JP2008539795A - Compositions and methods for treating symptoms related to ephrin signaling using cupredoxins - Google Patents

Abstract

The present invention relates to compositions of cupredoxins that interfere with the ephrin signaling system in mammalian cells, and variants, derivatives or structural equivalents of cupredoxins, and methods of use. In particular, the present invention relates to compositions and methods that use cupredoxins such as azurin, rusticyanin and plastocyanin, and variants, derivatives and structural equivalents thereof to treat cancer in mammals.
[Selection figure] None

Description

Related applications

  This application includes US Provisional Patent Application No. 60 / 764,749 filed February 3, 2006, US Provisional Patent Application No. 60 / 682,812 filed May 20, 2005, and October 6, 2005. Claims priority under US Patents 119 and 120 for US patent application No. 11 / 244,105 filed in US; this last basic application was filed on October 7, 2004 Provisional Patent Application No. 60 / 616,782 and U.S. Provisional Patent Application No. 60 / 680,500 filed May 13, 2005, and US Patent Application filed November 11, 2003 No. 10 / 720,603, which is a continuation-in-part application, claiming priority of US Provisional Patent Application No. 60 / 414,550 filed on August 15, 2003, and on January 15, 2002 This is a continuation-in-part of US patent application Ser. No. 10 / 047,710, which claims the priority of US Provisional Patent Application No. 60 / 269,133, filed on Feb. 15, 2001. The entire contents of these earlier applications are hereby incorporated by reference.

Statement of government interest

  The subject matter of this application has been supported by a research grant from the National Institutes of Health (NIH) in Bethesda, Maryland, USA (Grant numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 and N01- CM97567). The government may have certain rights in this invention.

Field of Invention

  The present invention relates to cupredoxins and their use in the regulation of cellular functions including ephrins and ephrin receptors. The present invention also relates to a method of treating ephrin related symptoms. In particular, the invention relates to methods of delaying cancer cell growth and metastasis and pathological conditions, particularly those associated with ephrin / ephrin receptor signaling, and other therapeutics associated with ephrin / ephrin receptor signaling. It relates to the use of substantially pure cupredoxin in the process. The invention also relates to variants, derivatives and structural equivalents of cupredoxins that retain the ability to interfere with the ephrin signaling system in cells.

background

  The ephrin receptors (Eph receptors) are a large family of receptor tyrosine kinases that regulate numerous processes in developing and adult tissues by binding to a family of ligands called ephrins. Eph receptors are classified as type A or B along with ephrin ligands. There are currently nine known type A members (EphA1-8 and EphA10), and four known type B members (EphB1-4 and EphB6). In general, class A receptors preferentially bind to type A ligands, while class B receptors preferentially bind to type B ligands. The Eph receptor is a single transmembrane domain, a glycosylated extracellular region with a ligand binding domain with an immunoglobulin-like motif, a cysteine-rich region, and other fibronectin type III repeats for other Similar to tyrosine kinase receptor. (Surawska et al., Cytokine & Growth Factor Reviews 15: 419-433 (2004)). These ephrin ligands are divided into A and B classifications based on their sequence conservation. Ephrin A ligands are glycosylphosphatidylinositols that are normally anchored and bound by Eph-A type receptors, whereas ephrin B ligands contain a transmembrane domain and a short cytoplasmic region, usually EphB type Bound by the receptor. (See the same literature as above).

  The signaling process is initiated when the Eph receptor dimerizes with the ephrin ligand, leading to phosphorylation of the receptor. Higher order clustering forms aggregates of ephrin-Eph receptor complexes. Receptor activation appears to be dependent on the degree of multimerization, but receptor phosphorylation is observed in both lower and higher order forms and thus is not limited to tetrameric forms It is. Depending on the state of multimerization, different Eph receptor complexes can induce biological effects. In addition to “forward” signaling through the Eph receptor to receptor-expressing cells, there is also “reverse” signaling through ephrin to ephrin-expressing cells. For example, the cytoplasmic tail on B-ephrin can be phosphorylated leading to recruitment of signaling effectors and signaling cascades within ephrin signaling cells. (See the same literature as above).

Currently, ephrins are known to have a role in many cell / cell interactions, including axonal pathway exploration, neuronal cell migration, and interactions in vascular endothelial cells and specialized epithelia. (Flanagan & Vanderhaeghen, Annu. Rev. Neurosci. 21: 309-345 (1998); Frisen et al., EMBO J. 18: 5159-5165 (1999)). Eph receptors are also involved in a variety of pathological processes, including tumor progression, pathological forms of angiogenesis, chronic pain after tissue injury, inhibition of neurodegeneration after spinal injury, and congenital human malformations. It has been said that (Koolpe et al., J Biol Chem. 280: 17301-17311 (2005)). Eph receptors have also been reported to play a role in the balance between hepatocyte self-renewal vs cell fate determination and differentiation (see above).
Overexpression of Eph receptors and ephrins can lead to tumor formation and is associated with angiogenesis and metastasis in many types of human cancer, including lung cancer, breast cancer and prostate cancer, and melanoma and leukemia Yes. (Surawska et al., Cytokine & Growth Factor Reviews 15: 419-433 (2004)). Eph receptor overexpression does not affect cell growth but appears to change its invasion behavior. According to one theory, in malignant cells with high levels of EphA2, the receptor mislocalizes and cannot bind their ephrin ligands, and thus is not phosphorylated, increased extracellular matrix attachment and high Brings transfer ability. (Ruoslahti, Adv. Cancer Res. 76: 1-20 (1999)). Angiogenesis is the formation of new blood vessels and capillaries from already existing blood vessels and is an essential process for tumors to survive and grow. There is evidence of upregulation of Eph receptors / ephrins during tumor vascular invasion. Type A ephrin is particularly associated with tumor angiogenesis, and EphA2-Fc and EphA3-Fc fusion proteins decreased tumor vascular density, tumor volume and cell proliferation, and increased apoptosis. (Brantley et al., Oncogene 21: 7011-7026 (2002)).

The crystal structure of the EphB2 receptor-ephrin B2 complex is an 8-chain barrel in which the ectodomain of the ephrin B2 folding topology is a variant of the normal Greek key 6-barrel fold, and is prominent with the copper-binding protein cuvredoxin family Ephrin B2 does not bind copper, though sharing similar homology. The major difference between ephrin and cupredoxin folding proteins is the unusual length of the ephrin GH _L and CD _L loops, which are part of the dimerization and tetramerization ligand receptor interface, respectively.

Crystallization studies further showed that the GH _L loop is involved in receptor binding. (Himanen et al., Nature 414: 933-938 (2001)). The extracellular domain of mouse ephrin B2 also has topological similarity to plant nodulin and phytocyanin. (Toth et al., Developmental Cell, 1: 83-92 (2001)).

  Reports on cancer degeneration in humans and animals infected with microbial pathogens go back more than 100 years, beginning with the first report by Coley (Clin. Orthop. Relat. Res. 262: 3-12 (1891)). To do. Several subsequent reports have shown that microbial pathogens replicate under hypoxic conditions at the tumor site, stimulating the host immune system during infection, leading to inhibition of cancer progression. (Alexandrof et al., Lancet 353: 1689-1694 (1999); Paglia & Guzman, Cancer Immunol. Immunother. 46: 88-92 (1998); Pawelek et al., Cancer Res. 57: 4537-4544 (1997) ). Pseudomonas aeruginosa and many other bacterial pathogens produce a range of virulence factors that allow bacteria to escape host defenses and cause disease. (Tang et al., Infect. Immun. 64: 37-43 (1996); Clark and Bavoil, Methods in Enzymology, vol. 235, Bacterial Pathogenesis, Academic Press, Inc. San Diego Calif. (1994); Salyers and Whitt , Bacterial Pathogenesis: A Molecular Approach, ASM Press, Washington DC (1994)). Some virulence factors induce apoptosis in phagocytic cells such as macrophages and weaken host defenses. (Monack et al., Proc. Natl. Acad. Sci. USA 94: 10385-10390 (1997); Zychlinsky and Sansonetti, J. Clin. Investig. 100: 493-495 (1997)).

Two redox proteins produced by P. aeruginosa, namely cupredoxin azurin and cytochrome c ₅₅₁ (Cyt c ₅₅₁ ), both invade J774 cells and are found against human cancer cells when compared to normal cells. Shows significant cytotoxic activity. (Zaborina et al., Microbiology 146: 2521-2530 (2000)). Azurin can also enter human melanoma UISO-Mel-2 cells or human breast cancer MCF-7 cells. (Yamada et al., PNAS 99: 14098-14103 (2002); Punj et al., Oncogene 23: 2367-2378 (2004); Yamada et al., Cell. Biol. 7: 14181431 (2005)). In addition, azurin from P. aeruginosa preferentially invades J774 mouse reticulosarcoma cells, forming a complex with the tumor suppressor protein p53 and stabilizing it, increasing the intracellular concentration of p53 Induces apoptosis. (Yamada et al., Infection and Immunity, 70: 7054-7062 (2002)). Azurin also causes a marked increase in apoptosis in human osteosarcoma cells compared to non-cancerous cells. (Ye et al., Ai Zheng 24: 298-304 (2003)).

Cytochrome C ₅₅₁ (Cyt C ₅₅₁ ) from P. aeruginosa increases the level of the tumor suppressor protein p16 ^Ink4a and inhibits cell cycle progression in J774 cells. (Hiraoka et al., PNAS 101: 6427-6432 (2004)). However, when colon cancer cells such as HCT 116 cells, or p53-zero lung cancer H1299 cells were grown for 3 days in the presence of wild-type azurin or wild-type cytochrome c ₅₅₁ , they were H1299 cells (> 20 HCT 116 cell growth was inhibited at a concentration much lower than that of (μg / ml) (IC50 = 17 μg / ml for azurin; 12 μg / ml for Cyt C). See the above reference.

  Cancer is a malignant tumor with potentially unlimited proliferation. It is primarily pathogenic replication (loss of normal regulatory control) in various cell types found in the human body. Initial treatment for this disease is often surgery, radiation therapy, or a combination of these treatments, but local recurrence and metastatic disease are frequent. Chemotherapy is available for some cancers, but they induce little long-term remission. Therefore, they are often not curative. In general, tumors and their metastases become resistant to chemotherapy, as is known as the development of multidrug resistance. In many cases, tumors are inherently resistant to certain chemotherapeutic agents. In addition, such treatments threaten non-cancerous cells and put stress on the human body, causing many side effects. Therefore, there is a need for improved drugs to prevent the spread of cancer cells.

Summary of the Invention

  The present invention relates to cupredoxins and compositions of variants, derivatives and structural equivalents of cupredoxins that interfere with the ephrin signaling system in mammalian cells, and methods of use thereof. Specifically, the present invention relates to compositions and methods that use cupredoxins such as azurin and plastocyanin, and variants, derivatives and structural equivalents thereof to treat cancer in mammals.

  One aspect of the invention relates to an isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin and that can inhibit the growth of cancer in mammalian cells or tissues. This peptide may be azurin, plastocyanin, pseudoazurin, plastocyanin, rusticyanin or aurecyanin, in particular azurin, plastocyanin and rusticyanin. In some embodiments, the cupredoxin is Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum, Alcaligenes faecalis, Axiomos (Achromobacter xylosoxidan), Bordetella bronchiseptica, Methylomonas sp. (Methylomonas sp.), Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis yl, fast moss・ Derived from Cucumis sativus, Chloroflexus aurantiacus, Vibrio parahaemolyticus or Ulva pertusa, especially Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormil Is. This isolated peptide may be part of SEQ ID NOs: 1-17 and 22-23. In addition, SEQ ID NOs: 1-17 and 22-23 may have at least about 90% amino acid sequence identity to the peptide.

In some embodiments, the isolated peptide may be a truncated form of a cupredoxin. In certain embodiments, the peptide is about 10 residues or more and 100 residues or less. The peptide comprises P. aeruginosa azurin residues 96-113, P. aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulva pertusa residues 57-98, or SEQ ID NOs 22-30 Or may consist of these. In some embodiments, the isolated peptide comprises P. aeruginosa azurin residues 96-113, P. aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulva pertusa residues 57- 98, or an equivalent residue of the subject cupredoxin as the region of the target cupredoxin selected from the group consisting of 98 or SEQ ID NOs: 22-30. A composition comprising a variant, derivative or structural equivalent of a cupredoxin capable of inhibiting the growth of cancer in mammalian cells or tissues. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. The cupredoxins include Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum, Alcaligenes faecalis, Alcaligenes faecalis, Achromobacter oxidobylacter (Bordetella bronchiseptica), Methylomonas sp. (Methylomonas sp.), Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis yl, fast moss • It may be derived from Cucumis sativus, Chloroflexus aurantiacus, Vibrio parahaemolyticus or Ulva pertusa. In some embodiments, the cupredoxin may be that of SEQ ID NOs: 1-17 and 22-23.

  Another aspect of the present invention is a method of treating a mammalian patient suffering from a pathological condition associated with the ephrin signaling system or suffering from cancer, wherein said patient is in a pharmaceutical composition. Administering a therapeutically effective amount of a composition comprising at least one cupredoxin, or a variant, derivative or structural equivalent of a cupredoxin. In some embodiments, the patient has interstitial cystitis (IC), a foci associated with inflammatory bowel disease, HIV infection, cardiovascular disease, central nervous system disorder, peripheral disagreement disease, viral disease, Suffers from central nervous system degeneration (Christopher Reeve's disease) or Alzheimer's disease. In another embodiment, the patient has cancer, such as breast cancer, liver cancer, gastrointestinal cancer, neuroblastoma, neuronal cancer, leukemia, lymphoma, prostate cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, endometrial tumor, villi It has cancer, teratocarcinoma, thyroid cancer, all sarcomas, including those arising from soft tissue and bone, renal cancer, epidermoid cancer, or non-small cell lung cancer. In some embodiments, the patient is a human.

  Another aspect of the present invention is a composition comprising at least two isolated polypeptides that are cupredoxins or variants, derivatives or structural equivalents of cupredoxins that are capable of inhibiting cancer growth in mammalian cells or tissues. It is. In some embodiments, the composition is in the form of a pharmaceutical composition.

  Another aspect of the invention is a kit comprising a composition comprising at least one cupredoxin, or a variant, derivative or structural equivalent of a cupredoxin, in the form of a pharmaceutical composition in a vial. The kit may be designed for intravenous administration.

  Another aspect of the invention is a method comprising contacting a mammalian cancer cell with a cupredoxin or variant, derivative or structural equivalent thereof; and measuring the growth of said cancer cell. This cancer cell is breast cancer, liver cancer, gastrointestinal cancer, neuroblastoma, neuronal cancer, leukemia, lymphoma, prostate cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, endometrial tumor, choriocarcinoma, teratocarcinoma, thyroid cancer, It can be any sarcoma, including those arising from soft tissue and bone, renal cancer, epidermoid cancer, or non-small cell lung cancer. In some embodiments, the cancer cell is in vivo.

  Another aspect of the invention is an expression vector encoding a variant, derivative or structural equivalent of a cupredoxin capable of inhibiting cancer growth in mammalian cells or tissues.

  Another aspect of the present invention is a cupredoxin variant, derivative, or structural equivalent; and an ephrin receptor such as EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, An isolated peptide capable of binding to EphB2, EphB3, EphB4 and EphB6. In some embodiments, the peptide can also bind to ephrins, such as ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3 and ephrin B4. In other embodiments, the isolated peptide binds to ephrin and its receptor, particularly ephrin 2B and Eph2B. In a related aspect, the isolated peptide is a variant, derivative or structural equivalent of a cupredoxin; and ephrins, such as ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, It can bind to ephrin B3 and ephrin B4.

  Another aspect of the invention is a method comprising administering to a patient at least one cupredoxin or a peptide of the invention to guide blood vessel growth in a mammalian patient.

  Another aspect of the invention is a method comprising administering to a patient a pharmaceutical composition comprising at least one cupredoxin or a peptide of the invention in order to reduce vascular growth in a mammalian patient. is there.

  Another aspect of the invention is a method comprising administering to a patient a pharmaceutical composition comprising at least one cupredoxin or a peptide of the invention to guide nerve growth in a mammalian patient. is there. Another aspect of the present invention is a method comprising administering to said patient a pharmaceutical composition comprising at least one cupredoxin or a peptide of the present invention to promote bone formation in a mammalian patient. .

  Another aspect of the invention is a method of treatment requiring the use of ephrin, comprising using an effective amount of at least one cupredoxin or a peptide of the invention to a mammalian patient instead of ephrin. It is.

  Another aspect of the present invention is to administer to a mammalian cell a pharmaceutical composition comprising at least one cupredoxin or a peptide of the present invention in order to inhibit the activity of an ephrin receptor associated with the mammalian cell. It is a method comprising.

  Another aspect of the present invention is a method comprising administering a pharmaceutical composition comprising at least one cuvredoxin or a peptide of the present invention to increase the activity of ephrin receptors associated with said cells. is there. Another aspect of the present invention relates to at least one cupredoxin fused to a pharmaceutical substance, or a derivative, variant, derivative or structural equivalent of at least one cupredoxin fused to a pharmaceutical substance for a human patient having a tissue expressing an ephrin receptor. A method comprising administering a pharmaceutical composition comprising a peptide of the present invention comprising the product. In some embodiments, the tissue expressing the ephrin receptor is cancer.

