# Patent

## Title
Compositions and methods to prevent cancer with cupredoxins

## Abstract

The present invention relates to compositions and methods comprising chemopreventive agents that may be cupredoxin(s) or variants, derivatives and structural equivalents of cupredoxins, and at least one other chemopreventive agent. Specifically, these compositions may comprise azurin from Pseudomonas aeruginosa, and/or the 50-77 residue region of azurin (p28). The at least one other chemopreventive agent may comprise an antiestrogen such as Tamoxifen. The compositions of the invention may be used to prevent the development of premalignant lesions in mammalian cells, tissues and animals, and thus prevent cancer.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §§119 and 120 to, and is a continuation in part of, U.S. patent application Ser. No. 11/854,654, filed on Sep. 13, 2007, now U.S. Pat. No. 7,618,939, which claims priority to Provisional U.S. Application Ser. No. 60/844,358, filed Sep. 14, 2006; and is a continuation in part of U.S. patent application Ser. No. 11/244,105, filed Oct. 6, 2005, now U.S. Pat. No. 7,691,383, which claims priority to U.S. Provisional Patent Application Ser. No. 60/680,500, filed May 13, 2005, and U.S. Provisional Patent Application Ser. No. 60/616,782, filed Oct. 7, 2004. The entire content of these prior applications is fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to compositions comprising variants, derivatives and structural equivalents of cupredoxins and at least one other chemopreventive agent for chemopreventive therapy which are used for cancer-related therapy, and specifically for inhibiting the development of premalignant lesions in mammalian cells, tissues and animals. The invention also relates to the use and/or administration of cupredoxins, and variants, derivatives and structural equivalents of cupredoxins, and at least one other chemopreventive agent in chemopreventive therapy and/or as chemopreventive agents in mammals to inhibit the development of premalignant lesions, and ultimately cancer.
BACKGROUND
Cancer chemoprevention is the use of natural, synthetic or biologic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. Recent clinical trials in preventing cancer in high-risk populations suggest that chemopreventive therapy is a realistic treatment for high-risk patients. Chemopreventive therapy is based on the concepts of multifocal field carcinogenesis and multistep carcinogenesis. In field carcinogenesis, generalized carcinogen exposure throughout the tissue field results in diffuse epithelial injury in tissue and clonal proliferation of the mutated cells. These genetic mutations throughout the field increase the likelihood that one or more premalignant or malignant lesions may develop in the field. Multistep carcinogenesis in the stepwise accumulation of these genetic and phenotypic alterations. Arresting one or more steps in the multistep carcinogenesis may impede or prevent the development of cancer. See generally Tsao et al., CA Cancer J Clin 54:150-180 (2004).
Azurin, and other cupredoxins, are cytotoxic specifically towards cancer cells Azurin induces apoptosis in J774 lung cancer cells. Yamada et al., PNAS 99(22):14098-14103 (2002). On entry into J774 lung cancer cells, azurin localizes in the cytosol and nuclear fractions, and forms a complex with tumor suppressor protein p53, thereby stabilizing it and enhancing its intracellular level. Id. The induction of azurin-mediated apoptosis is not limited to J774 cells. Azurin can also enter cancer cells such as human melanoma UISO-Mel-2, human breast cancer MCF-7 cells, and osteosarcoma. Yamada et al., Infect Immun. 70:7054-7062 (2002); Punj et al., Oncogene. 23:2367-2378 (2004). Azurin allowed the elevation of the intracellular p53 levels, leading to enhanced Bax formation and induction of apoptosis in such cells. Most interestingly, intraperitoneal injection of azurin in nude mice harboring xenografted Mel-2 or MCF-7 human cancers led to statistically significant regression of such cancers. Id.
The mouse mammary gland organ culture (MMOC) model has been fairly well established to study effects of hormonal regulation of mammary gland development and epithelial cell transformation. The MMOC assay may be used to evaluate the inhibitory effects of potential chemopreventive agents on both hormone-induced structural differentiation of mammary glands and on the development of DMBA-induced preneoplastic hyperplastic alveolar nodule-like lesions in the gland. Mammary glands respond to hormones in organ cultures to differentiate into alveolar structures or for inducing expression of casein and a-lactalbumin. Mammary glands from young, virgin animals, when incubated for 6 days in the presence of insulin (I)+prolactin (P)+aldosterone (A), can differentiate into fully-grown glands. These glands morphologically resemble the glands obtained from pregnant mice. Aldosterone can be replaced by estrogen (E)+progesterone (Pg). Inclusion of hydrocortisone (H) to the medium stimulates the functional differentiation of the mammary glands. Mehta and Banejee, Acta Endocrinol. 80:501 (1975); Mehta and Moon, Breast Cancer: Treatment and Prognosis 300, 300 (Basil A Stoll ed., Blackwell Press 1986). Thus, the hormone-induced structural and functional differentiation, observed in this culture system, mimics the responses to hormones observed during various physiological stages of the animal.
Mice exhibit a distinct preneoplastic stage prior to cancer formation in MMOC. Such preneoplastic lesions in C3H mice are induced by murine mammary tumor virus or in BALB/c mice by DMBA. Exposure of the glands to 2 μg/ml DMBA between days 3 and 4 of growth phases followed by regression of the glands for 2-3 weeks in the medium containing only insulin, results in the formation of mammary alveolar lesions (MAL). Hawthorne et al., Pharmaceutical Biology 40:70-74 (2002); Mehta et al., Methods in Cell Science 19:19-24 (1997). Furthermore, transplantation of epithelial cells, prepared from glands containing the DMBA-induced mammary lesions, into syngeneic host resulted in the development of mammary adenocarcinoma. Telang et al., PNAS 76:5886-5890 (1979). Pathologically, these tumors were similar to those observed in vivo when mice of the same strain are administered DMBA. Id.
DMBA-induced mammary lesion formation in MMOC can be inhibited by a variety of classes of chemopreventive agents such as retinoids. These agents include chemopreventive agents derived from the natural products such as brassinin and resveretrol, thiols, antioxidants, inhibitors of ornithine decarboxylase such as OFMO and deguelin, inhibitors of prostaglandin synthesis, Ca regulators, etc. Jang et al., Science 275:218-220 (1997); Mehta, Eur. J. Cancer 36:1275-1282 (2000); Metha et al., J. Natl. Cancer Inst. 89:212-219 (1997). These studies clearly demonstrate that this organ culture system offers a unique model to determine the effectiveness of compounds against mammary carcinogenesis. The results can be expected to closely correlate to the inhibition obtained by in vivo administration of such compounds.
In the presence of aldosterone and hydrocortisone, and in the absence of estrogen and progesterone, the MMOC can be induced to develop estrogen independent MAL. The MMOC may also be induced to form mammary ductal lesions (MDL). The MDL can be induced if estrogen and progesterone instead of aldosterone and hydrocortisone are included in the medium. The alveolar structures in the presence of ovarian steroids are very small but the intraductal lesions are observed in histopathological sections. Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001). The antiestrogens, which selectively work on ovarian hormone dependent ER+ breast cancers such as Tamoxifen, inhibited MDL formation and not MAL. Thus, this modified culture model in addition to conventional MAL induction protocol now can be used to evaluate effects of chemopreventive agents on both MAL and MOL.
In chemically transformed experimental MMOC models, p53 is rarely mutated or altered, but inactivation of p53 leads to increased aneuploidy in tumors. Further studies in mice bearing C3(1)-Tag transgene suggested that abrogation of p53 function can be a critical event in tumor formation even in the presence of oncogenes. Studies have shown that p53 is inactivated or suppressed by estrogen. Thus, this model can be used to evaluate the effects of azurin on the development of premalignant lesions and the ability of azurin to modulate p53 expression in mammary epithelial cells. This model can also be used to evaluate the effect of estrogen receptor (ER) inhibiting compounds in combination with azurin.
SUMMARY OF THE EMBODIMENTS
The present invention relates to compositions comprising peptides that may be cupredoxin(s) or variants, derivatives, truncations, or structural equivalents of cupredoxins that have chemopreventive effects on premalignant lesions and/or cancer. Specifically, these compositions may comprise azurin from Pseudomonas aeruginosa, and/or the 50-77 residue region of azurin (p28, SEQ ID NO: 2) and/or the 50-67 residue region of azurin (p18 SEQ ID NO: 25).
The present invention further relates to compositions that may comprise cupredoxin(s), and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, that have chemopreventive effects. These compositions may be isolated peptides or pharmaceutical compositions, among others. The compositions of the invention may be used in methods to prevent, inhibit, prevent, suppress, reverse, or delay the development of premalignant lesions and/or cancer in mammalian patients.
The present invention further relates to pharmaceutical compositions that comprise one or more chemopreventive agents including cupredoxins and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, and at least one other chemopreventive agent.
In some embodiments of the present invention, the cupredoxin is selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein. In further embodiments, the cupredoxin is azurin. In other embodiments, the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. In certain embodiments, the cupredoxin is from Pseudomonas aeruginosa. In other embodiments, the cupredoxin is azurin from Pseudomonas aeruginosa. 
In some embodiments of the pharmaceutical composition of the invention, the first chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2. In other embodiments, the truncation comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In other embodiments, the truncation is more than about 10 amino acids and not more than about 100 amino acids. In further embodiments, the truncation is from 18 to 28 amino acids, from 28 to 50 amino acids, from 18 to 100 amino acids, or from 28 to 100 amino acids. In some embodiments, the truncation binds to one or more binding sites of p53 selected from the group consisting of amino acids 1-17, 24-31, 80-276, and 297-305. In other embodiments, the truncation binds to the N-terminal binding domain of p53 or the DNA binding domain of p53.
In some embodiments of the pharmaceutical composition of the invention, the at least one other chemopreventive agent (or “second chemopreventive agent”) is an antiestrogen that prevents the binding of estrogen and/or progesterone, or prevents the production of estrogen and/or progesterone. In further embodiments, the antiestrogen may be Tamoxifen, fenretinide, or an aromatase inhibitor.
The present invention also relates to pharmaceutical compositions comprising one or more chemopreventive agents including cupredoxins and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, and at least one other chemopreventive compound, that further comprise a pharmaceutically acceptable carrier. In a further embodiment, the pharmaceutically acceptable carrier is suitable for intravenous administration. In a further embodiment, the first chemopreventive agent of the composition is a cupredoxin from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseprica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaernolyticus. In some embodiments, the cupredoxin is from Pseudomonas aeruginosa. In other embodiments, the chemopreventive agent is a truncation of azurin that comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2.
The present invention further relates to methods of treating a mammalian patient, which may comprise administering to the patient a therapeutically effective amount of the composition described above comprising a first chemopreventive agent that is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25, a second chemopreventive agent, and a pharmaceutically acceptable carrier. In a further embodiment, the mammalian patient is human. In another embodiment, the mammalian patient is at a higher risk to develop cancer than the general population. In further embodiments, the cancer is selected from melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin, and cervical cancer. In another embodiment, the patient has at least one high risk feature selected from the group consisting of smoking, poor diet, alcohol consumption, hormone replacement therapy, higher body mass index, nulliparity, betal nut use, frequent mouthwash use, exposure to human papillomavirus, childhood and chronic sun exposure, early age of first intercourse, multiple sexual partners, oral contraceptive use, a family history of cancer, gene carrier status of BRCA1 and BRCA2, prior history of breast neoplasia, familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), red or blond hair and fair-skinned phenotype, xeroderma pigmentosum, exposure to radon, polycyclic aromatic hydrocarbons, nickel, chromate, arsenic, asbestos, chloromethyl ethers, benzo[a]pyrene, radiation, or aromatic amines from rubber or paint occupational exposure, chronic obstructive pulmonary disease with airflow obstruction, chronic bladder infections, schistosomiasis, older age, and immunocompromised status. In other embodiments, the patient has premalignant lesions or has been cured of cancer or premalignant lesions. In yet another embodiment, a pharmaceutical composition is administered by a mode selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral. In a further embodiment, the mode of administration is by intravenous injection.
The present invention further relates to a kit comprising one or more chemopreventive agents that are cupredoxins and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, at least one other chemopreventive compound, and a pharmaceutically effective carrier in a vial. In one embodiment, the chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the kit further comprises a pharmaceutically acceptable carrier suitable for intravenous administration.
The present invention also relates to a method comprising treating a mammalian patient by administering a first chemopreventive agent that is a cupredoxin or a truncation, derivative or structural equivalent of a cupredoxin, and administering a second chemopreventive agent. In some embodiments, the first chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2. In further embodiments, the truncation comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In some embodiments, the truncation binds to one or more binding sites of p53 selected from the group consisting of amino acids 1-17, 24-31, 80-276, and 297-305. In other embodiments, the truncation binds to the N-terminal binding domain of p53 or the DNA binding domain of p53. In some embodiments, the other chemopreventive agent is an antiestrogen, such as Tamoxifen fenretinide, or an aromatase inhibitor. In other embodiments, the chemopreventive agent is a cupredoxin selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein. In a further embodiment, the cupredoxin is azurin. In other embodiments, the chemopreventive agent is a cupredoxin from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. In one embodiment the chemopreventive agent is from Pseudomonas aeruginosa. 
The present invention also relates to a method comprising increasing the expression of p53 in mammalian cells, comprising administering at least one chemopreventive agent, which may be a cupredoxin or a truncation, derivative or structural equivalent of a cupredoxin, and administering an antiestrogen, in any order. In some embodiments, the chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2. In further embodiments, the truncation comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In some embodiments, the truncation binds to one or more binding sites of p53 selected from the group consisting of amino acids 1-17, 24-31, 80-276, and 297-305. In other embodiments, the truncation binds to the N-terminal binding domain of p53 or the DNA binding domain of p53. In some embodiments, the antiestrogen is Tamoxifen, fenretinide, or an aromatase inhibitor. In some embodiments, the chemopreventive agent is a cupredoxin selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein. In a further embodiment, the cupredoxin is azurin. In other embodiments, the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. In a further embodiment, the cupredoxin is from Pseudomonas aeruginosa. 
The present invention also relates to a method comprising reducing the dose-related toxicity of Tamoxifen without decreasing its chemopreventive effects by administering a therapeutically effective dose of Tamoxifen and administering a therapeutically effective dose of at least one chemopreventive agent, such as a cupredoxin or a variant, truncation, derivative, or structural equivalent of a cupredoxin, in any order. In some embodiments, the chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2. In further embodiments, the truncation comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In some embodiments, the truncation binds to one or more binding sites of p53 selected from the group consisting of amino acids 1-17, 24-31, 80-276, and 297-305. In other embodiments, the truncation binds to the N-terminal binding domain of p53 or the DNA binding domain of p53. In another embodiment, the dose of Tamoxifen and dose of the chemopreventive agent are administered at or around the same time.
In other embodiments, the chemopreventive agent is a cupredoxin selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein. In some embodiments, the cupredoxin is azurin. The cupredoxin may be from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. In some embodiments, the cupredoxin is from Pseudomonas aeruginosa. 
The present invention also relates to a method comprising decreasing estrogen receptor binding to p53 in mammalian cells by administering to the mammalian cells at least one chemopreventive agent that is a cupredoxin or a variant, truncation, derivative, or structural equivalent of a cupredoxin, and administering an antiestrogen, in any order. In some embodiments, the chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. In a further embodiment, the truncation comprises SEQ ID NO: 2. In another embodiment, the truncation consists of SEQ ID NO: 2. In further embodiments, the truncation comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In some embodiments, the truncation binds to one or more binding sites of p53 selected from the group consisting of amino acids 1-17, 24-31, 80-276, and 297-305. In other embodiments, the truncation binds to the N-terminal binding domain of p53 or the DNA binding domain of p53. In other embodiments, the antiestrogen is Tamoxifen, fenretinide, or an aromatase inhibitor. In some embodiments, the chemopreventive agent is a cupredoxin selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein. In further embodiments, the cupredoxin is azurin. In other embodiments, the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. In a further embodiment, the cupredoxin is from Pseudomonas aeruginosa. 
