient's behavioral, genetic, environmental, or physiological factors. Behavioral factors that make the patient more prone to various forms of cancer include smoking, diet, alcohol consumption, hormone replacement therapy, high body mass index, Nulliparity, betel nut use, frequent mouthwash use, exposure to human papillomavirus, early childhood chronic sun exposure, first sex at young age, complex sexual partners, Oral contraceptive use is included, but is not limited to these. Genetic factors that make patients more susceptible to various forms of cancer include family history of cancer, gene carrier status of BRCA1 and BRCA2 , prior history of breast neoplasia, and familiality. Amilial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), red or blonde and fair-skinned phenotype, xeroderma pigmentosum, race (ethnicity) is included, but not limited to these. Environmental features that make patients more susceptible to various forms of cancer include radon, polycyclic aromatic hydrocarbons, nickel, chromate, arsenic, asbestos, Exposure to aromatic amines from, but not limited to, chloromethyl ether, benzo [a] pyrene, radiation, rubber or paint job exposures. Other factors that make patients more susceptible to various forms of cancer include chronic obstructive pulmonary disease with airflow obstruction, chronic bladder infection, schistosomiasis, and old age. older age, immunocompromised status, but not limited to these.

Additionally, patients at higher risk of developing cancer can be determined by using various risk models developed for certain types of cancer. For example, patients susceptible to breasts can be determined, in particular, using the Gail risk model, or Claus model (Gail et al. , J Natl Cancer Inst 81: 1879-1886 (1989); Cuzick, Breast 12: 405-411 (2003); Huang et al. , Am J Epidemiol 151: 703-714 (2000).

Patients with premalignant lesions are at higher risk of developing cancer than the general population. The presence of premalignant lesions in a patient can be determined by various methods well known to those skilled in the art. To determine if a patient has premalignant lesions, an intermediate marker or biomarker originating from the premalignant lesion may be measured in the patient. Chromosomal abnormalities occur in tumor cells and adjacent histologically normal tissues in most cancer patients. Progression in chromosomal abnormalities is accompanied by phenotypic progression from premalignant lesions to invasive cancer (Thiberville et al. , Cancer Res. 55: 5133-5139 (1995)). For this reason, chromosomal abnormalities associated with cancer can be used as intermediate markers for detecting premalignant lesions in patients. Common chromosomal abnormalities associated with cancer include tumor suppressor genes such as 3p (FHIT, etc.), 9p ( p16 ^INK4 , p15 ^INK4B and 9p21 for p19 ^ARF ), 17p ( p53 genes, etc.). And allelic deletion or loss of heterozygosity (LOH) in 17p13 ) and 13q ( 13q14 in the case of the retinoblastoma gene Rb, etc.). Deletion at 3p and 9p is associated with early stages of smoking and lung cancer (Mao et al. , J. Natl. Cancer Inst. 89: 857-862 (1997)). Deletions affecting 3p, 5q, 8p, 17p and 18q are common changes in epithelial cancer (Tsao et al ., CA Clin. Cancer J. Clin. 54: 153 (2004)). Other chromosomal mutations associated with cancer are mutations that activate oncogenes. Oncogenes that can be used as intermediate markers include, but are not limited to, Ras, c-myc, epidermal growth factor, erb-B2, cyclin E, D1 and B1 ( id. 154).

Another intermediate marker is the product of genes that are up-regulated in premalignant and cancer cells. The products of genes that are up-regulated in premalignant cells include, but are not limited to, cyclooxygenase COX-1 and COX-2, telomerase. Other biomarkers of cancer cells and some premalignant cells include p53, epidermal growth factor receptor (GFR), proliferating cell nuclear antigen (PCNA), RAS, COX-2, Ki-67, DNA aneuploidy, DNA polymerase-α, ER, Her2 neu , E-cadherin, RARβ, hTERT, p16 ^INK4a , FHIT ( 3p14 ), Bcl-2, VEGF-R, HPV infection, LOH 9p21 , LOH 17p , p-AKT , hn RNP A2 / B1, RAF , Myc, c-KIT, cyclin D1, E and B1, IGF1, bcl-2, p16, LOH 3p21.3, LOH 3p25, LOH 9p21, LOH 17p13, LOH 13q, LOH 8p, h MSH2, APC, DCC, DPC4, JV18, BAX, PSA, GSTP1, NF-kB, AP1, D3S2, HPV infection, LOH 3p14 , LOH 4q , LOH 5p , bladder tumor antigen (BTA), BTK TRAK (Alidex, Inc., Redmond WA), urinary tract matrix protein 22, fibrin degradation product, autocrine motility factor receptor, BCLA-4, cytokeratin ( cytokeratin) 20, hyaluronic acid, CYFRA 21-1 , BCA, beta-human chorionic gonadotropin, tissue polypeptide antigen (TPA), but are not limited to these ( id. 155-157).

Patients treated with early cancer or clearly treated premalignant tumors are also at higher risk of developing cancer than the general population. Secondary primary tumor refers to a new primary cancer in individuals with a history of cancer. Secondary primary tumors are the leading cause of death in head and neck cancer ( Id . 150). Secondary primary tumors are distinguished from metastasis, the former of de novo origin, while the latter of origin. Patients who have been treated for breast cancer, head and neck cancer, lung and skin cancer, or premalignant lesions of these cancers, are particularly at risk of developing secondary primary tumors.

Cupredoxin, or a composition containing a variant, derivative or structural equivalent of cupredoxin, can be administered to a patient in a variety of regimens by a variety of routes well known to those skilled in the art. In certain embodiments, the cupredoxin, or variant, derivative or functional equivalent of cupredoxin is administered intravenously, intramuscularly, subcutaneously, topically, orally, or by inhalation. These compositions can be administered to the patient by any means of delivering these peptides to a site at risk of developing cancer in the patient. In certain embodiments, the cupredoxin, or variant, derivative or structural equivalent of cupredoxin is administered intravenously.

In one embodiment, these methods sequentially comprise one unit dose of a cupredoxin or a composition containing a variant, derivative or structural equivalent of cupredoxin and one unit dose of a composition containing another chemopreventive agent. Co-administered to a patient at about the same time, or within a certain time after administration of another agent, for example, within approximately 1 to 60 minutes after administration of another agent, or approximately 1 to 12 hours after administration of another agent. Steps. Purpose chemical prophylactic agent which is tamoxifen (tamoxifen), aromatic other inhibitory substance (aromatase inhibitor), for example, for letrozole (letrozole), and anastrozole (anastrozole) (Arimidex ^ㄾ), retinoids (retinoid), for example, N- [4-hydroxyphenyl] retinamide (4-HPR, fenretinide), nonsteriodal antiinflammatory agents (NSAIDs) such as aspirin and sulindac ), Celecoxib (COX-2 inhibitor), defluoromethylornithin (DFMO), ursodeoxycholic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, EKI-785 (EGFR inhibitor), bevacizumab (antibody to VEGF-receptor), cetuximab (antibody to EGFR), retinol, for example vitamin A, beta-carotene, 13-cis retinoic acid, isotretinoin and Retinyl palmitate, α-tocopherol, interferon, oncolytic adenovirus dl1520 (ONYX-015), gefitinib, etretinate, finasteride, indole- 3-carbinol (indole-3-carbinol), resveratrol, chlorogenic acid, raloxifene, oltipraz are included, but are not limited to these.