  Another aspect of the present invention is a method for detecting tissue with an ephrin receptor, comprising at least one cupredoxin or peptide of the present invention fused to a detectable probe for a human patient. A method comprising administering a pharmaceutical composition and detecting a distribution of probes within the patient.

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

<Brief description of sequence>
SEQ ID NO: 1: Amino acid sequence of azurin from Pseudomonas aeruginosa SEQ ID NO: 2: Amino acid sequence of plastocyanin from Phormidium laminosum.

  SEQ ID NO: 3: amino acid sequence of rusticyanin derived from Thiobacillus ferrooxidans.

  SEQ ID NO: 4: Amino acid sequence of pseudoazurin from Achromobacter cycloclastes.

  SEQ ID NO: 5: Amino acid sequence of azurin from Alcaligenes faecalis.

  SEQ ID NO: 6: Amino acid sequence of azurin from Achromobacter xylosoxidans ssp. Denitrificans I.

  SEQ ID NO: 7: Amino acid sequence of azurin from Bordetella bronchiseptica.

  SEQ ID NO: 8: Amino acid sequence of azurin from Methylomonas sp. J.

  SEQ ID NO: 9: Amino acid sequence of azurin from Neisseria meningitidis Z2491.

  SEQ ID NO: 10: Amino acid sequence of azurin from Neisseria gonorrhoeae

  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 auracyanin A derived from Chloroflexus aurantiacus.

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

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

  SEQ ID NO: 18: 18-mer azurin peptide, ie the amino acid sequence of Pseudomonas aeruginosa azurin residues 96-113.

  SEQ ID NO: 19: Amino acid sequence of Pseudomonas aeruginosa azurin residues 88-113.

  SEQ ID NO: 20: amino acid sequence of Ulva pertusa plastocyanin plastocyanin residues 88-113.

  SEQ ID NO: 21: Amino acid sequence of Vibrio parahaemolyticus azurin.

  SEQ ID NO: 22: Amino acid sequence of Ulva pertusa plastocyanin.

  SEQ ID NO: 23: Amino acid sequence of ephrin B2 ectodomain derived from human.

  SEQ ID NO: 24: amino acid sequence of the GH loop region of human ephrin B2.

  SEQ ID NO: 25: Amino acid sequence of P. aeruginosa azurin structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 26: Amino acid sequence of Thiobacillus (Acidithiobacillus) ferrooxidans rusticyanin region structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 27: amino acid sequence of Chloroflexus aurantiacus auracyanin B region structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 28: Amino acid sequence of Ulva pertusa plastocyanin region structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 29: Amino acid sequence of Cucumis sativus cucumber basic protein region structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 30: Amino acid sequence of Cucumis sativus stellacyanin region structurally similar to the GH loop region of ephrin B2.

  SEQ ID NO: 31: Amino acid sequence of human ephrin B2 region residues 69-138.

  SEQ ID NO: 32: amino acid sequence of Ulva pertusa plastocyanin residues 57-98.

  SEQ ID NO: 33: Amino acid sequence of human ephrin B2 region residues 68-138.

  SEQ ID NO: 34: Amino acid sequence of P. aeruginosa azurin residues 76-128.

Detailed Description of the Invention

<Definition>
As used herein, the term “cell” includes one or more of the terms, unless specifically described as “single cell”.

  As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably to mean a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids. “Polypeptides”, “peptides”, and “proteins” also include, but are not limited to, glycosylation, lipid binding, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Including modifications are also included. It will be appreciated that polypeptides are not always linear in their entirety. For example, polypeptides can branch as a result of ubiquitin binding and are generally cyclic as a result of post-translational events, including those caused by natural processing events and non-naturally occurring human manipulations. (With or without branching). Cyclic, branched, and branched cyclic polypeptides can be synthesized by untranslated natural processes as well as by total synthetic methods. The present invention further contemplates the use of both methionine-containing and methionine-free amino terminal variants of the proteins of the invention.

  As used herein, the term “pathological condition” includes anatomical and physiological deviations from the normal state that constitute a normal state dysfunction of a living animal or part thereof; The disorder interferes with or alters the characteristics of bodily function, and against a particular infectious agent (insect, parasitic protozoa, bacteria, or virus) against various factors (malnutrition, industrial hazards, or climate), It may be a response to an organism's inherent defects (gene abnormalities) or to a combination of these factors.

  As used herein, the term “condition” is intended to include anatomical and physiological deviations from the normal state, which refers to the dysfunction of one normal state of a living animal or part thereof. Construct and disturb or change the characteristics of physical function.

  The term “inhibit cell growth” as used herein means slowing or stopping cell division and / or cell growth. The term also includes inhibiting cell growth or increasing cell death.

  As used herein, the term “affected by” refers to presenting a syndrome of a pathological condition, having a pathological condition without an observable syndrome, or a process of recovery from a pathological condition And recovery from a pathological condition.

  As used herein, the term “treatment” is intended to include preventing, reducing, stopping or reversing the progression or severity of a condition or syndrome associated with the condition being treated. Thus, “treatment” includes medical, therapeutic and / or prophylactic administration as appropriate.

  A “therapeutically effective amount” is an amount effective to prevent or delay the occurrence of symptoms present in a particular condition of the patient being treated or to partially or totally alleviate. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

  As used herein, the term “underexpression of p53 tumor suppressor gene” means a cell with a p53 tumor suppressor gene that is inactivated, mutated, lost or underproduced. For example, such deficiencies may result from genetic abnormalities within the p53 gene or for epigenetic reasons, such as hypermethylation or virality of C residues in the CG island upstream of the tumor suppressor gene and cellularity. May result from interaction with sex oncogenes.

  As used herein, the term “substantially pure”, when used to modify the term “cupredoxin”, refers to cupredoxins, eg, other proteins isolated from growth media or cell contents. And / or a cupredoxin that is substantially free of or free of active inhibitory compounds. The term “substantially pure” means at least about 75%, or at least “75% substantially pure” factor by dry weight of the isolated fraction. More specifically, the term “substantially pure” means at least about 85% active compound or at least “85% substantially pure” compound by dry weight. Most particularly, the term “substantially pure” means at least about 95% active compound, or at least “95% substantially pure” compound by dry weight. A substantially pure cupredoxin can be used in combination with one or more other substantially pure compounds or isolated cupredoxins.

  The phrase “isolated”, “pure”, or “biologically pure” refers to components that are normally associated with a substance, substantially or essentially, when the substance is present in its natural state. Means a substance not included in Thus, an isolated peptide according to the present invention preferably does not contain substances normally associated with the peptide in its in situ environment. An “isolated” region refers to a region that does not include the entire sequence of the polypeptide from which the region is derived. “Isolated” nucleic acids, proteins, or respective fragments thereof, include but are not limited to nucleic acid sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for nucleic acid fragments. As well as being able to be manipulated by those skilled in the art, such as obtaining the protein or protein fragment in a substantially pure amount, etc. Has been removed.

  The term “variant” as used herein with respect to a peptide means an amino acid sequence variant that may have an amino acid substituted, deleted or inserted when compared to the wild-type polypeptide. The mutant may be a shortened version of the wild-type peptide. Accordingly, mutant peptides may be produced by manipulation of the gene encoding the polypeptide. Variants may be made by changing the base composition or characteristics of the polypeptide but not changing at least some of its basic activities. For example, a “variant” of azurin can be a mutated azurin that retains its ability to inhibit the growth of mammalian cancer cells. In some cases, variant peptides are synthesized using unnatural amino acids such as ε- (3,5-dinitrobenzoyl) -Lys residues. (Ghadiri & Fernholz, J. Am. Chem. Soc., 112: 9633-9635 (1990)). In some embodiments, the variant has no more than 20, 19, 18, 17 or 16 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 15, 14, 13, 12, or 11 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 10, 9, 8, or 7 amino acids substituted, deleted, or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 6 amino acid substitutions, deletions, or insertions compared to the wild-type peptide.

  As used herein, the term “amino acid” refers to any naturally occurring amino acid or amino acid moiety comprising non-naturally occurring or synthetic amino acid residues, ie, one, two or more carbons. It means any moiety comprising at least one carbonyl residue and at least one amino acid residue bonded directly by an atom, typically one (α) carbon atom.

  The term “derivative” as used herein with respect to a peptide means a peptide derived from the peptide of interest. Derivatization involves chemical modification of the peptide such that the peptide still retains some of its basic activity. For example, a “derivative” of azurin can be a chemically modified azurin that retains its ability to inhibit the growth of mammalian cancer cells. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation or glycosylation of the peptide. In addition, a derivative peptide can be a polypeptide or fragment thereof to another chemical compound such as, but not limited to, another peptide, drug molecule, other therapeutic or pharmaceutical substance or detectable probe. It may be a fusion of

  The term “percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a polypeptide that are identical to amino acid residues in a candidate sequence when the two sequences are aligned. To determine% amino acid identity, sequences are aligned and, if necessary, gaps are introduced to achieve maximum% sequence identity; conservative substitutions are not considered part of sequence identity . Amino acid sequence alignment procedures for determining percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. In certain embodiments, Blastp (available from the National Center for Biotechnology Information in Bethesda, Maryland) using the default parameters for long complexity filters (expected value 10, word size 3, presence 11, and extension 1) Is used.

When the amino acid sequences are aligned, the% amino acid sequence identity of the given amino acid sequence A to the given amino acid sequence B (or it has a certain% amino acid sequence identity to the given amino acid sequence B) Or a predetermined amino acid sequence A comprising) can be calculated as follows:
% Amino acid sequence identity = X / Y * 100
here,
X is the number of amino acid residues evaluated by the sequence alignment program or algorithm as the same string by alignment of sequences A and B;
Y is the total number of amino acid residues in B

  If the length of amino acid sequence A is equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B is not equal to the% amino acid sequence identity of B to A.

  When comparing a longer sequence to a shorter sequence, the shorter sequence will be the “B” sequence. For example, when comparing a truncated peptide to a wild-type polypeptide, the truncated peptide will be a “B” sequence.

<General>
Some aspects of the present invention use cupredoxins that have structural similarity to ephrin to interfere with the ephrin signaling system in various mammalian cells and tissues and to inhibit the growth of mammalian cancer cells. Compositions and methods are provided. That is, the present invention relates to cupredoxins, and variants, derivatives and structural thereof, in vitro and in vivo, to interfere with the ephrin signaling system in various mammalian cells and tissues and to inhibit the growth of mammalian cancer cells. Compositions and methods are provided that use equivalents.

  The inventors have previously described that pathogenic microorganisms secrete redox proteins such as azurin from ATP-independent cytotoxic factors such as azurin from P. aeruginosa, and such factors are We found that apoptosis caused death of J774 cells in cells. Azurin also has a domain of about amino acid residues 50-77, which promotes the protein to preferentially enter cancer cells and induce cytotoxicity.

  Surprisingly, the inventor has now discovered that there is a C-terminal domain in azurin and other cupredoxins that exhibits structural similarity to ephrin. For example, see Experimental Example 2, Experimental Example 5 and Experimental Example 9. It has also been found that azurin and plastocyanin, as well as special regions of these peptides, are structurally homologous to the ephrin GH loop and competitively bind to the ephrin receptor in a 1: 1 ratio. It was. See Experimental Examples 6-8. In particular, P. aeruginosa azurin binds to the ephrin receptors EphB2 and EphA6. See Experimental Example 6. Phormidium laminosum plastocyanin exhibits specificity for binding the ephrin receptors EphA1, A3 and B2, and also to a lesser extent ephrin receptors EphA2 and A6. For example, see Experimental Example 6. Finally, rusticyanin shows weak binding to the ephrin receptors EphA8 and B1. See Experimental Example 6. In addition, it is now known that amino acid residues 88 to 113 of the azurin region containing structural homology to the ephrin GH loop region bind to the ephrin receptor EphB2. See Experimental Example 7. Finally, it is now known that azurin and the 88-113 region of azurin bind to ephrin B2 and the ephrin receptor EphB2 and can compete with ephrin B2 for the ephrin receptor EphB2. See Experimental Example 8.

  The inventors have also discovered that cupredoxins with regions having structural similarity to the ephrin G-H region inhibit the growth of mammalian cancer cells in vitro. In general, Phormidium laminosum plastocyanin, Thiobacillus ferrooxidans rustocyanin, and P. aeruginosa azurin inhibit in vitro growth of Mel-2 human melanoma cells and MCF-7 human breast cancer cells in a trypan blue assay. See Experimental Example 4. Furthermore, the 88-113 residue region of azurin is now known to inhibit cell proliferation of MCF-7 breast cancer cells. See Experimental Example 10. Finally, the 18-mer azurin peptide and 15-mer Ulva pertusa plastocyanin peptide, which correspond to regions of structural similarity to the GH loop region of ephrin B2, are now MCF-7 human breast cancer cells, CCF-STTG1 brain tumor stellate cells It is known to inhibit the growth of cells of tumors and LN-229 glioblastoma in vitro. See Experimental Example 3 and Experimental Example 9.

  Surprisingly, cupredoxins with structural similarity to the GH loop of ephrin B2 also affect ephrin-related development in vivo. In vivo studies of ephrin-related development in C. elegans now know that cupredoxin rusticyanin interferes with tail muscle formation while cupredoxin azurin interferes with embryogenesis, both of which are ephrin-related outbreaks Is a process. See Experimental Example 1.

  In addition to azurin, cupredoxins, rusticyanins and plastocyanins also share structural homology with the G and H regions of ephrin B2. See Experimental Example 2 and Experimental Example 5. In addition, azurin and phytocyanin, stellacyanin and cucumber basic proteins also share significant structural homology with ephrin. Many other cupredoxins and cupredoxin-like proteins also exhibit significant structural homology to ephrins, generally because of the conservation of structure in proteins of the cupredoxin family. See Experimental Example 2. Thus, it is generally believed that cupredoxin family proteins can be used to treat conditions and cancers associated with ephrin signaling using the compositions and orientations of the present invention. Special cupredoxins of interest include, but are not limited to, azurin, rusticyanin, plastocyanin, stellacyanin, auracyanin, pseudoazurin, and cucumber basic protein. Examples of protein sequences are plastocyanin (SEQ ID NO: 2 and 22), rusticyanin (SEQ ID NO: 3), pseudoazurin (SEQ ID NO: 4), stellacyanin (SEQ ID NO: 14), auracyanin (SEQ ID NO: 15 and 16), and Found here as a cucumber basic protein (SEQ ID NO: 17). As used herein, the term “cupredoxin” means any member of a protein of the cupredoxin family, including a cupredoxin-like protein such as Laz from Neisseria.

  Azurin is particularly specific, and the protein sequences of these cupredoxins are found here, including but not limited to those isolated from: Pseudomonas aeruginosa (SEQ ID NO: 1); Alcaligenes faecalis ( SEQ ID NO: 5); Achromobacter xylosoxidans ssp. Denitrificans I (SEQ ID NO: 6); Bordetella bronchiseptica (SEQ ID NO: 7); Methylomonas sp. J (SEQ ID NO: 8); Neisseria meningitidis (SEQ ID NO: 9); Neisseria gonnorrhoeae (SEQ ID NO: 10) Pseudomonas fluorescens (SEQ ID NO: 11); Pseudomonas chlororaphis (SEQ ID NO: 12); Xylella fastidiosa 9a5c (SEQ ID NO: 13) and Vibrio parahaemolyticus (SEQ ID NO: 21). In the most specific embodiment, the azurin is from Pseudomonas aeruginosa. In other specific embodiments, the cupredoxin is plastocyanin (SEQ ID NO: 2 and 22), rusticyanin (SEQ ID NO: 3), pseudoazurin (SEQ ID NO: 4), stellacyanin (SEQ ID NO: 14), auracyanin (SEQ ID NO: 14). 15 and 16), and cucumber basic protein (SEQ ID NO: 17).

  In some embodiments, the cupredoxin has a Greek key beta barrel structure. This Greek key beta-barrel structure is a well-known protein fold. See, for example, Zhang & Kim (Proteins 40: 409-419 (2000)). Greek key topology is named after a pattern common to Greek pottery. It has three upward and downward beta strands joined by a hairpin, followed by a long link to the fourth beta strand, which is adjacent to the first beta strand. In certain embodiments, cupredoxins, and variants, derivatives and structural equivalents of cupredoxins comprise at least one Greek key beta-barrel structure. In another specific embodiment, the cupredoxin, and variants, derivatives and structural equivalents of the cupredoxin comprise at least a 4 beta chain Greek key structure. In another more specific embodiment, cupredoxins, and variants, derivatives and structural equivalents of cupredoxins contain more than one Greek key beta-barrel structure.