These and other aspects, advantages, and features of the invention will become apparent from the following figures and detailed description of the specific embodiments.
Description of the Sequences
SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa (Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln Met Gln Phe Asn Thr Asn Ala Ile Thr Val Asp Lys Ser Cys Lys Gln Phe Thr Val Asn Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys).
SEQ ID NO: 2. Amino acid sequence of p28, Pseudomonas aeruginosa azurin residues 50-77 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidium laminosum (Glu Thr Phe Thr Val Lys Met Gly Ala Asp Ser Gly Leu Leu Gln Phe Glu Pro Ala Asn Val Thr Val His Pro Gly Asp Thr Val Lys Trp Val Asn Asn Lys Leu Pro Pro His Asn Ile Leu Phe Asp Asp Lys Gln Val Pro Gly Ala Ser Lys Glu Leu Ala Asp Lys Leu Ser His Ser Gln Leu Met Phe Ser Pro Gly Glu Ser Tyr Glu Ile Thr Phe Ser Ser Asp Phe Pro Ala Gly Thr Tyr Thr Tyr Tyr Cys Ala Pro His Arg Gly Ala Gly Met Val Gly Lys Ile Thr Val Glu Gly).
SEQ ID NO: 4. Amino acid sequence of rusticyanin from Thiobacillus ferrooxidans (Gly Thr Leu Asp Thr Thr Trp Lys Glu Ala Thr Leu Pro Gln Val Lys Ala Met Leu Glu Lys Asp Thr Gly Lys Val Ser Gly Asp Thr Val Thr Tyr Ser Gly Lys Thr Val His Val Val Ala Ala Ala Val Leu Pro Gly Phe Pro Phe Pro Ser Phe Glu Val His Asp Lys Lys Asn Pro Thr Leu Glu Ile Pro Ala Gly Ala Thr Val Asp Val Thr Phe Ile Asn Thr Asn Lys Gly Phe Gly His Ser Phe Asp Ile Thr Lys Lys Gly Pro Pro Tyr Ala Val Met Pro Val Ile Asp Pro Ile Val Ala Gly Thr Gly Phe Ser Pro Val Pro Lys Asp Gly Lys Phe Gly Tyr Thr Asp Phe Thr Trp His Pro Thr Ala Gly Thr Tyr Tyr Tyr Val Cys Gln Ile Pro Gly His Ala Ala Thr Gly Met Phe Gly Lys Ile Val Val Lys).
SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobacter cycloclastes (Ala Asp Phe Glu Val His Met Leu Asn Lys Gly Lys Asp Gly Ala Met Val Phe Glu Pro Ala Ser Leu Lys Val Ala Pro Gly Asp Thr Val Thr Phe Ile Pro Thr Asp Lys Gly His Asn Val Glu Thr Ile Lys Gly Met Ile Pro Asp Gly Ala Glu Ala Phe Lys Ser Lys Ile Asn Glu Asn Tyr Lys Val Thr Phe Thr Ala Pro Gly Val Tyr Gly Val Lys Cys Thr Pro His Tyr Gly Met Gly Met Val Gly Val Val Gln Val Gly Asp Ala Pro Ala Asn Leu Glu Ala Val Lys Gly Ala Lys Asn Pro Lys Lys Ala Gln Glu Arg Leu Asp Ala Ala Leu Ala Ala Leu Gly Asn).
SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis (Ala Cys Asp Val Ser Ile Glu Gly Asn Asp Ser Met Gln Phe Asn Thr Lys Ser Ile Val Val Asp Lys Thr Cys Lys Glu Phe Thr Ile Asn Leu Lys His Thr Gly Lys Leu Pro Lys Ala Ala Met Gly His Asn Val Val Val Ser Lys Lys Ser Asp Glu Ser Ala Val Ala Thr Asp Gly Met Lys Ala Gly Leu Asn Asn Asp Tyr Val Lys Ala Gly Asp Glu Arg Val Ile Ala His Thr Ser Val Ile Gly Gly Gly Glu Thr Asp Ser Val Thr Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Asp Tyr Ala Phe Phe Cys Ser Phe Pro Gly His Trp Ser Ile Met Lys Gly Thr Ile Glu Leu Gly Ser).
SEQ ID NO: 7. Amino acid sequence of azurin from Achromobacter xylosoxidans ssp. denitrificans I (Ala Gln Cys Glu Ala Thr Ile Glu Ser Asn Asp Ala Met Gln Tyr Asn Leu Lys Glu Met Val Val Asp Lys Ser Cys Lys Gln Phe Thr Val His Leu Lys His Val Gly Lys Met Ala Lys Val Ala Met Gly His Asn Trp Val Leu Thr Lys Glu Ala Asp Lys Gln Gly Val Ala Thr Asp Gly Met Asn Ala Gly Leu Ala Gln Asp Tyr Val Lys Ala Gly Asp Thr Arg Val Ile Ala His Thr Lys Val Ile Gly Gly Gly Glu Ser Asp Ser Val Thr Phe Asp Val Ser Lys Leu Thr Pro Gly Glu Ala Tyr Ala Tyr Phe Cys Ser Phe Pro Gly His Trp Ala Met Met Lys Gly Thr Leu Lys Leu Ser Asn).
SEQ ID NO: 8. Amino acid sequence of azurin from Bordetella bronchiseptica (Ala Glu Cys Ser Val Asp Ile Ala Gly Thr Asp Gln Met Gln Phe Asp Lys Lys Ala Ile Glu Val Ser Lys Ser Cys Lys Gln Phe Thr Val Asn Leu Lys His Thr Gly Lys Leu Pro Arg Asn Val Met Gly His Asn Trp Val Leu Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp Thr Arg Val Leu Ala His Thr Lys Val Leu Gly Gly Gly Glu Ser Asp Ser Val Thr Phe Asp Val Ala Lys Leu Ala Ala Gly Asp Asp Tyr Thr Phe Phe Cys Ser Phe Pro Gly His Gly Ala Leu Met Lys Gly Thr Leu Lys Leu Val Asp).
SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J (Ala Ser Cys Glu Thr Thr Val Thr Ser Gly Asp Thr Met Thr Tyr Ser Thr Arg Ser Ile Ser Val Pro Ala Ser Cys Ala Glu Phe Thr Val Asn Phe Glu His Lys Gly His Met Pro Lys Thr Gly Met Gly His Asn Trp Val Leu Ala Lys Ser Ala Asp Val Gly Asp Val Ala Lys Glu Gly Ala His Ala Gly Ala Asp Asn Asn Phe Val Thr Pro Gly Asp Lys Arg Val Ile Ala Phe Thr Pro Ile Ile Gly Gly Gly Glu Lys Thr Ser Val Lys Phe Lys Val Ser Ala Leu Ser Lys Asp Glu Ala Tyr Thr Tyr Phe Cys Ser Tyr Pro Gly His Phe Ser Met Met Arg Gly Thr Leu Lys Leu Glu Glu).
SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidis Z2491 (Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Ala Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys Thr Ser Met Gly His Asn Ile Val Ile Gly Lys Thr Glu Asp Met Asp Gly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Glu Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly Lys Val Thr Leu Val Asp).
SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonas fluorescen (Ala Glu Cys Lys Thr Thr Ile Asp Ser Thr Asp Gln Met Ser Phe Asn Thr Lys Ala Ile Glu Ile Asp Lys Ala Cys Lys Thr Phe Thr Val Glu Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Leu Val Ile Ser Lys Gln Ala Asp Met Gln Pro Ile Ala Thr Asp Gly Leu Ser Ala Gly Ile Asp Lys Asn Tyr Leu Lys Glu Gly Asp Thr Arg Val Ile Ala His Thr Lys Val Ile Gly Ala Gly Glu Lys Asp Ser Leu Thr Ile Asp Val Ser Lys Leu Asn Ala Ala Glu Lys Tyr Gly Phe Phe Cys Ser Phe Pro Gly His Ile Ser Met Met Lys Gly Thr Val Thr Leu Lys).
SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonas chlororaphis (Ala Glu Cys Lys Val Asp Val Asp Ser Thr Asp Gln Met Ser Phe Asn Thr Lys Glu Ile Thr Ile Asp Lys Ser Cys Lys Thr Phe Thr Val Asn Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Trp Val Leu Ser Lys Ser Ala Asp Met Ala Gly Ile Ala Thr Asp Gly Met Ala Ala Gly Ile Asp Lys Asp Tyr Leu Lys Pro Gly Asp Ser Arg Val Ile Ala His Thr Lys Ile Ile Gly Ser Gly Glu Lys Asp Ser Val Thr Phe Asp Val Ser Lys Leu Thr Ala Gly Glu Ser Tyr Glu Phe Phe Cys Ser Phe Pro Gly His Asn Ser Met Met Lys Gly Ala Val Val Leu Lys).
SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa 9a5c (Lys Thr Cys Ala Val Thr Ile Ser Ala Asn Asp Gln Met Lys Phe Asp Gln Asn Thr Ile Lys Ile Ala Ala Glu Cys Thr His Val Asn Leu Thr Leu Thr His Thr Gly Lys Lys Ser Ala Arg Val Met Gly His Asn Trp Val Leu Thr Lys Thr Thr Asp Met Gln Ala Val Ala Leu Ala Gly Leu His Ala Thr Leu Ala Asp Asn Tyr Val Pro Lys Ala Asp Pro Arg Val Ile Ala His Thr Ala Ile Ile Gly Gly Gly Glu Arg Thr Ser Ile Thr Phe Pro Thr Asn Thr Leu Ser Lys Asn Val Ser Tyr Thr Phe Phe Cys Ser Phe Pro Gly His Trp Ala Leu Met Lys Gly Thr Leu Asn Phe Gly Gly).
SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus (Met Gln Ser Thr Val His Ile Val Gly Asp Asn Thr Gly Trp Ser Val Pro Ser Ser Pro Asn Phe Tyr Ser Gln Trp Ala Ala Gly Lys Thr Phe Arg Val Gly Asp Ser Leu Gln Phe Asn Phe Pro Ala Asn Ala His Asn Val His Glu Met Glu Thr Lys Gln Ser Phe Asp Ala Cys Asn Phe Val Asn Ser Asp Asn Asp Val Glu Arg Thr Ser Pro Val Ile Glu Arg Leu Asp Glu Leu Gly Met His Tyr Phe Val Cys Thr Val Gly Thr His Cys Ser Asn Gly Gln Lys Leu Ser Ile Asn Val Val Ala Ala Asn Ala Thr Val Ser Met Pro Pro Pro Ser Ser Ser Pro Pro Ser Ser Val Met Pro Pro Pro Val Met Pro Pro Pro Ser Pro Ser).
SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexus aurantiacus (Met Lys Ile Thr Leu Arg Met Met Val Leu Ala Val Leu Thr Ala Met Ala Met Val Leu Ala Ala Cys Gly Gly Gly Gly Ser Ser Gly Gly Ser Thr Gly Gly Gly Ser Gly Ser Gly Pro Val Thr Ile Glu Ile Gly Ser Lys Gly Glu Glu Leu Ala Phe Asp Lys Thr Glu Leu Thr Val Ser Ala Gly Gln Thr Val Thr Ile Arg Phe Lys Asn Asn Ser Ala Val Gln Gln His Asn Trp Ile Leu Val Lys Gly Gly Glu Ala Glu Ala Ala Asn Ile Ala Asn Ala Gly Leu Ser Ala Gly Pro Ala Ala Asn Tyr Leu Pro Ala Asp Lys Ser Asn Ile Ile Ala Glu Ser Pro Leu Ala Asn Gly Asn Glu Thr Val Glu Val Thr Phe Thr Ala Pro Ala Ala Gly Thr Tyr Leu Tyr Ile Cys Thr Val Pro Gly His Tyr Pro Leu Met Gln Gly Lys Leu Val Val Asn).
SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexus auranliacus (Ala Ala Asn Ala Pro Gly Gly Ser Asn Val Val Asn Glu Thr Pro Ala Gln Thr Val Glu Val Arg Ala Ala Pro Asp Ala Leu Ala Phe Ala Gln Thr Ser Leu Ser Leu Pro Ala Asn Thr Val Val Arg Leu Asp Phe Val Asn Gln Asn Asn Leu Gly Val Gln His Asn Trp Val Leu Val Asn Gly Gly Asp Asp Val Ala Ala Ala Val Asn Thr Ala Ala Gln Asn Asn Ala Asp Ala Leu Phe Val Pro Pro Pro Asp Thr Pro Asn Ala Leu Ala Trp Thr Ala Met Leu Asn Ala Gly Glu Ser Gly Ser Val Thr Phe Arg Thr Pro Ala Pro Gly Thr Tyr Leu Tyr Ile Cys Thr Phe Pro Gly His Tyr Leu Ala Gly Met Lys Gly Thr Leu Thr Val Thr Pro).
SEQ ID NO: 17. Amino acid sequence of cucumber basic protein from Cucumis sativus (Ala Val Tyr Val Val Gly Gly Ser Gly Gly Trp Thr Phe Asn Thr Glu Ser Trp Pro Lys Gly Lys Arg Phe Arg Ala Gly Asp Ile Leu Leu Phe Asn Tyr Asn Pro Ser Met His Asn Val Val Val Val Asn Gln Gly Gly Phe Ser Thr Cys Asn Thr Pro Ala Gly Ala Lys Val Tyr Thr Ser Gly Arg Asp Gln Ile Lys Leu Pro Lys Gly Gln Ser Tyr Phe Ile Cys Asn Phe Pro Gly His Cys Gln Ser Gly Met Lys Ile Ala Val Asn Ala Leu).
SEQ ID NO: 18. Amino acid sequence of Laz from Neisseria gonorrhoeae F62 (Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Asp Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly Lys Val Thr Leu Val Asp).
SEQ ID NO: 19. Amino acid sequence of the azurin from Vibrio parahaemolyticus (Met Ser Leu Arg Ile Leu Ala Ala Thr Leu Ala Leu Ala Gly Leu Ser Phe Gly Ala Gln Ala Ser Ala Glu Cys Glu Val Ser Ile Asp Ala Asn Asp Met Met Gln Phe Ser Thr Lys Thr Leu Ser Val Pro Ala Thr Cys Lys Glu Val Thr Leu Thr Leu Asn His Thr Gly Lys Met Pro Ala Gln Ser Met Gly His Asn Val Val Ile Ala Asp Thr Ala Asn Ile Gln Ala Val Gly Thr Asp Gly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val Lys Pro Asp Asp Glu Arg Val Tyr Ala His Thr Lys Val Val Gly Gly Gly Glu Ser Thr Ser Ile Thr Phe Ser Thr Glu Lys Met Thr Ala Gly Gly Asp Tyr Ser Phe Phe Cys Ser Phe Pro Gly His Trp Ala Ile Met Gln Gly Lys Phe Glu Phe Lys).
SEQ ID NO: 20. Amino acid sequence of amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus (His Asn Trp Val Leu Val Asn Gly Gly Asp Asp Val Ala Ala Ala Val Asn Thr Ala Ala Gln Asn Asn Ala Asp Ala Leu Phe Val Pro Pro Pro Asp).
SEQ ID NO: 21. Amino acid sequence of amino acids 51-77 of Pseudomonas syringae azurin (Ser Lys Lys Ala Asp Ala Ser Ala Ile Thr Thr Asp Gly Met Ser Val Gly Ile Asp Lys Asp Tyr Val Lys Pro Asp Asp).
SEQ ID NO: 22. Amino acid sequence of amino acids 89-115 of Neisseria meningitidis Laz (Ile Gly Lys Thr Glu Asp Met Asp Gly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp).
SEQ ID NO: 23. Amino acid sequence of amino acids 52-78 of Vibrio parahaemolyticus azurin (Ala Asp Thr Ala Asn Ile Gln Ala Val Gly Thr Asp Gly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val Lys Pro Asp Asp).
SEQ ID NO: 24. Amino acid sequence of amino acids 51-77 of Bordetella bronchiseptica azurin (Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp).
SEQ ID NO: 25. Amino acid sequence of p18, Pseudomonas aeruginosa azurin residues 50-67 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly).
SEQ ID NO: 26. Amino acid sequence of p19, Pseudomonas aeruginosa azurin residues 50-68 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu).
SEQ ID NO: 27. Amino acid sequence of p20, Pseudomonas aeruginosa azurin residues 50-69 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp).
SEQ ID NO: 28. Amino acid sequence of p21, Pseudomonas aeruginosa azurin residues 50-70 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys).
SEQ ID NO: 29. Amino acid sequence of p22, Pseudomonas aeruginosa azurin residues 50-71 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp).
SEQ ID NO: 30. Amino acid sequence of p23, Pseudomonas aeruginosa azurin residues 50-72 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr)
SEQ ID NO: 31. Amino acid sequence of p24, Pseudomonas aeruginosa azurin residues 50-73 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu).
SEQ ID NO: 32. Amino acid sequence of p25, Pseudomonas aeruginosa azurin residues 50-74 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys).
SEQ ID NO: 33. Amino acid sequence of p26, Pseudomonas aeruginosa azurin residues 50-75 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro).
SEQ ID NO: 34. Amino acid sequence of p27, Pseudomonas aeruginosa azurin residues 50-76 (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp).
SEQ ID NO: 35. Amino acid sequence of Pseudomonas aeruginosa azurin residues 36-88 (Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala His Thr Lys Leu Ile Gly).
SEQ ID NO: 36 and 83. Nucleotide sequence of primer for p53 (forward: 5-GATGACTCCCATGGAGGAG-3′ reverse: ′-GTCCATCCAAGGTGATG
SEQ ID NO: 37 and 84. Nucleotide sequence of primer for 18S (forwards 5′-CATCCCCCTTCTTAGTTCGT-3′ reverse: 5′-GAACGCCACTTGTCCCTCTA-3′).
SEQ ID NO: 38. Amino acid sequence of p18b, Pseudomonas aeruginosa azurin residues 60-77 (Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 39. Sequence of C-terminal 12 amino acids of p28, Pseudomonas aeruginosa azurin residues 66-77 (Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 40. Sequence of C-terminal 10 amino acids of p28, Pseudomonas aeruginosa azurin residues 68-77 (Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 41. Sequence of C-terminal 11 amino acids of p28, Pseudomonas aeruginosa azurin residues [[66]]67-77 (Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 42 is the amino acid sequence of a variant of the azurin truncation p28 (Leu Ser Thr Ala Ala Asp Met Gln Ala Val Val Thr Asp Thr Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 43 is the amino acid sequence of a variant of the azurin truncation p28 (Leu Ser Thr Ala Ala Asp Leu Gln Gly Val Val Thr Asp Gly Leu Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 44 is the amino acid sequence of a variant of the azurin truncation p28 (Leu Ser Thr Ala Ala Asp Val Gln Gly Val Val Thr Asp Gly Val Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 45 is the amino acid sequence of a modified cupredoxin derived peptide (Asp Asp Pro Lys Leu Tyr Asp Lys Asp Leu Gly Ser Ala Met Gly Asp Thr Val Val Gly Gln Met Asp Ala Ala Thr Ser Leu).
SEQ ID NO: 46 is the amino acid sequence of a modified cupredoxin derived peptide (Acetylation-Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp-amidation).
SEQ ID NO: 47 is the amino acid sequence of a hexapeptide (Val Ser Pro Pro Ala Arg).
SEQ ID NO: 48 is the amino acid sequence of a hexapeptide (Tyr Thr Pro Pro Ala Leu).
SEQ ID NO: 49 is the amino acid sequence of a hexapeptide (Phe Ser Phe Phe Ala Phe).
SEQ ID NO: 50 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Thr Pro Gly Cys).
SEQ ID NO: 51 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Cys Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 52 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Cys Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 53 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Cys Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 54 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Thr Met Gln Cys Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 55 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Thr Met Gln Gly Cys Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 56 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asn Thr Gln Gly Cys Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 57 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asn Thr Gln Gly Val Cys Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 58 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Thr Ala Val Cys Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 59 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Thr Ala Val Val Cys Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 60 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Thr Val Val Cys Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 61 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Thr Val Val Thr Cys Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 62 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Ala Thr Val Thr Cys Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 63 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Ala Thr Val Thr Asp Cys Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 64 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Thr Ala Asp Cys Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 65 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Thr Ala Asp Gly Cys Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 66 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asn Gly Cys Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 67 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Ala Thr Met Gly Ser Gly Leu Cys Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 68 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Leu Thr Ala Ser Gly Leu Cys Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 69 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 70 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 71 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Trp Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 72 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 73 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Trp Ala Ala Asp Met Trp Gly Val Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 74 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val Trp Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 75 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val Thr Asp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 76 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val Trp Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 77 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val Thr Asp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 78 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Trp Asp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 79 is the amino acid sequence of a modified cupredoxin-derived peptide (Leu Ser Trp Ala Ala Asp Met Trp Gly Val Val Trp Asp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).
SEQ ID NO: 80 is the amino acid sequence of a modified cupredoxin-derived peptide (X1 Ser X2 Ala Ala Asp X3 X4 X5 Val Val X6 Asp X7 X8 Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp X9).
SEQ ID NO: 81 is the amino acid sequence of a modified cupredoxin-derived peptide (X1 Asp Pro Lys Leu Tyr Asp Lys Asp Leu Gly Ser Ala X2 X3 Asp X4 Val Val X5 X6 X7 Asp Ala Ala X8 Ser X9).
SEQ ID NO: 82 is the amino acid sequence of cationic peptide Arg8 (Arg Arg Arg Arg Arg Arg Arg Arg).
SEQ ID NO: 85. Sequence of amino acids that may interact with p53 (Ser Gly Leu Asp Lys Asp)