Pharmaceutical composition containing cupredoxin, or variant, derivative or structural equivalent of cupredoxin

A pharmaceutical composition containing cupredoxin, or a variant, derivative or structural equivalent of cupredoxin, may be prepared in any conventional manner, such as conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, Can be prepared by capture, or by freeze drying. Substantially pure or pharmaceutical grade cupredoxin, or variants, derivatives or structural equivalents of cupredoxin may be readily mixed with pharmaceutically acceptable carriers known in the art. These carriers allow the preparation of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Suitable carriers or excipients may include, for example, fillers and cellulose preparations. Other excipients may include, for example, fragrances, colorants, detackifiers, thickness and other acceptable additives, adjuvants or binders. In some embodiments, the pharmaceutical preparations are substantially free of preservatives. In another embodiment, the pharmaceutical preparation contains at least one preservative. Overall methods for pharmaceutical forms are found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore MD (1999)).

Compositions containing cupredoxin, or variants, derivatives or structural equivalents thereof, used in the present invention may be administered by injection (eg, intradermal, subcutaneous, intramuscular, intraperitoneal, etc.), inhalation, topical administration, suppositories, transdermal patches, or oral. It can be administered in a variety of ways, including. For general information about drug delivery systems, see Ansel et al., Id. See. In some embodiments, a composition containing cupredoxin, or a variant, derivative or structural equivalent thereof, may be prepared and directly used as an injectable, in particular for subcutaneous and intravenous injection. Such injectable preparations can be particularly useful for treating patients for whom chemoprevention therapy is suitable. Compositions containing cupredoxin, or variants, derivatives or structural equivalents thereof, can also be taken orally after mixing with a protective agent, such as polypropylene glycol, or a similar coating agent.

When administered by injection, cupredoxin, or variant, derivative or structural equivalent thereof, is prepared in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline. The solution may contain formulary agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the cupredoxin, or variant, derivative or structural equivalent thereof, is present in powder form for constitution with a suitable vehicle, eg pyrogen-free sterile water, prior to use. In some embodiments, the pharmaceutical composition does not contain an adjuvant or any other substance added to enhance the immune response stimulated by such peptide. In some embodiments, the pharmaceutical composition contains a substance that inhibits an immune response to such peptides.

When administered in an intravenous fluid, the intravenous fluid used to administer cupredoxin, or variants, derivatives or structural equivalents thereof, may consist of crystalloids or colloids. In this specification, the crystalline is an aqueous solution of an inorganic salt or other water soluble molecule. In the present specification, colloids include large insoluble molecules such as gelatin. Intravenous fluid is sterile.

Crystalloid fluids used for intravenous administration include, as described in Table 2, normal saline (sodium chloride solution at 0.9% concentration), Ringer's lactate or Ringer's solution, dissolved in water. % Dextrose solution (aka D5W) is included, but is not limited to these.

Composition of common crystalline solution solution Other designations [Na ⁺ ] [Cl ^- ] [Glucose] D5W 5% dextrose 0 0 252 2/3 & 1/3 3.3% dextrose / 0.3% brine 51 51 168 Semi-normal brine 0.45% NaCl 77 77 0 Normal saline 0.9% NaCl 154 154 0 Ringer's Lactate * Ringer's solution 130 109 0

^* Ringer's lactate is 28 mmol / ℓ lactate, 4 mmol / ℓ and K ^+, and also containing 3 mmol / ℓ Ca ^2+.

When administered by inhalation, the cupredoxin, or variant, derivative or structural equivalent thereof is pressurized with a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas. It is delivered in the form of an aerosol spray from the pack or sprayer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. By way of example, capsules and cartridges of gelatin used in an inhaler or blower are prepared to contain a powder mixture of protein and a suitable powder base, such as lactose or starch.

When administered topically, cupredoxin, or variant, derivative or structural equivalent thereof, is prepared as a solution, gel, ointment, cream, jelly, suspension, and the like, as is well known in the art. In some embodiments, administration is accomplished by transdermal patches. When administered as a suppository (eg rectal or vaginal), cupredoxin, or variant, derivative or structural equivalent thereof, may be prepared in the form of a composition containing a conventional suppository base.

When administered orally, the cupredoxin, or variant, derivative or structural equivalent thereof is readily prepared by mixing such cupredoxin, or variant, derivative or structural equivalent thereof with pharmaceutically acceptable carriers well known in the art. Can be prepared. Solid carriers such as mannitol, lactose, magnesium stearate and the like; These carriers allow the cupredoxin, or variant, derivative, or structural equivalent thereof, to be prepared in tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject to be treated. Suitable excipients for oral solid preparations such as powders, capsules and tablets include fillers (eg, sugars), cellulose preparations, granulating agents, binders and the like.

Other conventional carriers well known in the art also include multivalent carriers such as bacterial capsular polysaccharides, dextran or genetically engineered vectors. In addition, sustained release formulations containing cupredoxin, or variants, derivatives, or structural equivalents thereof, release such cupredoxins, or variants, derivatives, or structural equivalents thereof, for extended periods of time, Predoxin, or variant, derivative or structural equivalent thereof, is removed and / or degraded from the subject's tissue, for example by proteases and simple hydrolysis, prior to inducing or enhancing the therapeutic effect.

The half-life in the bloodstream of the peptides of the invention can be extended or optimized in several ways well known to those skilled in the art. Peptide variants of the invention maintain the ability of cupredoxin to inhibit the development of premalignant lesions in mammalian cells, tissues and animals, while maintaining the stability, specific activity, and lifetime of the peptide in the bloodstream. Various variants that increase longevity and / or reduce immunogenicity are included, but are not limited to these. Such variants include reduced hydrolysis of the peptide, reduced deamidation of the peptide, reduced oxidation, reduced immunogenicity, or structural stability of the peptide. Or variants that increase the size of the peptide or increase the size of the peptide. Such peptides include circularized peptides (Monk et al., BioDrugs 19 (4): 261-78, (2005); DeFreest et al., J. Pept. Res. 63 (5): 409-19 (2004)), D, L-peptide (diastereomer) (Futaki et al., J. Biol. Chem. Feb 23; 276 (8): 5836-40 (2001); Papo et al., Cancer Res. 64 (16): 5779-86 (2004); Miller et al, Biochem. Pharmacol. 36 (1): 169-76, (1987)), peptides with special amino acids (Lee et al. Res. 63 (2): 69-84 (2004)), N- and C-terminal modifications (Labrie et al., Clin. Invest. Med. 13 (5): 275-8, (1990)), hydrocarbon staples Hydrocarbon stapling (Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)), PEGylation Included.