<Composition of the present invention>
The present invention provides peptides that are variants, derivatives or structural equivalents of cupredoxins. In some embodiments, the peptide is substantially pure. In other embodiments, the peptide is in the form of a composition, the composition comprising, consisting of, or consisting essentially of the peptide. In other embodiments, the peptide is isolated. In some embodiments, the peptide is smaller than a full-length cupredoxin and retains some of the functional characteristics of the cupredoxin. In some embodiments, the peptides retain the ability to interfere with ephrin signaling and / or inhibit the growth of mammalian cancer cells in mammalian cells and tissues. In another particular embodiment, the peptide does not produce an immune response in mammals, particularly humans.

  The present invention also provides compositions containing at least one, at least two, or at least three cupredoxins, or variants, derivatives or structural equivalents of cupredoxins. The present invention also provides compositions containing at least one, at least two, or at least three cupredoxins, or variants, derivatives or structural equivalents of cupredoxins in a pharmaceutical composition.

  Because of the high structural homology between cupredoxins, it is envisaged that other families of cupredoxins could interfere with ephrin signaling and specifically inhibit cancer growth in mammalian cells and tissues.

  In some embodiments, the cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, pseudoazurin, rusticyanin, or auracyanin. In a particularly specific embodiment, the azurin is Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan ssp, denitrificans I, deceptificans I Bordetella bronchiseptica), Methylomonas sp. (Methylomonas sp.), Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis, ios5 Or derived from Vibrio parahaemolyticus. In a very specific embodiment, the azurin is derived from Pseudomonas aeruginosa. In another special embodiment, the cupredoxin is plastocyanin, more particularly plastocyanin from Phormidium laminosum or Ulva pertusa. In another special embodiment, the cupredoxin is rusticyanin, more particularly rusticyanin from Thiobacillus ferrooxidans. In another special embodiment, the cupredoxin comprises the amino acid sequence of SEQ ID NOs: 1-17, 21-22.

  The present invention provides amino acid sequence variants of cupredoxins that have substituted, deleted or inserted amino acids as compared to wild-type polypeptides. The mutant of the present invention may be a shortened form of the wild-type polypeptide. As used herein, a “truncated form” of a polypeptide is a peptide that results from the removal of at least one amino acid residue from at least one end of a polypeptide sequence. In some embodiments, the truncated peptide has a total of at least 1 amino acid residue, at least 5 amino acid residues, at least 10 amino acid residues, at least 50 amino acid residues, or at least 100 from one or both ends of the polypeptide sequence. Result from removal of amino acid residues. In some embodiments, the composition comprises a peptide consisting of a cupredoxin region that is smaller than the full length of the wild-type polypeptide. In some embodiments, the composition comprises a peptide consisting of a truncated cupredoxin of greater than about 10 residues, greater than about 15 residues, or greater than about 20 residues. In some embodiments, the composition comprises a peptide consisting of a truncated cupredoxin of about 100 residues or less, about 50 residues or less, about 40 residues or less, or about 30 residues or less. . In some embodiments, the variant is a cupredoxin, more particularly SEQ ID NOs: 1-17, 21-22, to which at least 90% amino acid sequence identity, at least about 91% amino acid sequence identity. Or a peptide having at least about 99% amino acid sequence identity.

  In certain embodiments, the variant of cupredoxin comprises P. aeruginosa azurin residues 96-113 (SEQ ID NO: 18), 88-113 (SEQ ID NO: 19) or SEQ ID NO: 25. In other embodiments, the variant of cupredoxin comprises residues 70-84 of Ulva pertusa (SEQ ID NO: 20), residues 57-98 of Ulva pertusa (SEQ ID NO: 32), or SEQ ID NO: 28 of Ulva pertusa It will be. In other specific embodiments, the cupredoxin variant consists of residues 70-84 of Ulva pertusa (SEQ ID NO: 20), residues 57-98 of Ulva pertusa (SEQ ID NO: 32), or Ulva pertusa SEQ ID NO: 28 Is. In another specific embodiment, the cupredoxin variant comprises the Thiobacillus ferrooxidans rusticyanin sequence (SEQ ID NO: 26), Chloroflexus aurantiacus auracyanin (SEQ ID NO: 27), Cucumis sativus sequence (SEQ ID NO: 29 and 30) It is. In another specific embodiment, the cupredoxin variant consists of the Thiobacillus ferrooxidans rusticyanin sequence (SEQ ID NO: 26), Chloroflexus aurantiacus auracyanin (SEQ ID NO: 27), and Cucumis sativus sequences (SEQ ID NOs: 29 and 30). In another specific embodiment, the variant consists of an equivalent sequence to the truncated sequence from another cupredoxin. It is envisioned that other cupredoxin variants can be designed to have similar activity to any of the above variants. To do this, the amino acid sequence of the subject cupredoxin is shortened using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the desired cupredoxin sequence containing the above-mentioned truncated variants, and a truncated located on the desired cupredoxin sequence. Related residues of type variants, and equivalent residues found on the subject cupredoxin sequences, and equivalent shortenings thus designed will be aligned to the variant.

  This variant also includes peptides produced using non-naturally occurring synthetic amino acids. For example, non-naturally occurring amino acids can be incorporated into the mutant peptide to extend or optimize the half-life of the composition in the bloodstream. Such mutants include D, L-peptides (diastereomers) (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 containing unusual amino acids (Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), and hydrocarbon stapling following olefin-containing unnatural amino acids (Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000) Walenski et al., Science 305: 1466-1470 (2004)), and ε- (3,5-dinitrobenzoyl) -Lys residues.

  In other embodiments, the peptides of the invention are derivatives of cupredoxins. This cupredoxin derivative is a chemical modification of the peptide such that the peptide still retains some of its basic activity. For example, a “derivative” of azurin can be a chemically modified azurin that interferes with ephrin signaling and retains the ability to specifically inhibit cancer growth in mammalian cells and tissues. Chemical modifications of interest include, but are not limited to, peptide amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation and glycosylation. In addition, a derivative peptide may be a chemical compound of a cupredoxin, or a variant, derivative or structural equivalent thereof (including but not limited to another peptide, drug molecule or other therapeutic or pharmaceutical substance or A fusion to a detectable probe). Derivatives of interest include chemical modifications that increase or optimize the half-life of the peptides and compositions of the present invention in the bloodstream, for example by several methods well known to those skilled in the art. Is a cyclized peptide (Monk et al., BioDrugs 19 (4): 261-78, (2005); DeFreest et al., J. Pept. Res. 63 (5): 409-19 (2004)) , N-terminal and C-terminal modifications (Labrie et al., Clin. Invest. Med. 13 (5): 275-8, (1990)), and hydrocarbon stapling following olefin-containing unnatural amino acids (Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al., Science 305: 1466-1470 (2004)).

  It is envisioned that the invention of the peptide may be a variant, derivative and / or structural equivalent of a cupredoxin. For example, the peptide may be a truncated form of azurin that is PEGylated (thus it is both a variant and a derivative). In one embodiment, the peptides of the present invention are synthesized using an alpha disubstituted unnatural amino acid comprising a tether containing an alpha olefin followed by an all hydrocarbon "staple" by ruthenium catalyzed olefin side cracking. (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 may be fused to other peptides, creating peptides that are both structural equivalents and derivatives. These examples are merely illustrative of the invention and do not limit the invention. The cupredoxin derivative or structural equivalent may or may not bind to copper.

In another embodiment, the peptide is a structural equivalent of a cupredoxin. Examples of studies that determine significant structural homology between cupredoxins and other proteins include Toth et al. (Developmental Cell 1: 82-92 (2001)). That is, significant homology between the cupredoxin and the structural equivalent is determined 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 P value of VAST from a structural comparison of cupredoxin to the structural equivalent is less than about 10 ^−3, less than about 10 ⁻⁵ , or less than about 10 ⁻⁷ . In other embodiments, significant structural homology between the cupredoxin and the structural equivalent is determined using the DALI algorithm (Holm & Sander, J. Mol. Biol. 233: 123-138 (1993)). Is done. In certain embodiments, the DALI Z score for pairwise structural comparisons is at least about 3.5, at least about 7.0, or at least about 10.0. Examples of these determinations can be found in Examples 2 and 9.

  In some embodiments, the cupredoxin, or variant, derivative or structural equivalent thereof has some of the functional characteristics of P. aeruginosa azurin, Ulva pertusa plastocyanin, Phormidium laminosum plastocyanin, or Thiobacillus ferrooxidans rusticyanin. . In certain embodiments, the cupredoxin, or a variant, derivative or structural equivalent thereof, inhibits ephrin signaling system interference and / or inhibits cancer growth in mammalian cells and tissues. Inhibition of mammalian cancer cell or tissue growth may or may not be related to any interference of the ephrin signaling system by cupredoxins, or variants, derivatives or structural equivalents thereof. Methods for determining whether a cupredoxin, or a variant, derivative or structural equivalent thereof interferes with an ephrin signaling system are well known in the art, and the cupredoxin, or a variant, derivative or structural equivalent thereof is ephrin Determining whether it binds to a component of the signal transduction pathway, including but not limited to ephrin and / or ephrin receptors. The ephrin signaling system, in its broadest sense, includes ephrin and related ephrin receptors, as well as any molecules (or “components”) required to transmit signals in the reverse and forward directions. To a lesser extent, the ephrin signaling system includes only the ephrin responsible for the signaling and the related ephrin receptors. The term “interference”, when used in the context of an ephrin signaling system, causes the associated ephrin signaling to either increase or decrease in “forward” and / or “reverse” directions. be able to. Methods for measuring interference in the ephrin signaling system are well known in the art. One method of determining ephrin signaling interference is peptide binding or competition with ephrin or ephrin receptors, or other components of the ephrin signaling system as shown in Examples 6-8. An in vivo system such as C. elegans can be used, where the cupredoxin now interferes with ephrin signaling to alter tail muscle formation and embryonic development as described in Example 1 It is known. Other methods include the Eph receptor in Himanen et al. (Nat. Neurosci. 7: 501-509 (2004)) and Koolpe et al. (J. Biol. Chem. 280: 17301-17311 (2005)). This includes but is not limited to phosphorylation assays.

  Now, cupredoxins, and variants of cupredoxins, are known to specifically interfere with ephrin signaling and inhibit cancer growth in mammalian cells and tissues, for example, variants, derivatives of cupredoxins that maintain this activity And structural equivalents can be designed. Such variants, derivatives and structural equivalents, for example, create a “library” of various variants, derivatives and structural equivalents, and then, for example, the exemplary methods in Examples 3, 9 and 10 Can be prepared by testing each for anti-cancer activity using one of many methods known in the art such as: The resulting variants, derivatives and structural equivalents of cupredoxins with anticancer activity can be used in place of or in addition to cupredoxins in the methods of the invention.

In other embodiments, the cupredoxin, or a variant, derivative or structural equivalent thereof, may have a significant structural ancestry with ephrin. In certain embodiments, cupredoxins, and variants and derivatives of cupredoxins, have significant structural homology around the GH loop region of ephrin. Examples of studies that determine significant structural homology between cupredoxins and ephrins include Toth et al. (Developmental Cell 1: 82-92 (2001)), and Example 2 herein. More specifically, significant structural homology between cupredoxins and ephrins is found in 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 P value of VAST derived from a structural comparison of cupredoxin to ephrin is less than about 10 ^−3, less than about 10 ⁻⁵ , or less than about 10 ⁻⁷ . In other embodiments, significant structural homology between cupredoxin and ephrin is determined using the DALI algorithm (Holm & Sander, J. Mol. Biol. 233: 123-138 (1993)). In certain embodiments, the DALI Z score for pairwise structural comparisons is at least about 3.5, at least about 7.0, or at least about 10.0.

  In another embodiment, the cupredoxin, or variant, derivative or structural equivalent thereof, binds to either ephrin and / or Eph receptor. It is now known that some cupredoxins have a C-terminal region that is structurally similar to the ephrin B2 ectodomain. See Examples 2, 5 and 9. In certain embodiments, the cupredoxin, or a variant, derivative or structural equivalent thereof, binds to ephrin. In particularly specific embodiments, the ephrins are, but are not limited to, ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3 and ephrin B4. In particularly specific embodiments, the ephrins are, but are not limited to, ephrin B1, ephrin B2, ephrin B3 and ephrin B4. In another specific embodiment, the cupredoxin, or a variant, derivative or structural equivalent thereof binds to the Eph receptor. In particularly specific embodiments, the Eph receptor is, but is not limited to, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4 and EphB6 . In some embodiments, the cupredoxin, or a variant, derivative or structural equivalent thereof, binds to ephrin and ephrin receptor. In some embodiments, the cupredoxin, or variant, derivative or structural equivalent thereof, binds to ephrins and receptors, particularly ephrin B2 and EphB2. Methods for determining the binding of proteins to other proteins are well known in the art. Examples of methods for determining binding to the Eph receptor include Examples 6-8 and Koolpe et al. (J. Biol. Chem. 280: 17301-17311 (2005)), and Himanen et al. (Nat. Neurosci. 7: 501-509 (2004)).

In some specific embodiments, the cupredoxin, or a variant, derivative or structural equivalent thereof, induces apoptosis in mammalian cancer cells, more particularly J774 cells. The ability of cupredoxins or other polypeptides to induce apoptosis was determined by ApoAlert confocal microscopy of mitotic sensors using the MITOSENSOR ^™ APOLERT ^™ mitochondrial membrane sensor kit (Clontech Laboratories, Inc., Palo Alto, California, USA); By measuring the activity of caspase 8, caspase 9 and caspase 3 using the method described in Zou et al. (J. Biol. Chem. 274: 11549-11556 (1999)); and, for example, APOLERT It may be observed by detecting apoptosis-induced nuclear DNA degradation using a ^TM DNA degradation kit (Clontech Laboratories, Inc., Palo Alto, California, USA).

  In another specific embodiment, the cupredoxin, or variant, derivative or structural equivalent thereof, induces cell growth arrest in mammalian cancer cells, more specifically J774 cells. Arrest of cell proliferation can be determined by measuring the extent of inhibition of cell cycle progression, for example by the method found in Yamada et al. (PNAS 101: 4770-4775 (2004)). In another specific embodiment, the cupredoxin, or variant, derivative or structural equivalent thereof, inhibits cell cycle progression in mammalian cancer cells, more particularly J774 cells.

<Cupredoxin>
These small blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or have unknown functions. Copper ions are bound by a protein matrix alone. The special distorted triangular planar configuration for two histidine ligands and one cysteine ligand around copper results in very strange electronic properties of the metal sites and a strong blue color. Many cupredoxins have been crystallographically characterized from medium to high resolution.

Cupredoxins generally have 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 auracyanin B sequence, 16.3% to the rusticyanin sequence, 20.3% to the plastocyanin sequence, and 17.3% to the pseudoazurin sequence. have. See Table 1. However, the structural similarity of these proteins is even more pronounced. P values VAST for structural comparison for azurin auracyanin B is 10 ^-7.4, the comparison value for rusticyanin of azurin is 10 ^-5, the comparison value for the plastocyanin azurin is 10 ^-5.6, the azurin The comparison value for ^Pseudoazurin is 10 ^-4.1 .

All cupredoxins have an eight-stranded Greek key beta barrel or beta sandwich fold and have a highly conserved site structure. (De Rienzo et al., Protein Science 9: 1439-1454 (2000)). Remarkably hydrophobic patches are due to many long-chain aliphatic residues such as methionine and leucine, azurin, amicyanine, cyanobacterial plastocyanin, cucumber basic protein, and to a lesser extent pseudoazurin, and It exists around copper in eukaryotic plastocyanin. Same source. Hydrophobic patches are also found to a lesser extent in the copper sites of stellacyanin and rusticyanin, but have different characteristics. Same source.

¹ Alignment length: the number of equivalent pairs of C-alpha atoms superimposed between the two structures, ie how many residues were used to calculate the 3D superposition.

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

³ score: VAST structural similarity score. This number is related to the number of secondary structural elements superimposed and the quality of the overlay. A higher VAST score is associated with higher similarity.

⁴ RMSD: The root mean square overlap residue in angstroms. This number is calculated as the square root of the root mean square distance between equivalent C-alpha atoms after optimal overlap of the two structures. It should be noted that the RMSD value is proportional to the range of structure alignment, and this size must be taken into account when using RMSD as a descriptor of overall structure similarity.

⁵ C. elegans major sperm protein has been proven to be an ephrin antagonist in oocyte maturation (Kuwabara, 2003 “The multifaceted C. elegans major sperm protein: an ephrin signaling antagonist in oocyte maturation” Genes and Development, 17 : 155-161).

<Azurin>
Azurin is a 123 amino acid copper-containing protein belonging to the cupredoxin family that is involved in electron transfer in certain bacteria. The azurin includes those derived from P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans. (Murphy et al., J. Mol. Biol. 315: 859-871 (2002)). Amino acid sequence identity between azurins varied between 60-90% and these proteins showed strong structural homology. All azurins have a characteristic β-sandwich with a Greek key motif and the copper atom is always located in the same region of the protein. In addition, azurins have an essentially neutral hydrophobic patch surrounding the copper site. Same source.