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts photographs of the glands evaluated for the efficacy of p28 and azurin. FIG. 1A shows a representative photograph of alveolar lesions in a DMBA-treated gland and its comparison with a gland that was treated with DMBA along with a chemopreventive agent. FIGS. 1B-1G show representative photographs of the effects of p28 on the development of alveolar lesions.
FIG. 2 depicts a graph showing the efficacy of p28 against DMBA-induced mammary alveolar lesions.
FIG. 3 depicts photographs of representative sections of ductal lesions and effect of p28.
FIG. 4 depicts a graph showing the efficacy of p28 against DMBA-induced ductal lesions.
FIG. 5 depicts the overall 3D structure of azurin, with the p28 peptide region bolded (SEQ ID NO: 2).
FIG. 6A depicts a photograph of representative sections of mammary alveolar lesions and effects of p28 on the development of mammary alveolar lesions.
FIG. 6B depicts photographs of representative sections of mammary lesions and effects of p28 on said lesions.
FIG. 6C depicts a graph showing the effects of p28 and Tamoxifen on mammary lesions.
FIG. 7A depicts a graph comparing the expression of p53 in mammary glands incubated in the presence or absence of estrogen and progesterone.
FIG. 7B depicts a graph showing the effects of p28, Tamoxifen, and a combination of the two on p53 expression.
FIG. 7C depicts photographs of immunohistochemical staining of p53 and Ki67 in representative sections of mammary glands of MMOC.
FIG. 7D depicts photographs of representative sections of mouse mammary glands and effects of azurin, p28, and Tamoxifen on p53 and Ki-67 in said mammary glands.
FIG. 8 depicts a graph showing the effects of combinations of fenretinide and p28 on MAL development in the MMOC model.
FIG. 9 depicts a graph showing the effects of combinations of Tamoxifen and p28 on MDL development in the MMOC model
FIGS. 10 A-L. Depict photographs of representative sections of mouse mammary glands and the effects of fenretinide and p28, alone and in combination, in these mammary glands. FIG. 1A shows a representative photograph of alveolar lesions in a DMBA-treated gland and its comparison with a gland that was treated with DMBA along with a chemopreventive agent. FIGS. 10B-10L show representative photographs of the effects of p28 on the development of alveolar lesions.
FIGS. 11 A-C. Depict photographs showing penetration of azurin derived peptides, p18 and p28, into cancer cell lines of diverse histogenesis and their normal counterparts. (A) Photos showing penetration of Alexafluor 568 labeled p28 or p18 after 2 hrs at 37° C. The cationic Arg8 (SEQ ID NO: 82) was used as a control. (B) Graphs depicting flow cytometric analysis of the penetration of Alexafluor 568 labeled p28 or p18 into the same cell lines after 2 hrs at 37° C. (C) Graphs depicting fold increase over fluorescence from normal cells. Similar observations of p28 or p18 entry into 4 melanoma cell lines show a several fold increase over fluorescence from normal cells.
FIGS. 12 A and B. Depict photographs showing entry of aza 60-77 (p18b) and azu 66-77 (p12) into cancer and normal cells. Cells were incubated with alexafluor 568 labeled p18b (A) or p12 (B) at 37° C. for 2 hrs and images recorded by confocal microscopy.
FIGS. 13 A and B. Graphs depicting cellular membrane toxicity of azurin and its peptides. (A) LDH leakage assay of UISOMel-2 cells exposure for 10 min to different concentrations of p28, p18 and azurin at 37° C. A standard lysis buffer (cytotox-one reagent) was included as a positive control. Changes in fluorescence following exposure were measured at k,x 560 nm and kem 590 nm. Lysis buffer was defined as 100% LDH release. Data represent % of positive fluorescence of control. Data are shown as mean±SEM. (B) Hemoglobin leakage from human erythrocytes incubated with p28, p18 and azurin. Human erythrocytes were incubated with peptide for 30 min at 37° C. and absorbance at 540 nm determined. Hemoglobin release following 0.1% Triton X-100 was defined as 100% hemoglobin release. Data represent mean±SEM of triplicate determinations.
FIGS. 14 A-D. Depict photographs showing temperature dependent and competitive internalization of p28 and p18 into UISO-Mel-2 cells. Penetration of Alexafluor 568 labeled p28 (A) or p18 (B) at 2011M was evaluated by confocal microscopy at different temperatures. (C) and (D) Confocal analysis of entry of Alexafluor 568 labeled p28 (C) or p18 (D) at 5 μM into UISO-Mel-2 cells after 30 min at 37° C. in the presence/absence of unlabeled peptide (200 fold excess).
FIGS. 15 A-D. (A) Depicts photographs showing confocal analysis of 28, p18 (20 μM) and Arg8 (SEQ ID NO: 82) (10 μM) entry into UISO-Mel-2 cells after 1 hr at 37° C. in the presence/absence of heparin sulfate (100 μg/ml). (B) Graphs showing flow cytometric analysis of p28 or p18 entry in the presence of inhibitors. Cell fluorescence intensity in the absence of inhibitor (control) was considered as 100%. (C) Graphs depicting FRCS analysis of p28 and p18 entry into fibroblasts in presence of inhibitors. (D) Depicts photographs showing colocalization of p18 and p28 with caveolin I (Panel 1). UISO-Mel-2 cells were incubated with Alexafluor 568 labeled p18 or p28 (20 μM) or media for 2 hrs at 37° C. Cells were fixed and processed for anti-caveolin 1 immunostaining. Confocal analysis of entry of Alexafluor 568 labeled p18 or p28 (20 μM) into UISO-Mel-2 cells after 2 hrs at 37° C. followed by antigolgin 97 antibodies (Panel 2). Colocalization of Alexafluor 568 labeled azurin, p28 and p18 (red) with mitotracker (green) (Panel 3) and Lysotracker (green) (Panel 4) dyes in UISO-Mel-2 cells. Cells were incubated at 37° C. with 20 μM azurin, p28, p18 or media only. After 90 min incubation, mitotracker/lysotracker probes were added and cells incubated for 30 min. Cells were counterstained with DAPI (blue). Colocalization of azurin, p28 or p18 appears as a yellow florescence.
FIGS. 16 A and B. Graphs depicting UISO-Mel-2 cells that were incubated with increasing concentrations of azurin, p28, or p18 at 37° C. for 72 hrs. MTT (A); Direct cell count (B). Cell viability (MTT) or cell number in control wells were considered as 100%. Data represent mean±SEM.
FIG. 17. Graphs depicting growth inhibition of human breast cancer cells by p28. MCF-7 cells were incubated with p28 (0-200 μM) at 37° C. for 24, 48 and 72 h. Cell count (A) and MTT assays (B). Doxorubicin (10 μM) was used as a positive control. Cell number or viability of control wells were considered as 100%. Data represent mean % of control±SEM. *, p<0.05. (C) Inhibition of MCF-7 xenograft growth by p28. A minimum of 10 mice per group were treated with paclitaxel 15 μmol/kg i.p. on days 10, 14, 21 and 25 or 5 or 10 mg/kg p28 i.p. daily for 30 days. Bars represent Mean±SEM. *, p<0.05.
FIG. 18. (A) and (B) are graphs depicting FACS analyses of cell cycle and penetration of breast cancer cells by p28. MCF-7 (A) and MDD2 cells (B) were treated with p28 (50 μM) for 48 and 72 hr. Cells were stained with propidium iodide and analyzed by flow cytometry as described in Yamada, et al., Proc Natl Acad Sci USA, 101:4770-4775 (2004). The percentage of cells in the G1, S, G2/M and sub-G1 (apoptosis) phases are indicated. (C) contains photographs depicting MCF-7 and MDD2 cells cultured on cover slips overnight in phenol-red free MEM, which were treated with 20 μM p28 or 10 μM of the cationic (positive control) peptide, octaarginine (Arg8, SEQ ID NO: 82), for 2 hr at 37° C. Red-Alexa fluor 568 labeled p28, Blue-DAPI (nucleus).
FIG. 19. (A)-(D) are photographs depicting p53 and p28 levels in cells. (A) p53 levels in MCF-7 cells with time after incubation with p28. (%) increase relative to p53 level immediately prior to treatment (0 hr as 100%). (B) GST pull-down assay demonstrating complex formation between GST-p28 and p53. Left to right GST-p28 (10 and 20 μg/reaction), GST-MDM2 and GST alone. p53 was detected by immunoblotting (IB) using anti-p53 antibody. (C) p53 was pulled down by GST-MDM2 in the presence of a molar excess of p28 (upper). Three different anti-p53 antibodies, Pab 1801 (32-79 aa), ab 2433 (277-296aa) and Pab1802 (306-393aa) reacted with GST-p53 immobilized beads in the presence of p28. p28 detected by IB using an anti-p28 antibody (lower). (D) Competition for p28 binding to GST-p53 by a molar excess of p28 fragments p12, p18 and p18b. Relative amount of binding (p28 alone expressed as 100%). M: p28 marker. (E) is a graph depicting p53 DNA-binding in MCF-7 nuclear extracts after exposure to p28 or azurin. Nuclear extracts of H2O2-treated MCF-7 cells served as an internal control. The p53-oligonucleotide complex was quantified with a monoclonal antibody to p53. Data are expressed as Mean±SEM of triplicates.
FIG. 20. Photographs depicting induction of the cyclin (CDK and CDKI) cascade by p28. MCF-7 (A) and MDD2 cells (B) were exposed to p28 (50 μM) for 24, 48 and 72 hr and protein levels determined by immunoblotting. Intracellular localization and relative level of p21 (C) and cyclin B1 (D) MCF-7 cells were cultured on cover slips with p28 for 72 h. p21 and cyclin B were stained with corresponding to the specific antibodies. (E) Phosphorylated cdc2 was estimated with an anti p-cdc2 antibody (Santa Cruz Biotechnology, CA). All results normalized by actin as an internal control.
FIG. 21 Pictures depicting protein structures. A) Azurin truncation with alpha-helical structure; B) Result of 70 ns simulation; C) Measurement of thioether bridge positions based on distances between Cα atoms in a simulated structure.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, the term “cell” includes either the singular or the plural of the term, unless specifically described as a “single cell.”
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid. The terms also apply to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination and they may be circular (with or without branching), generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods as well.
As used herein, the term “pharmacologic activity” means the effect of a drug or other chemical on a biological system. The effect of chemical may be beneficial (therapeutic) or harmful (toxic). The pure chemicals or mixtures may be of natural origin (plant, animal, or mineral) or may be synthetic compounds.
As used herein, the term “premalignant” means precancerous, or before abnormal cells divide without control.
As used herein, the term “antiestrogen” means a substance that blocks the activity of estrogens, or otherwise prevents cells from making or using estrogen.
As used herein, the term “ER” means estrogen receptor.
As used herein, the term “lesion” means an area of abnormal tissue.
As used herein, the term “pathological condition” includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions, and is a response to various factors (as malnutrition, industrial hazards, or climate), to specific infective agents (as worms, parasitic protozoa, bacteria, or viruses), to inherent defects of the organism (as genetic anomalies), or to combinations of these factors.
As used herein, the term “condition” includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.
As used herein, the term “suffering from” includes presently exhibiting the symptoms of a pathological condition, having a pathological condition even without observable symptoms, in recovery from a pathological condition, or recovered from a pathological condition.
As used herein, the term “chemoprevention” is the use of drugs, vitamins, or other natural or synthetic agents, which may be biologic or chemical, to try to reduce the risk of, prevent, suppress, reverse, or delay the development, or recurrence of, premalignant lesions and/or cancer.
As used herein, the term “second chemopreventive agent” refers to drugs, vitamins, or other natural or synthetic agents, which may be biologic or chemical, which reduce the risk of, prevent, suppress, reverse, or delay the development of, or recurrence of, premalignant lesions and/or cancer, other than truncations of azurin.
A used herein, the term “treatment” includes preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms associated with a condition being treated. As such, the term “treatment” includes medical and/or prophylactic administration, as appropriate. Treatment may also include preventing or lessening the development of a condition, such as premalignant lesions or cancer.
As used herein, the term “inhibit cell growth” means the slowing or ceasing of cell division and/or cell expansion. This term also includes the inhibition of cell development or increases in cell death.
A “therapeutically effective amount” is an amount effective to prevent, lower, stop or reverse the development of, or to partially or totally alleviate the existing symptoms of a particular condition for which the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
The term “substantially pure,” as used herein, when used to modify a protein or other cellular product of the invention, refers to, for example, a protein isolated from the growth medium or cellular contents, in a form substantially free of; or unadulterated by, other proteins and/or other compounds. The term “substantially pure” refers to a factor in an amount of at least about 75%, by dry weight, of isolated fraction, or at least “75% substantially pure.” More specifically, the term “substantially pure” refers to a compound of at least about 85%, by dry weight, of isolated fraction, or at least “85% substantially pure.” Most specifically, the term “substantially pure” refers to a compound of at least about 95%, by dry weight, of isolated fraction, or at least “95% substantially pure.” The term “substantially pure” may also be used to modify a synthetically-made protein or compound of the invention, where, for example, the synthetic protein is isolated from the reagents and by-products of the synthesis reaction(s).
The term “pharmaceutical grade,” as used herein, when referring to a peptide or compound of the invention, is a peptide or compound that is isolated substantially or essentially from components which normally accompany the material as it is found in its natural state, including synthesis reagents and by-products, and substantially or essentially isolated from components that would impair its use as a pharmaceutical. For example, a “pharmaceutical grade” peptide may be isolated from any carcinogen. In some instances, “pharmaceutical grade” may be modified by the intended method of administration, such as “intravenous pharmaceutical grade,” in order to specify a peptide or compound that is substantially or essentially isolated from any substance that would render the composition unsuitable for intravenous administration to a patient. For example, an “intravenous pharmaceutical grade” peptide may be isolated from detergents, such as SDS, and anti-bacterial agents, such as azide.
The terms “isolated,” “purified” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. An “isolated” region of a polypeptide refers to a region that does not include the whole sequence of the polypeptide from which the region was derived. An “isolated” nucleic acid, protein, or respective fragment thereof has been substantially removed from its in vivo environment so that it may be manipulated by the skilled artisan, such as but not limited to, nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in substantially pure quantities.
The term “variant” as used herein with respect to a peptide, refers to amino acid sequence variants which may have amino acids replaced, deleted, or inserted as compared to the wild-type polypeptide. Variants may be truncations of the wild-type peptide. A “deletion” is the removal of one or more amino acids from within the polypeptide, which a “truncation” is the removal of one or more amino acids from one or both ends of the polypeptide. Thus, a variant peptide may be made by manipulation of genes encoding the polypeptide. A variant may be made by altering the basic composition or characteristics of the polypeptide, but not at least some of its pharmacologic activities. For example, a “variant” of azurin can be a mutated azurin that retains its ability to inhibit the development of premalignant mammalian cells. In some cases, a variant peptide is synthesized with non-natural amino acids, such as e-(3,5-dinitrobenzoyl)-Lys residues. Ghadiri & Fernholz, J. Am. Chem. Soc., 112:9633-9635 (1990). In some embodiments, the variant has not more than 20 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 15 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 10 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 6 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 5 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 3 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof.
The term “amino acid,” as used herein, means an amino acid moiety that comprises any naturally-occurring or non-naturally occurring or synthetic amino acid residue, i.e., any moiety comprising at least one carboxyl and at least one amino residue directly linked by one, two three or more carbon atoms, typically one (a) carbon atom.
The term “derivative” as used herein with respect to a peptide refers to a peptide that is derived from the subject peptide. A derivation includes chemical modifications of the peptide such that the peptide still retains some of its fundamental activities. For example, a “derivative” of azurin can, for example, be a chemically modified azurin that retains its ability to inhibit angiogenesis in mammalian 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 may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.
The term “percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a polypeptide that are identical with 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 the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine 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 a specific embodiment, Blastp (available from the National Center for Biotechnology Information, Bethesda Md.) is used using the default parameters of long complexity filter, expect 10, word size 3, existence 11 and extension 1.
When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:

% amino acid sequence identity=X/Y*100

where

    
    
        X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and
        Y is the total number of amino acid residues in B.
    
    


If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. When comparing longer sequences to shorter sequences, the shorter sequence will be the “B” sequence. For example, when comparing truncated peptides to the corresponding wild-type polypeptide, the truncated peptide will be the “B” sequence.
General
The present invention provides compositions comprising cupredoxin, and variants, derivatives, truncations, and structural equivalents of cupredoxins, and methods to prevent the development of cancer in mammals. The invention also provides compositions comprising chemopreventive cupredoxins or variants, derivatives, truncations, and structural equivalents thereof and other chemopreventive agents.
The invention also provides variants, derivatives, truncations, and structural equivalents of cupredoxin that retain the ability to prevent the development of cancer or the re-occurrence of cancer in mammals. The invention also provides compositions comprising Pseudomonas aeruginosa azurin, variants, derivatives, truncations, and structural equivalents of azurin, and their use to treat patients, and particularly patients at a higher risk of developing cancer than the general population. Finally, the invention provides methods to study the development of cancer in mammalian cells, tissues and animals by contacting the cells with a cupredoxin, or variant, derivative, truncation, or structural equivalent thereof, before or after inducing premalignant lesions, and observing the development of premalignant and/or malignant cells. For each of these embodiments, the invention provides the combination or co-administration of the cupredoxins or variants, derivatives, truncations, and structural equivalents thereof with another chemopreventive agent.
Preferential Entry into Cells
A redox protein elaborated by Pseudomonas aerugisnosa, the cupredoxin azurin, selectively enters J774 lung cancer cells but not normal cells, and induces apoptosis. Zaborina et al, Microbiology 146:2521-2530 (2000). Azurin can also selectively enter and kill human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells. Yamada et al., PNAS 99:14098-14103 (2002); Punj et al., Oncogene 23:2367-2378 (2004). Azurin thus targets cancer cells but not normal cells because of its inability to enter normal cells. Fialho et al., Recent Patents Anticancer Drug Discov. 2; 224-234 (2007).
Azurin from P. aeruginosa preferentially enters J774 murine reticulum cell sarcoma cells, forms a complex with and stabilizes the tumor suppressor protein p53, enhances the intracellular concentration of p53, and induces apoptosis. Yamada et al., Infection and Immunity 70:7054-7062 (2002). Detailed studies of various domains of the azurin molecule showed that amino acids 50-77 (p28) (SEQ ID NO: 2) represented a protein transduction domain (PTD) critical for internalization and subsequent apoptotic activity. Yamada et al., Cell. Microbial. 7:1418-1431 (2005).
The temperature dependent entry of cationic cell penetrating peptides (“CPPs”), which supports an endocytotic component to cell penetration, is reflected in the entry of azurin and aa fragment 50-77 (p28, SEQ ID NO: 2). Yamada, T., et al., Cell Microbiol 7: 1418-1431 (2005). The entry of 50-67 of azurin (p18, SEQ ID NO: 25) into normal and malignant cells appears accelerated relative to p28. The lower Km and higher Vmax of p18 suggest that aa 50-67 define an amphipathic structure when associated with phospholipid membranes that more closely represents the actual PTD of azurin. However, an energy dependent endocytotic or pore related process is not the only entry mechanism available to these peptides. For example, the metabolic and membrane potential inhibitors sodium azide and ouabain (Na+ K+ ATPase inhibitor), which inhibit the entry- of cationic peptides, did not impair the entry of either p18 or p28 into UISO-Mel-2 cells or fibroblasts (FIG. 15 B,C), suggesting that either peptide may penetrate the cell membrane directly.
p18, p28 and azurin penetrate the plasma membrane and reach late endosomes, lysosomes and the golgi associated with caveolae in what has been described as a dynamin-independent clathrinin dependent carrier mediated manner. Kirkham, M. and Parton, R. G., Biochem Biophys Acta 1746: 349-363 (2005). The striking inhibition of penetration by nocodazole and relative lack of inhibition by cytochalasin-D, which disrupts actin filaments, supports caveolae mediated entry. This route of entry has been described for integral cell surface components and seemingly disparate molecules, i.e., dextran, and a broad range of pathogens or their products that also utilize caveolae to bypass classic endocytic pathways. Depletion of cholesterol from the plasma membrane with β-methylcylodextran, filipin or nystatin to disrupt lipid rafts, plasma membrane domains that provide fluid platforms to segregate membrane components and compartmentalize membranes, significantly inhibited the penetration of p18 (50%) and p28 ( ̃60%) into UISO-Mel-2 cells and fibroblasts (35% and 42%, respectively) demonstrating that a significant percentage ( ̃60%) of p18 and p28 penetrates the plasma membrane via caveolac. Caveolae are a 50- to 100-mm omega-shaped subset of lipid raft invaginations of the plasma membrane defined by the presence of caveolin specific proteins (caveolin-1, -2, or -3) that function as regulators of signal transduction.
Brefeldin A disrupts the Golgi apparatus and inhibited p18 accumulation, so it follows that this pathway is also utilized in p18 and p28 entry and intracellular transport. Cell penetration of p18 and p28 via caveolae comports with the evidence that inhibitors of N-glycosylation reduce cell entry by 60% in UISO-Mel-2 cells and 25% and 35% respectively in fibroblasts. The percentile differences between p18 and p28 entry relate to the numbers of N-glycosylation membrane structures in cancer vs normal cells and the relative route of entry of p28 and p18 via this mechanism. FIG. 15 B, C.
Azurin, p28, and p18 all bind to cancer cells with high affinity and high capacity relative to many other potential anti-cancer peptides. It is believed that, after binding, this protein/receptor complex localizes in caveolae and is internalized, eventually moving (via caveosomes) to the golgi, ER, and nucleus. In addition to caveolar-mediated entry, kinetic analysis also demonstrates that p28 and p18 penetrate the plasma membrane via a non-clathrin caveolae mediated process. A clathrin- and caveolin-independent pathway can exist as a constitutive internalization mechanism, such as for the interleukin 2 receptor and for certain glycosyl-phosphatidylinositol (GPI)-anchored proteins. Lamaze, C., et al, Mol Cell 7: 661-671 (2001); Sabharanjak, S., et al, Dev Cell, 2: 411-423 (2002).
An increase in caveolin-1 expression in cancer cells over normal cells is not likely to be the sole basis for the preferential entry of azurin, p28 and p18 into cancer cells. Fibroblasts and a number of other normal cells also have significant numbers of caveolae on their surface. The findings reflected in Examples 6-12 demonstrate that the cellular penetration of aa 50-67 and 50-77 of azurin is unique relative to all current CCPs in its preference for cancer cells, and that the C-terminal 10-12 amino acids of p28, aa 50-77 of azurin, contain the domain responsible for cell cycle inhibition and apoptitic activity.
Chemoprevention
Azurin, and peptides derived from azurin, such as p28 (SEQ ID NO: 2), have chemopreventive properties. Azurin and p28 prevent the formation of premalignant preneoplastic lesions in mouse mammary gland organ culture. In a mouse mammary gland organ culture model, azurin at 50 μg/ml was found to inhibit the formation of alveolar lesions by 67%. Likewise, p28 at 25 μg/ml was found to inhibit the formation of alveolar lesions by 67%. See Example 1. Further, azurin at 50 μg/ml was found to inhibit the formation of ductal lesions by 79%, and p28 at 25 μg/ml inhibited the formation of ductal lesions by 71%. See Example 1. Confocal microscopy and FAC showed that azurin and p28 entered normal murine mammary epithelial cells (MM3MG) and mammary cancer cells (4T1). p28 also entered human umbilical vein endothelial cells (HUVEC) in a temperature, time and concentration dependent manner and inhibited capillary tube formation of HUVEC plated on Matrigel® in a dose dependent manner.
Further support for the efficacy of variants, derivatives, and structural equivalents of cupredoxins and specifically azurin and p28 in preventing the formation of premalignant preneoplastic lesions is found in Examples 2 through 4. Both azurin and p28 inhibited the development of mammary alveolar lesions as well as in ovarian hormone dependent mammary ductal lesions in MMOC. p28 inhibited the development and multiplicity of MAL and MDL in dose dependent manner. The inhibition was due to antiproliferative effects, as evident from reduced Ki67 expression in these glands. Estrogen and progesterone treated glands expressed ER and PR, however the glands incubated with aldosterone and hydrocortisone did not stain for steroid receptors when analyzed with immunohistochemistry. Since p28 and azurin inhibited development of both hormone independent MAL and estrogen progesterone dependent MDL, it can be concluded that azurin and p28 action are not totally dependent on the estrogen and progesterone receptors and may be efficacious for both hormone independent and dependent lesions.
The most likely mechanism of azurin and p28 in inhibiting preneoplastic lesions is disclosed in the Examples below. Using computational procedures of rigid-body protein docking and cluster analyses, the binding of p53 and azurin has been extensively studied and the structural results have been validated using computational mutagenesis approaches. See Apivo et al., Biochem Biophys Res Commun. 332: 965-968 (2005). The later reports strongly suggest that the mechanism of azurin action in suppressing tumor cell growth is mediated by physical interactions with p53, as discussed herein. The status of p53 expression in breast cancer suggests that nearly 50-60% of the breast cancers contain wild type p53. Junk et al., Neoplasia. 10: 450-461 (2008); Gasco et al., Breast Cancer Res. 4(2):70-76 (2002). It has also been reported by several investigators that inactivation of p53 is one of the frequent and significant events in human cancer. Kastan, Cell. 128; 837-840 (2007).
The inactivation of p53 or its suppression of action is observed in estrogen receptor (ER) positive breast cancer cells. In MCF 7 cells, ERα directly binds to p53 and inactivates its function. Sengupta et al., Ann N Y Acad. Sci. 1024: 54-71 (2004); Konduri et al, Cancer Res. 2007; 67: 7746-7755 (2007). These studies address a clear possibility of a mechanism of ERα-mediated cell proliferation and suppression of p53 mediated apoptosis. This interaction of ERα and p53 has been demonstrated by ChIP assays. Radiation is shown to be effective in restoring the stable p53 and suppresses surviving expression in these cells. The mechanism by which radiation mediates its effects is not well understood, however it is postulated that this may be direct effect of disruption of ERα-p53 association by radiotherapy. Liu et al., J. Biol. Chem. 281: 9837-9340 (2006).
It is now known that p28 not only increases accumulation of nuclear p53 in mammary cells, but also induces expression of p53 mRNA. The results disclosed in Examples 2-4 herein indicate that the interference by estrogen with p53 may result in decreased p53 expression in mammalian cells. However, inhibition of ductal lesions induced by DMBA in the presence of estradiol and increased nuclear accumulation of p53 following p28 treatment shows that p53 stabilization by p28 may be responsible for the suppression of MDL formation in these glands.
It is also now known that p28 enhances the expression of p53 and thus has an inhibitory effect on the formation of premalignant preneoplastic lesions. See, for example, Examples 13-17. The results discussed in Example 4 show that the presence of estrogen interferes with p28-mediated induction of p53 mRNA, but that the p53/p28 interaction is unaffected likely due to different binding sites on p53 protein for ER and p28. Another interesting observation was the difference in azurin and p28 action on p53 mRNA expression.
It is therefore now known that cupredoxins and variants, derivatives, truncations, and structural equivalents of cupredoxins and specifically azurin and variants, derivatives, truncations, and structural equivalents of azurin, including p28, may be used to inhibit, suppress, delay, prevent, and/or reverse the formation of premalignant preneoplastic lesions, and thus the development of cancer, and specifically breast cancer, in mammalian patients.
As shown by Examples 3 and 4, p28 or azurin, in combination with Tamoxifen, has an unexpected, increased inhibitory effect on the formation of premalignant preneoplastic lesions, and thus the development of cancer. Tamoxifen has been successfully used in the treatment of breast cancer patients that are ER positive. It has also been approved for the use in breast cancer prevention trials. Tamoxifen inhibits development of mammary ductal lesions (MDL). It is established that the action of Tamoxifen is mediated via the estrogen receptor pathway. It was not expected that Tamoxifen, in combination with a cupredoxin and/or variants, derivatives, or structural equivalents of cupredoxins, such as azurin, would have a synergistic effect on the formation of premalignant preneoplastic lesions.
The ER-p53 complex prevents the expression of p53. Tamoxifen treatment prevents ER from binding to p53, so incubation of glands with p28 in the presence of Tamoxifen enhances or increases the expression of p53 in these glands. As shown in Example 4 and FIG. 7A, incubation of glands with Tamoxifen and p28 resulted in an unexpected 120 fold increase in p53 mRNA expression as compared to the glands treated with p28 alone.
Due to the high degree of structural similarity between cupredoxins, it is likely that other cupredoxins and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, will inhibit the formation of premalignant lesions in mammals as well as azurin and p28, and would, in combination with Tamoxifen, will have synergistic effects on the formation of premalignant lesions and cancer. Such cupredoxins may be found in, for example, bacteria or plants. Several cupredoxins are known to have pharmacokinetic activities similar to those of azurin from Pseudomonas aeruginosa. For example, rusticyanin from Thiobacillus ferrooxidans can also enter macrophages and induce apoptosis. Yamada et al., Cell Cycle 3:1182-1187 (2004); Yamada et al., Cell. Micro. 7:1418-1431 (2005). Plastocyanin from Phormidium laminosum and pseudoazurin form Achromobacter cycloclastes also are cytotoxic towards macrophages. U.S. Pat. Pub. No. 20060040269, published Feb. 23, 2006. It is therefore contemplated that other cupredoxins may be used in the compositions and methods of the invention. Further, variants, derivatives, truncations, and structural equivalents of cupredoxins that retain the ability to inhibit the formation of cancer in mammals may also be used in the compositions and methods of the invention. These variants and derivatives may include, but are not limited to, truncations of a cupredoxin, conservative substitutions of amino acids and proteins modifications such as PEGylation and all-hydrocarbon stabling of α-helices.
Inhibition of Cancer Growth Through p53
The interaction of amino acids 50-77 of azurin (p28, SEQ ID NO: 2) and p53 was studied and is described in Examples 13-17 below. As disclosed herein, p28 penetrates and exhibits an anti-proliferative effect on human breast cancer cells that is mediated by p53, a tumor suppressor protein that becomes functionally active in response to stress and triggers either cell cycle arrest or cell death. Experiments using a series of GST-pull down assays, glycerol gradient centrifugation, microcalorimetric experiments, single molecule force spectroscopy, and computer modeling show that azurin binds within either the N-terminal or DNA binding domains of p53 and increases its intracellular levels. The results disclosed in Examples 13-17 herein refine the binding site(s) for p28 to within aa 1-17, 24-31, 80-276 or 297-305, the N-terminal and DNA binding domains of p53.
Suggestions that the azurin binding domain for p53 includes a hydrophobic patch described by azurin Met44 and Met64 are supported by evidence that a disrupted hydrophobic patch mutant (mutant azurin M44KM64E) is less cytotoxic to human melanoma (Mel-2) cells than wt azurin. This shows that the p53 binding domain of the azurin molecule surrounds the hydrophobic patch. A recent docking simulation study demonstrated a significant loss of 75 kJ/mol in the interaction free energy of the mutant complex with respect to wild type azurin, again indicating that the hydrophobic patch of azurin surrounding residues Met44 and Met64 is important for interaction with p53. As Met64 resides within the p53 binding site of p28 (aa 15 of p28), competition assays, mutant studies, and docking experiments clearly show that this is the azurin domain that binds to p53.
The tumor suppressor protein p53 is a predominantly nuclear protein that acts as a transcriptional regulator for many genes, including the 21 kDa protein p21/Waf1/Cip1, an inhibitor of cell cycle progression. Treatment of MCF-7 cells with p28 increased p53 levels, leading to higher intracellular levels of p21, a strong inhibitor of cyclin dependent kinase (CDK) activity, especially cdc2 and CDK2 that regulate cell cycle progression at G1 and G2/M, respectively. In the progression through the G2/M phase, cdc2 and CDK2 kinases are activated primarily in association with cyclin B and cyclin A, respectively. The CDK inhibitor p21 associates efficiently with cyclin A in G2/M arrested cells, although under the same conditions, cyclin B1 does not associate with p21 and the level of cyclin B1 increases continuously. This shows that the p28 induced G2/M arrest in MCF-7 cells is associated with inhibition of CDK2 and cyclin A (FIG. 20 A).
The p28-induced increase in p21 in MCF-7 cells was also accompanied by a time-dependent increase in p27, another member of the Cip/Kip CDKI family. Hsu et al., 2008 recently demonstrated that induction of p53 increased both p21 and p27 promoter activity as determined by luciferase assay. Cellular and Molecular Life Sciences (CMLS), 2008. In addition, p53 DNA binding activity of the p21 and p27 promoters is activated by the p53 inducer, progesterone, which means that not only p21, but also p27 is transcriptionally regulated by p53. Collectively, data shows that p28 enhancement of p53 levels subsequently up-regulates p21 and p27, inducing a significant decrease in intracellular CDK2 and cyclin A levels in MCF-7 cells and inhibition of the cell cycle at G2/M (FIG. 20 A). The reported lack of or inefficient association between cyclin B1 and p21, suggests the increase in cyclin B/cdc2 activity following exposure to p28 may reflect a similar pattern following a p28 induced increase in p21. An increase in phosphorylated cdc2 (inactive form) following exposure to p28 accompanied the increased cellular level of cyclin B1, suggesting the increase in the cdc2-cyclin B complex is reflected by the increase in cdc2 phosphorylation. A similar G2 arrest in MCF-7 and MDA-MB-468 human breast cancer cells, accompanied by high levels of cytoplasmic cyclin B1 is induced by nocodazole, a known disrupter of microtubules, and transcriptional and translational activator of p21. Differentiation agents such as all-trans retinoic acid (ATRA) and sodium butyrate (SB) produce a similar phenomenon of growth inhibition and G1 arrest in oral squamous carcinoma cells that correlates with the induction of G1 phase cell cycle regulatory proteins CDK6, p21 and p27, and the inhibition of the G2 phase cell cycle regulatory protein CDK2. Since p28 did not enhance p21 in MDD2 cells, and p27 appears absent in these cells, the levels of CDK2 and cyclin A were not significantly altered (FIG. 20 B) and no inhibition of cell cycle occurred. Additional evidence for a p28 induced decrease in the CDK2 and cyclin A complex, a key regulator of cdc2 activity in human cells, causing a G1 and G2/M arrest is found in the G2 delay that follows cyclin A RNAi introduction to HeLa cells, which inactivates the CDK2-cyclin A complex causing cell cycle arrest in G2/M.
Although Cip/Kip family proteins such as p21 and p27 are potent inhibitors of cyclin A dependent CDK2, they also act as positive regulators of cyclin D-dependent kinases. Cip/Kip family proteins can stabilize CDK4 and CDK6. CDK4 is amplified and overexpressed in wide variety of tumors including breast, gliomas, sarcomas and carcinomas of the uterine cervix, whereas the CDK6 gene is amplified in certain type of malignancies including squamous cell carcinomas, gliomas and lymphoid tumors. Although, the initial or control level of CDK6 is lower than CDK4, CDK6, but not CDK4 levels are continuously elevated in MCF-7 cells exposed to p28. Again, there was no alteration in CDK4 and CDK6 in MDD2 cells where p53 and p21 did not increase in response to p28 (FIG. 20 B). The Ink4 group, p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d of CDKIs specifically associates with and inhibits CDK4 and CDK6 which regulate cell cycle progression at G1. Since the p16Ink4a gene is homozygously deleted in MCF-7 cells, Ink4 CDKI proteins should exhibit less of an inhibitory effect on CDK6 than CDK4, providing a rationale for the increase on CDK6 observed in CDK6 in the presence of essentially stable CDK4 levels.
Collectively, these results demonstrate that p28 binds to p53, increasing p53 levels that subsequently amplify anti-proliferative activity through p21 and p27 inactivation of the CDK2-cyclin A complex, causing a G2/M cell cycle arrest in MCF-7 breast cancer cells in vitro and inhibition MCF-7 xenograft growth in athymic mice.
Compositions of the Invention
The invention provides for chemopreventive agents such as cupredoxins and/or variants, derivatives, truncations, or structural equivalents of cupredoxins, alone or in combination with at least one other chemopreventive agent. In some embodiments, the first chemopreventive agent is a cupredoxin or a truncation thereof. In some such embodiments, the first chemopreventive agent is a truncation of azurin, wherein the truncation comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25. The at least one other chemopreventive agent may be an antiestrogen, including one that blocks the activity of estrogen or progesterone, such as Tamoxifen or fenretinide, or one that prevents the production of estrogen or progesterone, such as aromatase inhibitors like exemestane, anastrozole, and letrozole, or other compounds with similar properties.
The invention further provides for chemopreventive agents that are variants, derivatives, truncations or structural equivalents of cupredoxin. In some embodiments, the chemopreventive agent is isolated. In some embodiments, the chemopreventive agent is substantially pure or pharmaceutical grade. In other embodiments, the chemopreventive agent is in a composition that comprises, or consists essentially of, the chemopreventive agent. In other embodiments, the chemopreventive agent is in a composition that comprises both the chemopreventive agent and at least one other chemopreventive agent. In another specific embodiment, the chemopreventive agent is non-antigenic and does not raise an immune response in a mammal, and more specifically a human. In some embodiments, the chemopreventive agent is less that a full-length cupredoxin, and retains some of the pharmacologic activities of the cupredoxins. Specifically, in some embodiments, the chemopreventive agent may retain the ability to inhibit the development of premalignant lesions in the mouse mammary gland organ culture.
The invention also provides compositions comprising at least one chemopreventive agent that is a cupredoxin, or variant, derivative, truncation, or structural equivalent of a cupredoxin, specifically in a pharmaceutical composition, alone or in combination with at least one other chemopreventive agent. In specific embodiments, the pharmaceutical composition is designed for a particular mode of administration, for example, but not limited to, oral, intraperitoneal, or intravenous. Such compositions may be hydrated in water, or may be dried (such as by lyophilization) for later hydration. Such compositions may be in solvents other than water, such as but not limited to, alcohol.
Because of the high structural homology between the cupredoxins, it is contemplated that cupredoxins will have the same chemopreventive properties as azurin and p28. In some embodiments, the cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin, auracyanin, stellacyanin, cucumber basic protein or Laz. In particularly specific embodiments, the azurin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa, Ulva pertussis or Vibrio parahaemolyticus. In a specific embodiment, the azurin is from Pseudomonas aeruginosa. In other specific embodiments, the cupredoxin comprises an amino acid sequence that is SEQ ID NOS: 1-19 and 25.
The invention provides chemopreventive agents that are amino acid sequence variants of cupredoxins which have amino acids replaced, deleted, or inserted as compared to the wild-type cupredoxin. Variants of the invention may be truncations of the wild-type cupredoxin. In some embodiments, the chemopreventive agent of the invention comprises a region of a cupredoxin that is less that the full length wild-type polypeptide. In some embodiments, the chemopreventive agent of the invention comprises more than about 10 residues, more than about 15 residues or more than about 20 residues of a truncated cupredoxin. In some embodiments, the chemopreventive agent comprises not more than about 100 residues, not more than about 50 residues, not more than about 40 residues, not more than about 30 residues or not more than about 20 residues of a truncated cupredoxin. In some embodiments, a cupredoxin has to chemopreventive agent, and more specifically SEQ ID NOS: 1-19 and 25 has to the chemopreventive agent of the invention, at least about 70% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity or at least about 99% amino acid sequence identity.
In specific embodiments, the chemopreventive agent comprises P. aeruginosa azurin residues 50-77 (p28, SEQ ID NO: 2), azurin residues 50-67 (p18, SEQ ID NO: 25), azurin residues 50-68 (p19, SEQ ID NO: 26), azurin residues 50-69 (p20, SEQ ID NO: 27), azurin residues 50-70 (p21, SEQ ID NO: 28), azurin residues 50-71 (p22, SEQ ID NO: 29), azurin residues 50-72 (p23, SEQ ID NO: 30), azurin residues 50-73 (p24, SEQ ID NO: 31), azurin residues 50-74 (p25, SEQ ID NO: 32), azurin residues 50-75 (p26, SEQ ID NO: 33), azurin residues 50-76 (p27, SEQ ID NO: 34) or azurin residues 36-88 (SEQ ID NO: 35). In other embodiments, the chemopreventive agent consists of P. aeruginosa azurin residues 50-77 (SEQ ID NO: 2), azurin residues 50-67 (SEQ ID NO: 25), azurin residues 50-68 (SEQ ID NO: 26), azurin residues 50-69 (SEQ ID NO: 27), azurin residues 50-70 (SEQ ID NO: 28), azurin residues 50-71 (SEQ ID NO: 29), azurin residues 50-72 (SEQ ID NO: 30), azurin residues 50-73 (SEQ ID NO: 31), azurin residues 50-74 (SEQ ID NO: 32), azurin residues 50-75 (SEQ ID NO: 33), azurin residues 50-76 (SEQ ID NO: 34), or azurin residues 36-88 (SEQ ID NO: 35). In other specific embodiments, the chemopreventive agent consists of the equivalent residues of a cupredoxin other that azurin. It is also contemplated that other cupredoxin variants can be designed that have a similar pharmacologic activity to azurin residues 50-77 (SEQ ID NO: 2), or azurin residues 36-88 (SEQ ID NO: 35). To do this, the subject cupredoxin amino acid sequence will be aligned to the Pseudomonas aeruginosa azurin sequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located on the P. aeruginosa azurin amino acid sequence, and the equivalent residues found on the subject cupredoxin sequence, and the equivalent peptide thus designed.
In one embodiment of the invention, the chemopreventive agent contains at least amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus (SEQ ID NO: 20). In another embodiment of the invention, the chemopreventive agent contains at least amino acids 51-77 of Pseudomonas syringae azurin (SEQ ID NO: 21). In another embodiment of the invention, the chemopreventive agent contains at least amino acids 89-115 of Neisseria meningitidis Laz (SEQ ID NO: 22). In another embodiment of the invention, the c chemopreventive agent contains at least amino acids 52-78 of Vibrio parahaemolyticus azurin (SEQ ID NO: 23). In another embodiment of the invention, the chemopreventive agent contains at least amino acids 51-77 of Bordetella bronchiseptica azurin (SEQ ID NO: 24).
The chemopreventive agents may also include peptides made with synthetic amino acids not naturally occurring. For example, non-naturally occurring amino acids may be integrated into the chemopreventive agent to extend or optimize the half-life of the composition in the bloodstream. Such chemopreventive agents include, but are not limited to, D,L-peptides (diastereomer), (for example 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 (for example Lee et al., J. Pept. Res. 63(2):69-84 (2004)), olefin-containing non-natural amino acid followed by hydrocarbon stapling (for example Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)), and peptides comprising ε-(3,5-dinitrobenzoyl)-Lys residues.
In other embodiments, the chemopreventive agent of the invention is a derivative of a cupredoxin. The derivatives of cupredoxin are chemical modifications of the peptide such that the peptide still retains some of its fundamental activities. For example, a “derivative” of azurin can be a chemically modified azurin that retains its ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. Chemical modifications of interest include, but are not limited to, hydrocarbon stabling, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation and glycosylation of the peptide, and the methods and techniques disclosed herein. In addition, a derivative peptide maybe a fusion of a cupredoxin, or variant, derivative or structural equivalent thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe. Derivatives of interest include chemical modifications by which the half-life in the bloodstream of the peptides and compositions of the invention can be extended or optimized, such as by several methods well known to those in the art, including but not limited to, circularized peptides (for example Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), N- and C-terminal modifications (for example Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), and olefin-containing non-natural amino acid followed by hydrocarbon stapling (for example Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)).
In some embodiments, the cupredoxin may be varied using methods that include, but are not limited to, those which decrease the hydrolysis of the peptide, decrease the deamidation of the peptide, decrease the oxidation, decrease the immunogenicity and/or increase the structural stability of the peptide. It is contemplated that two or more of the modifications described herein may be combined in one modified cupredoxin derived peptide, as well as combinations of one or more modifications described herein with other modification to improve pharmacokinetic properties that are well know to those in the art. Many methods to design such variants and derivatives are well known in the art.
Biotransformation
One approach to improving the pharmacokinetic properties of chemopreventive agents, particularly cupredoxin-derived peptides such as truncations of azurin, is to create variants and derivatives of the cupredoxin derived peptides that are less susceptible to biotransformation. Biotransformation may decrease the pharmacologic activity of the peptide as well as increase the rate at which it is eliminated from the patient's body. One way of achieving this is to determine the amino acids and/or amino acid sequences that are most likely to be biotransformed and to replace these amino acids with ones that are not susceptible to that particular transformative process.
In some embodiments, the cupredoxin derived peptides may include unnatural amino acids or modified amino acids. In some embodiments, the introduction of certain unnatural amino acids enhances the pharmacokinetic properties of the cupredoxin derived peptide. Such introduction may be site-specific and may be done to avoid certain biochemical modifications in vivo. Exemplary unnatural amino acids include b-amino acids (e.g., b3 and b2), homo-amino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3-substituted Alanine derivatives, Glycine derivatives, Ring-substituted Phe and Tyr Derivatives, Linear Core Amino Acids and Diamino Acids. Such unnatural amino acids may be incorporated into peptides by site directed modification, ribosomal translation, or by chemical synthesis of the peptide. Each of these methods may be applied in synthesizing cupredoxin derived peptides.
For example, modified cupredoxin derived peptides may be synthesized by the use of wild-type Aminoacyl-tRNA synthetases (AARSs) with unnatural amino acids building for the production of unnatural cupredoxin variants. See Hartman, et al., PLoS One, 2(10): e972 (2007); Miranda, et al., J. Am. Chem. Soc. 129: 13153-13159 (2007). The specificity of the ribosomal translation apparatus limits the diversity of unnatural amino acids that may be incorporated into peptides using ribosomal translation. Over ninety unnatural building blocks that are AARS substrates have been uncovered including side chain and backbone analogs. Hartman, et al., PLoS One, 2(10): e972 (2007). Over fifty unnatural amino acids may be incorporated into peptides with high efficiency using an all-enzymatic translation system, with peptides containing up to thirteen different unnatural amino acids. Hartman, et al., PLoS One, 2(10): e972 (2007). In some embodiments, such amino acids may be incorporated in cupredoxin derived peptides.
Other modifications may include the use of optically active α-amino acids. The use of optically active α-amino acids and their derivatives is being expanded for their use in pharmaceuticals, agrochemicals and as chiral ligands. In particular, chiral glycine and alanine equivalents plan an important role. At least one stereoselective strategy for constructing α-amino acids has been proposed, allowing for enantiopure α-amino acids in predetermined stereochemistry. Lu, et al. “Asymmetric Synthesis of α-amino acids: Preparation and alkylation of monocyclic iminolactones derived from α-Methyl trans-cinnamaldehyde” published on Internet on Sep. 11, 2008 (to be published in J. Org. Chem.), the disclosure of which is incorporated by reference herein. The modified cupredoxin derived peptides may be synthesized using the optically active α-amino acids to produce enantiomerically enriched iterations.
Hydrolysis is generally a problem in peptides containing aspartate. Aspartate is susceptible to dehydration to form a cyclic imide intermediate, causing the aspartate to be converted to the potentially inactive iso-aspartate analog, and ultimately cleaving the peptide chain. For example, in the presence of aspartic acid-proline in the peptide sequence, the acid catalyzed formation of cyclic imide intermediate can result to cleavage of the peptide chain. Similarly, in the presence of aspartic acid-glycine in the peptide sequence, the cyclic intermediate can be hydrolyzed either into the original aspartate form (harmless) or into the iso-aspartate analog. Eventually, all of the aspartate form can be completely converted into the iso-aspartate analog. Similarly sequences with serine can also be dehydrated to form a cyclic imide intermediate that can cleave the peptide chain. Cleavage of the peptide may result in reduced plasma half-life as well as reduced specific pharmacologic activity of the peptide.
It is contemplated that substituting other amino acids for asparagine and/or serine in the sequence of the cupredoxin derived peptide may result in a peptide with improved pharmacokinetic properties such as a longer plasma half-life and increased specific activity of a pharmacologic activity of the peptide. In one contemplated variant, at one or more asparagine residues of the cupredoxin derived peptide may be replaced with another amino acid residue, and specifically a glutamic acid residue. In another contemplated variant, one or more serine residues of the cupredoxin derived peptide may be replaced with another amino acid residue, and specifically a threonine residue. In some variants of cupredoxin derived peptide, one or more asparagine residues and one or more serine residues are substituted. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made.
Deamidation of amino acid residues is a particular problem in biotransformation. This base-catalyzed reaction frequently occurs in sequences containing asparagine-glycine or glutamine-glycine and follows a mechanism analogous to the aspartic acid-glycine sequence above. The de-amidation of the asparagine-glycine sequence forms a cyclic imide intermediate that is subsequently hydrolyzed to form the aspartate or iso-asparate analog of asparagine. In addition, the cyclic imide intermediate can lead to racemization into D-aspartic acid or D-iso-aspartic acid analogs of asparagine, all of which can potentially lead to inactive forms of the peptide.
It is contemplated that deamidation in the cupredoxin peptides may be prevented by replacing a glycine, asparagine and/or glutamine of the asparagine-glycine or glutamine-glycine sequences of the cupredoxin with another amino acid and may result in a peptide with improved pharmacokinetic properties, such as a longer plasma half-life and increased specific activity of a pharmacologic activity of the peptide. In some embodiments, the one or more glycine residues of the cupredoxin derived peptide are replaced by another amino acid residue. In specific embodiments, one or more glycine residues of the cupredoxin derived peptide are replaced with a threonine or an alanine residue. In some embodiments, the one or more asparagine or glutamine residues of the cupredoxin derived peptide are replaced by another amino acid residue. In specific embodiments, one or more asparagine or glutamine residues of the cupredoxin derived peptide are replaced with an alanine residue. In other specific embodiments, the glycine at residues 58 and/or 63 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent glycines of other cupredoxins, are replaced with an alanine or a threonine. In other specific embodiments, the methionine at residue 59 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent methionine residue of another cupredoxin derived peptide, is replaced by an alanine residue. In other specific embodiments, the glycine at residue 63 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent glycine residue of another cupredoxin derived peptide, is replaced by a threonine residue. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made. In specific embodiments, the modified cupredoxin derived peptide of the invention comprises the following sequence, wherein the underlined amino acids are substituted into the wildtype Pseudomonas aeruginosa p28 sequence LSTAADMQAVVTDTMASGLDKDYLKPDD (SEQ ID NO: 42).
Reversible and irreversible oxidation of amino acids are other biotransformative processes that may also pose a problem that may reduce the pharmacologic activity, and/or plasma half-life of cupredoxin derived peptides. The cysteine and methionine residues are the predominant residues that undergo reversible oxidation. Oxidation of cysteine is accelerated at higher pH, where the thiol is more easily deprotonated and readily forms intra-chain or inter-chain disulfide bonds. These disulfide bonds can be readily reversed in vitro by treatment with dithiothreitol (DTT) or tris(2-carboxyethylphosphine) hydrochloride (TCEP). Methionine oxidizes by both chemical and photochemical pathways to form methionine sulfoxide and further into methionine sulfone, both of which are almost impossible to reverse.
It is contemplated that oxidation in the cupredoxin derived peptides may be prevented by replacing methionine and/or cysteine residues with other residues. In some embodiments, one or more methionine and/or cysteine residues of the cupredoxin derived peptide are replaced by another amino acid residue. In specific embodiments, the methionine residue is replaced with a leucine or valine residue. In other specific embodiments, one or more of the methionines at residues 56 and 64 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent methionine residues in other cupredoxin derived peptides, are replaced with leucine or valine. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made. In specific embodiments, the cupredoxin peptides of the invention comprise one of the following sequences, wherein the underlined amino acid is substituted into the wildtype Pseudomonas aeruginosa p28 sequence:








(SEQ ID NO: 43)











LSTAADLQGVVTDGLASGLDKDYLKPDD




or



 








(SEQ ID NO: 44)











LSTAADVQGVVTDGVASGLDKDYLKPDD.