In various embodiments, the pharmaceutical composition comprises a carrier and excipients (buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids (eg glycine), antioxidants, bacteriostatic agents, chelating agents, suspending agents, thickening agents and / or preservatives). ), Water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliaries required to approximate physiological conditions (e.g. buffers, tonicity adjusting agents, wetting agents) ), Etc.). Although any carrier known to those skilled in the art can be used to administer the compositions of the present invention, the type of carrier will vary depending upon the mode of administration. The compound may be encapsulated in liposomes using well known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in U.S. Patent No. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,942,252.

These pharmaceutical compositions may be sterilized by conventional well known sterilization techniques, or may be sterile filtered. The resulting aqueous solution is packaged intact or lyophilized, which freeze-dried preparation is mixed with sterile solution prior to administration.

Administration of cupredoxin, or variant, derivative or structural equivalent thereof

Cupredoxin, or variant, derivative or structural equivalent thereof, is prepared as a pharmaceutical composition and may be, by any suitable route, e.g. oral, buccal, inhaled, sublingual, rectal, vaginal, transurethral, nasal It may be administered by topical, transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, coronary), or vitreous administration. Pharmaceutical formulations thereof may be administered in an effective amount to achieve the intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is generally approximately 0.01-20 mg / kg per kg body weight.

Compounds comprising cupredoxin, or variants, derivatives or structural equivalents thereof, alone or in combination with other active agents, are useful for the prevention of cancer. Appropriate doses will vary depending, for example, on the compound of the cupredoxin, or variant, derivative or structural equivalent thereof used, the host, the mode of administration, and the nature and severity of the potential cancer. However, in general, satisfactory results in humans are designated as being achieved at a daily dosage of approximately 0.01-20 mg / kg body weight. The designated daily dose in humans is, for example, cupredoxin in the range of approximately 0.7 mg to 1400 mg, or a variant thereof, conveniently administered in a daily, weekly, monthly, and / or continuous dosing regimen, Compounds of derivatives or structural equivalents. Daily doses may be administered separately from 1 to 12 times per day. Alternatively, the dose may be administered in increments of one every two days, every three days, every four days, every five days, every six days, every week, and similarly up to 31 days or more. Alternatively, the dosage may be a patch, i.v. Continuous administration using administration and the like.

The exact formulation, route of administration, and dose are determined by the attending physician, taking into account the patient's condition. Dosage and resting periods may be individually adjusted to provide an active cupredoxin, or variant, derivative or structural equivalent thereof at a plasma level sufficient to maintain the therapeutic effect. In general, the desired cupredoxin, or variant, derivative or structural equivalent thereof is administered in a mixture with a pharmaceutical carrier selected according to the intended route of administration and standard pharmaceutical practice.

In one aspect, the cupredoxin, or variant, derivative or structural equivalent thereof is delivered as DNA and such polypeptides are produced in situ . In one embodiment, DNA is described by way of example in Ulmer et al., Science, Vol. 259, pp-1745-49 (1993) and reviewed in Cohen, Science, vol. 259, pp-1691-92 (1993). As it is, it is "naked". Uptake of naked DNA can be increased by coating the DNA onto a carrier, eg, a biodegradable bead that is effectively carried into a cell. In this method, DNA is present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial expression systems, viral expression systems. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art (WO90 / 11092, WO93 / 24640, WO93 / 17706, US Pat. No. 5,736,524).

Vectors used to transfer genetic material from organisms to organisms can be broadly divided into two classes: Cloning vectors are replicating plasmids or phages that are essential for proliferation in suitable host cells and that contain regions in which foreign DNA can be inserted. to be; Foreign DNA replicates and multiplies as if it is a component of the vector. Expression vectors (eg, plasmids, yeast or animal viral genomes) are used to introduce foreign genetic material into host cells or tissues for the transcription and translation of foreign DNA, eg, the DNA of cupredoxin. In expression vectors, the introduced DNA is operably linked to elements such as promoters that signal the host cell to highly transcribe the inserted DNA. Some promoters are particularly useful, such as inducible promoters that regulate gene transcription in response to certain factors. By operably linking the cupredoxin, or variant, derivative, or structural equivalent polynucleotide thereof to an inducible promoter, the expression of the cupredoxin and its variants and derivatives can be controlled in response to certain factors. Examples of classical inducible promoters are promoters that respond to α-interferon, heat shock, heavy metal ions and steroids such as glucocorticoids (Kaufman, Methods Enzymol. 185: 487-511 (1990)) and tetracycline. Other preferred inducible promoters are those which are not endogenous to the cells into which these constructs are introduced but which react in these cells when induction agents are supplied from outside. In general, useful expression vectors are mainly plasmids. However, other forms of expression vectors such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) are also contemplated.

Vector selection depends on the organism or cell employed and the desired purpose of the vector. Generally, vectors include signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters and transcription termination sequences.

Kits comprising cupredoxin, or variants, derivatives or structural equivalents thereof

In one aspect, the invention provides a biologically active composition containing (1) at least one cupredoxin, or variant, derivative or structural equivalent thereof in a package or container; (2) additional chemopreventive agents; (3) Provides a regimen or kit comprising one or more of the instruments for administering a biologically active composition to a patient, such as a syringe, nebulizer, and the like.

When a kit is supplied, different components of the composition can be packaged in separate containers and mixed just prior to use. Such individual packaging of these components allows for long term storage without loss of function of the active component.

The reactants included in the kit are supplied in a constant container so that the life of different components is preserved and not absorbed or changed by the container material. For example, sealed glass ampoules include lyophilized cupredoxin, or variants, derivatives or structural equivalents thereof, or buffers packaged under a neutral, non-active gas such as nitrogen. The ampoule consists of any suitable material, such as glass, organic polymers (eg, polycarbonates, polystyrenes, etc.), ceramics, metals, or any other material typically used to hold similar reactants. Another example of a suitable container is a simple bottle made from an ampoule-like material and a lid holding a foiled interior, such as aluminum or alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container holds a sterile access port, for example a bottle with a stopper through which a hypodermic needle can penetrate. The other container is easily removable and has two compartments separated by a membrane that allow components to be mixed after removal. Removable membranes are glass, plastic, rubber, and the like.

Instructions may also be provided with the kit. The instructions may be printed on paper or other substrate and / or provided on an electronically readable medium, such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape, flash memory device, or the like. Can be. Detailed instructions may not be physically attached to the kit; Instead, the user may be directed to an Internet web site specified by the manufacturer or distributor of such a kit, or provided with instructions for use as an e-mail.

Modification of cupredoxin and variants, derivatives or structural equivalents thereof

Cupredoxin, or variant, derivative or structural equivalent thereof, may be chemically modified or genetically modified to produce variants and derivatives, as described above. Such variants and derivatives can be synthesized by standard techniques.