<Plastocyanin>
Plastocyanin is a soluble protein of cyanobacteria, algae, and plants, contains one molecule of copper per molecule, and is blue in its oxidized form. They exist in chloroplasts, where they function as electron carriers. Since the structure of polar plastocyanin was determined in 1978, the structure of plastocyanins in algae (Scenedesmus, Enteromorpha, Chlamydomonas) and plants (kidney beans) has been determined by crystallographic or NMR methods. Refined to resolution. SEQ ID NO: 2 shows the amino acid sequence of plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium. SEQ ID NO: 22 shows the amino acid sequence of plastocyanin from Ulva petrusa.

  Despite sequence diversity between algae and vascular plant plastocyanins (eg, 62% sequence identity between Chlamydomonas and poplar proteins), the three-dimensional structure is preserved (eg, Chlamydomonas and poplar (0.76 Årms deviation in C alpha position between proteins). Structural features include a twisted tetrahedral copper binding site at one end of an eight-stranded anti-equilibrium β-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site has been optimized for its electron transfer function, and it has been proposed that negative and hydrophobic patches are involved in the recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments confirmed the importance of the negative hydrophobic patch in the binding interaction with cytochrome f, and two functionally important electrons including plastocyanin A travel path model was demonstrated. One putative electron transfer path is relatively short (about 4cm) and contains the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is longer (about 12-15cm), in the negative patch. Contains the nearly conserved residue Tyr-83. (Redinbo et al., J. Bioenerg. Biomembr. 26: 49-66 (1994)).

<Rusticyanin>
Rusticyanin is a single-chain polypeptide containing blue copper obtained from Thiobacillus (now called Acidithiobacillus). A very stable and highly oxidative cupredoxin, Thiobacillus ferrooxidans-derived rusticyanin (SEQ ID NO: 3) was determined by multiple wavelength anomalous diffraction and was purified to a resolution of 1.9 Å. The rusticyanin is composed of a core beta sandwich fold composed of 6-strand and 7-strand β sheets. Like other cupredoxins, copper ions are coordinated by clusters of four conserved residues (His 85, Cys138, His143, Met148) arranged in a twisted tetrahedron. (Walter et al., J. Mol. Biol. 263: 730-51 (1996)).

<Pseudoazurin>
Pseudoazurins are a family of single-chain polypeptides that contain blue copper. The amino acid sequence of pseudoazurin obtained from Achromobacter cycloclastes is shown in SEQ ID NO: 4. X-ray structural analysis of pseudoazurin showed that it has a similar structure to azurin, but there is low sequence homology between these proteins. There are two major differences between the overall structure of pseudoazurin and azurin. Pseudoazurin has a carboxy terminal extension consisting of two alpha helices compared to azurin. In the intermediate peptide region, azurin contains an extended loop, which is shortened in pseudoazurin to form a flap containing a short alpha helix. The only major differences in the copper atom sites are the MET side chain conformation and the Met-S copper bond length, which is significantly shorter in pseudoazurin than in azurin.

<Phytocyanin>
Proteins that can be identified as phytocyanin include cucumber basic protein, stellacyanin, mavicyanin, umecyanin, cucumber exfoliated cupredoxin, putative blue copper protein in pea pods, and blue copper protein from Arabidopsis thaliana. It is not limited. In all but cucumber basic protein and pea pod protein, the axial methionine ligand normally found at the blue copper site is replaced with glutamine.

<Auracyanin>
Three small blue copper proteins termed auracyanin A, auracyanin B-1 and auracyanin B-2 were isolated from Chloroflexus aurantiacus, a thermophilic green gliding photosynthetic bacterium. The two types B are glycoproteins and have almost the same properties as each other, but are different from type A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the apparent monomer molecular weights were 14 kDa (A), 18 kDa (B-2) and 22 kDa (B-1).

  The amino acid sequence of auracyanin A was determined and shown to be a 139 residue polypeptide. (Van Dreissche et al ;. Protein Science 8: 947-957 (1999)). His58, Cys123, His128, and Met132 are spaced in the expected manner if they are evolutionarily conserved metal ligands such as in the known small copper proteins plastocyanin and azurin. Secondary structure predictions also indicate that auracyanin has a general beta-barrel structure similar to azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classifications. The overall similarity to the azurin consensus sequence is roughly the same as the plastocyanin consensus sequence, ie 30.5%. The auracyanin N-terminal sequence regions 1-18 are significantly rich in glycine and hydroxy amino acids. Same source. For the A chain of auracyanin from Chloroflexus aurantiacus (NCBI protein data bank access number No. AAM12874), see the exemplified amino acid sequence (SEQ ID NO: 15).

  The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of auracyanin B from Chloroflexus aurantiacus has been studied. (Bond et al., J. Mol. Biol. 306: 47-67 (2001); the contents of which are incorporated herein by reference for all purposes). Except for the additional N-terminal chain, the molecule is very similar to that of azurin, a bacterial cupredoxin. As in other cupredoxins, one of the Cu ligands is in polypeptide chain 4 and the other three are along a large loop between chains 7 and 8. The geometric positional relationship of the Cu site is described with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B probably has a cytoplasmic membrane with an N-terminal tail that shows significant sequence identity with known tethers in several other membrane-related electron transfer proteins. Moored on the peripheries side. The amino acid sequence of type B is presented in McManus et al. (J Biol Chem. 267: 6531-6540 (1992)). For the B chain of auracyanin (NCBI protein data bank access number No. 1QHQA) from Chloroflexus aurantiacus, see the exemplified amino acid sequence (SEQ ID NO: 16).

<Stellacyanin>
Stellacyanin is a subclass of phytocyanin, a universal family of plant cupredoxins. An exemplary sequence of stellacyanin is included herein as SEQ ID NO: 14. Umecyanin, stellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc. 127: 158-166 (2005)), and cuylesteracyanin (Hart el al., Protein Science 5: 2175-2183 (1996)) is also known. The protein has an overall fold similar to other phytocyanins. The tertiary structure of the ephrin B2 protein ectodomain has significant similarity to stellacyanin. (Toth et al., Developmental Cell 1: 83-92 (2001)). An exemplary amino acid sequence of stellacyanin can be found at Access Number 1JER (SEQ ID NO: 14) of the National Biotechnology Information Center Protein Data Bank.

<Cucumber basic protein>
An exemplary amino acid sequence derived from cucumber basic protein is included herein as SEQ ID NO: 17. The crystal structure of cucumber basic protein (CBP), a type 1 blue copper protein, has been purified with a resolution of 1.8Å. This molecule is similar to other blue copper proteins in that it has a Greek key beta barrel structure, but this barrel is better described as a “beta sandwich” or “beta taco” because it is open on one side. The (Guss et al., J. Mol. Biol. 262: 686-705 (1996)). The tertiary structure of this ephrin B2 protein ectodomain has a high similarity to the cucumber basic protein (1.5Å rms excursion for 50 α carbons). (Toth et al., Developmental Cell 1: 83-92 (2001)).

  Cu atom is normal with bond length Cu-N (His39) = 1.931.9, Cu-S (Cys79) = 2.16Å, Cu-N (His84) = 1.95Å, Cu-S (Met89) = 2.61Å It has a good blue copper NNSS 'coordination. The disulfide bond (Cys52) -S-S- (Cys85) appears to play an important role in stabilizing this molecular structure. The folding of the polypeptide is typical of the blue copper protein (phytocyanin) subfamily, as well as the non-metal binding protein, ragweed allergen Ra3, where CBP has a high degree of sequence identity. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, cucumber exfoliated cupredoxin, putative blue copper protein in pea pods, and blue copper protein from Arabidopsis thaliana. In all but CBP and pea pod protein, the axial methionine ligand normally found at the blue copper site is replaced by glutamine. An exemplary sequence of a cucumber basic protein is found in NCBI protein data bank access number 2CBP (SEQ ID NO: 17).

<Method of the present invention>
Another aspect of the invention relates to the use of one or more cupredoxins, and variants, derivatives and structural equivalents thereof in a method of treating a mammalian patient suffering from a pathological condition. More specifically, the state is related to the ephrin signaling system. In addition, the mammalian patient may suffer from cancer.

  In general, cupredoxins and their variants, derivatives and structural equivalents are currently known to interfere with the activity of the ephrin signaling system in cells or tissues. In addition, cupredoxins and their variants, derivatives and structural equivalents are currently known to inhibit the growth of cancer in cells and tissues. In a specific embodiment, the cell or tissue is a mammal. In a more specific embodiment, the cell is a human. In some embodiments, the cell is not human. In one embodiment, cupredoxins, and variants, derivatives and structural equivalents thereof are administered to cells to inhibit the activity of ephrin receptors on the cell surface. In another embodiment, cupredoxins and their variants, derivatives and structural equivalents are administered to increase the activity of ephrin receptors on the cell surface. In other specific embodiments, the cupredoxins, and variants and derivatives of cupredoxins, inhibit and / or increase the forward and / or backward of ephrin signaling. Furthermore, for example, it is possible to inhibit the forward signal while increasing the backward signal.

  The pathological condition affecting mammalian patients is specifically related to the activity of the ephrin signaling system. It is now also recognized that cupredoxins contain certain structural homology to ephrins and can act like ephrins in vivo with respect to the ephrin signaling system. It is believed that cupredoxins can be used to treat pathological conditions related to the ephrin signaling system. In a specific embodiment, the pathological condition is accompanied by higher or lower than normal concentrations of components of the ephrin signaling system. In other specific embodiments, the pathological condition is caused by overexpression or underexpression of a component of the ephrin signaling system. In other specific embodiments, the pathological condition results from excessive turnover or lack of turnover of components of the ephrin signaling system. In other specific embodiments, the pathological condition results in overexpression or underexpression of components of the ephrin signaling system. In another specific embodiment, the pathological condition results in excessive turnover or lack of turnover of components of the ephrin signaling system. The “component” of the ephrin signaling system includes any molecule that transmits or renders signals generated by ephrin binding to the ephrin receptor. In a more specific embodiment, the component is ephrin or ephrin receptor.

  In addition, pathological conditions can involve abnormal distribution within cells, between cells, or tissue-specific of components of the ephrin signaling system. In a specific embodiment, ephrin receptors are found at higher concentrations on the cell or cell surface. In more specific embodiments, ephrin receptors are upregulated or downregulated in tissue. In another more specific embodiment, ephrin is upregulated or downregulated in the tissue.

  In another aspect of the invention, the cupredoxin or a variant, derivative or structural equivalent thereof is administered to a patient with cancer. In some embodiments, the patient is a mammal, particularly a human. In other embodiments, the patient is not a human. Without limiting the mechanism of action of this treatment method, many cancers are associated with increased or decreased concentrations of components of the ephrin signaling system. Many ephrins and Eph receptors have been shown to be up-regulated or down-regulated in tumors, particularly in the more aggressive stages of tumor progression. For example, EphA2 is upregulated in breast cancer, liver cancer and prostate cancer, as well as glioblastoma, esophageal squamous cell carcinoma, ovarian cancer and melanoma. In a specific embodiment, the cancer is associated with upregulation of the EphA2 receptor. However, in many cancers, it is known that various components of the ephrin signaling system, in particular ephrin and Eph receptors, are up-regulated or down-regulated. Examples of aberrant expression of ephrin and ephrin receptors in various tumors are well known in the art and are described in Surawska et al. (Cytokine & Growth Factor Reviews 15: 419-433 (2004)). In other embodiments, the cancer is not associated with an abnormal amount or activity of a component of the ephrin signaling system. In a specific embodiment, the cancer is breast cancer, liver cancer, gastrointestinal cancer, neuroblastoma, neuronal cancer, leukemia, lymphoma, prostate cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, endometrial tumor, villi All, including but not limited to cancer, teratocarcinoma, thyroid cancer, sarcomas, including those arising from soft tissue and bone, renal cancer, squamous cell carcinoma, and non-small cell lung cancer. In a specific embodiment, the cancer is deficient in the expression of the p53 tumor suppressor gene.

  In other embodiments, the cancer has various properties associated with the stage of tumor growth. An early stage in tumor development is angiogenesis, where arteries are replenished to supply blood to the tumor. Although the working mechanism is not limited to any one means, the ephrin signaling system is known to be associated with angiogenesis. In one embodiment, the cancer is associated with angiogenesis. Another stage of tumor development is metastasis, defined as discontinuous tumor transplantation with the primary tumor, where the tumor forms a metastasis. Although the mechanism of action is not limited to any one means, metastasis is thought to be related to the ephrin signaling system. In one embodiment, the cancer is premetastatic. In another embodiment, the cancer is metastatic. Methods for measuring the effects of compounds on angiogenesis are well known in the art. Specific methods for measuring angiogenesis include, but are not limited to, the assay methods found in the following articles, the contents of which are incorporated by reference for all purposes: Daniel et al. , Kidney Int. Suppl. 57: S73-S81 (1996); Myers et al., J. Cell Biol. 148: 343-351 (2000); Pandey et al., Science 268: 567-569 (1995); Brantley et al., Oncogene 21: 7011-7026 (2002).

  In addition to tumors, there are many pathological conditions associated with the ephrin signaling pathway. For example, pathological forms of angiogenesis (Adams & Klein, Trends Cardiov. Medicine 10: 183-188 (2000); Brantley-Sieders & Chen, Angiogenesis 7: 17-28 (2004); Noren et al., Proc. Natl. Acad. Sci. USA 101: 5583-558 (2004)), chronic pain after tissue injury (Battaglia et al., Nat Neurosci. 6: 339-340 (2003)), nerve regeneration after spinal injury. Inhibition (Goldscmit et al., J. Neurosci. 6: 339-340 (2003)) and human congenital malformations (Twigg et al. Proc. Natl. Acad. Sci. USA 101: 8652-8657 (2004); Wieland et al., Am. J. Hum. Genet. 74: 1209-1215 (2004)), and in particular embodiments of the present invention, to treat these pathological conditions, cupredoxins or variants, derivatives or Use structural equivalents. In a specific embodiment, the pathological condition is interstitial cystitis (IC) inflammatory bowel disease (IBD) associated lesions, HIV infection, cardiovascular disease, central nervous system disorder, peripheral vascular disease, viral disease, Central nervous system degeneration (Christopher Reeve disease) and Alzheimer's disease. For a methodology related to the treatment of patients with interstitial cystitis and inflammatory bowel disease, see, for example, US Patent Application Publication No. 20050049176 (published March 3, 2005, which is incorporated herein by reference in its entirety) See incorporated by reference). For methodologies relating to the inhibition or stimulation of angiogenesis, see, for example, US Patent Application Publication No. 20040136983 (published July 15, 2004, the contents of which are incorporated by reference for all purposes). Please refer. For methodologies related to therapy for bone formation, see, for example, US Patent Application Publication No. 20040265808 (published July 15, 2004, the contents of which are incorporated by reference for all purposes). Please refer.

  A cupredoxin or variant, derivative or structural equivalent of a cupredoxin can be administered to a patient by a number of routes and in a number of regimens that would be well known to those skilled in the art. In a specific embodiment, the cupredoxin or variant or derivative of cupredoxin is administered intravenously, intramuscularly, subcutaneously, or by injection into a tumor. In a specific embodiment, the cupredoxin or a variant, derivative or structural equivalent of a cupredoxin is administered intravenously. In particularly specific embodiments, the cupredoxin or variant or derivative thereof is administered in chemotherapy to a patient who has cancer or who recovers from cancer.

  In addition to pathological conditions, cupredoxins or variants, derivatives or structural equivalents of cupredoxins can be used in treatment methods related to other conditions in which the patient is affected. Such a condition may be the result of an accident that damages the nerve or vasculature, recovery from other therapies such as surgery, especially a condition related to old age. In one embodiment, the patient is in need of blood vessel growth or regrowth and the cupredoxin or a variant, derivative or structural equivalent of the cupredoxin is administered to direct blood vessel growth. In another embodiment, the patient requires a decrease in blood vessel growth and the cupredoxin or variant, derivative or structural equivalent of the cupredoxin is administered to inhibit blood vessel growth. In another embodiment, a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin is administered to a patient in need of neuronal development or regeneration to direct neuronal growth. In another embodiment, a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin is administered to a patient to promote bone formation.

  Another aspect of the invention is a method for detecting cells that exhibit specific ephrin receptors in vivo. It is now known that cupredoxins contain regions of high structural homology to ephrin B2 and other ephrins, and that cupredoxins can specifically bind to ephrin receptors in vitro. Thus, cupredoxins will localize to the surface of cells expressing ephrin receptors. Thus, in some embodiments, cupredoxins or variants, derivatives or structural equivalents of cupredoxins are among the cells and tissues that express non-Eph receptors, and the location of cells or tissues that express these Eph receptors. Can be used to determine. In addition, a cupredoxin or variant, derivative or structural equivalent of a particular type of Eph receptor binding preference positions a cell or tissue that specifically expresses that type of Eph receptor. Can be used for. In one embodiment, a cupredoxin or variant or derivative thereof is linked to a detectable probe and administered to a human patient, and the localization of the detectable probe is measured in the patient to express an Eph receptor. The position is determined. In particularly specific embodiments, the cell is a cancer cell. The detectable probe may be many currently known in the art. Probes of particular interest include, but are not limited to, fluorescent probes, radioactive probes, and iodine, gadolinium and gold.