Another biotransformative process that may affect the pharmacologic activity, plasma half-life and/or immunogenicity of the cupredoxin derived peptides is diketopiperazine and pyroglutamic acid formation. Diketopiperazine formation usually occurs when glycine is in the third position from the N-terminus, and more especially if proline or glycine is in position 1 or 2. The reaction involves nucleophilic attack of the N-terminal nitrogen on the amide carbonyl between the second and third amino acid, which leads to the cleavage of the first two amino acids in the form of a diketopiperazine. On the other hand, pyroglutamic acid formation may be almost inevitable if glutamine is in the N-terminus. This is an analogous reaction where the N-terminal nitrogen attacks the side chain carbonyl carbon of glutamine to form a deaminated pyroglutamayl peptide analog. This conversion also occurs in peptide containing asparagine in the N-terminus, but to a much lesser extent.
It is contemplated that diketopiperazine and pyroglutamic acid formation may be decreased in cupredoxin derived peptides by replacing glycine in position 1, 2, or 3 from the N-terminus, proline in position 3 from the N-terminus, or asparagine at the N-terminus of the peptide with another amino acid residue. In some embodiments, a glycine in positions 1, 2, or 3 from the N-terminus of the cupredoxin derived peptide is replaced with another amino acid residue. In specific embodiments, the glycine residue is replaced by a threonine or alanine residue. In another embodiment, a proline at position 3 from the N-terminus of the cupredoxin derived peptide is replaced with another amino acid residue. In specific embodiments, the proline is replaced by an alanine residue. In another embodiment, an asparagine at the N-terminus is replaced with another amino acid residue. In specific embodiments, the asparagine residue is replaced by a glutamine residue. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made.
Another biotransformative process that may affect the pharmacologic activity, plasma half-life and/or immunigenicity of the cupredoxin derived peptide is racemization. This term is loosely used to refer to the overall loss of chiral integrity of the amino acid or peptide. Racemization involves the base-catalyzed conversion of one enantiomer (usually the L-form) of an amino acid into a 1:1 mixture of L- and D-enantiomers. One way to improve stability of the peptide in general is by making a retro-inverso (D-isomer) peptide. The double inversion of peptide structure often leaves the surface topology of the side-chain intact and has been used extensively to stabilize biologically active peptides. Snyder et al., PLoS Biol. 2:0186-0193 (2004). A D-amino acid substituted Tat is internalized into cells as well as the L-amino acid peptide. Futaki et al., J. Biol. Chem. 276:5836-5840 (2001); Huq et al., Biochemistry 38:5172-5177 (1999). In some embodiments, one or more amino acid residues of the cupredoxin derived peptide are replaced by the D-isomer of that amino acid residue. In other embodiments, all of the amino acid residues of the cupredoxin derived peptide are replaced with D-isomers of those residues. In one embodiment, the modified cupredoxin derived peptide is a retro-inverso (D-isomer) version of the cupredoxin derived peptide. In a specific embodiment, the modified cupredoxin derived peptide is








(SEQ ID NO: 45)











DDPKLYDKDLGSAMGDTVVGQMDAATSL.







Other methods to protect a cupredoxin derived peptide from biotransformative degradation are N-acetylation and C-amidation. These derivatives may protect the peptide from degradation and may make the cupredoxin derived peptide more closely mimic the charge state of the alpha amino and carboxyl groups in the native protein. Peptides with the N-acetylation and/or C-amidation can be provided by commercial suppliers. In one embodiment of the invention, the N-terminus of the cupredoxin derived peptide may be acetylated. In another embodiment of the invention, the C-terminus of the cupredoxin derived peptides may be amidated. In one specific embodiment, the modified cupredoxin derived peptide is








(SEQ ID NO: 46)











Acetylation-LSTAADMQGVVTDGMASGLDKDYLKPDD-




amidation.






Cyclization is an additional manner of biotransformation that may be beneficial to therapeutic peptides including the cupredoxins as described herein. Cyclization may stabilize therapeutic peptides, allowing them to be stored longer, be administered at lower doses and be administered less frequently. Cyclization has been shown to protect peptides against peptidase and protease degradation. Cyclization can be done chemically or enzymatically. Enzymatic cyclization is generally less problematic than chemical cyclization, as chemical cyclization can lack in regio- and stereospecificity, can lead to multimerization in lieu of cyclization and can require complicated multistep processes. Indeed, it has been shown that thioether cyclization is more protective and stable than a disulfide bond against proteolytic enzymes.
Enzymatic cyclization has been shown in lantibiotics—(mehtlyl)lanthionine-containing bacterial peptides. E.g., R. Rink, et al., “Lantibiotic Structures as Guidelines for the Design of Peptides That Can Be Modified by Lantibioitic Enzymes” 44 Biochem., 8873-82 (2005); R. Rink, et al., “Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis” 73:6 Applied and Environmental Microbiology, 1792-96 (2007); R. Rink, et al., “NisC, the Cylcase of the Lantibiotic Nisin, Can Catalyze Cyclization of Designed Nonlantibiotic Peptides” 46 Biochem., 13179-89 (2007) (each of which is hereby incorporated by reference in its entirety). Lantibiotics are produced by and inhibit the growth of gram-positive bacteria. In lantibiotics, dehydroalanine and dehydrobutyrine are created by enzyme mediated dehydration of serine and threonine residues. Cysteines are then enzymatically coupled to the dehydrated serine and threonine residues to form thioether cyclizations. Naturally occurring lantibiotics show such couplings via thioether bonds between residues that are up to 19 residues apart. Thioether ring formation depends upon the leader peptide. The location of the cyclization depends upon the cyclase mediated regio- and stereospecific ring closure and the positions of the dehydratable serine and threonine residues.
The best characterized of the lantibiotics is nisin—a pentacyclic peptide antibiotic produced by Lactococcus lactis. Nisin is composed of four methyllanthionines, one lanthionine, two dehydroalanines, one dehydrobutyrine, and twenty-six unmodified amino acids. Nisin's five thioether cross-links are formed by the addition of cysteine residues to dehydroalanine and dehydrobutyrine residues that originate from serine and threonine. Nisin contains thioether-containing amino acids that are posttranslationally introduced by a membrane-associated enzyme complex. This enzyme complex includes: transporter NisT, serine and threonine dehydratase NisB, and cyclase NisC. NisB dehydrates serine and threonine residues, converting them into dehydroalanine and dehydrobutyrine, respectively. This is followed by NisC catalyzed enantioselective coupling of cysteines to the formed dehydroresidues. NisT facilitates the export of the modified prenisin. Another enzyme, NisP cleaves the nisin leader peptide from prenisin.
The cyclase NisC has been well characterized. Li et al, “Structure and Mechanism of the Lantibiotic Cylclase Involved in Nisin Biosynthesis” 311 Science, 1464-67 (2006) (hereby incorporated by reference in its entirety).
An analysis of cyclization in lantibiotics has led to the identification of amino acid sequences and characteristics in peptides that favor cyclization. It has been shown that the NisB enzyme dehydrates more often where certain amino acids flank the serine and threonine residues. It has been shown that cyclization occurs more often in lantibiotic propeptides where hydrophobic, nonaromatic residues are in proximity to the serine and threonine residues. The flanking residues of the modified cysteines are typically less hydrophobic than the flanking residues of the modified threonines and serines. Exceptions have been found, including hexapeptides VSPPAR (SEQ ID NO: 47), YTPPAL (SEQ ID NO: 48) and FSFFAF (SEQ ID NO: 49). The hexapeptides suggest that the presence of a proline at position 3 or 4 or having phenylalanine flanking both sides may prohibit dehydration. The rings are typically formed by coupling a dehydrated residue to a C-terminally located cysteine. However, rings may be formed by coupling a dehydrate residue to a N-terminally located cysteine.
It has also been shown that the nisin dehydrating and transport enzymes are not specific to nisin and may, in fact, be used to modify non-nisin peptides (and non-lantibiotic peptides). NisB has been shown to dehydrate serine and threonine residues in peptides such as human peptide hormones when such peptides are N-terminally fused to the lantibiotic leader peptide. On non-lantibiotic peptides, similar ring formation characteristics apply; namely, the extent of dehydration can be controlled by the amino acid context of the flanking region of the dehydratable serine and threonine residues. The presence of hydrophobic flanking residues (e.g., alanine and valine) around the serines and threonines allowed full dehydration and therefore enhanced thioether ring formation. The presence of an N-terminal aspartate and C-terminally flanked arginine prevented dehydration. It also shown that the presence of proline residues and phenylalanine residues is disfavorable for dehydration. Generally, the presence of hydrophilic flanking residues prevented dehydration of the serine and threonine residues. Hydrophobic flanking favors dehydration; hydrophilic flanking disfavors dehydration. Studies have shown that where dehydration does occur, the average hydrophobicity of the flanking residues of serines and threonine is positive—0.40 on the N-terminal side and 0.13 on the C-terminal side. Also, the average hydrophobicity of the residues flanking serines and threonines that are not dehydrated is negative—−0.36 on the N-terminal side and −1.03 on the C-terminal side. Dehydration is not restricted by the presence of a series of flanking threonine residues and is not restricted by the distance between the nisin leader peptide and the residue to be dehydrated.
NisC has been shown to catalyze the regiospecific formation of thioether rings in peptides unrelated to naturally occurring lantibiotics. Generally, such peptides must be fused to the nisin leader peptide. In some cases, thioether rings may form spontaneously, for example where a dehydroalanine is spaced by two amino acids from a cysteine. Unlike spontaneous cyclization, NisC catalyzed cyclization is stereospecific for dehydrated pre-nisin. Consequently, the methyllanthionines and lanthionine in nisin are in the DL configuration. It is thought that cyclization in nonlantibiotic peptides will also be stereospecific
These principles can be applied to the compounds described herein, including cupredoxins and variants and truncations thereof.
Thioether Bridges
In nature, lantibiotic-enzyme-induced thioether bridges occur with up to 19 amino acids under the bridge. Thioether bridges with 2 to 4 amino acids under the bridge are abundant.
In some embodiments, the chemopreventive agents of the invention, specifically cupredoxins and derivatives, variants, truncations, or structural equivalents of cupredoxins, such as truncated azurin, may be modified by introducing thioether bridges into the structure. The azurin truncation p28 (SEQ ID NO: 2), for example, may be modified using this method. Extended molecular dynamics simulations (70 ns) using software package GROMACS (www.gromacs.org) suggest that, at 37° C., the region of the p28 alpha helix from position 6 to 16 is unstable, and that the peptide tends to adopt a beta sheet conformation. This, together with the fact that the part of the molecule presumed to be responsible for interaction with p53 remains solvent exposed, suggests that introduction of a thioether bridge in this region of the p28 peptide may not affect its functionality.
The amino acid sequence of p28 is SEQ ID NO: 2 (LSTAADMQGVVIDGMASGLDKDYLKPDD). The amino acid sequence known as p18 is SEQ ID NO: 25 (LSTAADMQGVVTDGMASG). The sequence SGLDKD (SEQ ID NO: 85) may interact with p53. Thioether bridges can be formed between Ser/Thr on the N-side to Cys on the C-side. The serine/threonine is dehydrated and subsequently coupled to the cysteine. Threonines are preferred since they are more easily dehydrated than serines. Generally, hydrophobic flanking residues (at least one) to the threonine are preferred since they enhance the extent of dehydration. Negatively charged amino acids, glutamate and aspartate, that are flanking residues have a strong negative effect on dehydration. Generally, hydrophilic flanking residues, especially glycin, do not favor dehydration. Preceding the Cys there is a slight preference for charged hydrophilic residues, especially glutamate/aspartate. Depending on the size of the thioether ring, the bulkiness of the amino acids that participate in the ring matters.
In one embodiment, the truncated azurin sequence is LSTAADMQGVVTDGMASGLDKDYLTPGC (SEQ ID NO: 50). A thioether bridge is formed between positions 25 and 28 of p28, and will be fully protected against carboxyetidases. Positions 2, 3 and 25 will be dehydrated, but neither the import sequence, nor the sequence thought to be relevant for interaction with p53, is altered by thioether ring introduction. As such, peptide activity should not be altered. The threonine is between two hydrophobic amino acids and hence is expected to be fully dehydrated by dehydratase, NisB, according to specific guidelines. See Rink et al., Biochemistry 2005. The same guidelines also predict cyclization involving positions 25 and 28 by cyclase NisC, especially because of the aspartate located before the cysteine.
In another embodiment, the truncated azurin sequence is LSTAADCQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 51) and the thioether bridge is formed between positions 3 and 7. The ring between position 3 and 7 mimics ring A of nisin and makes use of the existing threonine at position 2. The aspartate at position 6 will favor cyclization.
In another embodiment, the truncated azurin sequence is LSTAACMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 52), and the threonine in position 2 is utilized to form a thioether bridge.
In another embodiment, two or more of the thioether rings in the truncated azurins described in the paragraphs above are combined into one peptide.
In another embodiment, many truncated azurin sequences can be created and screened for threonine rings by analyzing the peptides with a ring of one lanthionine and two to three additional amino acids under the sulfur bridge. This might involve one or combinations of the sequences below:










LSTACDMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 53)




 



LSTAATMQCVVTDGMASGLDKDYLKPDD (SEQ ID NO: 54)



 



LSTAATMQGCVTDGMASGLDKDYLKPDD (SEQ ID NO: 55)



 



LSTAANTQGCVTDGMASGLDKDYLKPDD (SEQ ID NO: 56)



 



LSTAANTQGVCTDGMASGLDKDYLKPDD (SEQ ID NO: 57)



 



LSTAADMTAVCTDGMASGLDKDYLKPDD (SEQ ID NO: 58)



 



LSTAADMTAVVCDGMASGLDKDYLKPDD (SEQ ID NO: 59)



 



LSTAADMQTVVCDGMASGLDKDYLKPDD (SEQ ID NO: 60)



 



LSTAADMQTVVTCGMASGLDKDYLKPDD (SEQ ID NO: 61)



 



LSTAADMQATVTCGMASGLDKDYLKPDD (SEQ ID NO: 62)



 



LSTAADMQATVTDCMASGLDKDYLKPDD (SEQ ID NO: 63)



 



LSTAADMQGVTADCMASGLDKDYLKPDD (SEQ ID NO: 64)



 



LSTAADMQGVTADGCASGLDKDYLKPDD (SEQ ID NO: 65)



 



LSTAADMQGVVTNGCASGLDKDYLKPDD (SEQ ID NO: 66)






A practical approach would be to genetically make a large number of such sequences and select a group for purification on the basis of extent of modification and level of production.
In another embodiment, a thioether bridge is formed between a threonine at position 12 in p28 (SEQ ID NO: 2) and the c-terminus of the peptide. The distance between the Cα of position 13 and the aspartate at position 28 might be 17.52 angstroms, larger than 1.5 nanometers, implying significant alteration of the structure of the peptide.
In another embodiment, the peptide sequence is LSTAADMQGVVTATMGSGLCKDYLKPDD (SEQ ID NO: 67), with a thioether bridge from position 14 to position 2 at a distance of 4.38 angstroms. The mutation of aspartate at position 13 to alanine favors dehydration of threonine at position 14. Mutation of alanine at position 16 to glycine completely prevents dehydration of serine at position 17 and enhances cyclization.
In another embodiment, the peptide sequence is LSTAADMQGVVTDLTASGLCKDYLKPDD (SEQ ID NO: 68), with the thioether bridge from position 15 to position 20 at a distance of 5.83 angstroms. In this situation, mutation of glycine at position 14 to leucine favors dehydration of threonine at position 15.
Tertiary Structure Stabilization
The stability of the tertiary structure of the cupredoxin derived peptide will affect most aspects of the pharmacokinetics, including the pharmacologic activity, plasma half-life, and/or immunogenicity among others. See Kanovsky et al., Cancer Chemother. Pharmacol. 52:202-208 (2003); Kanovsky et al., PNAS 23:12438-12443 (2001). Peptide helices often fall apart into random coils, becoming more susceptible to protease attack and may not penetrate cell membrane well Schafmeister et al, J. Am. Chem. Soc. 122:5891-5892 (2000). Therefore, one way to stabilize the overall structure of the peptide is to stabilize the α-helix structure of the peptide. The intra-molecular hydrogen bonding associated with helix formation reduces the exposure of the polar amide backbone, thereby reducing the barrier to membrane penetration in a transport peptide, and thus increasing related pharmacologic activities and increasing the resistance of the peptide to protease cleavage. Id. Pseudomonas aeruginosa azurin (SEQ ID NO: 1) has α-helices at residues 53-56, 58-64 and 68-70.
One method to stabilize an α-helix is to replace in the α-helix helix breaking amino acid residues such as glycine, proline, serine and aspartic acid, or helix neutral amino acid residues such as alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine or arginine, with helix forming residues, such as leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, tryptophan and methionine or helix favoring amino acid residue substitutions, for example α-amino-isobutyric acid (Aib). See Miranda et al., J. Med. Chem., 51, 2758-2765 (2008), the disclosure of which is incorporated by reference herein. It is contemplated that the α-helix of cupredoxin derived peptides may be stabilized by replacing one or more glycine, proline, serine and/or aspartic acid residues with other amino acids. In specific embodiments, the glycine, proline, serine, aspartic acid, alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine and/or arginine residues are replaced by leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, tryptophan, Aib and/or methionine residues. See Lee et al., Cancer Cell Intl. 11:21 (2005). In other specific embodiments, one or more serine or glutamine residues in the α-helices of a cupredoxin derived peptide may be substituted. In still more specific embodiments, the serine and/or glutamine residues in residues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent residues of other cupredoxin derived peptides, may be replaced. In another specific embodiment, the glutamine residue at amino acid residue 57 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue of another cupredoxin derived peptide, may be replaced, more specifically replaced with tryptophan. In another specific embodiment, the threonine residue at amino acid residue 52 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue of another cupredoxin derived peptide, may be replaced, more specifically replaced with tryptophan. In another specific embodiment, the threonine residue at amino acid residue 61 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue of another cupredoxin derived peptide, may be replaced, more specifically replaced with tryptophan. In another specific embodiment, the glycine residue at amino acid residue 63 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue of another cupredoxin derived peptide, may be replaced, more specifically replaced with tryptophan. In another specific embodiment, one or more threonine, glutamine or glycine residues at amino acid residues 52, 57, 61 or 63 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue of another cupredoxin derived peptide, may be replaced, more specifically replaced with tryptophan. In specific embodiments, the cupredoxin peptide comprises one of the following sequences wherein the underlined amino acid is substituted into the wildtype Pseudomonas aeruginosa p28 sequence:








(SEQ ID NO: 69)











LSWAADMQGVVTDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 70)











LSTAADMWGVVTDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 71)











LSTAADMQGVVWDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 72)











LSTAADMQGVVTDWMASGLDKDYLKPDD;




 








(SEQ ID NO: 73)











LSWAADMWGVVTDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 74)











LSWAADMQGVVWDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 75)











LSWAADMQGVVTDWMASGLDKDYLKPDD;




 








(SEQ ID NO: 76)











LSTAADMWGVVWDGMASGLDKDYLKPDD;




 








(SEQ ID NO: 77)











LSTAADMWGVVTDWMASGLDKDYLKPDD;




 








(SEQ ID NO: 78)











LSTAADMQGVVWDWMASGLDKDYLKPDD;




or



 








(SEQ ID NO: 79)











LSWAADMWGVVWDWMASGLDKDYLKPDD.







In other embodiments, equivalent amino acids in other cupredoxin derived peptides are substituted with tryptophan.