In addition to the naturally-occurring allelic variants of cupredoxin, changes that induce modifications within the amino acid sequence of encoded cupredoxin that do not significantly change the ability of cupredoxin to inhibit the development of premalignant lesions may result in mutations. By the cupredoxin coding sequence. "Non-essential" amino acid residues are those residues that can be modified from the wild-type sequence of cupredoxin without a change in pharmacological activity, while "essential" amino acid residues are required for such pharmacological activity. For example, amino acid residues that are conserved between cupredoxins are, in particular, unmodified and are therefore expected to be "essential".

Amino acids for which conservative substitutions can be achieved that do not alter the pharmacological activity of the polypeptide are well known in the art. Useful conservative substitutions are shown in Table 3, “Preferred Substitutions”. Conservative substitutions in which one class of amino acids are substituted with other amino acids of the same type are within the scope of the present invention unless such substitutions physically alter the pharmacological activity of the compound.

Preferred Substitution Initial residue Typical substitution Preferred Substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norleucine Leu

Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as the β-sheet or α-helix structure, (2) charge, (3) hydrophobicity, or (4) the size of the target site side chain are pharmacological It can change activity. The residues are divided into several groups based on common side chain properties, as shown in Table 4. Non-conservative substitutions involve the replacement of members of one class with members of another class. Substitutions can be introduced into conservative substitution sites, or more specifically, into non-conserved sites.

Amino acid class Bracket amino acid Hydrophobic Norleucine, Met, Ala, Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr acid Asp, Glu Basic Asn, Gln, His, Lys, Arg Disturbed chain structure Gly, Pro Aromatic Trp, Tyr, Phe

Variant polypeptides can be made using methods known in the art, such as oligonucleotide-mediated (specific site) mutagenesis, alanine scanning and PCR mutagenesis. Specific site mutagenesis in cloned DNA (Carter, Biochem J. 237: 1-7 (1986); Zoller and Smith, Methods Enzymol., 154: 329-50 (1987)), cassette mutagenesis, restriction Restriction selection mutagenesis (Wells et al., Gene 34: 315-23 (1985)), or other known techniques, can be performed to yield cupredoxin variant DNA.

Known mutations of cupredoxin can also be used to yield variant cupredoxins for use in the methods of the invention. For example, the C112D and M44KM64E mutants of azurin are known to exhibit different cytotoxic and growth arrest activity than native azurin, which may be useful in the therapeutic methods of the invention.

A more complete understanding of the invention may be achieved by reference to the specific examples below. These examples are described for illustrative purposes only and do not limit the scope of the invention. Changes in form and substitution of equivalents are considered to be shortcuts. Although specific terms are used herein, these terms are for illustrative purposes and do not limit the invention. Modifications of the invention are possible without departing from the spirit and scope of the invention, and therefore the invention is limited only by the appended claims.

Example 1 Effect of Peptide P-28 on DMBA-Induced Breast Lesions in MMOC Model

The mouse mammary gland organ culture (MMOC) model enables the evaluation of the efficacy of potential chemopreventive agents against the development of breast alveolar lesions (MAL) or breast duct lesions (MDL) due to DMBA. Under appropriate culture conditions, DMBA forms MAL or MDL based on the hormonal milieu in the medium (Hawthorne et al. , Pharmaceutical Biology 40: 70-74 (2002); Mehta et al. , J. Natl. Cancer Inst. 93: 1103-1106 (2001). Estrogen and progesterone-treated mammary glands develop mammary duct lesions in culture, whereas aldosterone and hydrocortisone-treated mammary glands form estrogen and progesterone-independent alveolar lesions. Mammary glands not exposed to carcinogens or chemopreventive agents undergo structural regression in the absence of growth-promoting hormones, whereas treatment with DMBA for 24 hours between days 3 and 4 is deficiency of hormones. To prevent degeneration of the structure caused by This is presumably because the mammary glands lose normal hormonal responsiveness and their developmental processes change. The production of mammary adenocarcinoma by transplanting transformed cells into syngeneic mice demonstrates the premalignant precancerous nature of these uninhibited sites.

Thoracic pairs of mammary glands were excised from each Balb / c mouse aseptically and these mammary glands were divided into several groups. The effect of p28 was evaluated at four different dilutions in the medium. Carcinogen-treated mammary gland without test agent served as a measure to determine percentage incidence in the absence of chemopreventive agents. Additional controls were included to serve as positive controls for chemoprevention. Azurin was included in the medium at a concentration of 50 μg / ml. In the case of alveolar lesions (MAL), the stained mammary gland assessed the incidence of lesions (mammary gland with any lesion compared to the total number of mammary glands in certain treatment groups). In the case of mammary duct lesions (MDL), a similar protocol was adopted, except that the hormonal combinations differ in alveolar lesions and mammary duct lesions as indicated in the Methods section below. These mammary glands were fixed in formalin and then processed for histopathology. These sections were stained with eosin and hematoxelene and evaluated under the microscope. Here, the multiplicity of breast duct lesions is compared between the control and treatment groups.

Long term culture procedure. The experimental animals used in the study were 3-4 week-old young virgin BALB / c female mice (Charles River, Wilmington, Mass.). These mice were treated daily with subcutaneous infusion of 1 μg estradiol-17β + 1 mg progesterone for 9 days. This treatment is necessary because animals that are not pretreated with steroids do not respond to hormones in vitro. The entire culture procedure is described in detail in the literature (Jang et al. , Science 275: 218-220 (1997); Mehta, Eu. J. Cancer 36: 1275-1282 (2000); Mehta et al. , J. Natl. Cancer Inst. 89: 212-219 (1997); Mehta et al ., J. Natl. Cancer Inst. 93: 1103-1106 (2001)).

In short, these animals are sacrificed with cervical dislocations, thorax pairs of mammary glands are excised on silk rafts, and in serum-free Waymouth MB752 / 1 medium (5-wire / 5 mL / dish). Incubated for days. The medium is glutamine, antibiotic (penicillin and streptomycin 100 unit / ml medium) and growth-promoting hormone per ml medium, in the case of the protocol for inducing breast alveolar lesions (MAL). Supplemented with μg insulin (I), 5 μg prolactin (P), 1 μg aldosterone (A) and 1 μg hydrocortisone (H). For induction of mammary duct lesions (MDL), the medium contained 5 μg / ml, 5 μg / ml P, 0.001 μg / ml estradiol 17β and 1 μg / ml progesterone (Pg) (Mehta et al ., J Natl. Cancer Inst. 93: 1103-1106 (2001). Carcinogen, DMBA (2 μg / ml) was added to the medium between 3 and 4 days. In this study, DMBA was dissolved in DMSO at a final concentration of 4 mg / ml and 50 μg I was added to 100 ml medium to yield a 2 μg / ml final concentration. The control dish contained DMSO as vehicle.