  In another embodiment of the invention, a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin or linked to a drug is administered to a human patient for delivery of the drug to a cell or tissue that expresses an Eph receptor. To do. In another specific embodiment, the tissue is a tumor. In particularly specific embodiments, the cell is a cancer cell. The drug may be any type of chemical that has an effect on cells that express the Eph receptor. The drug may be an organic or inorganic compound, including but not limited to peptides, DNA molecules, RNA molecules, pharmaceutical compositions and derivatives of any of these. In a specific embodiment, the drug is a toxin such as Pseudomonas exotoxin A domain III or a chemical such as taxol or other drug that kills cancer cells.

  A cupredoxin or a variant, derivative or structural equivalent of a cupredoxin is a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin or cupredoxin operably linked to a promoter region expressed in a desired human patient cell or tissue. It can be administered to cells or human patients by administering DNA containing the coding region that it encodes. In a specific embodiment, the DNA encoding a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin is used to treat a condition that is amenable to treatment with the cupredoxin or a variant, derivative or structural equivalent of a cupredoxin. Administered to patients. Suitable vectors and methods for administering them to cells and human patients are well known in the art. Methodologies for expressing foreign proteins in human subjects are well known in the art and can be adapted to express cupredoxins or variants, derivatives or structural equivalents of cupredoxins in patients. Exemplary protocols are found in the following US patents, among other sources: US Patent No. 6,339,068 issued on January 15, 2002; US Patent No. issued on March 15, 2005 US Patent No. 6,821,957 issued on November 23, 2004; US Patent No. 6,821,955 issued on November 23, 2004; US Patent No. 6,562,376 issued on May 13, 2003 All of these contents are hereby incorporated by reference for all purposes. In a more specific embodiment, the DNA encoding cupredoxin or a variant, derivative or structural equivalent of cupredoxin is injected into the tumor of a patient suffering from cancer. In a more specific embodiment, the DNA encoding cupredoxin or a variant, derivative or structural equivalent of cupredoxin is injected into the tumor of a patient suffering from cancer.

  In some embodiments, the pharmaceutical composition is administered to a patient by intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository or oral, and particularly intravenous injection. The pharmaceutical composition may be administered to the patient concurrently with administration of another drug known to treat a particular pathological condition associated with ephrin signaling or cancer, or within 1 minute to 1 week or more. The pharmaceutical composition may be administered at about the same time as another anticancer agent.

Pharmaceutical compositions comprising cupredoxins and variants, derivatives and structural equivalents of cupredoxins A pharmaceutical composition comprising at least one cupredoxin or a variant, derivative or structural equivalent of a cupredoxin may be used in any conventional manner, for example conventional It can be produced by a mixing method, a dissolution method, a granulation method, a draze preparation method, an emulsification method, an encapsulation method, an encapsulation method or a freeze-drying method. Substantially pure cupredoxins or variants, derivatives or structural equivalents of cupredoxins can be readily combined with pharmaceutically acceptable carriers well known in the art. With such a carrier, the formulation can be formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. Suitable excipients can also include, for example, fillers and cellulose preparations. Other excipients can include, for example, flavors, colorants, non-tackifiers, thickeners and other acceptable additives, adjuvants or binders. General methodologies for pharmaceutical dosage forms are found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore MD (1999)).

  A composition comprising a cupredoxin or a variant, derivative or structural equivalent thereof used in the present invention can be administered by injection (eg, intradermal, subcutaneous, intramuscular, intraperitoneal, etc.) or by topical inhalation. It may be administered in a variety of ways, including by administration, by suppository, by using a transdermal patch, or by mouth. General information about drug delivery systems can be found in Ansel et al., Ibid. In some embodiments, the compositions comprising cupredoxins or variants, derivatives or structural equivalents thereof are formulated and used directly as injectibles, especially for subcutaneous and intravenous injection. be able to. Also, a composition comprising cupredoxin or a variant, derivative or structural equivalent thereof can be taken orally after mixing with a protective agent such as polypropylene glycol or similar coating agent.

  When administration is by injection, the cupredoxin or a variant, derivative or structural equivalent thereof is formulated in an aqueous solution, particularly in a physiologically compatible buffer such as Hanks's solution, Ringer's solution or saline buffer. May be used. The solution may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the cupredoxin or variant, derivative or structural equivalent thereof may be in powder form for constitution with a suitable medium, such as sterile pyrogen-free water, prior to use. In some embodiments, the pharmaceutical composition does not include an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition comprises a substance that inhibits an immune response against the peptide.

*リンゲル乳酸塩は、28mmol/lの乳酸塩、4mmol/lのK⁺および3mmol/lのCa²⁺も有する。 When administration is by intravenous fluid replacement, the intravenous fluid replacement for use in administering cupredoxin or a variant, derivative or structural equivalent thereof may be composed of crystalloids or colloids. As used herein, crystalloid is an aqueous solution of a mineral salt or other water-soluble molecule. As used herein, colloids include larger insoluble molecules such as gelatin. Intravenous fluid replacement may be sterile. Crystalloid fluids that may be used for intravenous administration include normal saline (0.9% strength sodium chloride solution), Ringer's lactate or Ringer's solution, as described in Table 2 Including, but not limited to, 5% dextrose in water, sometimes referred to as D5W.

* Ringer lactate also has 28 mmol / l lactate, 4 mmol / l K ⁺ and 3 mmol / l Ca ²⁺ .

  When administration is by inhalation, the cupredoxin or variant, derivative or structural equivalent thereof is pressurized with an appropriate propellant such as dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas. Delivery may be in the form of an aerosol spray from a pack or nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for metering. For example, gelatin capsules and cartridges for use in inhalers or ventilators may be formulated with a powder mixture of protein and a suitable powder base such as lactose or starch.

  When administration is by local administration, the cupredoxin or variant, derivative or structural equivalent thereof is formulated as a solution, gel, ointment, cream, jelly, suspension, etc., as is well known in the art. May be used. In some embodiments, administration is by a transdermal patch. When administration is by suppository (eg, rectal or vaginal), the composition of cupredoxin or variants, derivatives and structural equivalents thereof may be formulated into a composition containing a conventional suppository base. Good.

  When administered orally, the cupredoxin or a variant, derivative or structural equivalent thereof is obtained by combining the cupredoxin or a variant, derivative or structural equivalent thereof with a pharmaceutically acceptable carrier of the known art. Can be easily formulated. Solid carriers such as mannitol, lactose, magnesium stearate, etc. may be used; with such carriers, cupredoxins and their variants, derivatives and structural equivalents for oral ingestion by subjects to be treated The product can be formulated as a tablet, pill, draze, capsule, liquid, gel, syrup, slurry, suspension, etc. For example, for oral solid formulations such as powders, capsules and tablets, suitable excipients include fillers such as sugars, cellulose preparations, granulating agents, binders and the like.

  Other convenient carriers also include multivalent carriers such as bacterial capsular polysaccharides, dextrans or genetically engineered vectors, as is well known in the art. In addition, sustained release formulations containing cupredoxins or their variants, derivatives or structural equivalents allow the release of cupredoxins or their variants, derivatives or structural equivalents over an extended period of time Without a formulation, cupredoxins or their mutant derivatives or structural equivalents are cleared from the subject's system before eliciting or enhancing a therapeutic effect and / or, for example, proteases and simple hydrolysis Will be broken down by.

  The half-life in the bloodstream of the composition of the invention is determined by peptide cyclization (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 containing unusual amino acids (Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), N- and C-terminal modifications (Labrie et al., Clin. Invest. Med. 13 (5): 275-8, ( 1990)) and hydrocarbon stapling (Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al., Science 305: 1466-1470 (2004)) It can be extended or optimized by several methods well known to those skilled in the art without limitation: Modification of peptide stability via d-isomerization (substitution) and D-substitution or L-amino acid substitution is of particular interest Lean.

  In various embodiments, the pharmaceutical composition comprises carriers and excipients (buffers such as glycine, antioxidants, bacteriostatic agents, chelating agents, suspending agents, pastes and / or preservatives, carbohydrates, mannitol, proteins Including, but not limited to, polypeptides or amino acids), water, oil, saline, aqueous dextrose, and buffers, tonicity adjusting agents, wetting agents, etc., glycerin solutions, near physiological conditions Contains other pharmaceutically acceptable auxiliary substances as required. Although any suitable carrier known to those of skill in the art may be used to administer the composition of the invention, it will be appreciated that the type of carrier will vary depending on the mode of administration. The compound may also be encapsulated in liposomes using well known techniques. Biodegradable microparticles may also be used as a carrier for the pharmaceutical composition of the present invention. Suitable biodegradable microparticles are disclosed, for example, in US Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

  The pharmaceutical composition may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solution may be packaged for use as is, or lyophilized and the lyophilized preparation combined with a sterile solution prior to administration.

Administration of cupredoxin The cupredoxin or a variant, derivative or structural equivalent thereof can be administered as a pharmaceutical composition and can be administered by any suitable route, eg, oral, buccal Can be administered by sublingual, rectal, vaginal, urethral, nasal, topical, dermal, i.e., transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary) administration it can. These pharmaceutical formulations can be administered in any amount effective to achieve their intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In a specific embodiment, the therapeutically effective amount is generally about 0.01-20 mg / day / kg of body weight.

  Compounds including cupredoxins or variants, derivatives or structural equivalents thereof for treating conditions or cancers associated with ephrin-signaling in mammalian cells and tissues, alone or in combination with other active agents Useful for. Of course, the appropriate dosage will vary depending on, for example, the cupredoxin or variant, derivative or structural equivalent compound used, host, mode of administration, and the nature and severity of the condition being treated. However, in general, satisfactory results in humans are indicated to be obtained at daily dosages of about 0.01-20 mg / kg body weight. Daily doses shown in humans are cupredoxins in the range of about 0.7 mg to about 1400 mg, or a variant, derivative or structural equivalent compound thereof, conveniently for example daily doses, weekly doses. Administered in monthly doses and / or in continuous dosing. The daily dose can be 1 to 12 separate doses per day. Alternatively, the dose can be administered every other day, every 3 days, every 4 days, every 5 days, every 6 days, every week, and in increments of 1 day up to 31 days or more It is the same as above. Alternatively, the medication can be a continuous patch, i.v. administration, etc.

  Methods of introducing cupredoxins and / or variants, derivatives or structural equivalents thereof into a patient, in some embodiments, treat a particular pathological condition or cancer or other condition or disease associated with ephrin signaling For simultaneous administration with other known agents. Such methods are well known in the art. In a specific embodiment, the cupredoxin and / or variants, derivatives or structural equivalents thereof comprise or are part of a cocktail together with other pathological conditions or cancers associated with ephrin signaling Or co-medication. Drugs of interest include those used to treat inflammatory bowel disease, HIV infection, viral disease, cardiovascular disease, peripheral vascular disease, central nervous system disorders, central nervous system degeneration and Alzheimer's disease Including.

  Drugs for treating inflammatory bowel disease include sulfasalazine (azulfidine (registered trademark), oxalazine (Dipentum (registered trademark)), mesalamine (Asacol (registered trademark), pentasa (registered trademark)) And amino salicylates such as balsalazide (Corazal®); (adrenal corticosteroids such as prednisone, medrol®, methylprednisolone, hydrocortisone, budesonide (Encort EC); azathioprine (Imlan®) , Immunomodulators such as 6-mercaptopurine (6-MP, Prinasol®) and cyclosporin A (Sandimune®, Neoral®); metronidazole (Flagill®) and Profloxacin (Cipro) TM)) antibiotics such as; infliximab (Remicade (R)) biotherapies like; including various therapies such as and tacrolimus (FK506) and mycophenolate mofetil, without limitation.

  Drugs for treating HIV infection include reverse transcriptase inhibitors: AXT (zidovudine [Retrovir®]), ddC (zarcitabine [Hivid®] dideoxyinosine), d4T (stavudine) [Zelit (R)]) and 3TC (Lamivudine [Epivir (R)]), non-nucleoside reverse transcriptase inhibitors (NNRTIS)): Delavirdine (Rescripter (R)) and Nevirapine (VilaMun (R)) ) (Protease inhibitor): Ritonavir (Norvir (registered trademark)), lopinavir and ritonavir combination (Kaletra (registered trademark)), saquinavir (inbilase (registered trademark)), indinavir sulfate (crixiban (registered trademark)), amprenavir (Agenerase®) and Nelfinavir, (Biracept®) Muga, but it is not limited. Drugs for treating viral diseases include acyclovir, varicella-zoster immunoglobulin (VZIG®), peginterferon (ribavirin), acyclovir (zobilax®), valacyclovir (valtrex®) fam Cyclovir (Famvir®) amantadine, rimantadine, zanamivir, oseltamivir and alpha interferon include but are not limited to.

  Drugs for treating cardiovascular disease disorders include blood coagulation inhibitors, antiplatelet agents, platelet lytic agents, adrenergic blockers, adrenergic stimulants, α / β-adrenergic blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin converting enzyme (ACE) inhibitors with calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors with diuretics, angiotensin II receptor antagonists, calcium channel blockers, diuretics (carbonic anhydrase) Inhibitors, loop diuretics, potassium-sparing diuretics, thiazide and related diuretics, vasodilators, blood pressure raising factors, etc.).

  Drugs for treating peripheral vascular disease include pentoxifylline (Trental®), oral methylxanthine derivatives and cilostazol, (Pretal®) phosphodiesterase III inhibitors; antiplatelet / antithrombotic such as aspirin Anticoagulants such as heparin and warfarin® (coumadin®); treatment of cholesterolemia such as niacin, statins, fibrate, lopid® tablets) (gemfibrozil; Parke-Davis) Drugs: Tricor tablets (fenofibrate; Abbott), bile acid sequestering agents, Colestid® tablets (fine particulate colestipol hydrochloride; Pharmacia and Upjohn); Welchol® tablets ( Colesevelam hydrochloride; Sankyo); calcium channel Vitamin and dietary supplements such as folate, B-6, B-12, L-arginine and omega-3 fatty acids; and HMG- such as Adobecol® tablets, (niacin / lovastatin; Kos) COA reductase inhibitors; Artocol® sustained-release tablets (lovastatin; Andryx labs); Rescol® capsules (fluvastatin sodium; Novartis &Reliant); Lipitol® tablets (atorvastatin; Parke-Davis and Mebacor® tablets (lovastatin; Merck); Prabacol® tablets (pravastatin sodium; Bristol-Myers Squibb), Pravigard® PAC tablets (buffered aspirin and pravastatin sodium; Bristol- Myers Squibb); Zokol® tablets (simvastatin; Merck); Nicotinic acid drugs such as niacin tablets (niacin / lovastatin; Kos (also listed as an HMG-COA reductase inhibitor)); niaspan® (niacin; Kos); and zetia® tablets (ezetimibe) Including, but not limited to, various drugs such as Merck / Schering Plow).

  Drugs for treating central nervous system disorders include psychotherapeutic drugs such as various benzodiazepine preparations and combinations, anxiolytics, antidepressants (monoamine oxidase inhibitors (MAO inhibitors), selective serotonin reuptake inhibitors) (Including SSRI), tricyclic antidepressants), antidepressants, anti-panic agents, psychotics, stimulants and obsessive compulsive disorders; migraine headaches such as beta-adrenergic inhibitor, isometheptene and serotonin receptor agonists Formulations and various migraine preparations including the active ingredients of Depacote® (divalproex sodium; Abbott) and Exedrine® migraine tablets (acetaminophen; BMS Products); sedatives and hypnotics; Including but not limited to anticonvulsants; as well as pimozide®. Drugs for treating Parkinson's disease include, but are not limited to, anticholinergics, catechol-o-methyltransferase inhibitors, dopamine drugs and monoamine oxidase (MAO) inhibitors.

  Drugs for treating CNS degenerative disorders include: Drugs for treating multiple sclerosis include Avonex® (interferon β-1a; Biogen Neurology) active ingredient; for SC injections Betacelon® (modified form of interferon β-1b; Berlex); Copaxone® for injection (glatilamarate acetate; Teva Neuroscience); Depot Medrol® injection suspension (methylprednisolone acetate; Pharmacia &Upjohn); Novantrone® for injection concentrate (mitoxantrone supplied as mitoxantrone hydrochloride; Serono); Orapred® oral solution (prednisolone sodium phosphate oral solution; Ascent); And Rebif® injection (interferon β-1a; Pfizer & Serono). Drugs for treating Huntington's disease include clonazepam (tranolizers such as Clonopin®; haloperidol (Haldol®) and clozapine (antipsychotics such as Clozaril®); fluoxetine (Prozac ( (Registered trademark), salafem (registered trademark), sertraline (Zoloft (registered trademark)), nortriptyline (Aventyl (registered trademark), pamelol (registered trademark)) and lithium (Escaris® (registered trademark), Lithobid) (Registered trademark)), but is not limited.

  Drugs for treating Alzheimer's disease include: Aricept® tablets (donepezil hydrochloride; Eisai or Pfizer); Exelon® capsules (rivastigmine (as hydrogen tartrate); Novartis); Exelon® Oral solutions (rivastigmine tartrate; Novartis); including, but not limited to, Reminil® oral solution (galantamine hydrobromide; Janssen) or Reminyl® tablets (galantamine hydrobromide; Janssen).