Another method to stabilize an α-helix tertiary structure involves using unnatural amino acid residues capable of π-stacking. For example, in Andrews and Tabor (Tetrahedron 55:11711-11743 (1999)), pairs of s-(3,5-dinitrobenzoyl)-Lys residues were substituted into the α-helix region of a peptide at different spacings. The overall results showed that the i,(i+4) spacing was the most effective stabilizing arrangement. Increasing the percentage of water, up to 90%, increased the helical content of the peptide. Pairs of ε-acyl-Lys residues in the same i,(i+4) spacing had no stabilizing effect, indicating that the majority of the stabilization arises from π-π interactions. In one embodiment, the cupredoxin derived peptide may be modified so that the lysine residues are substituted by ε-(3,5-dinitrobenzoyl)-Lys residues. In a specific embodiment, the lysine residues may be substituted by ε-(3,5-dinitrobenzoyl)-Lys in a i,(i+4) spacing.
Another method to stabilize an α-helix tertiary structure uses the electrostatic interactions between side-chains in the α-helix. When His-Cys or His-His residue pairs were substituted in into peptides in an i,(i+4) arrangement, the peptides changed from about 50% helical to about 90% helical on the addition of Cu, Zn or Cd ions. When ruthenium (Ru) salts were added to the His-His peptides, an exchange-inert complex was formed, a macrocyclic cis-[Ru—(NH3)4L2]3+ complex where L2 are the side chains of two histidines, which improved the helix stability. Ghadiri and Fernholz, J. Am. Chem. Soc. 112, 9633-9635 (1990). In some embodiments, the cupredoxin derived peptides may comprise macrocyclic cis-[Ru—(NH3)4L2]3+ complexes where L2 is the side chains of two histidines. In some embodiments, one or more histidine-cysteine or histidine-histidine residue pairs may be substituted an i,(i+4) arrangement into the α-helices of the cupredoxin derived peptide. In other embodiments, one or more histidine-cysteine or histidine-histidine residue pairs may be substituted an i,(i+4) arrangement in residues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent residues of other cupredoxin derived peptides. In some embodiments, the cupredoxin derived peptide may further comprise Cu, Zn, Cd and/or Ru ions.
Another method to stabilize an α-helix tertiary structure involves disulfide bond formation between side-chains of the α-helix. It is also possible to stabilize helical structures by means of formal covalent bonds between residues separated in the peptide sequence. The commonly employed natural method is to use disulfide bonds. Pierret et al., Intl. J. Pept. Prot. Res., 46:471-479 (1995). In some embodiments, one or more cysteine residue pairs are substituted into the α-helices of the cupredoxin derived peptide. In other embodiments, one or more cysteine residue pairs are substituted at residues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent residues of other cupredoxin derived peptides.
Another method to stabilize an α-helical tertiary structure involves the use of side chain lactam bridges. A lactam is a cyclic amide which can form from the cyclisation of amino acids. Side chain to side chain bridges have been successfully used as constraints in a variety of peptides and peptide analogues, such as amphipathic or model α-helical peptides, oxytocin antagonists, melanoptropin analogues, glucagon, and SDF-1 peptide analogues. For example, the Glucagon-like Peptide-1 (GLP-1) gradually assumes a helical conformation under certain helix-favoring conditions and can be stabilized using lactam bridging. Miranda et at, J. Med. Chem., 51, 2758-2765 (2008). These lactam bridges may be varied in size, effecting stability and binding affinity. Id. Such modifications improved the stability of the compounds in plasma. Id. Depending on the space between the cyclization sites and choice of residues, lactam bridges can be used to induce and stabilize turn or helical conformations. In some embodiments, one or more cupredoxin or variant analogues are prepared with lactam bridging between nearby amino acids (such as i to i+4 glutamic acid-lysine constraints). In some embodiments, the cupredoxin derived peptide may comprise such modifications to enhance α-helix content.
Another method to stabilize an α-helix tertiary structure is the all-carbon cross-link method. The all-hydrocarbon cross-link method is proven to increase the stabilization of helical structure, protease resistant and cell-permeability. Walensky et al., Science, 305, 1466-1470 (2004). α,α-disubstituted non-natural amino acids containing olefin-bearing tethers are incorporated into peptides. Ruthenium catalyzed olefin metathesis generates an all-hydrocarbon “staple” to cross-link the helix. Schafmeister et al., J. Am. Chem. Soc., 122, 5891-5892 (2000); Walensky et al., id. Non-natural amino acids containing olefin-bearing tethers may be synthesized according to methodology provided in Schafmeister et au (id.) and Williams and Im (J. Am. Chem. Soc., 113:9276-9286 (1991)). In some embodiments, the cupredoxin derived peptides are stabilized by all-hydrocarbon staples. In specific embodiments, one or more pairs of α,α-disubstituted non-natural amino acids containing olefin-bearing tethers corresponding to the native amino acids are substituted into the α-helices of the cupredoxin derived peptide. In other embodiments, one or more pairs of α,α-disubstituted non-natural amino acids containing olefin-bearing tethers corresponded to the native amino acids are substituted into residues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1), or equivalent residues of other cupredoxin derived peptides.
In some embodiments, the modified cupredoxin derived peptide may comprise X1SX2AADX3X4X5VVX6DX7X8ASGLDKDYLKPDX9 (SEQ ID NO: 80), where X1 is L or acetylated-L, X2 is T or W, X3 is M, L or V, X4 is Q or W, X5 is G or A, X6 is T or W, X7 is G, T or W, X8 is M, L or V, and X9 is D or amidated-D. In other embodiments, the modified cupredoxin derived peptide may consist of X1SX2AADX3X4X5VVX6DX7X8ASGLDKDYLKPDX9 (SEQ ID NO: 80), where X1 is L or acetylated-L, X2 is T or W, X3 is M, L or V, X4 is Q or W, Xs is G or A, X6 is T or W, X7 is G, T or W, X8 is M, L or V, and X9 is D or amidated-D. In other embodiments, the modified cupredoxin derived peptide may comprise X1DPKLYDKDLGSAX2X3DX4VVX5X6X7DAAX8 SX9 (SEQ ID NO: 81), where X1 is D or acetylated-D, X2 is M, L or V, X3 is G, T or W, X4 is T or W, X5 is G or A, X6 is Q or W, X7 is M, L or V, X8 is T or W, and X9 is L or amidated-L. In other embodiments, the modified cupredoxin derived peptide may consist of X1DPKLYDKDLGSAX2X3DX4VVX5X6X7DAAX8SX9 (SEQ ID NO: 81), where X1 is D or acetylated-D, X2 is M, L or V, X3 is G, T or W, X4 is T or W, X5 is G or A, X6 is Q or W, X7 is M, L or V, X8 is T or W, and X9 is L or amidated-L. Specific peptides of interest are listed in Table 3.
PEGylation
Covalent attachment of PEG to drugs of therapeutic and diagnostic importance has extended the plasma half-life of the drug in vivo, and/or reduced their immunogenicity and antigenicity. Harris and Chess, Nature Reviews Drug Discovery 2:214-221 (2003). For example, PEG attachment has improved the pharmacokinetic properties of many therapeutic proteins, including interleukins (Kaufman et al., J. Biol. Chem. 263:15064 (1988); Tsutsumi et al., J. Controlled Release 33:447 (1995)), interferons (Kita et al., Drug Des. Delivery 6:157 (1990)), catalase (Abuchowski et al., J. Biol. Chem. 252:3582 (1977)), superoxide dismutase (Beauchamp et al, Anal. Biochem. 131:25 (1983)), and adenosine deanimase (Chen et al., Biochem. Biophys. Acta 660:293 (1981)), among others. The FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations. PEG shows little toxicity, and is eliminated from the body intact by either the kidneys (for PEGs <30 kDa) or in the feces (for PEGs >20 kDa). PEG is highly soluble in water.
PEGylation of a chemopreventive agents, particularly cupredoxin-derived peptides such as truncations of azurin, may be used to increase the lifetime of the peptide in the bloodstream of the patient by reducing renal ultrafiltration, and thus reduce elimination of the drug from the body. Charge masking may affect renal permeation. Charge masking may be a consequence of the paramchemical modification of protein ionizable functional group, namely amines or carboxyls. In particular, the most common procedures for producing protein-PEG derivatives involves the conversion of protein amino groups into amides with the consequent loss of positive charges, and this can alter protein ultrafiltration. Since anionic macromolecules have been found to be cleared by renal ultrafiltration more slowly than neutral or positive ones, it could be expected that PEG conjugation to amino groups prolongs the permanence of the PEGylated peptide in the bloodstream.
Molecular size and globular ultrafiltration may also affect renal ultrafiltration of therapeutic peptides. The molecular weight cut off for kidney elimination of native globular proteins is considered to be about 70 kDa, which is close to the molecular weight of serum albumin. Thus, proteins with molecular weight exceeding 70 kDa are mainly eliminated from the body by pathways other than renal ultrafiltration, such as liver uptake, proteolytic digestion and clearance by the immune system. Therefore, increasing the size of a therapeutic peptide by PEGylation may decrease renal ultrafiltration of that peptide form the bloodstream of the patient.
Additionally, PEGylation of a peptide may decrease the immunogenicity of that peptide, as well as protect the peptide from proteolytic enzymes, phagocytic cells, and other factors that require direct contact with the therapeutic peptide. The umbrella-like structure of branched PEG in particular has been found to give better protection than linear PEG towards approaching proteolytic enzymes, antibodies, phagocytic cells, etc. Caliceti and Veronese, Adv. Drug. Deliv. Rev. 55:1261-12778 (2003).
In some embodiments, the cupredoxin derived peptides are modified to have one or more PEG molecules covalently bonded to a cysteine molecule. The covalent bonding does not necessarily need to be a covalent bond directly from the PEG molecule to the cupredoxin derived peptide, but may be covalently bonded to one or more linker molecules which in turn are covalently bonded to each other and/or the cupredoxin derived peptide. In some embodiments, the cupredoxin derived peptide have site-specific PEGylation. In specific embodiments, the PEG molecule(s) may be covalently bonded to the cysteine residues 3, 26 and/or 112 of P. aeruginosa azurin (SEQ ID NO: 1). In other embodiments, one or more cysteine residues may be substituted into the cupredoxin derived peptide and is PEGylated. In some embodiments, the method to PEGylate the cupredoxin derived peptide may be NHS, reductive animation, malimid or epoxid, among others. In other embodiments, the cupredoxin derived peptides may be PEGylated on one or more lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine, or the N-terminal amino group or the C-terminal carboxylic acid. In more specific embodiments, the cupredoxin derived peptides may be PEGylated on one or more lysines or N-terminal amino groups. In other embodiments, one or more lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine residue are substituted into the cupredoxin derived peptides and are PEGylated. In other embodiments, the cupredoxin derived peptides may be PEGylated on one or more amino groups. In other embodiments, the cupredoxin derived peptides may be PEGylated in a random, non-site specific manner. In some embodiments, the cupredoxin derived peptides may have an average molecular weight of PEG-based polymers of about 200 daltons to about 100,000 daltons, about 2,000 daltons to about 20,000 daltons, or about 2,000 daltons to about 5,000 daltons. In other embodiments, the cupredoxin derived peptides may be comprised of one or more PEG molecules that is branched, specifically a branched PEG molecule that is about 50 kDa. In other embodiments, the cupredoxin derived peptides may comprise one or more linear PEG molecules, specifically a linear PEG molecule that is about 5 kDa.
In another embodiment, the chemopreventive agent is a peptide that is a cupredoxin, or variant, structural equivalent, or derivative thereof that is a conjugate of Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and is internalized into cancer cells. The cupredoxin or variant, structural equivalent, or derivative of cupredoxin may be conjugated with Pep42 pursuant to the synthesis methods disclosed in Yoneda et al, “A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy,” Bioorganic & Medicinal Chemistry Letters 18: 1632-1636 (2008), the disclosure of which is incorporated in its entirety herein.
In another embodiment, the chemopreventive agent is a peptide that 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)). Specifically, significant structural homology between a cupredoxin and the structural equivalent may be determined by using the VAST algorithm. Gibrat et al., Curr Opin Struct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995). In specific embodiments, the VAST p value from a structural comparison of a cupredoxin to the structural equivalent may be less than about 10−3, less than about 10−5, or less than about 10−7. In other embodiments, significant structural homology between a cupredoxin and the structural equivalent may be determined by using the DALI algorithm. Holm & Sander, J. Mol. Biol. 233:123-138 (1993). In specific embodiments, the DALI Z score for a pairwise structural comparison is at least about 3.5, at least about 7.0, or at least about 10.0.
It is contemplated that the chemopreventive agents, particularly the peptides, of the composition of invention may be more than one of a variant, derivative and/or structural equivalent of a cupredoxin. For example, the peptides may be a truncation of azurin that has been PEGylated, thus making it both a variant and a derivative. In one embodiment, the peptides of the invention are synthesized with α,α-disubstituted non-natural amino acids containing olefin-bearing tethers, followed by an all-hydrocarbon “staple” by ruthenium catalyzed olefin metathesis. Scharmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walensky et al., Science 305:1466-1470 (2004). Additionally, peptides that are structural equivalents of azurin may be fused to other peptides, thus making a peptide that is both a structural equivalent and a derivative. These examples are merely to illustrate and not to limit the invention. Variants, derivatives or structural equivalents of cupredoxin may or may not bind copper.
In some embodiments, the chemopreventive agent that is a cupredoxin, or variant, derivative or structural equivalent thereof has some of the pharmacologic activities of the P. aeruginosa azurin, and specifically p28. In a specific embodiment, the cupredoxins and variants, derivatives and structural equivalents of cupredoxins may inhibit or prevent the development of premalignant lesions in mammalian cells, tissues or animals, and specifically but not limited to, mammary gland cells. The invention also provides for the cupredoxins and variants, derivatives and structural equivalents of cupredoxins that may have the ability to inhibit the development of mammalian premalignant lesions, and specifically but not limited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin and cervical cancer cells. Inhibition of the development of cancer cells is any decrease, or lessening of the rate of increase, of the development of premalignant lesions that is statistically significant as compared to control treatments.
Because it is now known that cupredoxins can inhibit the development of premalignant lesions and ultimately cancer in mammalian cells, tissues or animals, and specifically breast cells, and more specifically, mouse mammary gland cells, it is now possible to design variants and derivatives of cupredoxins that retain this chemopreventive activity. Such variants, derivatives and structural equivalents can be made by, for example, creating a “library” of various variants, derivatives and structural equivalents of cupredoxins and cupredoxin derived peptides and then testing each for chemopreventive activity, and specifically chemopreventive activity in the mouse mammary gland organ culture using one of many methods known in the art, such the exemplary method in Example 1. It is contemplated that the resulting variants, derivatives and structural equivalents of cupredoxins with chemopreventive activity may be used in the methods of the invention, in place of or in addition to azurin or p28.
In some specific embodiments, the variant, derivative or structural equivalent of cupredoxin may inhibit the development of 7,12-dimethylbenz (a) anthracene (DMBA) induced premalignant lesions in a mouse mammary gland organ culture (MMOC) to a degree that is statistically different from a non-treated control. A peptide can be tested for this activity by using the MMOC model system is described in Example 1, or as in Mehta et al (J Natl Cancer Inst 93:1103-1106 (2001)) and Mehta et al. (Meth Cell Sci 19:19-24 (1997)). Other methods to determine whether cancer development is inhibited another are well known in the art and may be used as well.
In some specific embodiments, the variant, derivative or structural equivalent of cupredoxin inhibits the development of mammary alveolar lesions (MAL) in the a MMOC model to a degree that is statistically different from a non-treated control. In some specific embodiments, the variant, derivative or structural equivalent of cupredoxin inhibits the development of mammary ductal lesions (MDL) in the a MMOC model to a degree that is statistically different from a non-treated control. A peptide can be tested for these activities by using the MMOC model system induced to form premalignant lesions by DMBA, as described in Example 1. Evaluation of development of premalignant lesions in a MMOC model system may be determined by morphometic analysis, or histopathological analysis, as provided in Example 1.
Cupredoxins
These small blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or are of unknown function. The copper ion is solely bound by the protein matrix. A special distorted trigonal planar arrangement to two histidine and one cysteine ligands around the copper gives rise to very peculiar electronic properties of the metal site and an intense blue color. A number of cupredoxins have been crystallographically characterized at medium to high resolution.
The cupredoxins in general have a 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 that of auracyanin B, 16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3% to that of pseudoazurin. See, Table 1. However, the structural similarity of these proteins is more pronounced. The VAST p value for the comparison of the structure of azurin to auracyanin B is 10−7.4, azurin to rusticyanin is 10−5, azurin to plastocyanin is 10−5.6, and azurin to psuedoazurin is 10−4.1.
All of the cupredoxins possess an eight-stranded Greek key beta-barrel or beta-sandwich fold and have a highly conserved site architecture. De Rienzo et al., Protein Science 9:1439-1454 (2000). A prominent hydrophobic patch, due to the presence of many long chain aliphatic residues such as methionines and leucines, is present around the copper site in azurins, amicyanins, cyanobacterial plastocyanins, cucumber basic protein and to a lesser extent, pseudoazurin and eukaryotic plastocyanins. Id. Hydrophobic patches are also found to a lesser extent in stellacyanin and rusticyanin copper sites, but have different features. Id.







TABLE 1







Sequence and structure alignment of azurin (1JZG) from P. aeruginosa to other


proteins using VAST algorithm.














Alignment
% aa






PDB
length1
identity
P-value2
Score3
RMSD4
Description
















1AOZ A 2
82
18.3
10e−7
12.2
1.9
Ascorbate oxidase


1QHQ_A
113
31
10e−7.4
12.1
1.9
AuracyaninB


1V54 B 1
79
20.3
10e−6.0
11.2
2.1
Cytocrome c oxidase


1GY2 A
92
16.3
10e−5.0
11.1
1.8
Rusticyanin


3MSP A
74
8.1
10e−6.7
10.9
2.5
Motile Major Sperm








Protein5


1IUZ
74
20.3
10e−5.6
10.3
2.3
Plastocyanin


1KGY E
90
5.6
10e−4.6
10.1
3.4
Ephrinb2


1PMY
75
17.3
10e−4.1
9.8
2.3
Pseudoazurin





1Aligned Length: The number of equivalent pairs of C-alpha atoms superimposed between the two structures, i.e. how many residues have been used to calculate the 3D superposition.


2P-VAL: The VAST p value is a measure of the significance of the comparison, expressed as a probability. For example, if the p value is 0.001, then the odds are 1000 to 1 against seeing a match of this quality by pure chance. 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 p value shown thus corresponds to the p value for the pairwise comparison of each domain pair, divided by 500.


3Score: The VAST structure-similarity score. This number is related to the number of secondary structure elements superimposed and the quality of that superposition. Higher VAST scores correlate with higher similarity.


4RMSD: The root mean square superposition residual in Angstroms. This number is calculated after optimal superposition of two structures, as the square root of the mean square distances between equivalent C-alpha atoms. Note that the RMSD value scales with the extent of the structural alignments and that this size must be taken into consideration when using RMSD as a descriptor of overall structural similarity.