On day 4, DMBA was removed from the medium by washing the mammary glands in fresh medium and transferring them to a new dish containing fresh medium without DMBA. After incubation for 10 days, these mammary glands were maintained for an additional 14 days in medium containing I (5 μg / ml) alone. During the entire incubation period, these mammary glands were maintained at 37 ° C. under 95% O ₂ and 5% CO ₂ environments. Chemoprophylactic agents were included in the medium during the first 10 days of the growth-promoting phase. Test peptide p28 was evaluated at four concentrations in the range of 12.5 μg / ml to 100 μg / ml. Azurin was assessed at 50 μg / ml in medium. The peptide was dissolved in sterile water and filtered prior to use. The badge was replaced three times a week (Monday, Wednesday and Friday). At the end of the exposure, these mammary glands were fixed in formalin.

Results were analyzed by Chi-square analysis and Fisher's Exact Test.

Structural Measurement Analysis of MAL. For examination of MAL, mammary glands were stained with alum carmine and the presence of lesions was assessed. These mammary glands recorded scores for the presence or absence of breast lesions, the severity of the lesions and drug toxicity in each gland. Mammary glands stored in xylene were evaluated for the presence or absence, incidence and severity of breast lesions for each gland under a dissecting microscope. Mammary glands scored positive or negative for breast lesions, and the incidence was determined as the ratio of mammary glands representing lesions to the total number of mammary glands in each group. Dilation of the ducts or disruption of breast structure due to treatment with chemopreventive agents was considered a toxic effect. Data on incidence was performed by statistical analysis to determine the efficacy of potential chemopreventive agents.

FIG. 1A shows a representative photograph of a comparison with mammary gland treated with DMBA with alveolar lesions and chemoprophylaxis in a DMBA treated mammary gland. The effect of p28 on the development of alveolar lesions is shown in FIGS. 1B-1G and summarized in FIG. 2. Peptide p28 inhibited 67% MAL formation at 25 μg / ml concentration. Increasing concentrations up to 100 μg / ml did not enhance the efficacy of the peptide. Comparison of the peptide with azurin suggested that p28 was as effective as azurin for MAL development. Azurin at 67 μg / ml resulted in 67% inhibition. In statistical analysis, the effect of p28 was found to be statistically significant compared to the DMBA control at concentrations above 12.5 μg / ml (p <0.01, Fisher's exact test; chi-square analysis).

Histological Evaluation of MDL. In the case of MDL, mammary glands were processed for histopathological evaluation. These mammary glands were cut longitudinally into 5-micron sections and stained with eosin hematoxeline. Cell sections of each mammary gland were divided into several fields, each field assessing breast duct lesions (Mehta et al ., J. Natl. Cancer Inst. 93: 1103-1106 (2001)). In short, the entire streamline is evaluated under scope; Smaller streamlines will have fewer total fields compared to larger streamlines. Thus each wire will have a variable number of fields. Often, the number of segments through the canal also varies significantly from streamline to streamline. This results in variable numbers from each group. Fields containing hyperplasia or atypia were determined and compared to the total number of fields evaluated in each mammary gland. There is no distinction between these hyperplasia or atypical and severely occluded mammary glands. Fields containing this histological pattern were considered positive for the lesion. The treatment group was compared with the control group for severity and the percentage inhibition was calculated.

3 shows representative breast duct lesions. DMBA induces a variety of breast duct lesions from aberrant proliferation, atypical to complete occlusion of the ducts. The ratio of the total number of mammary duct lesions / mammary segment was determined. Again, 12.5 μg / ml p28 inhibited MDL formation by 15%. However, significant inhibition of lesions was observed comparable to that observed at 25 μg / ml at 50 μg / ml azurin. The efficacy of p28 at concentrations above 12.5 μg / ml was statistically significant (p <0.01, Fisher's exact validation). These results are summarized in FIG. 4. Often, the effectiveness of chemopreventive agents can be differentiated between MAL and MDL. For example, tamoxifen inhibited the development of MDL but did not inhibit the development of MAL. Interestingly, azurin and p28 inhibited estrogen and progesterone-dependent breast duct lesions as well as independent alveolar lesions.

This example demonstrates that both p28 and azurin can prevent the development of precancerous lesions in breast tissue. Thus p28 and azurin can be used as chemopreventive agents in mammalian patients.