  The method of introducing a compound comprising a cupredoxin or a variant, derivative or structural equivalent thereof into a patient is, in some embodiments, co-administration of other agents known to treat cancer. Such methods are well known in the art. In a specific embodiment, the compound comprising cupredoxin or a variant, derivative or structural equivalent thereof includes or is part of a cocktail or co-medication with other agents for treating cancer. Such drugs include, for example, those listed herein, specifically 5-fluorouracil; interferon alpha; methotrexate; tamoxifen; and vincristine. The above examples are provided for illustration only, and many other such compounds are known to those skilled in the art.

  Other agents suitable for treating cancer include alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethyleneimines and triazenes; antimetabolites such as folic acid antagonists, purine analogs and pyrimidine analogs; Antibiotics such as anthracycline, bleomycin, mitomycin, dactinomycin and pricamycin; enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; inhibitors of 5α-reductase; inhibitors of 17β-hydroxysteroid dehydrogenase type 3; Hormonal agents such as glucocorticoids, estrogens / antiestrogens, androgens / antiandrogens, progestins and luteinizing hormone-releasing hormone antagonists, octreotide acetate; Or microtubule disruptors such as analogs and derivatives thereof; taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®) and analogs thereof, and epothilones such as epothilone AF and analogs thereof Microtubule-stabilizers such as; plant-derived products such as vinca alkaloids, epipodophyllotoxins, taxanes; and topoisomerase inhibitors; prenyl-protein transferase inhibitors; and hydroxyurea, procarbazine, mitotane, hexamethyl Various drugs such as platinum coordination compounds such as melamine, cisplatin and carboplatin; and other drugs used as anti-cancer and cytotoxic agents such as biological response modifiers, growth factors; immunomodulating components and monoclonal antibodies Including, but not limited to. The compounds of the present invention may also be used in combination with radiation therapy and surgery.

  Representatives of these classes of anticancer and cytotoxic agents include mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustine, lomustine, semustine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, Mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracin, saframycin, quinocarcins, discodermolide, vincristine, vinblastine liquor, vinblastine liquor Vinorelbine, etoposide, etoposide phosphate, teniposide, paclitaxel, tamoxifen, Stramstin, sodium estramustine phosphate, flutamide, buserelin, leuprolide, pteridine, diyneses, levamisole, aflacon, interferon, interleukin, aldesleukin, filgrastim, sargramostim, rituximab, hydrochloric acid Including but not limited to irinotecan, betamethosone, gemcitabine hydrochloride, altretamine and topoteca and any analogs or derivatives thereof.

  Preferred members of these classes include paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ectenacidin 743 or pofiromycin, 5-fluorouracil, 6-mercaptopurine , Gemcitabine, cytosine arabinoside, podophyllotoxin or etoposide, podophyllotoxin derivatives such as etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine and leucine.

  Examples of other anticancer and cytotoxic agents useful for co-administration with the compositions of the present invention include: German Patent No. 4138042.8; WO 97/19086, WO 98/22461 International Publication No.98 / 25929, International Publication No.98 / 38192, International Publication No.99 / 01124, International Publication No.99 / 02224, International Publication No.99 / 02514, International Publication No.99 / 03848, International Publication No. Publication No. 99/07692, International Publication No.99 / 27890, International Publication No.99 / 28324, International Publication No.99 / 43653, International Publication No.99 / 54330, International Publication No.99 / 54318, International Publication No. Epothilone derivatives as found in 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; WO 99/24416 (Also see US Pat. No. 6,040,321); cyclin-dependent kinase inhibitors; and WO 97/30992 and Prenyl-protein transferase inhibitors as found in International Publication No. 98/54966; and agents such as those generally and specifically described in US Pat. No. 6,011,029 (compounds of the US patent include AR modulators, Can be used with any NHR modulator (including but not limited to the present invention) such as an ER modulator, with an LHRH modulator, or with surgical techniques, particularly in the treatment of cancer.

  The exact formulation, route of administration and dosage will be determined by the attending physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide plasma levels of the active cupredoxins or variants, derivatives or structural equivalents thereof which are sufficient to maintain therapeutic effect. In general, the desired cupredoxin or variant, derivative or structural equivalent thereof is administered in a mixture with a drug carrier selected in view of 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 such that the polypeptide is produced in situ. In one embodiment, the DNA is `` naked '' as described, for example, in Ulmer et al., (Science 259: 1745-1749 (1993)) and outlined by Cohen (Science 259: 1691-1692 (1993)). It is. Naked DNA uptake may be increased by coating the DNA on a carrier, such as a biodegradable bead, which is then efficiently transported into the cell. In such methods, the DNA may be present in any of a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial, and viral expression systems. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. See, for example, WO 90/11092, WO 93/24640, WO 93/17706, and US Pat. No. 5,736,524.

  Vectors used to transfer genetic material between organisms can be divided into two general classifications: Cloning vectors have regions that are essential for transmission within the appropriate host cell. A replicating plasmid or phage into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector. Expression vectors (such as plasmids, yeast or animal viral genomes) are used to introduce foreign genetic material into host cells or tissues to transcribe and translate foreign DNA such as cupredoxin DNA . In an expression vector, the introduced DNA is operably linked to an element such as a promoter that highly transcribes the inserted DNA by transmitting a signal to the host cell. Several other promoters are also useful, such as inducible promoters that control gene transcription in response to specific factors. Expression of cupredoxin and / or cytochrome c and their variants and derivatives in response to specific factors by operably linking the cupredoxins and their variants, derivatives or structural equivalent polynucleotides to inducible promoters Can be controlled. Examples of classical inducible promoters include those that respond to steroids such as α-interferon, heat shock, heavy metal ions and glucocorticoids, and tetracycline. Other desirable inducible promoters include those that are not endogenous to the cells into which the construct is introduced, but are responsive in these cells when the inducer is supplied exogenously. In general, useful expression vectors are often plasmids. However, other forms of expression vectors are also envisioned, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

  Vector selection is required by the organism or cell used and the fate of the desired vector. In general, a vector includes a signal sequence, an origin of replication, a marker gene, a polylinker site, an enhancer element, a promoter, and a transcription termination sequence.

Kits comprising cupredoxins or variants, derivatives or structural equivalents thereof In one aspect, the invention provides a kit comprising one or more of the following in a package or container: (1) at least one cupredoxin Or a biologically active composition comprising these variants, derivatives or structural equivalents; (2) a biologically active composition comprising a co-administered drug; (3) pharmaceutically acceptable. (4) a medium for administration such as a syringe; (5) instructions for administration. Also contemplated are embodiments in which two or more of components (1)-(5) are found in the same packaging or container. Co-administered drugs may be selected from those previously mentioned.

  When the kit is supplied, different components of the components may be packaged in separate containers and mixed immediately before use. Such packaging of the components may be stored separately for long periods without losing the function of the active ingredient.

  The reagents included in the kit can be supplied in any type of container where the lifetime of the different components is maintained and is not adsorbed or altered by the material of the container. For example, a sealed glass ampoule may contain lyophilized cupredoxins and their variants, derivatives or structural equivalents, or buffers packed under a neutral non-reactive gas such as nitrogen. Good. The ampoule is made of any suitable material such as organic polymers such as glass, polycarbonate, polystyrene, etc., ceramics, metals or any other material typically used to hold similar reagents. It may be. Other examples of suitable containers include a simple bottle that would be manufactured from a similar material as an ampoule and an envelope that would contain a lined interior such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes or others. The container may have a sterile access port such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by a removable membrane from which the components can be easily mixed by removal. The removable film may be glass, plastic, rubber, or the like.

  Kits may also be supplied with instructional materials. The instructions may be printed on paper or other substrate and / or floppy disk, CD-ROM, DVD-ROM, zip disk, video tape, recording tape, flash memory device, etc. It may be supplied as an electronically readable medium. The detailed instructions may not be physically attached to the kit; instead, the user may be identified by the kit manufacturer or distributor, or provided on the Internet via e-mail You may teach a website.

Modification of cupredoxins and their variants, derivatives or structural equivalents Cupredoxins or their variants, derivatives or their structural equivalents are chemically modified to produce variants and derivatives as described above. Or may be genetically modified. Such variants and derivatives may be synthesized by standard techniques.

  In addition to naturally occurring allelic variants of cupredoxins, the amino acid sequence of the encoded cupredoxin is altered without significantly changing the ability of the cupredoxin to interfere with ephrin signaling or inhibit cancer growth. Changes can be introduced by mutations within the predoxin coding sequence. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a cupredoxin without altering the biological activity, whereas an “essential” amino acid residue is Needed. For example, amino acid residues conserved among cupredoxins are not particularly amenable to change and are therefore expected to be “essential”.

（1）β-シートまたはαらせん高次構造などのポリペプチドバックボーンの構造、（2）電荷、（3）疎水性または（4）標的部位の側鎖のかさに影響を及ぼす非保存的置換は、細胞障害性因子機能を修飾することができる。残基は、表4に示したように、共通の側鎖特性に基づいてグループに分けられる。非保存的置換では、これらのクラスの1つのメンバーを別のクラスに交換することを必要とする。置換は、保存的置換部位に、またはより具体的には非保存的部位に導入されてもよい。

変種ポリペプチドは、オリゴヌクレオチドを媒介した（部位特異的）突然変異誘発、アラニン走査およびPCR突然変異誘発などの当該技術分野において公知の方法を使用して作製することができる。部位特異的変異誘発、カセット突然変異誘発（制限選択突然変異誘発））またはその他の公知の技術をクローン化DNAに対して行って、キュプレドキシン変異体DNAを産生することができる。 Amino acids for which conservative substitutions can be made that do not alter the 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 type of amino acid is replaced with another amino acid of the same form falls within the scope of the invention, as long as the substitution does not specifically alter the biological activity of the compound.

Non-conservative substitutions that affect the structure of the polypeptide backbone, such as (1) β-sheet or α-helical conformation, (2) charge, (3) hydrophobicity, or (4) the side chain size of the target site Can modify the cytotoxic factor function. Residues are grouped based on common side chain properties as shown in Table 4. Non-conservative substitutions require exchanging one member of these classes for another class. Substitutions may be introduced at conservative substitution sites, or more specifically at non-conservative sites.

Variant polypeptides can be made using methods known in the art, such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis, cassette mutagenesis (restriction selective mutagenesis)) or other known techniques can be performed on the cloned DNA to produce cupredoxin mutant DNA.

  Also, known mutations of cupredoxins can be used to create mutant cupredoxins used in the methods of the invention. For example, the C112D and M44KM64E mutants of Pseudomonas aeruginosa azurin are known to have different cytotoxic and growth-inhibitory activities than natural azurin, and such altered activity is It may be useful in the inventive therapeutic methods. One embodiment of the method of the present invention utilizes cupredoxins and their variants and derivatives that retain the ability to prevent ephrin signaling or inhibit cancer growth in mammalian cells. In another embodiment, the methods of the invention utilize a cupredoxin variant such as the M44KM64E mutant that has the ability to cause cell growth arrest.

  A more complete understanding of the present invention can be obtained by reference to the following specific examples. The examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in shape and replacement of equivalents are envisaged as suggested or convenient by the circumstances. Although specific terms are used herein, such terms are intended to be descriptive and not limiting. Modifications of the invention as set forth above may be made without departing from the spirit and scope thereof, and therefore only limitations as set forth by the appended claims should be imposed. It is.

Example 1: In vivo study of C. elefans development In Caenorhabditis elegans, several ephrins are known to be involved in tail muscle formation and embryogenesis. Our preliminary experiments show that rusticyanin inhibits tail muscle formation (causing tail paralysis), while azurin inhibits offspring birth (embryogenesis). In these experiments, the azurin and rusticyanin genes were overexpressed in E. coli. Control E. coli containing no azurin or rusticyanin genes was maintained. When nematodes were given control E. coli for up to 3 days, they were healthy and gave birth to offspring. When nematodes were given E. coli expressing azurin, they looked healthy, but produced few offspring. When the nematode is given E. coli expressing rusticyanin, only about 30% can move its head or upper part of the body, but the tail or lower part of the body cannot move and shows paralysis It was.

Example 2: Analysis of structurally similar azurin neighbors Analysis of protein structural neighbors involves directly comparing 3D protein structures in a molecular modeling database using the Vector Alignment Search Tool (VAST) algorithm. (Gibrat et al., Curr Opin Struct Biol 6: 377-385 (1996);. Madej et al., Proteins 23: 356-3690 (1995)). The molecular modeling database includes crystallographic and NMR structure determinations and is maintained by the National Center for Biotechnology Information (Bethesda, MD, USA). A program for performing VAST protein structural neighbor analysis is available through the National Center for Biotechnology Information.

  Azurin is significant (Vast P value: 10 e -4.6; alignment length: 90 aa;% identity) using MMDB (Molecular Modeling Database) entry 1 KGY E (Crystal structure of EphB2-Ephrin B complex) : 6) (Fig. 1) showing structural superposition. The superposition alignment of azurin using the crystal structure is related to proteins called chain E (138aa) and ephrin (ephrin-B2). Computational analysis follows the structural data published in Himanen et al. (Nature, vol 414: 933-938 (2001)).

  Further VAST analysis shows that the cupredoxins rusticyanin, auracyanin, plastocyanin, cucumber basic protein, and stellacyanin have significant structural homology to ephrin B2 in the GH loop region (Figure 2) .

Example 3: Efficacy of synthetic peptides derived from azurin and plastocyanin The efficacy of synthetic peptides derived from azurin and plastocyanin, which are structurally similar to the human ephrin B-2 GH loop, was analyzed. An 18-mer azurin peptide having the following sequence was synthesized by standard techniques:
TFDVSKLKEGEQYMFFCT SEQ ID NO: 18.

  MCF-7 breast cancer cells were incubated with 5 and 50 μg / ml 18-mer azurin peptide in 16-well plates for 0, 24, 48, and 72 hours. The peptide at 50 μg / ml was found to inhibit MCF-7 cell growth by 50% between 48 and 72 hours compared to cells not treated with synthetic peptide. The degree of cell growth inhibition was about 25% at 5 μg / ml 18-mer synthetic peptide compared to untreated control. This experiment shows that a synthetic peptide constructed solely based on structural homology to B-2 ephrin actually inhibits the development of cancer cells promoted by B-2 ephrin.

Example 4: In vitro measurement of the effect of cupredoxin on the proliferation of Mel-2 and MCF-7 cells The proliferation of cells treated with cupredoxin was measured using 16-well plates. Mel-2 or MCF-7 cells (5 × 10 ⁵ cells per well) were allowed to attach to multiwell (16 wells in this example) plates for 24 hours. After attachment, the medium was aspirated. PBS (phosphate buffered saline) or various cupredoxins / cytochromes were added to wells containing fresh media at concentrations of 0.1-10 μM in PBS and cancer cell growth was observed for 24, 48, and 72 hours. After incubation, trypan blue was added to the medium and the number of dead and floating cells was counted. Both live and dead floating cells were counted to determine the IC50 at various cupredoxin doses. IC50 is the concentration of protein that inhibits cell culture growth by 50%.

  In a 24-hour culture of 500,000 cells per well, there were sufficient cells for a reproducible count. In the cupredoxin (−) control cell culture, as the cells grew, the space to attach to the bottom of the wells decreased, began to die and became floating cells. In plastocyanin-treated or rusticyanin-treated cell cultures, the cells overproliferated on the surface of the wells and began to die and float, but the number was less than the controls. However, in cell cultures treated with azurin, cultures of the Mel-2 and MCF-7 cell lines showed little development of floating cells.

Example 5: Structural similarity between ephrin B2 and cupredoxin Structural similarity between ephrin B2 ectodomain and cupredoxin was determined using the VAST and DALI algorithms (Holm and Sander, J. Mol. Biol. 233: 123-138 (1993); Gibrat et al., Curr. Opin. Biol. 6 ”377-385 (1996)), available through the US National Institutes of Health and European Bioinformatics research, respectively. Structure-based pairwise sequence alignments were calculated using the VAST algorithm, and protein structure diagrams are made in two dimensions using TOPS (Protein Structure Topology) comics (Torrance et al., Bioinformatics 21: 2537-2538 (2005)). Structural evaluation was performed using the program Mol Mol (Koradi et al., J. Mol. Graphics 14: 51-55 (1996)).

Some homology was found using the two programs (data not shown) and includes a small subset of plastocyanin, azurin, and rusticyanin, monomeric cupredoxin proteins. In particular, the structural comparison between the ephrin B2 ectodomain and these three cupredoxins (selected as representative proteins of the cupredoxin family) provides a significant and substantial similarity score with VAST and DALI alignments, They ranged from 11.0 to 9.7 (outside the maximum possible 15.7) and 6.7 to 6.4, respectively. A DALI Z score <2.0 is structurally dissimilar (Table 5). The most striking structural conservation exists between the ephrin B2 ectodomain and the plastocyanin, and was overlaid using a mean square deviation (rmsd) of 1.8 angstroms (all 67 structurally equivalent Cα atoms (Table 5). In contrast, azurin, plastocyanin, and rusticyanin show low primary sequence identity (less than 10%) to the ephrin B2 ectodomain (Table 5).

  a-VAST score-VAST structural similarity score; this number relates to the number of secondary structural elements superimposed and the quality of the overlay. A higher VAST score correlates with a higher similarity.

  b-DALI Z score-The Z score is calculated using a pair-wise comparison of the ephrin ectodomain with other structures in the DALI database. The higher the Z score, the less likely the similarity between 3D structures is random (Z <2.0 pairs are structurally dissimilar).