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






Azurin

The azurins are copper containing proteins of 128 amino acid residues which belong to the family of cupredoxins involved in electron transfer in certain bacteria. The azurins include those from P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans. Murphy et al., J. Mol. Biol. 315:859-871 (2002). The amino acid sequence identity between the azurins varies between 60-90%, these proteins showed a strong structural homology. All azurins have a characteristic β-sandwich with Greek key motif and the single copper atom is always placed at the same region of the protein. In addition, azurins possess an essentially neutral hydrophobic patch surrounding the copper site. Id.
Plastocyanins
The plastocyanins are soluble proteins of cyanobacteria, algae and plants that contain one molecule of copper per molecule and are blue in their oxidized form. They occur in the chloroplast, where they function as electron carriers. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 Å resolution. SEQ ID NO: 3 shows the amino acid sequence of plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium. Another plastocyanin of interest is from Ulva pertussis. 
Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between the Chlamydomonas and poplar proteins), the three-dimensional structures are conserved (e.g., 0.76 Å rms deviation in the C alpha positions between the Chlamydomonas and Poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochrome f, and validated the model of two functionally significant electron transfer paths involving plastocyanin. One putative electron transfer path is relatively short (approximately 4 Å) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (approximately 12-15 Å) and involves the nearly conserved residue Tyr-83 in the negative patch. Redinbo et al., J. Bioenerg. Biomembr. 26:49-66 (1994).
Rusticyanins
Rusticyanins are blue-copper containing single-chain polypeptides obtained from a Thiobacillus (now called Acidithiobacillus). The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 4) has been determined by multiwavelength anomalous diffraction and refined to 1.9 Å resolution. The rusticyanins are composed of a core beta-sandwich fold composed of a six- and a seven-stranded b-sheet. Like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His 85, Cys138, His143, Met148) arranged in a distorted tetrahedron. Walter, R. L. et al., J. Mol. Biol. 263:730-51 (1996).
Pseudoazurins
The pseudoazurins are a family of blue-copper containing single-chain polypeptide. The amino acid sequence of pseudoazurin obtained from Achromobacter cycloclastes is shown in SEQ ID NO: 5. The X-ray structure analysis of pseudoazurin shows that it has a similar structure to the azurins although there is low sequence homology between these proteins. Two main differences exist between the overall structure of the pseudoazurins and azurins. There is a carboxy terminus extension in the pseudoazurins, relative to the azurins, consisting of two alpha-helices. In the mid-peptide region azurins contain an extended loop, shortened in the pseudoazurins, which forms a flap containing a short α-helix. The only major differences at the copper atom site are the conformation of the MET side-chain and the Met-S copper bond length, which is significantly shorter in pseudoazurin than in azurin.
Phytocyanins
The proteins identifiable as phytocyanins include, but are not limited to, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except cucumber basic protein and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine.
Auracyanin
Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates apparent monomer molecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.
The amino acid sequence of auracyanin A has been determined and showed auracyanin A to be a polypeptide of 139 residues. Van Dreissche et al., Protein Science 8:947-957 (1999). His58, Cys123, His128, and Met132 are spaced in away to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general beta-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids. Id. See exemplary amino acid sequence SEQ ID NO: 15 for chain A of auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Accession No. AAM12874).
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). With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins. The amino acid sequences of the B forms are presented in McManus et al. J. Biol. Chem. 267:6531-6540 (1992). See exemplary amino acid sequence SEQ ID NO: 16 for chain B of auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Accession No. 1QHQA).
Stellacyanin
Stellacyanins are a subclass of phytocyanins, a ubiquitous family of plant cupredoxins. An exemplary sequence of a stellacyanin is included herein as SEQ ID NO: 14. The crystal structure of umecyanin, a stellacyanin from horseradish root (Koch et al, J. Am. Chem. Soc. 127:158-166 (2005)) and cucumber stellacyanin (Hart et al., Protein Science 5:2175-2183 (1996)) is also known. The protein has an overall fold similar to the other phytocyanins. The ephrin B2 protein ectodomain tertiary structure bears a significant similarity to stellacyanin. Toth et al., Developmental Cell 1:83-92 (2001). An exemplary amino acid sequence of a stellacyanin is found in the National Center for Biotechnology Information Protein Data Bank as Accession No. 1JER, SEQ ID NO: 14.
Cucumber Basic Protein
An exemplary amino acid sequence from a cucumber basic protein is included herein as SEQ ID NO: 17. The crystal structure of the cucumber basic protein (CBP), a type 1 blue copper protein, has been refined at 1.8 Å resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a “beta-sandwich” or “beta-taco”. Guss et al., J. Mol. Biol. 262:686-705 (1996). The ephrinB2 protein ectodomian tertiary structure bears a high similarity (rms deviation 1.5 Å for the 50 α carbons) to the cucumber basic protein. Toth et al., Developmental Cell 1:83-92 (2001).
The Cu atom has the normal blue copper NNSS′ co-ordination with bond lengths Cu—N(His39)=1.93 A, Cu—S(Cys79)=2.16 A, Cu—N(His84)=1.95 A, Cu—S(Met89)=2.61 A. A disulphide link, (Cys52)-S—S-(Cys85), appears to play an important role in stabilizing the molecular structure. The polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identity. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. An exemplary sequence for cucumber basic protein is found in NCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.
Antiestrogens
Antiestrogens that may be used in compositions and methods of the invention include, but are not limited to, compounds that prevent estrogen or progesterone from binding to receptors, such as Tamoxifen, toremifene, Estradiol, raloxifene, EM-652, arzoxifene, lasofoxifene, ICI-164,384, GW7604, and Tocris (ICI-182,780). Compounds that target or block the activity of estrogens and estrogen receptors may also include fulvestrant, as well as other compounds discussed in Katzenellenbogen, B. S. and Frasor, J., Seminars in Oncology, Vol 31, No. 1, Suppl. 3 28-38 (2004), the disclosure of which is incorporated in its entirety herein.
Antiestrogens that may be used in compositions and methods of the invention also may include, but are not limited to, compounds that prevent production of estrogen or progesterone, such as aromatase inhibitors, which may be steroidal or non-steroidal, including but not limited to anastrazole, exemestane, and letrozole, and other compounds disclosed or discussed in Eisen, A., et al., Cancer Treatment Reviews, 34:157-174 (2008), the disclosure of which is incorporated in its entirety herein. The dosages disclosed in the Eisen article, such as 20 mg daily of Tamoxifen, 1 mg daily of anastrozole, 2.5 mg daily of letrozole, and 25 mg daily of exemestane, may also be used in the compositions and methods of the invention disclosed herein.
Methods of Use
Chemopreventive therapies are based on evidence that the interruption of processes involved in cancergenesis will prevent or at least slow the development of cancer. The cupredoxin Pseudomonas aeruginosa azurin (SEQ ID NO: 1), the truncated azurin peptide p28, representing amino acids 50-77 of azurin (SEQ ID NO: 2), and the truncated azurin peptide p18, representing amino acids 50-57 of azurin (SEQ ID NO: 25), are now known to inhibit the development of premalignant lesions, either by inhibiting the initial formation of premalignant lesions, or killing or inhibiting the growth of premalignant lesions that are present. It therefore contemplated that a cupredoxin, or variant, derivative, truncation, or structural equivalent thereof, as described herein, with the ability to inhibit, suppress, prevent, reverse, delay or reduce the risk of the development of premalignant lesions, may be used in chemopreventive therapies alone or in combination with one or more other chemopreventive agents in patients suffering from premalignant lesions, patients suffering from cancer, or otherwise healthy patients. Such otherwise healthy patients are, in some embodiments, patients at a higher risk to develop cancer than those in the general population. Cancers that may be treated or prevented by treatment with the compositions of the invention include, but are not limited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin, and cervical cancer. In some embodiments, the patient may be human. In other embodiments, the patient is not human, including a non-human mammal.
The invention provides methods to have chemopreventive effects on premalignant lesions and cancers in mammalian cells and/or patients comprising administering to the patient a pharmaceutical composition comprising a first chemopreventive agent, which may be a truncation of azurin such as p18 (SEQ ID NO: 25) or p28 (SEQ ID NO: 2), a second chemopreventive agent, such as an antiestrogen, and a pharmaceutically acceptable carrier.
The invention also provides methods to treat mammalian patients, or otherwise have chemopreventive effects in mammalian cells and/or patients, by administering at least one chemopreventive agent that is a cupredoxin, or variant, derivative, truncation, or structural equivalent thereof, as described above, alone or in combination with one or more other chemopreventive agents, such as antiestrogens. Antiestrogens used in the methods of the invention may include Tamoxifen, fenretinide, and aromatase inhibitors. As demonstrated in Examples 3 and 4, Tamoxifen and cupredoxins, such as the azurin truncation p28, have a synergistic inhibitory effect on cancerous lesions.
The invention further includes methods of increasing expression of p53 in mammalian cells and mammalian patients by administering to the cells or the patient at least one chemopreventive agent, which may be, for example, a truncation of azurin such as p18 (SEQ ID NO: 25) or p28 (SEQ ID NO: 2), and administering an antiestrogen, in any order. As demonstrated in Example 4, p28 can increase the expression of p53 in mammalian cells if Tamoxifen or another antiestrogen is present to block the activity of estrogens.
The invention further includes methods of decreasing estrogen receptor binding to p53 in mammalian cells or a mammalian patient by administering to the mammalian cells or patient at least one chemopreventive agent, which may be, for example, a truncation of azurin such as p18 (SEQ ID NO: 25) or p28 (SEQ ID NO: 2), and administering an antiestrogen, in any order. As discussed herein, the estrogen receptor (“ER”)-p53 complex prevents the expression of p53. Treatment with Tamoxifen prevents ER from binding to p53. As demonstrated in Examples 3 and 4, incubation of glands with p28 in the presence of Tamoxifen enhances or increases the expression of p53 in mammalian cells.
The invention further includes methods of reducing the dose-related toxicity of Tamoxifen by administering to a mammalian patient or mammalian cells a chemopreventive agent, which may be, for example, a truncated azurin such as p18 (SEQ ID NO: 25) or p28 (SEQ ID NO: 2), in combination with a dose of Tamoxifen. As demonstrated by Example 3, inhibition of premalignant lesions and cancer can be achieved by combining a low dose of Tamoxifen with a low concentration of p28. In some embodiments, the chemopreventive agent, such as p28, and the antiestrogen, such as Tamoxifen, may be administered at or around the same time, in any order.
The invention further includes methods to study the development of cancer comprising contacting mammalian cells before or after induction with a carcinogen with a composition comprising cupredoxin, or a variant, derivative, truncation, or structural equivalent thereof, alone or in combination with one or more other chemopreventive agents, and observing the development of the cells. In some embodiments, the cells are mouse mammary gland cells, while in others they are other cells that may become malignant in mammals.
The methods disclosed herein may be used to treat patients with premalignant lesions, patients with cancer, and/or patients at a higher risk of developing cancer than the general population. Patients at a higher at risk to develop cancer than the general population may be patients with high risk features, patients with premalignant lesions, and patients that have been cured of their initial cancer or definitively treated for their premalignant lesions. See generally Tsao et al., CA Cancer J Clin 54:150-180 (2004). High risk features may be behavioral, genetic, environmental or physiological factors of the patient. Behavioral factors that predispose a patient to various forms of cancer include, but are not limited to, smoking, diet, alcohol consumption, hormone replacement therapy, higher body mass index, nulliparity, betal nut use, frequent mouthwash use, exposure to human papillomavirus, childhood and chronic sun exposure, early age of first intercourse, multiple sexual partners, and oral contraceptive use. Genetic factors that predispose a patient to various forms of cancer include, but are not limited to, a family history of cancer, gene carrier status of BRCA1 and BRCA2, prior history of breast neoplasia, familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), red or blond hair and fair-skinned phenotype, xeroderma pigmentosum, and ethnicity. Environmental features that predispose a patient to various forms of cancer include, but are not limited to, exposure to radon, polycyclic aromatic hydrocarbons, nickel, chromate, arsenic, asbestos, chloromethyl ethers, benzo[a]pyrene, radiation, and aromatic amines from rubber or paint occupational exposure. Other miscellaneous factors that predispose a patient to various forms of cancer include, but are not limited to, chronic obstructive pulmonary disease with airflow obstruction, chronic bladder infections, schistosomiasis, older age, and immunocompromised status.
Additionally, patients at a higher risk of developing cancer may be determined by the use of various risk models that have been developed for certain kinds of cancer. For example, patients predisposed to breast cancer may be determined using the Gail risk model, or the Claus model, among others. See Gail et al., J Natl Cancer Inst 81:1879-1886 (1989); Cuzick, Breast 12:405-411 (2003); Huang et al., Am J Epidemiol 151:703-714 (2000).
Patients with premalignant lesions are at a higher risk to develop cancer than the general population. The presence of premalignant lesions in or on a patient may be determined by many methods that are well known to those in the art. Intermediate markers or biomarkers that originate from premalignant lesions may be measured in a patient to determine if the patient harbors premalignant lesions. Chromosomal abnormalities occur in tumor cells and the adjacent histologically normal tissues in the majority of cancer patients. Progression in chromosomal abnormalities parallels the phenotypic progression from premalignant lesion to invasive cancer. Thiberville et al., Cancer Res. 55:5133-5139 (1995). Therefore, chromosomal abnormalities associated with cancer may be used as intermediate markers to detect premalignant lesions in a patient. Common chromosomal abnormalities associated with cancer include, but are not limited to, allelic deletions or loss of heterozygosity (LOH) in tumor suppressor genes such as 3p (FHIT and others), 9p (9p21 for p16INK4, p15INK4B, and p19ARF), 17p (17p13 for p53 gene and others) and 13q (13q14 for retinoblastoma gene Rb and others). Deletions in 3p and 9p are associated with smoking and the early stages of lung cancer. Mao et al., J. Natl. Cancer Inst. 89:857-862 (1997). Deletions affecting 3p, 5q, 8p, 17p and 18q are common change in epithelial cancers. See generally Tsao et al., CA Clin. Cancer J. Clin. 54:153 (2004). Other chromosomal mutations associated with cancer include those which activate oncogenes. Oncogenes whose presence may be used as intermediate markers include, but are not limited to, Ras, c-myc, epidermal growth factor, erb-B2 and cyclins E, D1 and B1. See generally id. at 154.
Other intermediate markers may be the products of genes up-regulated in premalignant cells and cancer cells. Genes that may be up-regulated in premalignant cells include, but are not limited to, cyclooxygenases COX-1 and COX-2, telomerase. Other biomarkers of cancer cells, and some premalignant cells, include, but are not limited to, p53, epidermal growth factor receptor (GFR), proliferating cell nuclear antigen (PCNA), RAS, COX-2, Ki-67, DNA aneuploidy, DNA polymerase-α, ER, Her2neu, E-cadherin, RARβ, hTERT, p16INK4a, FHIT (3p14), Bcl-2, VEGF-R, HPV infection, LOH 9p21, LOH 17p, p-AKT, hnRNP A2/B1, RAF, Myc, c-KIT, cyclin D1, E and B1, IGF1, bcl-2, p16, LOH 3p21.3, LOH 3p25, LOH 9p21, LOH 17p13, LOH 13q, LOH 8p, hMSH2, APC, DCC, DPC4, JV18, BAX, PSA, GSTP1, NF-kB, AP1, D3S2, HPV infection, LOH 3p14, LOH 4q, LOH 5p, bladder tumor antigen (BTA), BTK TRAK (Alidex, Inc., Redmond Wash.), urinary tract matrix protein 22, fibrin degradation product, autodrine motility factor receptor, BCLA-4, cytokeratin 20, hyaluronic acid, CYFRA 21-1, BCA, beta-human chorionic gonadotropin, and tissue polypeptide antigen (TPA). See generally id. at 155-157.
Patients that have been cured of their initial cancers or have been definitively treated for their premalignant lesions are also at a higher risk to develop cancer than the general population. A second primary tumor refers to a new primary cancer in a person with a history of cancer. Second primary tumors are the leading cause of mortality in head and neck cancer. Id. at 150. A second primary tumor is distinct from a metastasis in that the former originates de novo while the later originates from an existing tumor. Patients that have been cured of cancer or premalignant lesions of the breast, head and neck, lung, and skin are at a particularly high risk to develop second primary tumors.
The compositions comprising a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents can be administered to the patient by many routes and in many regimens that will be well known to those in the art. In specific embodiments, the cupredoxin, or variant, derivative, truncation, or structural equivalent thereof and the one or more other chemopreventive agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, or by inhalation. The compositions may be administered to the patient by any means that delivers the composition to the site in the patient that is at risk of developing cancer. In specific embodiments, the cupredoxin or variant, derivative, truncation or structural equivalent thereof and the one or more other chemopreventive agents are administered intravenously.
In one embodiment, the methods may comprise co-administering to a patient with, for example, premalignant lesions or a risk of developing premalignant lesions, one unit dose of a composition comprising a cupredoxin or a variant, derivative, truncation, or structural equivalent of cupredoxin and one unit dose of a composition comprising another chemopreventive drug, in either order, administered at about the same time, or within about a given time following the administration of the other, for example, about one minute to about 60 minutes following the administration of the other drug, or about 1 hour to about 12 hours following the administration of the other drug. Chemopreventive drugs of interest include, but are not limited to, antiestrogens such as Tamoxifen and aromatase inhibitors such as letrozole, exemestane, and anastrozole (Arimidex®), retinoids such as N-[4-hydroxyphenyl]retinamide (4-HPR, fenretinide), nonsteriodal antiinflammatory agents (NSAIDs) such as aspirin and sulindac, celecoxib (COX-2 inhibitor), defluoromethylornithing (DFMO), ursodeoxycholic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, EKI-785 (EGFR inhibitor), bevacizumab (antibody to VEGF-receptor), cetuximab (antibody to EGFR), retinol such as vitamin A, beta-carotene, 13-cis retinoic acid, isotretinoin and retinyl palmitate, α-tocopherol, interferon, oncolytic adenovirus dl1520 (ONYX-015), gefitinib, etretinate, finasteride, indole-3-carbinol, resveratrol, chlorogenic acid, raloxifene, and oltipraz.
Pharmaceutical Compositions Comprising Cupredoxin, or a Variant, Derivative, Truncation, or Structural Equivalent Thereof and One or More Other Chemopreventive Agents
Pharmaceutical compositions comprising cupredoxin or variant, derivative, truncation, or structural equivalents thereof and one or more other chemopreventive agents can be manufactured in any conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The substantially pure or pharmaceutical grade cupredoxin or variants, derivatives, truncations, and structural equivalents thereof and one or more other chemopreventive agents can be readily combined with a pharmaceutically acceptable carrier well-known in the art. Such carriers enable the preparation to be formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, and the like. Suitable carriers or excipients can also include, for example, fillers and cellulose preparations. Other excipients can include, for example, flavoring agents, coloring agents, detackifiers, thickeners, and other acceptable additives, adjuvants, or binders. In some embodiments, the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation may contain at least one preservative. General methodology on pharmaceutical dosage forms is found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999)).
The composition comprising a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents used in the invention may be administered in a variety of ways, including by injection (e.g., intradermal, subcutaneous, intramuscular, intraperitoneal and the like), by inhalation, by topical administration, by suppository, by using a transdermal patch or by mouth. General information on drug delivery systems can be found in Ansel et al., id. In some embodiments, the composition comprising a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents can be formulated and used directly as injectables, for subcutaneous and intravenous injection, among others. The injectable formulation, in particular, can advantageously be used to treat patients that are appropriate for chemopreventive therapy. The composition comprising a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents can also be taken orally after mixing with protective agents such as polypropylene glycols or similar coating agents.
When administration is by injection, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents may be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, the pharmaceutical composition does not comprise 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 to the peptide.
When administration is by intravenous fluids, the intravenous fluids for use administering the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents may be composed of crystalloids or colloids. Crystalloids as used herein are aqueous solutions of mineral salts or other water-soluble molecules. Colloids as used herein contain larger insoluble molecules, such as gelatin. Intravenous fluids may be sterile.
Crystalloid fluids that may be used for intravenous administration include but are not limited to, normal saline (a solution of sodium chloride at 0.9% concentration), Ringer's lactate or Ringer's solution, and a solution of 5% dextrose in water sometimes called D5W, as described in Table 2.







TABLE 2







Composition of Common Crystalloid Solutions











Solution
Other Name
[Na÷]
[CI−]
[Glucose]














D5W
5% Dextrose
0
0
252


2/3 & 1/3
3.3% Dextrose/
51
51
168



0.3% saline


Half-normal
0.45% NaCl
77
77
0


saline


Normal saline
0.9% NaCl
154
154
0


Ringer's
Ringer's
130
109
0


lactate*
solution





*Ringer's lactate also has 28 mmol/L lactate, 4 mmol/L K÷ and 3 mmol/L Ca2+.






When administration is by inhalation, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents may be delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the proteins and a suitable powder base such as lactose or starch.
When administration is by topical administration, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents may be formulated as solutions, gels, ointments, creams, jellies, suspensions, and the like, as are well known in the art. In some embodiments, administration is by means of a transdermal patch. When administration is by suppository (e.g., rectal or vaginal), cupredoxin or variants, truncations, and derivatives thereof and one or more other chemopreventive agents compositions may also be formulated in compositions containing conventional suppository bases.
When administration is oral, a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents can be readily formulated by combining the cupredoxin or variant, derivative, truncation or structural equivalent thereof and one or more other chemopreventive agents with pharmaceutically acceptable carriers well known in the art. A solid carrier, such as mannitol, lactose, magnesium stearate, and the like may be employed; such carriers enable the cupredoxin and variants, derivatives or structural equivalent thereof to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, cellulose preparation, granulating agents, and binding agents.
Other convenient carriers, as well-known in the art, also include muitivalent carriers, such as bacterial capsular polysaccharide, a dextran or a genetically engineered vector. In addition, sustained-release formulations that include a cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents allow for the release of cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents over extended periods of time, such that without the sustained release formulation, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents would be cleared from a subject's system, and/or degraded by, for example, proteases and simple hydrolysis before eliciting or enhancing a therapeutic effect.
The half-life in the bloodstream of the peptides and one or more other chemopreventive agents of the invention can be extended or optimized by several methods well known to those in the art. The peptide variants and one or more other chemopreventive agents of the invention may include, but are not limited to, various variants that may increase their stability, specific activity, longevity in the bloodstream, and/or decrease immunogenicity of the cupredoxin, while retaining the ability of the peptide and one or more other chemopreventive agents to inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Such variants include, but are not limited to, those which decrease the hydrolysis of the peptide and one or more other chemopreventive agents, decrease the deamidation of the peptide and one or more other chemopreventive agents, decrease the oxidation, decrease the immunogenicity, increase the structural stability of the peptide or increase the size of the peptide and one or more other chemopreventive agents. Such peptides also include circularized peptides (see Monk et al, BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), D,L-peptides (diastereomer), Futaki et al., J. Biol. Chem. February 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 (see Lee et al., J. Pept. Res. 63(2):69-84 (2004)), N- and C-terminal modifications (see Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), hydrocarbon stapling (see Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)) and PEGylation.
In various embodiments, the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like. It will be recognized that, while any suitable carrier known to those of ordinary skill in the art may be employed to administer the compositions of this invention, the type of carrier will vary depending on the mode of administration. Compounds may also be encapsulated within liposomes using well-known technology. Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. 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 compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
Administration of Cupredoxin or Variant, Derivative, Truncation, or Structural Equivalent Thereof and One or More Chemopreventive Agents
The cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents can be administered formulated as pharmaceutical compositions and administered by any suitable route, for example, by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical, percutaneous, i.e., transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary) or vitreous administration. The pharmaceutical formulations thereof can be administered in any amount effective to achieve its intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In specific embodiments, the therapeutically effective amount is generally from about 0.01-20 mg/day/kg of body weight.
The compounds comprising cupredoxin or variant, derivative, truncation, or structural equivalent thereof are useful for the prevention of cancer, alone or in combination with other chemopreventive agents. The appropriate dosage will, of course, vary depending upon, for example, the compound of cupredoxin or variant, derivative or structural equivalent thereof employed, the type of other chemopreventive agent selected for co-administration, if any, the host, the mode of administration and the nature and severity of the potential cancer. However, in general, satisfactory results in humans are indicated to be obtained at daily dosages from about 0.01-20 mg/kg of body weight. An indicated daily dosage in humans is in the range from about 0.7 mg to about 1400 mg of a compound of cupredoxin or variant, derivative or structural equivalent thereof conveniently administered, for example, in daily doses, weekly doses, monthly doses, and/or continuous dosing. Daily doses can be in discrete dosages from 1 to 12 times per day. Alternatively, doses can be administered every other day, every third day, every fourth day, every fifth day, every sixth day, every week, and similarly in day increments up to 31 days or over. Alternatively, dosing can be continuous using patches, i.v. administration and the like.
The exact formulation, route of administration, and dosage is 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 cupredoxin or variant, derivative or structural equivalent thereof and one or more other chemopreventive agents which are sufficient to maintain therapeutic effect. Generally, the desired cupredoxin or variant, derivative, truncation, or structural equivalent thereof and one or more other chemopreventive agents is administered in an admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
In one aspect, the cupredoxin or variant, derivative, truncation, or structural equivalent thereof and, if possible, one or more other chemopreventive agents, is delivered as DNA such that the polypeptide is generated in situ. In one embodiment, the DNA is “naked,” as described, for example, in Ulmer et al., (Science 259:1745-1749 (1993)) and reviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of naked DNA may be increased by coating the DNA onto a carrier, e.g., biodegradable beads, which are then efficiently transported into the cells. In such methods, the DNA may be present within any of a variety of delivery systems known to those of ordinary 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 of ordinary skill in the art. See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No. 5,736,524.
Vectors, used to shuttle genetic material from organism to organism, can be divided into two general classes: Cloning vectors are replicating plasmid or phage with regions that are essential for propagation in an appropriate host cell and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector. An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA, such as the DNA of a cupredoxin. In expression vectors, the introduced DNA is operably-linked to elements such as promoters that signal to the host cell to highly transcribe the inserted DNA. Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. Operably-linking a cupredoxin and variants and derivatives thereof and one or more other chemopreventive agents to an inducible promoter can control the expression of the cupredoxin and variants, truncations, and derivatives thereof and one or more other chemopreventive agents in response to specific factors. Examples of classic inducible promoters include those that are responsive to α-interferon, heat shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511 (1990)) and tetracycline. Other desirable inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but, are responsive in those cells when the induction agent is exogenously supplied. In general, useful expression vectors are often plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) are contemplated.
Vector choice is dictated by the organism or cells being used and the desired fate of the vector. In general, vectors comprise signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters, and transcription termination sequences.
Kits Comprising Cupredoxin, or Variant, Derivative, Truncation, or Structural Equivalent Thereof and One or More Chemopreventive Agents
In one aspect, the invention provides regimens or kits comprising one or more of the following in a package or container: (1) a pharmacologically active composition comprising at least one cupredoxin or variant, derivative, truncation, or structural equivalent thereof; (2) one or more other chemopreventive agents, and (3) apparatus to administer the biologically active composition to the patient, such as a syringe, nebulizer etc.
When a kit is supplied, the different components of the composition may be packaged in separate containers, if appropriate, and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.
The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampoules may contain lyophilized cupredoxin and variants, derivatives and structural equivalents thereof, or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampoules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold similar reagents. Other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampoules, and envelopes, that may comprise foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to be mixed. Removable membranes may be glass, plastic, rubber, etc.
Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, flash memory device etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
Modification of Cupredoxin and Variants, Derivatives, Truncations, and Structural Equivalents Thereof
Cupredoxin or variants, derivatives, truncations, or structural equivalents thereof may be chemically modified or genetically altered to produce variants and derivatives as explained above. Such variants, truncations, and derivatives may be synthesized by standard techniques.
In addition to naturally-occurring allelic variants of cupredoxin, changes can be introduced by mutation into cupredoxin coding sequence that incur alterations in the amino acid sequences of the encoded cupredoxin that do not significantly alter the ability of cupredoxin to inhibit the development of premalignant lesions. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequences of the cupredoxin without altering pharmacologic activity, whereas an “essential” amino acid residue is required for such pharmacologic activity. For example, amino acid residues that are conserved among the cupredoxins are predicted to be particularly non-amenable to alteration, and thus “essential.”
Amino acids for which conservative substitutions that do not change the pharmacologic activity of the polypeptide can be made are well known in the art. Useful conservative substitutions are shown in Table 3, “Preferred substitutions.” Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the invention so long as the substitution does not materially alter the pharmacologic activity of the compound.