                         SEQUENCE LISTING <110> Chakrabarty, Ananda        Das Gupta, Tapas   <120> Composition and Methods to Prevent Cancer with Cupredoxins <130> 14PR-132884 <140> PCT / US07 / 78371 <141> 2007-09-13 <150> 60 / 844,358 <151> 2006-09-14 <150> 11 / 244,105 <151> 2005-10-06 <150> 60 / 680,500 <151> 2005-05-13 <150> 60 / 616,782 <151> 2004-10-07 <150> 10 / 720,603 <151> 2003-11-11 <150> 60 / 414,550 <151> 2003-08-15 <150> 10 / 047,710 <151> 2002-01-15 <150> 60 / 269,133 <151> 2001-02-15 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 128 <212> PRT <213> Pseudomonas aeruginosa <400> 1 Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln Met Gln Phe Asn 1 5 10 15 Thr Asn Ala Ile Thr Val Asp Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met     50 55 60 Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys             100 105 110 Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys         115 120 125 <210> 2 <211> 28 <212> PRT <213> Pseudomonas aeruginosa <400> 2 Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala 1 5 10 15 Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp             20 25 <210> 3 <211> 105 <212> PRT <213> Phormidium laminosum <400> 3 Glu Thr Phe Thr Val Lys Met Gly Ala Asp Ser Gly Leu Leu Gln Phe 1 5 10 15 Glu Pro Ala Asn Val Thr Val His Pro Gly Asp Thr Val Lys Trp Val             20 25 30 Asn Asn Lys Leu Pro Pro His Asn Ile Leu Phe Asp Asp Lys Gln Val         35 40 45 Pro Gly Ala Ser Lys Glu Leu Ala Asp Lys Leu Ser His Ser Gln Leu     50 55 60 Met Phe Ser Pro Gly Glu Ser Tyr Glu Ile Thr Phe Ser Ser Asp Phe 65 70 75 80 Pro Ala Gly Thr Tyr Thr Tyr Tyr Cys Ala Pro His Arg Gly Ala Gly                 85 90 95 Met Val Gly Lys Ile Thr Val Glu Gly             100 105 <210> 4 <211> 155 <212> PRT <213> Thiobacillus ferrooxidans <400> 4 Gly Thr Leu Asp Thr Thr Trp Lys Glu Ala Thr Leu Pro Gln Val Lys 1 5 10 15 Ala Met Leu Glu Lys Asp Thr Gly Lys Val Ser Gly Asp Thr Val Thr             20 25 30 Tyr Ser Gly Lys Thr Val His Val Val Ala Ala Ala Val Leu Pro Gly         35 40 45 Phe Pro Phe Pro Ser Phe Glu Val His Asp Lys Lys Asn Pro Thr Leu     50 55 60 Glu Ile Pro Ala Gly Ala Thr Val Asp Val Thr Phe Ile Asn Thr Asn 65 70 75 80 Lys Gly Phe Gly His Ser Phe Asp Ile Thr Lys Lys Gly Pro Pro Tyr                 85 90 95 Ala Val Met Pro Val Ile Asp Pro Ile Val Ala Gly Thr Gly Phe Ser             100 105 110 Pro Val Pro Lys Asp Gly Lys Phe Gly Tyr Thr Asp Phe Thr Trp His         115 120 125 Pro Thr Ala Gly Thr Tyr Tyr Tyr Val Cys Gln Ile Pro Gly His Ala     130 135 140 Ala Thr Gly Met Phe Gly Lys Ile Val Val Lys 145 150 155 <210> 5 <211> 124 <212> PRT <213> Achromabacter cycloclastes <400> 5 Ala Asp Phe Glu Val His Met Leu Asn Lys Gly Lys Asp Gly Ala Met 1 5 10 15 Val Phe Glu Pro Ala Ser Leu Lys Val Ala Pro Gly Asp Thr Val Thr             20 25 30 Phe Ile Pro Thr Asp Lys Gly His Asn Val Glu Thr Ile Lys Gly Met         35 40 45 Ile Pro Asp Gly Ala Glu Ala Phe Lys Ser Lys Ile Asn Glu Asn Tyr     50 55 60 Lys Val Thr Phe Thr Ala Pro Gly Val Tyr Gly Val Lys Cys Thr Pro 65 70 75 80 His Tyr Gly Met Gly Met Val Gly Val Val Gln Val Gly Asp Ala Pro                 85 90 95 Ala Asn Leu Glu Ala Val Lys Gly Ala Lys Asn Pro Lys Lys Ala Gln             100 105 110 Glu Arg Leu Asp Ala Ala Leu Ala Ala Leu Gly Asn         115 120 <210> 6 <211> 128 <212> PRT <213> Alcaligenes faecalis <400> 6 Ala Cys Asp Val Ser Ile Glu Gly Asn Asp Ser Met Gln Phe Asn Thr 1 5 10 15 Lys Ser Ile Val Val Asp Lys Thr Cys Lys Glu Phe Thr Ile Asn Leu             20 25 30 Lys His Thr Gly Lys Leu Pro Lys Ala Ala Met Gly His Asn Val Val         35 40 45 Val Ser Lys Lys Ser Asp Glu Ser Ala Val Ala Thr Asp Gly Met Lys     50 55 60 Ala Gly Leu Asn Asn Asp Tyr Val Lys Ala Gly Asp Glu Arg Val Ile 65 70 75 80 Ala His Thr Ser Val Ile Gly Gly Gly Glu Thr Asp Ser Val Thr Phe                 85 90 95 Asp Val Ser Lys Leu Lys Glu Gly Glu Asp Tyr Ala Phe Phe Cys Ser             100 105 110 Phe Pro Gly His Trp Ser Ile Met Lys Gly Thr Ile Glu Leu Gly Ser         115 120 125 <210> 7 <211> 129 <212> PRT <213> Achromobacter xylosoxidans ssp. denitrificans I <400> 7 Ala Gln Cys Glu Ala Thr Ile Glu Ser Asn Asp Ala Met Gln Tyr Asn 1 5 10 15 Leu Lys Glu Met Val Val Asp Lys Ser Cys Lys Gln Phe Thr Val His             20 25 30 Leu Lys His Val Gly Lys Met Ala Lys Val Ala Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Glu Ala Asp Lys Gln Gly Val Ala Thr Asp Gly Met     50 55 60 Asn Ala Gly Leu Ala Gln Asp Tyr Val Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Pro Gly Glu Ala Tyr Ala Tyr Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Met Met Lys Gly Thr Leu Lys Leu Ser         115 120 125 Asn      <210> 8 <211> 129 <212> PRT <213> Bordetella bronchiseptica <400> 8 Ala Glu Cys Ser Val Asp Ile Ala Gly Thr Asp Gln Met Gln Phe Asp 1 5 10 15 Lys Lys Ala Ile Glu Val Ser Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Lys His Thr Gly Lys Leu Pro Arg Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile     50 55 60 Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Leu Ala His Thr Lys Val Leu Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ala Lys Leu Ala Gly Asp Asp Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Gly Ala Leu Met Lys Gly Thr Leu Lys Leu Val         115 120 125 Asp      <210> 9 <211> 129 <212> PRT <213> Methylomonas sp. J. <400> 9 Ala Ser Cys Glu Thr Thr Val Thr Ser Gly Asp Thr Met Thr Tyr Ser 1 5 10 15 Thr Arg Ser Ile Ser Val Pro Ala Ser Cys Ala Glu Phe Thr Val Asn             20 25 30 Phe Glu His Lys Gly His Met Pro Lys Thr Gly Met Gly His Asn Trp         35 40 45 Val Leu Ala Lys Ser Ala Asp Val Gly Asp Val Ala Lys Glu Gly Ala     50 55 60 His Ala Gly Ala Asp Asn Asn Phe Val Thr Pro Gly Asp Lys Arg Val 65 70 75 80 Ile Ala Phe Thr Pro Ile Ile Gly Gly Gly Glu Lys Thr Ser Val Lys                 85 90 95 Phe Lys Val Ser Ala Leu Ser Lys Asp Glu Ala Tyr Thr Tyr Phe Cys             100 105 110 Ser Tyr Pro Gly His Phe Ser Met Met Arg Gly Thr Leu Lys Leu Glu         115 120 125 Glu      <210> 10 <211> 166 <212> PRT <213> Neisseria meningitidis <400> 10 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Ala 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Thr Ser Met Gly His Asn Ile Val Ile Gly Lys Thr Glu Asp Met Asp                 85 90 95 Gly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Le Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Glu     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 11 <211> 128 <212> PRT <213> Pseudomomas fluorescens <400> 11 Ala Glu Cys Lys Thr Thr Ile Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Ala Ile Glu Ile Asp Lys Ala Cys Lys Thr Phe Thr Val Glu             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Leu         35 40 45 Val Ile Ser Lys Gln Ala Asp Met Gln Pro Ile Ala Thr Asp Gly Leu     50 55 60 Ser