  Mean squared deviation of backbone residues in angstroms between aligned pairs of c-RMSD-structure pairs.

  FIG. 3 shows TOPS cartoon (A) and MolMol photograph (B) of ephrin B2 ectodomain. The morphological description showed that the protein took the form of a sandwich of two β-sheets derived from the core of the Greek key fold structure and had significant structural similarity in terms of the number and orientation of β-strands 1KGY_E (ephrin B2), 1IUZ (plastocyanin), and 1JZG_A (azurin) (FIG. 3). In comparison, the conservation of α-helix number and configuration is much less. In JZGA, the helical structure is characteristic compared to the other proteins shown here. As expected for proteins with different sizes, the loops connecting the elements of the secondary structure showed differences in length and conformation. 1RCY (lasticyanin) exhibits the lowest structural homology, has substantial differences in the length of shared secondary structural elements (SSEs), and there are non-shared SSEs.

Example 6: Analysis of Eph-Fc receptor-cupredoxin interaction by surface plasmon resonance Specific interaction between cupredoxin and Eph receptor was measured by surface plasmon resonance (SPR) analysis. Human astrocytoma CCF-STTG1, glioblastoma LN 229, and MCF-7 (breast cancer) cells, 2 mM L-glutamine, 10 mM hepes, 10% (vol / vol) heat inactivated FBS, 100 units cultured in a humidified incubator with 5% CO ₂ as described above in RPMI medium 1640 containing 1 / ml penicillin and 100 μg / ml streptomycin (Yamada et al., Cell Microbiol. 7: 1418- 1431 (2005); Hiraoka et al., Proc. Natl. Acad. Sci. USA 101: 6427-6432 (2004)). UISO-Mel-2 human melanoma cells (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002)) were developed in MEM with Hank's medium supplemented with 10% FBS. Incubated. E. coli JM109 and BL21 (DE3) were used as host strains for azurin, rusticyanin, and plastocyanin. Azurin and rusticyanin were purified as described above (Yamada et al., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Yamada et al., Cell Cycle 3: 1182-1187 (2004 )). The construction and purification of GST-azurin fusion derivatives has been reported previously (Yamada et al., Cell Microbiol. 7: 1418-1431 (2005)). Purification and expression of plastocyanin from Phormidium laminosum is essentially the same as before except that E. coli strain BL21 (DE3) Cd + (RIL) was used instead of BL21 (DE3). It was carried out as described. The concentration of fully oxidized protein was measured spectrophotometrically at 598 nm using an extinction coefficient of 4700 M ⁻¹ cm ⁻¹ .

  Eph ectodomain Fc fusion protein and ephrin were purchased as lyophilized powders from R & D Systems, Minneapolis, MN. All other chemicals used for surface plasmon resonance and proliferation experiments were purchased from Biacore AB International or Sigma and were of high analytical grade. Direct protein-protein interaction between cupredoxin or GST-peptide and Eph-Fc or ephrin B2-Fc is measured using Biacore X biosensor system (Biacore AB) based on surface plasmon resonance (SPR) technology It was done.

  In initial screening experiments, immobilization of azurin, rusticyanin, or plastocyanin to a single channel on a CM5 sensor chip was achieved using an amine coupling method. N-hydroxysuccinimide / N- (3-dimethylaminopropyl) -N-ethylcarbodiimide (0.05M / 0.2M, 35μL), cupredoxin protein (255μM, 50μL), 1M ethanolamine (50μL, pH 8.8), and Injection over time of 100 mM NaOH (10 μL) covalently bound the protein to the CM5 sensor chip with an increase in the 300 RU resonance signal. The binding test consists of HBS-EP running with intermediate administration of 100 mM NaOH (10 μL pulse) to regenerate the cupredoxin-CM5 surface on the sensor surface for 2.3 minutes (70 μl injection) at a flow rate of 5 and 30 μ1 / min. This was done by sequential administration of 100 nM Eph-Fc protein in buffer (0.01 M Hepes, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20). All binding studies were performed on the bare Au CM5 sensor surface (negative channel) to correct for nonspecific binding.

  Binding screening was performed on a cupredoxin-modified sensor by sequential injection of 100 nM Eph-Fc receptor at a flow rate of 30 μL / min for 2.3 minutes. The curve signifies the initiation of the association phase for the interaction of Eph-Fc with azurin, plastocyanin, and rusticyanin. Relative binding affinity is expressed as a function of saturation resonance (Req) and varies from 79 RU for the binding of rusticyanin to EphB1-Fc or EphA8-Fc to 1248 RU for the binding of azurin to EphB2-Fc did. The cross-selective binding of cupredoxin to specific EphA and EphB receptor proteins is noteworthy with the best interaction occurring between cupredoxin and EphB.

  SPR sensorgrams for the binding of immobilized cupredoxin to Eph-Fc showed selective recognition between the two subsets of these proteins. (Figure 4A-C). In these measurements, the Eph-Fc concentration is kept constant at 100 nM, and the difference in the degree of association represented by Req indicates a difference in affinity between the Eph-Fc receptor protein and the cupredoxin. Azurin showed the highest affinity for EphB2-Fc and A6 and also tightly bound to A4 and A7 (FIG. 4A). On the other hand, plastocyanin showed selectivity for EphA1-Fc, A3, and B2, and to a lesser extent A2 and A6 (FIG. 4B). Finally, rusticyanin recognized EphA8-Fc and weakly recognized B1 (FIG. 4C). The association interaction between azurin and EphB2-Fc and EphA6-Fc was the highest, with Req values of 1248 and 1200 RU, respectively.

Example 7: Analysis of binding affinity of azurin and GST-Azu using EphB2-Fc-SPR binding assay
The binding affinity of azurin and GST-azu peptides using the EphB2-Fc-SPR binding assay is fixed to assess the relative binding affinity of full-length azurin or its various domains to the EphB2-Fc receptor. EphB2-Fc was used for azurin or GST-azurin. Azurin or GST-Azu peptide was injected at increasing concentrations (0.05-100 nM) onto EphB2-Fc or Ephrin B2-Fc immobilized CM5 chips using a 100 mM NaOH pulse during injection. The data was fitted to the Langmuir (1: 1) binding model [Req = Rmax / (1 + Kd / C)] and extrapolated to determine the equilibrium binding constant (Kd).

The experimental strategy to elucidate the structural determinants of the binding of azurin to EphB2-Fc is shown in FIG. 5 and, as mentioned earlier, the primary / secondary structure of the azurin and GST-Azu constructs A diagram of the parameters is shown (Yamada et al., Cell Microbiol. 7: 1418-1431 (2005)). In particular, GST-Azu 88-113 perfectly matched the GH loop region (natural ligand) of ephrin B2, and therefore its binding power to EphB2-Fc was particularly interesting. Initial binding measurements of azurin and various GST-Azu constructs (all 100 nM) to EphB2-Fc revealed their relative binding affinity: azurin> GST-Azu 88-113> GST- Azu 36-128> Ephrin B2-Fc> GST-Azu 36-89 >> GST (FIG. 6A). Azurin and GST-labeled azurin showed comparable affinity (data not shown). To quantify the binding affinity, saturation reaction values (Req) for azurin or GST-Azu were measured as a function of their concentration (0-100 nM) (FIG. 6B). The binding data fit the simple Langmuir (1: 1) binding equation and determined the equilibrium dissociation constant (Kd). In particular, azurin (Kd = 6 nM) and GST-Azu 88-113 (Kd = 12 nM) are 5 and 2.5 times greater than EphB2-Fc, respectively, than the natural ephrin B2-Fc ligand (Kd = 30 nM). High affinity was shown. GST-Azu 36-128 (Table 6) showed slightly higher affinity (Kd = 23 nM) than ephrin B2-Fc (Kd = 30 nM). In contrast, GST-Azu 36-89 and GST had negligible affinity (Table 6). These data show the significance of the azurin Azu 88-113 region for high affinity EphB2-Fc interactions. The Azu 88-113 region consists of a GH loop domain that is structurally homologous to the GH loop found in ephrin B2.

The relative binding length of azurin and GST-Azu constructs to EphB2-Fc was measured by SPR binding titration test, extrapolated data set was aggregated, and natural ephrin B2-Fc ligand and GST binding Compared with gender. Data from the initial screening experiment is generated by injection of 100 nM analyte and the saturation signal (R _eq ) is tabulated in resonance units. Saturation in binding was achieved at each titration point in the binding curve shown in FIG. 6b, and these values plotted against [analyte] were used to calculate the equilibrium dissociation constant (K _d ). . While K _d values for the interaction of azurin and GST-Azu ranged from 6 to 61 nM, EphB2-Fc with the natural ligand had an intermediate binding affinity within this range.

Example 8: Analysis of competitive binding studies of azurin / GST-Azu and ephrin B2-Fc with EphB2-Fc To better understand the physiological effects on high affinity azurin-EphB2-Fc binding, we Further binding measurements were made. Competitive binding titration is a binding constant test except that ephrin B2-Fc (246 nM) + competitor [azurin or GST-Azu (0-1020 nM)] is injected onto the EphB2-Fc CM5 sensor surface. The same was done.

  The azurin + ephrin B2-Fc sample was added to the sensor surface at different azurin concentrations (0 to 020 nM, [Ephrin B2-Fc] was 246 nM), the data show the percentage of resonance,% total response [Req (azurin + ephrin B2-Fc) / (Req / (Ephrin B2-Fc))]. GST-Azu 88-113 and GST-Azu 36-89 were titrated to EphB2-Fc immobilized with ephrin B2-Fc and analyzed in a similar manner. Competition data shows a 1: 1 stoichiometry of binding between azurin and GST-Azu 88-113 and immobilized EphB2-Fc.

  We first measured the binding of azurin and GST-Azu constructs to the EphB2-Fc ligand, ephrin B2-Fc. FIG. 7A shows that azurin actually binds ephrin B2-Fc with high affinity (Kd = 8.5 + 0.8 nM), indicating the structural similarity between these two proteins (Table 5). The relatively high binding of GST-Azu 88-113 to ephrin B2-Fc (Kd = 39 + 6.5 nM) indicates that the region between 88 and 113 is responsible for the binding to ephrin B2-Fc. Show. In contrast, GST and GST-Azu 36-89 showed no significant interaction with ephrin B2-Fc. Taken together, these data indicate that azurin can bind with high affinity to both EphB2-Fc and ephrin B2-Fc via the GH loop (88-113) region.

  In order to further test this view, we determined that ephrin B2-Fc against EphB2-Fc immobilized on a CM5 sensor chip after ephrin B2-Fc was incubated with various concentrations of azurin and GST-Azu constructs. An SPR analysis of the binding was performed. FIG. 7B shows the binding of ephrin B2-Fc ([Ephrin B2-Fc] = 246 nM) to EphB2-Fc by 0 to 120 nM azurin, GST-Azu 88-113, and GST-Azu 36-89, respectively. Inhibition is indicated. This inhibition is expressed as% total response (= [R eq (azurin + Ephrin B2-Fc) / Req (Ephrin B2-Fc alone)] * 100). This inhibition profile shows that when ephrin B2-Fc is incubated with azurin or GST-Azu 88-113, total protein binding to immobilized EphB2-Fc is reduced, It is a more potent inhibitor than 88-113. Preincubation of azurin or GST-Azu 88-113 with ephrin B2-Fc reduces the total protein bound to the surface by 60%. On the other hand, GST-Azu 36-89 does not affect the binding of all proteins (FIG. 7B), which is consistent with weak binding to ephrin B2-Fc or EphB2-Fc. Because maximal inhibition is achieved at a 1: 1 ratio of ephrin B2-Fc to azurin (or GST-Azu 88-113), azurin (or GST-Azu 88-113) is stoichiometric with ephrin B2-Fc. It is thought that a complex is formed. The fact that inhibition by azurin or GST-Azu 88-113 is uniform at 40-50% indicates that the putative azurin-ephrin B2-Fc complex has some affinity for the EphB2-Fc receptor Indicates. Collectively, these binding data indicate that azurin has a high affinity for both ephrin B2-Fc and EphB2-Fc, and ephrin B2-Fc due to the dual mechanism of ligand sequestration and receptor occupancy. -Shows that EphB2-Fc binding can be inhibited.

Example 9: Analysis of cytotoxic activity of Azu 96-113 and Plc70-84 synthetic peptides and GST-Azu fusion derivatives against various cancer cell lines Based on sequence alignment based on the structure of azurin and plastocyanin and human ephrin B2 ectodomain We constructed peptides corresponding to the GH loop region of ephrin B2 (Azu 96-113 (SEQ ID NO: 18) and Plc 70-84 (SEQ ID NO: 20)), the main region of which is the ephrin Eph receptor Mediates high binding to (Himanen et al., Nature 414: 933-938 (2001); Toth et al., Dev. Cell. 1: 83-92 (2001)). In FIG. 8, there is a structural overlap of the C-terminal segments of ephrin B2 ectodomain, azurin, and plastocyanin. Sequence alignment based on the structure of azurin and plastocyanin and human ephrin B2 ectodomain was used to construct a region-based peptide that mediates high affinity binding of ephrin B2 to the EphB2 receptor (ephrin B2-Fc G strand-loop-H strand of ectodomain), Azu 96-113 (96-TFDVSKLKEGEQYMFFCT-113) for azurin and Plc 70-84 (70-VRKLSTPGVYGVYCE-84) for plastocyanin (Fig. 8). The peptide was purchased from GenScript Corporation (Piscataway, NJ) with 99% purity. They were purified by reverse phase high pressure liquid chromatography and their identity was verified by mass spectrometry. Peptides were dissolved in phosphate buffered saline (PBS) (1 ×) and aliquots stored at −20 ° C. until use.

  To see whether such azurin domains serve as antagonists for Eph signaling in cancer progression, we quantify a number of cancer cell lines commonly known as overexpressed EphB receptors / ephrin ligands MTT analysis was performed. During a 24 hour incubation at 37 ° C., 75 μM azurin and plastocyanin GH loop peptide showed induction of cell death in brain tumor astrocytoma CCF-STTG1 and glioblastoma LN-229 (FIG. 9A) . The plastocyanin peptide Plc 70-84 showed somewhat higher cytotoxic activity than the azurin peptide Azu 96-113. To determine whether such cytotoxicity is dependent on and effective for other cancer cells, we have several concentrations of these peptides in melanoma (Figure 9B) or glioblastoma cells ( The impact on Fig. 9C) was evaluated. In both cases, increasing peptide concentration led to increased cytotoxicity (FIGS. 9B and 9C).

Example 10: Analysis of the ability of GST-Azu fusion protein to cause cell death in breast cancer MCF-7 cells We tested the ability of GST-Azu fusion protein to cause cell death in breast cancer MCF-7 cells. 3- (4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) (MTT) (Sigma) analysis (Mosmann, J) for the measurement of cytotoxicity of azurin and plastocyanin synthetic proteins Immunol. Methods 65: 55-63 (1983)). Approximately 2 × 10 ⁴ cells / well were seeded in a 96-well culture plate containing 100 μl RPMI 1640 medium. After overnight culture, the supernatant was removed and fresh medium containing azurin or plastocyanin synthetic protein at various specific concentrations was added to the attached cells. After 24 or 48 hours of treatment, 10 μl of 5 mg / ml MTT solution was added to the medium and incubated at 37 ° C. for 1 hour. The MTT reaction was terminated by the addition of 40 mM HCl in isopropanol. The MTT formazan formed was measured spectrophotometrically as previously described (Mosmann, 1983). Untreated control cells were compared to treated cells to determine viability and thereby make a cytotoxicity measurement.

  Similar to the control (no protein treatment), there was little cytotoxicity triggered by GST or GST-Azu 36-89 (FIG. 10). However, GST-Azu 36-128 or GST-Azu 88-113, which has a region of azurin capable of inhibiting ephrin B2 / EphB2 binding, showed significant cytotoxicity in a dose-dependent manner, resulting in MCF-7 cell death. The role of Azu 88-113 in triggering was confirmed.

Example 11: Treatment of a patient suffering from a condition related to ephrin signaling A phase I / II clinical trial of a cupredoxin composition (study drug) was conducted in a patient suffering from cancer. In particular, the cupredoxin compound is Plc 70-84 (SEQ ID NO: 20).

  Histologically breast and colon cancer, showing progression as clinical and radiographs, or recurrence after receiving appropriate treatment with currently useful FDA-approved chemotherapeutic agents and measures, and Forty-nine adult patients identified as melanoma are enrolled in an open-label prospective study that administers study drug. To be eligible for enrollment in the trial, all patients show an increase in measurable tumor volume after completing the approved course of chemotherapy. Evidence for persistent metastatic deposits and / or continued increase in size and volume must be established histologically. This histological evidence can be obtained by fine needle aspiration biopsy.

  Treatment planning begins after the institutional review board at the University of Chicago, Illinois and the FDA, and informed consent from all patients. Patients may have other diseases, such as other malignancies, a history of malignancy, blood disorders, insulin-dependent diabetes or other serious cardiovascular diseases that interfere with the appropriate assessment of the effectiveness of the proposed treatment. I don't have it. Baseline blood tests (whole blood count [CBC] and serum chemistry), including a liver function test (LFT), were performed before treatment began. All eligible patients should not receive any cancer chemotherapy at the same time for the duration of the trial.

  The study drug is administered for 12 weeks by daily intravenous injection of a pharmaceutically acceptable formulation of the study drug, and the subject is observed for any dose that limits toxicity. There are seven dose levels starting with 10 mg / kg / day and increasing by 5 mg / kg / day, up to a dose of 50 mg / kg / day. The effect of nuclear dose levels is recorded in 7 patients with advanced measurable cancer (breast cancer, colon cancer, and melanoma).

  Response is assessed by measuring tumors that can be measured in two dimensions (a and b). 1) Complete disappearance of the target metastatic tumor is considered a complete response (CR); 2) 75% reduction is considered a very good partial response (PR), 3) Good Response (PR) is a 50% decrease in size after treatment, 4) a 25% decrease in size is considered a stable disease (SD), and 5) less than 25% are unresponsive Possible (NR). Patients with disease progression will discontinue such treatment but will be followed for an additional 12 weeks.

  The complete disappearance and some decrease in the size of the target metastatic tumor indicates that azurin treatment is effective for cancer treatment. Other indications that Plc 70-84 treatment is effective are to reduce the rate of development of new metastatic tumors and to reduce angiogenesis associated with the tumors.

FIG. 1 depicts the structural alignment of azurin and ephrin B2. The G-H loop of ephrin B2, the region that mediates high affinity interactions with the EphB receptor, is indicated by a box. 1JZG_A is the amino acid sequence of azurin derived from Pseudomonas aeruginosa (SEQ ID NO: 1). 1KGY_E is the amino acid sequence of the human-derived ephrin B2 ectodomain (SEQ ID NO: 23). FIG. 2 depicts the structural alignment of cupredoxin and ephrin B2. The box indicates the GH loop (15 aa) of ephrin B2 involved in Eph receptor binding. Conserved residues are shown in bold and underlined. Capital letters indicate overlapping positions. A dash is a place where there is no alignment. The ephrin B2 G-H loop region is SEQ ID NO: 24. The P. aeruginosa azurin region similar to the GH loop region of ephrin B2 is SEQ ID NO: 25. A Thiobacillus (Acidithiobacillus) ferrooxidans rusticyanin region similar to the GH loop region of ephrin B2 is SEQ ID NO: 26. A Chloroflexus aurantiacus auracyanin region similar to the GH loop region of ephrin B2 is SEQ ID NO: 27. A Phormidium laminosum plastocyanin region similar to the GH loop region of ephrin B2 is SEQ ID NO: 28. A Cucumis sativus cucumber basic protein similar to the GH loop region of ephrin B2 is SEQ ID NO: 29. The Cucumis sativus stellacyanin region similar to the GH loop region of ephrin B2 is SEQ ID NO: 30. Figure 3 shows the structure of human ephrin B2 ectodomain (1kgy_E), plastocyanin from Ulva pertusa (1iuz), azurin from Pseudomonas aeruginosa (1jzg_A), and rusticyanin from Thiobacillus (Acidithiobacillus) ferrooxidans (1rcy) The comparison of is drawn. FIG. 3 shows the topology of each protein using TOPS comics. The TOPS cartoon represents the structure as an array of secondary structure elements (SSEs): β-chains (drawn as triangles) and spirals (α and 310) (represented by circles), Are linked in a sequence from the amino terminus to the carboxy terminus, and their relative spatial position and orientation. The orientation of these elements can be inferred from the connecting line. The “upward” chain is indicated by an upward-pointing triangle, and the “downward” chain is indicated by a downward-pointing triangle. The picture in FIG. 3B was drawn using the MolMlo program (Koradi et al., J. Mol. Graphics 14: 51-55 (1996)). FIG. 4 depicts a surface plasmon resonance sensor diagram for the binding of cupredoxin and conjugated Eph-Fc. The selective binding of azurin (FIG. 4A), plastocyanin (FIG. 4B), and rusticyanin (FIG. 4C) to EphA-Fc and EphB-Fc proteins is represented. FIG. 5 represents a schematic representation of various truncated azurin constructs derived from full-length azurin (SEQ ID NO: 1). Secondary structural elements are indicated by arrows for β sheets and rectangles for spirals (α and 310). Various sections of the 128 amino acid azurin-encoding gene were fused in-frame to the 3′-end of the gst gene (encoding glutamine S transferase), cloned into E. coli, overexpressed, The fusion protein is purified as described in (Yamada et al., Cell Micro. 7: 1418-1431 (2005)). FIG. 6 depicts the relative binding affinity of azurin and a selectively constructed GST-Azu fusion for EphB2-Fc as determined by surface plasmon resonance studies. In FIG. 6A, an initial screening experiment was performed to determine the relative binding strength of azurin or GST-Azu, where a cupredoxin (100 nM) on an EphB2-Fc-modified CM5 sensor chip. SPR and race were recorded after injection. In particular, azurin, GST-Azu 88-113, and GST-Azu 36-128 bind more strongly than the natural ligand for EphB2-Fc (ephrin B2-Fc). FIG. 6B shows azurin, GST-Azu 88-113, ephrin B2-Fc, and GST-Azu 36-89 against immobilized EphB2-Fc after titration with increasing cupredoxin concentration (0.05-100 nM). The binding affinity curves for the interactions are shown. To construct these curves, the balanced resonance signal (Req) was extrapolated from the individual sensor diagrams. The bond dissociation constant (Kd) was calculated after fitting the data to the Langmuir (1: 1) binding model using the formula Req = Rmax / (1 + Kd / C) (Table 6) and also the titration curve A curve fit connecting the data points at is shown. The quantitative data set is consistent with that from the initial binding screen. FIG. 7 depicts the binding interaction of azurin and GST-Azu with ephrin B2-Fc and EphB2-Fc as determined in binding titration and competition studies. FIG. 7A shows an SPR binding curve for the interaction of azurin and GST-Azu with ephrin B2-Fc, whose binding affinity was determined as described above. FIG. 7B shows the results of an SPR binding antagonism experiment using EphB2-Fc immobilized on a CM5 sensor chip. FIG. 8 shows plastocyanin from Ulva pertusa (1IUZ) and azurin from P. aeruginosa (1JZG_A) (FIGS. 8A and 8B, respectively) and human ephrin B2 exterior when computerized by the VAST algorithm. Draws a structural alignment comprising the domain (1KGY_E). Overlaid secondary structure elements are indicated by bold capital letters. Dash and lower-case lettering are places where there is no alignment. Secondary structural elements following this structure are shown as arrows on the β sheet and white rectangles on the 310 helix. Identical amino acids are indicated with an asterisk. Amino acids highlighted in dark gray and light gray in the ephrin B2 sequence indicate the interaction between the ephrin B2 and EphB2 receptors and the residues involved in ligand dimerization, respectively (Himanen et al., Nature 414: 933-938 (2001); Toth et al., Dev. Cell. 1: 83-92 (2001)). Plastocyanin and azurin peptides corresponding to the GH loop region of ephrin B2 (Plc 70-84 (SEQ ID NO: 20) and Azu 96-113 (referred to as SEQ ID NO: 18), respectively, show high-affinity binding of ephrin to the Eph receptor. The major region to mediate, boxed and presented under each alignment, G and H loop regions are marked with a thick arrow at the top of the ephrin B2 amino acid sequence. 138 and 68-138 are found in SEQ ID NOs: 31 and 33, respectively. 1 IUZ residues 57-98 are found in SEQ ID NO: 32. 1JZG_A residues 76-128 are found in SEQ ID NO: 34. FIG. 9 depicts the effect of a cupredoxin peptide on cancer cell survival. FIG. 9A shows the effect of azurin (Azu 96-113) and plastocyanin (Plc 70-84) synthetic peptides on the survival of astrocytoma CCF-STTG1 and glioblastoma LN-229 cancer cell lines. Yes. FIG. 9B shows the effect of different concentrations of plastocyanin (Plc 70-84) synthetic peptide on the survival of melanoma UISO-Mel-2 cells. Cell viability was determined by the MTT assay described in Example 10. Cancer cells (2 × 10 ⁴ cells per well in 96 wells) were treated with different concentrations of synthetic peptides at 37 ° C. for 24 hours. Data are presented as a percentage of cell survival as compared to untreated control (100% survival). FIG. 9C shows the cytotoxic activity of the glial Azu 96-113 synthetic peptide against blastoma. Cytotoxic effects were determined by MTT assay. Cancers (2 × 10 ⁴ cells per well in 96 wells) were treated with various concentrations of Azu 96-113 (10, 25, 50, 75, 100 μM) at 37 ° C. for 24 hours. Data are presented as a percentage of cell survival as compared to untreated control (100% survival). Percent cytotoxicity is expressed as a percentage of cell death compared to the untreated control (0% cytotoxicity). Effect of GST-Azu 36-128 and GST-Azu 88-113 on cell viability of MCF-7 cells. GST-Azu peptide was added in increasing concentrations (1.25, 6.25 and 12.5 μM) to 96 well plates containing 8 × 10 ³ cancer cells per well and incubated at 37 ° C. for 48 hours, followed by MTT assay Was used for analysis. As a control, GST and GST-Azu 36-89 cells at the same concentration and untreated were also tested in parallel with GST-Azu 36-128 and GST-Azu 88-113.

Claims (50)

  An isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin and that can inhibit the growth of mammalian cancer cells.   2. The isolated peptide of claim 1, wherein the cupredoxin is selected from the group consisting of azurin, plastocyanin, pseudoazurin, plastocyanin, rusticyanin and aurecyanin.   3. An isolated peptide according to claim 2, wherein the cupredoxin is selected from the group consisting of azurin, plastocyanin and rusticyanin.   2. The isolated peptide of claim 1, wherein the cupredoxin comprises Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum, Alcaligenes fecalis. faecalis), Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. (Methylomonas sp.), Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis yl, fast moss -Isolated from a microorganism selected from the group consisting of Sachibus (Cucumis sativus), Chloroflexus aurantiacus, Vibrio parahaemolyticus and Ulva pertusa Peptide.   5. The isolated peptide of claim 4, wherein the peptide is derived from a microorganism selected from the group consisting of Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum and Ulva pertusa.   2. The isolated peptide of claim 1, wherein the isolated peptide is part of a peptide selected from the group consisting of SEQ ID NOs: 1-17 and 22-23.   2. The isolated peptide of claim 1, wherein the peptide selected from the group consisting of SEQ ID NOs: 1-17 and 22-23 has at least about 90% amino acid sequence identity to the peptide. Isolated peptide.   2. The isolated peptide of claim 1, wherein the isolated peptide is a truncated form of a cupredoxin.   2. The isolated peptide of claim 1, wherein the peptide is about 10 residues or more and about 100 residues or less.   10. The isolated peptide of claim 9, wherein the peptide comprises P. aeruginosa azurin residues 96-113, P. aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulva. An isolated peptide comprising a region of cupredoxin selected from the group consisting of pertusa residues 57-98 and SEQ ID NOs: 22-30.   11. The isolated peptide of claim 10, wherein the peptide comprises P. aeruginosa azurin residues 96-113, P. aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulva. An isolated peptide consisting of a region of cupredoxin selected from the group consisting of pertusa residues 57-98 and SEQ ID NOs: 22-30.   2. The isolated peptide of claim 1, wherein the peptide comprises P. aeruginosa azurin residues 96-113, P. aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulva. A peptide comprising an equivalent residue of the subject cupredoxin as a region of the target cupredoxin selected from the group consisting of pertusa residues 57 to 98 and SEQ ID NOs: 22 to 30.   A composition comprising at least one cupredoxin or the peptide of claim 1 in a pharmaceutical composition.   14. A composition according to claim 13, wherein the pharmaceutical composition is formulated for intravenous administration.   14.The composition according to claim 13, wherein the cupredoxin is Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitisese A composition that is derived from a microorganism selected from the group consisting of Xylella fastidiosa, Cucumis sativus, Chloroflexus aurantiacus, Vibrio parahaemolyticus and Ulva pertusa.   16. The composition of claim 15, wherein the cupredoxin is derived from a microorganism selected from the group consisting of Pseudomonas aeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum and Ulva pertusa.   The composition according to claim 13, wherein the cupredoxin is selected from the group consisting of SEQ ID NOs: 1-17 and 22-23.   14. A method for treating a mammalian patient suffering from a pathological condition associated with the ephrin signaling system or suffering from cancer, wherein the composition comprises a therapeutically effective amount of said patient. Administering a product.   19. The method of claim 18, wherein the patient is interstitial cystitis (IC), a lesion associated with inflammatory bowel disease (IBD), HIV infection, cardiovascular disease, central nervous system disease (IBD). A method of suffering from a pathological disorder selected from the group consisting of peripheral vascular disease, viral disease, central nervous system degeneration (Christopher Reeve's disease) and Alzheimer's disease.   20. The method of claim 19, wherein the patient is breast cancer, liver cancer, gastrointestinal cancer, neuroblastoma, neuronal cancer, leukemia, lymphoma, prostate cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, endometrium Suffers from any sarcoma, renal, epidermoid, or non-small cell lung cancer, including those arising from tumors, choriocarcinoma, teratocarcinoma, thyroid cancer, soft tissue and bone   19. The method of claim 18, wherein the patient is a human.   19. The method of claim 18, wherein the composition is administered in a mode selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, and oral. Method.   24. The method of claim 22, wherein the mode of administration is by intravenous injection.   19. The method of claim 18, wherein the composition is administered within 0 minutes to 1 week of administration of another drug known to treat the pathological condition or cancer. .   15. The method of claim 14, wherein the composition is administered at about the same time as another anticancer drug.   A composition comprising at least two isolated polypeptides selected from the group consisting of a cupredoxin and the isolated peptide of claim 1.   27. A composition according to claim 26, in a pharmaceutical composition.   A kit comprising the composition of claim 13 in a vial.   30. The kit of claim 28, wherein the kit is designed for intravenous administration.   14. A method comprising contacting a mammalian cancer cell with the composition of claim 13; and measuring the growth of the cancer cell.   31. The method of claim 30, wherein the cancer cells are breast cancer, liver cancer, gastrointestinal cancer, neuroblastoma, neuronal cancer, leukemia, lymphoma, prostate cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, intrauterine A method selected from the group consisting of membrane tumors, choriocarcinoma, teratocarcinoma, thyroid cancer, all sarcomas, including those arising from soft tissue and bone, renal cancer, epidermoid cancer, or non-small cell lung cancer.   32. The method of claim 30, wherein the cancer cell is in vivo.   An expression vector encoding the peptide of claim 1.   An isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin and binds to an ephrin receptor.   35. The isolated peptide of claim 34, wherein the ephrin receptor consists of EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4 and EphB6 An isolated peptide selected from the group.   36. The isolated peptide of claim 35, which also binds to ephrin.   37. The isolated peptide of claim 36, wherein said ephrin is selected from the group consisting of ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3 and ephrin B4. Isolated peptides   37. The isolated peptide of claim 36, wherein the isolated peptide binds to ephrin and its receptor.   40. The isolated peptide of claim 38, wherein the peptide binds to ephrin 2B and Eph2B.   An ephrin selected from the group consisting of a variant, derivative or structural equivalent of a cupredoxin and ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3 and ephrin B4 An isolated peptide that binds.   14. A method comprising administering to a mammalian patient a composition according to claim 13 to guide blood vessel growth in the patient.   14. A method comprising administering to a mammalian patient a composition according to claim 13 to reduce blood vessel growth in the patient.   14. A method comprising administering to a mammalian patient a composition according to claim 13 for guiding the growth of neurons in the patient.   14. A method comprising administering to a mammalian patient a composition according to claim 13 to promote bone formation in the patient.   14. A method of treatment requiring the use of ephrin, comprising administering to a mammalian patient an effective amount of the composition of claim 13 instead of ephrin.   14. A method comprising administering to a mammalian patient an effective amount of the composition of claim 13 to inhibit ephrin receptor activity associated with cells.   14. A method comprising administering to a mammalian patient an effective amount of the composition of claim 13 to increase the activity of ephrin receptors associated with the cells.   Peptides of the invention comprising at least one cupredoxin fused to a pharmaceutical substance or a derivative, variant, derivative or structural equivalent of a cupredoxin fused to a pharmaceutical substance for a human patient having a tissue expressing an ephrin receptor A method comprising administering a pharmaceutical composition comprising   49. The method of claim 48, wherein the tissue is cancer. A method for detecting tissue with an ephrin receptor in a mammalian patient comprising:
(A) administering to a human patient a pharmaceutical composition comprising at least one cupredoxin or a peptide of the invention fused to a detectable probe;
(B) a method comprising detecting a distribution of probes in the patient

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