Ala Gly Ile Asp Lys Asn Tyr Leu Lys Glu Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Ala Gly Glu Lys Asp Ser Leu Thr                 85 90 95 Ile Asp Val Ser Lys Leu Asn Ala Ala Glu Lys Tyr Gly Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Ile Ser Met Met Lys Gly Thr Val Thr Leu Lys         115 120 125 <210> 12 <211> 128 <212> PRT <213> Pseudomonas chlororaphis <400> 12 Ala Glu Cys Lys Val Asp Val Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Glu Ile Thr Ile Asp Lys Ser Cys Lys Thr Phe Thr Val Asn             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Lys Ser Ala Asp Met Ala Gly Ile Ala Thr Asp Gly Met     50 55 60 Ala Ala Gly Ile Asp Lys Asp Tyr Leu Lys Pro Gly Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Ile Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Ala Gly Glu Ser Tyr Glu Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Asn Ser Met Met Lys Gly Ala Val Val Leu Lys         115 120 125 <210> 13 <211> 129 <212> PRT <213> Xylella fastidiosa 9a5c <400> 13 Lys Thr Cys Ala Val Thr Ile Ser Ala Asn Asp Gln Met Lys Phe Asp 1 5 10 15 Gln Asn Thr Ile Lys Ile Ala Ala Glu Cys Thr His Val Asn Leu Thr             20 25 30 Leu Thr His Thr Gly Lys Lys Ser Ala Arg Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Thr Asp Met Gln Ala Val Ala Leu Ala Gly Leu     50 55 60 His Ala Thr Leu Ala Asp Asn Tyr Val Pro Lys Ala Asp Pro Arg Val 65 70 75 80 Ile Ala His Thr Ala Ile Ile Gly Gly Gly Glu Arg Thr Ser Ile Thr                 85 90 95 Phe Pro Thr Asn Thr Leu Ser Lys Asn Val Ser Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Leu Met Lys Gly Thr Leu Asn Phe Gly         115 120 125 Gly      <210> 14 <211> 138 <212> PRT Cucumis sativus <400> 14 Met Gln Ser Thr Val His Ile Val Gly Asp Asn Thr Gly Trp Ser Val 1 5 10 15 Pro Ser Ser Pro Asn Phe Tyr Ser Gln Trp Ala Ala Gly Lys Thr Phe             20 25 30 Arg Val Gly Asp Ser Leu Gln Phe Asn Phe Pro Ala Asn Ala His Asn         35 40 45 Val His Glu Met Glu Thr Lys Gln Ser Phe Asp Ala Cys Asn Phe Val     50 55 60 Asn Ser Asp Asn Asp Val Glu Arg Thr Ser Pro Val Ile Glu Arg Leu 65 70 75 80 Asp Glu Leu Gly Met His Tyr Phe Val Cys Thr Val Gly Thr His Cys                 85 90 95 Ser Asn Gly Gln Lys Leu Ser Ile Asn Val Val Ala Ala Asn Ala Thr             100 105 110 Val Ser Met Pro Pro Pro Ser Ser Pro Pro Ser Ser Val Met Pro         115 120 125 Pro Pro Val Met Pro Pro Pro Ser Pro Ser     130 135 <210> 15 <211> 162 <212> PRT <213> Chloroflexus aurantiacus <400> 15 Met Lys Ile Thr Leu Arg Met Met Val Leu Ala Val Leu Thr Ala Met 1 5 10 15 Ala Met Val Leu Ala Ala Cys Gly Gly Gly Gly Ser Ser Gly Gly Ser             20 25 30 Thr Gly Gly Gly Ser Gly Ser Gly Pro Val Thr Ile Glu Ile Gly Ser         35 40 45 Lys Gly Glu Glu Leu Ala Phe Asp Lys Thr Glu Leu Thr Val Ser Ala     50 55 60 Gly Gln Thr Val Thr Ile Arg Phe Lys Asn Asn Ser Ala Val Gln Gln 65 70 75 80 His Asn Trp Ile Leu Val Lys Gly Gly Glu Ala Glu Ala Ala Asn Ile                 85 90 95 Ala Asn Ala Gly Leu Ser Ala Gly Pro Ala Ala Asn Tyr Leu Pro Ala             100 105 110 Asp Lys Ser Asn Ile Ile Ala Glu Ser Pro Leu Ala Asn Gly Asn Glu         115 120 125 Thr Val Glu Val Thr Phe Thr Ala Pro Ala Ala Gly Thr Tyr Leu Tyr     130 135 140 Ile Cys Thr Val Pro Gly His Tyr Pro Leu Met Gln Gly Lys Leu Val 145 150 155 160 Val asn          <210> 16 <211> 140 <212> PRT <213> Chloroflexus aurantiacus <400> 16 Ala Ala Asn Ala Pro Gly Gly Ser Asn Val Val Asn Glu Thr Pro Ala 1 5 10 15 Gln Thr Val Glu Val Arg Ala Ala Pro Asp Ala Leu Ala Phe Ala Gln             20 25 30 Thr Ser Leu Ser Leu Pro Ala Asn Thr Val Val Arg Leu Asp Phe Val         35 40 45 Asn Gln Asn Asn Leu Gly Val Gln His Asn Trp Val Leu Val Asn Gly     50 55 60 Gly Asp Asp Val Ala Ala Ala Val Asn Thr Ala Ala Gln Asn Asn Ala 65 70 75 80 Asp Ala Leu Phe Val Pro Pro Pro Asp Thr Pro Asn Ala Leu Ala Trp                 85 90 95 Thr Ala Met Leu Asn Ala Gly Glu Ser Gly Ser Val Thr Phe Arg Thr             100 105 110 Pro Ala Pro Gly Thr Tyr Leu Tyr Ile Cys Thr Phe Pro Gly His Tyr         115 120 125 Leu Ala Gly Met Lys Gly Thr Leu Thr Val Thr Pro     130 135 140 <210> 17 <211> 96 <212> PRT Cucumis sativus <400> 17 Ala Val Tyr Val Val Gly Gly Ser Gly Gly Trp Thr Phe Asn Thr Glu 1 5 10 15 Ser Trp Pro Lys Gly Lys Arg Phe Arg Ala Gly Asp Ile Leu Leu Phe             20 25 30 Asn Tyr Asn Pro Ser Met His Asn Val Val Val Val Asn Gln Gly Gly         35 40 45 Phe Ser Thr Cys Asn Thr Pro Ala Gly Ala Lys Val Tyr Thr Ser Gly     50 55 60 Arg Asp Gln Ile Lys Leu Pro Lys Gly Gln Ser Tyr Phe Ile Cys Asn 65 70 75 80 Phe Pro Gly His Cys Gln Ser Gly Met Lys Ile Ala Val Asn Ala Leu                 85 90 95 <210> 18 <211> 166 <212> PRT <213> Neisseria gonorrhoeae F62 <400> 18 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp                 85 90 95 Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Asp     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 19 <211> 150 <212> PRT <213> Vibrio parahaemolyticus <400> 19 Met Ser Leu Arg Ile Leu Ala Ala Thr Leu Ala Leu Ala Gly Leu Ser 1 5 10 15 Phe Gly Ala Gln Ala Ser Ala Glu Cys Glu Val Ser Ile Asp Ala Asn             20 25 30 Asp Met Met Gln Phe Ser Thr Lys Thr Leu Ser Val Pro Ala Thr Cys         35 40 45 Lys Glu Val Thr Leu Thr Leu Asn His Thr Gly Lys Met Pro Ala Gln     50 55 60 Ser Met Gly His Asn Val Val Ile Ala Asp Thr Ala Asn Ile Gln Ala 65 70 75 80 Val Gly Thr Asp Gly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val Lys                 85 90 95 Pro Asp Asp Glu Arg Val Tyr Ala His Thr Lys Val Val Gly Gly Gly             100 105 110 Glu Ser Thr Ser Ile Thr Phe Ser Thr Glu Lys Met Thr Ala Gly Gly         115 120 125 Asp Tyr Ser Phe Phe Cys Ser Phe Pro Gly His Trp Ala Ile Met Gln     130 135 140 Gly Lys Phe Glu Phe Lys 145 150 <210> 20 <211> 33 <212> PRT <213> Chloroflexus aurantiacus <400> 20 His Asn Trp Val Leu Val Asn Gly Gly Asp Asp Val Ala Ala Ala Val 1 5 10 15 Asn Thr Ala Ala Gln Asn Asn Ala Asp Ala Leu Phe Val Pro Pro Pro             20 25 30 Asp      <210> 21 <211> 27 <212> PRT <213> Pseudomonas syringae <400> 21 Ser Lys Lys Ala Asp Ala Ser Ala Ile Thr Thr Asp Gly Met Ser Val 1 5 10 15 Gly Ile Asp Lys Asp Tyr Val Lys Pro Asp Asp             20 25 <210> 22 <211> 27 <212> PRT <213> Neisseria meningitidis <400> 22 Ile Gly Lys Thr Glu Asp Met Asp Gly Ile Phe Lys Asp Gly Val Gly 1 5 10 15 Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp             20 25 <210> 23 <211> 27 <212> PRT <213> Vibrio parahaemolyticus <400> 23 Ala Asp Thr Ala Asn Ile Gln Ala Val Gly Thr Asp Gly Met Ser Ala 1 5 10 15 Gly Ala Asp Asn Ser Tyr Val Lys Pro Asp Asp             20 25 <210> 24 <211> 27 <212> PRT <213> Bordetella bronchiseptica <400> 24 Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile Ala Ala 1 5 10 15 Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp             20 25  

Claims (36)

An isolated peptide that is a variant, derivative or structural equivalent of cupredoxin, and which can inhibit the development of premalignant lesions in mammalian tissue. The method of claim 1, wherein the cupredoxin is azurin (pseudoazurin), platocyanin (plastocyanin), Rusticyanin, Laz, auracyanin (auracyanin), Stellacyanin ( stellacyanin) or cucumber based protein. The isolated peptide of claim 2, wherein the cupredoxin is azurin. The method of claim 1, wherein the cupredoxin is Pseudomonas aeruginosa , Alcaligenes faecalis , Achromobacter xylosoxidan , Bordetella bronchiseptica , Methylmonamonas species (Methylomonas sp.), meningitis bacteria (Neisseria meningitidis), gonorrhea (Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens), Pseudomonas chloro lapis (Pseudomonas chlororaphis), grape Pearson bacteria (Xylella fastidiosa), or Vibrio (Vibrio parahaemolyticus ) . An isolated peptide derived from an organism selected from parahaemolyticus . The isolated peptide of claim 4, wherein the peptide is derived from Pseudomonas aeruginosa . The isolated peptide of claim 1, wherein the peptide is part of a peptide selected from the group consisting of SEQ ID NO: 1, 3-19. The isolated peptide of claim 1, having at least 80% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, 3-19. The isolated peptide of claim 1, which is a truncation of cupredoxin. The isolated peptide of claim 8, having at least 10 residues and up to 100 residues. The method according to claim 8, Pseudomonas aeruginosa (Pseudomonas aeruginosa) very lean residues 50-77, Pseudomonas aeruginosa (Pseudomonas aeruginosa) very lean residues 50-67, Pseudomonas aeruginosa (Pseudomonas aeruginosa) very lean residues 36-88, or SEQ ID NO: 20 and 24 in An isolated peptide comprising the sequence of choice. The method according to claim 10, wherein Pseudomonas aeruginosa azurin residues 50-77, Pseudomonas aeruginosa azurin residues 50-67, Pseudomonas aeruginosa azurin residues 36-88, or SEQ ID NO: 20-24 An isolated peptide, consisting of the sequence selected. The isolated peptide of claim 1, comprising a cupredoxin residue equivalent to a region of Pseudomonas aeruginosa azurin selected from residues 50-77, residues 50-67 or residues 36-88. A pharmaceutical composition containing at least one cupredoxin or peptide of claim 1 in a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 13, containing at least two cupredoxins or peptides. The pharmaceutical composition of claim 13, which is prepared for intravenous administration. The method of claim 13, wherein the cupredoxin is Pseudomonas aeruginosa , Alcaligenes faecalis , Achromobacter xylosoxidan , Bordetella bronchiseptica , Methylmonamonas species (Methylomonas sp.), meningitis bacteria (Neisseria meningitidis), gonorrhea (Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens), Pseudomonas chloro lapis (Pseudomonas chlororaphis), grape Pearson bacteria (Xylella fastidiosa), or Vibrio (Vibrio parahaemolyticus ) pharmaceutical composition, characterized in that it is derived from an organism selected from . The pharmaceutical composition of claim 16, wherein the cupredoxin is derived from Pseudomonas aeruginosa . The pharmaceutical composition of claim 13, wherein the cupredoxin is selected from the group consisting of SEQ ID NOs: 1, 3-19. A method of treating a mammalian patient, the method comprising administering to the patient a therapeutically effective amount of the composition of claim 13. The method of claim 19, wherein the patient is a human. The method of claim 19, wherein the patient has a higher risk of developing cancer than the general population. The method of claim 21, wherein the cancer is selected from melanoma, breast cancer, pancreatic cancer, glioblastoma, astrocytoma, lung cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, skin cancer, or cervical cancer. The method of claim 21, wherein the patient has at least one high risk feature. The method of claim 19, wherein the patient has premalignant lesions. The method of claim 19, wherein the patient has been treated for cancer or premalignant lesion. The method of claim 19, wherein the pharmaceutical composition is administered in a manner selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous infusion, or oral. The method of claim 24, wherein the mode of administration is intravenous injection. The method of claim 21, wherein the pharmaceutical composition is co-administered with at least one other chemopreventive agent. The method of claim 26, wherein the pharmaceutical composition is administered concurrently with other chemopreventive agents. A kit comprising the composition of claim 13 in a vial. 31. The kit of claim 30, wherein the kit is designed for intravenous administration. A method of investigating the occurrence of cancer, the method comprising contacting a mammalian cell with the cupredoxin or peptide of claim 1 and measuring the extent of development of premalignant and malignant cells. The method of claim 32, wherein the cell is a human cell. The method of claim 32, wherein the cell is a mammary cell. The method of claim 32, wherein the cells are induced to develop cancer. An expression vector encoding the peptide of claim 1.

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