TABLE 3







Preferred substitutions











Preferred


Original residue
Exemplary substitutions
substitutions





Ala (A)
Val, Leu, Ile
Val


Arg (R)
Lys, Gln, Asn
Lys


Asn (N)
Gln, His, Lys, Arg
Gln


Asp (D)
Glu
Glu


Cys (C)
Ser
Ser


Gln (Q)
Asn
Asn


Glu (E)
Asp
Asp


Gly (G)
Pro, Ala
Ala


His (H)
Asn, Gln, Lys, Arg
Arg


Ile (I)
Leu, Val, Met, Ala, Phe,
Leu



Norleucine


Leu (L)
Norleucine, Ile, Val, Met, Ala,
Ile



Phe


Lys (K)
Arg, Gln, Asn
Arg


Met (M)
Leu, Phe, Ile
Leu


Phe (F)
Leu, Val, Ile, Ala, Tyr
Leu


Pro (P)
Ala
Ala


Ser (S)
Thr
Thr


Thr (T)
Ser
Ser


Trp (W)
Tyr, Phe
Tyr


Tyr (Y)
Trp, Phe, Thr, Ser
Phe


Val (V)
Ile, Leu, Met, Phe, Ala,
Leu



Norleucine









Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as a β-sheet or α-helical conformation, (2) the charge, (3) hydrophobicity, or (4) the bulk of the side chain of the target site can modify the pharmacologic activity. Residues are divided into groups based on common side-chain properties as denoted in Table 4. Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more specifically into non-conserved sites.







TABLE 4







Amino acid classes










Class
Amino acids







hydrophobic
Norleucine, Met, Ala, Val, Leu, Ile



neutral hydrophilic
Cys, Ser, Thr



acidic
Asp, Glu



basic
Asn, Gln, His, Lys, Arg



disrupt chain conformation
Gly, Pro



aromatic
Trp, Tyr, Phe










The variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol. 154:329-350 (1987)), cassette mutagenesis, restriction selection mutagenesis (Wells et al., Gene 34:315-323 (1985)) or other known techniques can be performed on the cloned DNA to produce the cupredoxin variant DNA.
Known mutations of cupredoxins can also be used to create variant cupredoxin to be used in the methods of the invention. For example, the C112D and M44KM64E mutants of azurin are known to have cytotoxic and growth arresting activity that is different from the native azurin, and such altered activity can be useful in the treatment methods of the present invention.
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 form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations. Modifications and variations of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof.
EXAMPLES
Example 1
Effect of Peptide p28 on DMBA-Induced Mammary Lesions in the MMOC Model
The mouse mammary gland organ (MMOC) culture model has been successfully used to identify potential chemopreventive agents for mammary carcinogenesis. There is a 75% positive correlation observed between the efficacy in vitro in MMOC and in vivo in experimental carcinogenesis models. Mehta and Pezzuto, Curr. Oncol. Rep 4: 478-486 (2002).
The MMOC model allows evaluating efficacy of potentially chemopreventive agents against development of mammary alveolar lesions (MAL) or mammary ductal lesions (MDL) in response to DMBA. DMBA under appropriate incubation conditions forms either MAL or MDL based on the hormonal milieu in the medium. Hawthorne et al., Pharmaceutical Biology 40: 70-74 (2002); Mehta et al., J. Natl. Cancer Inst. 93: 1103-1106 (2001). Estrogen and progesterone-treated glands in culture develop ductal lesions whereas aldosterone and hydrocortisone-treated glands form estrogen and progesterone-independent alveolar lesions. Mammary glands not exposed to a carcinogen or chemopreventive agent, undergo structural regression in the absence of growth-promoting hormones, whereas treatment with DMBA for the 24-hr period between days 3 and 4 prevents the regression of structures caused by deprivation of hormones. It is assumed that this is because the glands have lost normal hormonal responsiveness and now have altered their course of development. Generating mammary adenocarcinoma by transplanting transformed cells into syngeneic mice has proved the premalignant preneoplastic nature of these unrepressed areas.
The thoracic pair of mammary glands was excised aseptically from each Balb/c mouse, and the glands were divided into several groups. The effects of p28 were evaluated at 4 different dilutions in the medium. Carcinogen treated glands without the test agent served as a measure to determine percent incidence in the absence of a chemopreventive agent. An additional control was included to serve as a positive control for chemoprevention. Azurin was included in the medium at 50 μg/ml concentration. For alveolar lesions (MAL) stained glands were evaluated for the incidence of lesions (glands containing any lesions as compared to total number of glands in a given treatment group). For the ductal lesions (MDL) similar protocol was adapted, however, as indicated below in the methods section the hormonal combination is different for alveolar and ductal lesions. The glands were fixed in formalin and then processed for histopathology. The sections are stained with eosin and hematoxelene and evaluated under microscope. Here the multiplicity of ductal lesions between the control and the treatment groups are compared.
Organ Culture Procedure. The experimental animals used for the studies were young, virgin BALB/c female mice 3 to 4 weeks of age obtained from Charles River, Wilmington, Mass. The mice were treated daily by subcutaneous injections with 1 μg estradiol-17β+1 mg progesterone for 9 days. This treatment is a prerequisite inasmuch as animals not pretreated with steroids fail to respond to hormones in vitro. The entire culture procedure is described in detail. Jang et al., Science 275:218-220 (1997); Mehta, Eu. J. Cancer 36:1275-1282 (2000); Mehta et al., J. Natl. Cancer Inst. 89:212-219 (1997); Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001).
Briefly, the animals were killed by cervical dislocation, and the thoracic pair of mammary glands were dissected out on silk rafts and incubated for 10 days in serum free Waymouth MB752/1 medium (5-glands/5 ml/dish). The medium was supplemented with glutamine, antibiotics (penicillin and streptomycin 100 units/ml medium) and growth-promoting hormones, 5 μg insulin (I), 5 μg prolactin (P), 1 μg aldosterone (A) and 1 μg hydrocortisone (H) per ml of medium for the protocol to induce mammary alveolar lesions (MAL). For induction of ductal lesions (MDL), the medium contained 5 μg/ml, 5 μg/ml P, 0.001 μg/ml estradiol 17β and 1 μg/ml progesterone (Pg). Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001). The carcinogen, DMBA (2 μg/ml) was added to the medium between days 3 and 4. For the present study, DMBA was dissolved in DMSO at a final concentration of 4 mg/ml, and 50 μg I was added to 100 ml medium resulting in 2 μg/ml final concentrations. The control dishes contained DMSO as vehicle.
On day 4, DMBA is removed from the medium by rinsing the glands in fresh medium and transferring them to new dishes containing fresh medium without DMBA. After 10 days of incubation, the glands were maintained for another 14 days in the medium containing only I (5 μg/ml). During the entire culture period, the glands were maintained at 37° C. under 95% O2 and 5% CO2 environment. The chemopreventive agent was included in the medium during the first ten days of growth-promoting phase. The test peptide p28 was evaluated at 4 concentrations ranging from 12.5 μg/ml to 100 μg/ml. Azurin was evaluated at 50 μg/ml in the medium. The peptide was dissolved in sterile water and filtered prior to use. The medium was changed three times per week (Monday, Wednesday and Friday). At the end of the exposure, the glands were fixed in formalin.
Results were analyzed by Chi-square analysis and Fisher's Exact Test.
Morphometic Analysis of MAL. For examination of MAL, the glands were stained in alum carmine, and evaluated for the presence of the lesions. The glands were scored for the presence or absence of mammary lesions, severity of lesions per gland, and toxicity of the agent. The glands stored in xylene were evaluated for the presence or absence, incidence, and severity of mammary lesions for each gland under a dissecting microscope. Mammary glands were scored as positive or negative for mammary lesions, and the percent incidence was determined as a ratio of glands exhibiting lesions and the total number of glands in that group. Dilation of ducts or disintegration of mammary structure because of treatment with chemopreventive agent was considered a toxic effect. The data were subjected to statistical analysis for the incidence to determine the effectiveness of the potential chemopreventive agents.
FIG. 1A shows a representative photograph of alveolar lesions in a DMBA treated gland and its comparison with a gland that was treated with DMBA along with a chemopreventive agent. The effects of p28 on the development of alveolar lesion are shown in FIGS. 1B-1G and summarized in FIG. 2. The peptide p28 inhibited MAL formation by 67% at 25 μg/ml concentration. Increasing concentration further up to 100 μg/ml did not enhance the efficacy of the peptide. The comparison of the peptide with azurin indicated that p28 was as effective as azurin for MAL development. Azurin at 50 μg/ml concentration resulted in a 67% inhibition. Statistical analyses indicated that the effect of p28 was statistically significant compared to DMBA control at concentrations greater than 12.5 μg/ml (p<0.01, Fisher's Exact Test; Chi Square analysis).
Histopathological Evaluation of MDL. For MDL, the glands were processed for histopathological evaluations. The glands were sectioned longitudinally into 5-micron sections and stained with eosin hematoxeline. The longitudinal section of each gland was divided into several fields and each field was evaluated for ductal lesions. Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001). Briefly, the entire gland is evaluated under the scope; smaller glands will have fewer total fields as compared to larger glands. Thus, each gland will have variable number of fields. Often the number of sections through the ducts also varies greatly from gland to gland. This results in the variable number from group to group. Fields containing ductal hyperplasia or atypia were determined and were compared with total number of field evaluated for each gland. No discrimination is made between the hyperplasia or atypia and severely occluded glands. Any field containing any of these histological patterns was considered positive for the lesion. The treatment groups were compared with the controls for the severity and percent inhibition was calculated.
FIG. 3 shows a representative ductal lesion. DMBA induces ductal lesions varying from hyperplasia, atypia to complete occlusion of the ducts. A ratio of ductal lesions/total number of ductal sections was determined. Again, 12.5 μg/ml concentration of p28 suppressed only 15% of the MDL formation. However, at 25 μg/ml there was a significant inhibition of the lesions comparable to that observed with 50 μg/ml azurin. The efficacy of p28 at concentrations greater than 12.5 μg/ml was statistically significant (p<0.01, Fishers Exact Test). These results are summarized in FIG. 4. Often effects of chemopreventive agents can be differentiated between the MAL and MDL. For example Tamoxifen inhibited the development of MDL but not MAL. It is interesting to note that azurin and p28 inhibited both estrogen and progesterone-dependent ductal lesions as well as independent alveolar lesions.
This example indicates that both p28 and azurin can prevent the development of precancerous lesions in breast tissue. Thus, p28 and azurin may be used as chemopreventive agents in mammalian patients.
Example 2
Effects of Azurin and Peptide p28 on the Induction of Mammary Alveolar Lesions in the MMOC Model
Chemicals: Azurin and other synthetic peptides: Escherichia coli JM109 was used as a host strain for expression of the azurin-encoding gene (azuA) of P. aeruginosa strain PA01. The recombinant E. coli strain was cultivated in 2YT medium containing 50 μg of ampicillin/ml, 0.1 mM IPTG, and 0.5 mM CUSO4 for 16 h at 37° C. (31). Azurin (SEQ ID NO 1) was purified from periplasmic fraction of the recombinant E. coli JM109 strain to a single band as described previously in Wang et al., Biochem Biophys Res Commun. 349:1117-1124 (2006), and determined to be endotoxin free. The sequence of p28 is LSTAADMQGVVTDGMASG LDKDYLKPDD (SEQ ID NO 2), and has been chemically synthesized to >95% purity by C S Bio Inc. (Menlo Park, Calif.). Azurin and p28 were aliquoted, dried and stored in clean sterile vials at −20° C. in a desiccator. All compounds were dissolved in culture media and filter (0.22 μpm) sterilized immediately prior to use.
Mammary gland Organ Culture (MMOC): The procedure of MMOC has been described in detail previously. Mehta et al, J. Natl. Cancer Inst. 92: 418-423 (2000). Briefly, thoracic mammary glands are aseptically dissected from the estrogen and progesterone pretreated 4 weeks old female Balb/c mice and explanted into chemically defined Weymouth 752/MB culture medium containing growth promoting hormones. For ovarian hormone independent growth the glands were incubated with insulin (5 μg/ml), prolactin (5 μg/ml) aldeosterone (1 μg/ml) and hydrocortisone (1 μg/ml). For hormone dependent lesions the glands were incubated with insulin, prolactin and estradiol 17β (0.001 μg/ml) and progesterone (1 μg/ml) for 10 days. The carcinogen, 7,12 dimethylbenz(a)anthracene (DBMA) (2 μg/ml) was introduced for 24 hours on day 3. The glands were incubated for 10 days and then were regressed in the presence of only insulin for additional 14 days. Thus, typically for the development of mammary lesions the culture period is 24 days. The glands are stained with alum carmine for alveolar lesions (estrogen independent) and histopathologically identified ductal lesions (estrogen dependent lesions). Glands were treated with azurin, p28 or tamoxifen only during the first 10 days of culture. For determining the p28 and p53 interactions, the glands were incubated for four days with the hormones and p28. The expressions both by PCR and immunohistochemistry were measured after 48 hours.
Immunohistochemistry: Mammary glands were incubated with growth promoting hormones for 4 days in the presence or absence of azurin or p28. DMBA was included in the medium for 24 hours on day 3. Formalin fixed paraffin embedded tissue sections were deparaffinized, hydrated. For p53 antigen retrieval was performed by placing slides in 10 mM citrate buffer prewarmed (95° C.) and microwave for 3 min. Slides were then placed in oven at 95-1000 for 1 h in citrate buffer. For ER/PgR and Ki-67 immunostaining antigen retrieval was achieved using pressure chambers, in 10 mM citrate buffer for 6 min. After PBS rinse, nonspecific staining was blocked by incubating slides in 5% gelatin followed by avidin and biotin (Zymed Laboratories, South California, Calif.). Sections were incubated with primary antibody (p53, Vector/novacastra-VP-P956, PGR, ER and Ki-67 from Labvision, Freemont Calif., Ki-67 Santa Cruz Biotechnology (Santa Cruz, Calif.), preabsorbed with mouse serum) for 90 min at RT. Following PBS washes (3×) sections were incubated with appropriate biotinylated goat anti-rabbit/anti-mouse antibody, then with ABC reagent (Vectashield Elite ABC kit) DAB (Diaminobenzidine, Sigma, St Louis, Mo.) was used as chromogen to visualize immunoreactivity. Sections were counterstained using Gill's #1 Hematoxylin.
RNA extraction and real-time PCR: Individual mammary glands were homogenized by using Tissue Tearor (Biospec Products, Inc., Racine, Wis.). RNA was extracted using the phenol:chloroform extraction procedure, which separates out the nucleic acids in the aqueous phase and the proteins being eluted out in the lower organic phase. The aqueous phase is further purified by isopropanol and RNA precipitated by ice cold 70% ethanol prepared in DEPC water. RNA was dissolved in DEPC and analyzed by Spectrophotometry. RNA was converted to cDNA by using the Bio-Rad thermal cycler PCR machine.
Real-time PCR was performed as previously described using 1 μL reverse transcription product in a MyiQ real-time PCR detection system (Bio-Rad, Hercules, Calif.) by using iQ SYBR Green PCR Supermix (Bio-Rad) according to the manufacturer's guidelines. The expression of p53 was determined. Expression of two housekeeping genes 18S mitochondrial DNA and CAPM were used for data analyses. For p53 the Primers used for real time PCR were (forward: 5-GATGACTGCCATGGAGGAG-3′ reverse: ′-GTCCATGCAAGGTGATG-3′) (SEQ ID NO: 36 and 83), Ribosomal 18S (forwards 5′-CATGCCCGTTCTTAGTTGGT-3′ reverse: 5′-GAACGCCACTTGTCCCTCTA-3′) (SEQ ID NO 37 and 84).
Statistical analyses: Chi-square analyses were carried out to evaluate statistical significance between the incidence of MAL and MDL in control and chemopreventive agent treatment groups.
Azurin and p28: As shown in FIG. 5, Azurin is a 128 amino acid, 14 kD copper containing protein produced by aerobic bacteria. p28 was generated and evaluated for entry into the cells. The results indicated that a 28 amino acid peptide, azu 55-77 (FIG. 5) fused to GST could transport GST into the J774 cells; however, GST by itself could not be internalized. These results provided a sound rationale for selecting a 28 amino acid (aa 50-77, SEQ ID NO 2) construct of azurin as an active peptide for possible azurin action and was designated as p28. Therefore initial experiments were carried out using azurin and subsequent studies used p28, keeping azurin as a positive control.
Effects of azurin and p28 on the induction of mammary alveolar lesions (MAL): Effects of azurin at 3 different concentrations (10, 20 and 50 μg/ml) was evaluated, 15 glands were included in each group. The results showed that there was 71.4% incidence of MAL in the DMBA control group of glands and there was a 16, 20 and 63% inhibition of incidence was observed with azurin at three concentrations respectively. At 50 μg/ml concentration, azurin mediated inhibition of the MAL development was statistically significant (p<0.05). Efficacy of p28 on MAL development was also determined by evaluating p28 at concentration range of 10 to 100 μg/ml. The peptide was added in the medium during the initial 10 days of growth phase of the mammary gland development.
FIGS. 6A and 6B show representative whole mounts of glands with MAL in control and p28 (50 μg/ml) treatment groups. With regard to FIG. 6A, Mammary glands were either incubated with DMBA or DMBA and p28 for 10 days during the growth promoting phase of MAL development. Glands were stained after 24 days of culture period with alumn carmine. A reduced number of MAL is observed in p28 treated glands. Specifically, FIG. 6A shows histopathology of mammary alveolar lesions either in the absence or the presence of 50 μg p28. p28 at 25 μg resulted in >60% inhibition of MAL development (p<0.05), which is comparable to the inhibition obtained with azurin. There was no toxicity associated with any concentration of p28 as no dilation of ducts was observed in any of the treatment groups.
With regard to FIG. 6B, ductal lesions were identified in hematoxelene and eosin stained histopathological sections. Representative sections show mammary ducts are occluded in DMBA treated glands, whereas p28 treatment exhibits normal histology of the mammary ducts.
This example further indicates that both p28 and azurin can prevent the development of precancerous lesions in breast tissue. Thus, p28 and azurin may be used as chemopreventive agents in mammalian patients.
Example 3
Effect of Combination of Peptide p28 and Tamoxifen on DMBA-Induced Mammary Lesions in the MMOC Model
The MMOC system can discriminate between the lesions induced by DMBA that are estrogen and progesterone dependent and hormone independent. The lesions developed in the presence of estrogen and progesterone are MDL and they can be compared to human DCIS/ductal carcinoma. Unlike distinct alveolar lesions developed in the absence of estrogen, the alveolar structures formed in the presence of estrogen and absence of aldosterone are very small, which makes it difficult to measure the effects of chemopreventive agents on the alveolar structure of estrogen dependent lesions. Tamoxifen, an antiestrogen, which can selectively inhibit MDL, fails to inhibit MAL in this model.
The effects of p28 and Tamoxifen on MDL development were evaluated based on the experimental procedures discussed in Example 2 above. FIG. 6C shows the effects of either p28 on MAL and MDL or p28 with 1 μg/ml Tamoxifen on MDL. Ten glands per group were incubated with various treatments either with IPAF for MAL or with IPEPg for MDL. The glands were stained with alumn carmine for MAL or histologically processed for MDL. The effects of experimental agents were evaluated either on the incidence of MAL (number of glands with MAL in treatment group as compared to experimental group) or multiplicity (number of ducts with MDL per field in controls as compared to experimental groups) for MDL
Results showed that p28 inhibited the development of MDL at 50 μg/ml (FIG. 6C). At 12.5 μg/ml there was only 30% inhibition as compared to 70% inhibition of MDL at 25 μg/ml. Increasing concentrations to 100 μg/ml did not further enhance prevention of the MDL. Tamoxifen (1 μM) treatment alone inhibited MDL formation by about 50%. However the combination of ineffective concentration of p28 (12.5 μg/ml) with 1 μM Tamoxifen increased the effectiveness to 70% (FIG. 6C). These dramatic effects of combination can have clinical translational value because the dose-related toxicity of Tamoxifen can be significantly curtailed by combining the treatment of Tamoxifen with lower concentration of p28. Suppression of both MAL and MDL formation by p28 was due to inhibition of cell proliferation as reduced expression of Ki67 was observed in p28 treated mammary gland sections compared to control (FIG. 3).
These results provide a translational benefit of combining Tamoxifen and p28 at low concentrations for enhanced protection against the development of estrogen dependent precancerous mammary ductal lesions.
Example 4
Effects of Peptide p28 and Tamoxifen on Induction of p53 in the MMOC Model
Earlier studies provided some evidence for possible interactions between azurin and p53; however the functional significance of these interactions or stabilization of p53 by azurin have not been reported. Since the design of MMOC experiments can identify interactive role of estrogen and progesterone with p53 by comparing the p53 expression under two separate hormonal conditions, a set of studies were carried out to determine if the steroid hormones have a selective role on p53 expression. Aldosterone/hydrocortisone and estrogen/progesterone independently induce development of alveolar structures in mammary glands and yet the DMSA-induced lesions formed under these two hormonal environments are different. Moreover, the development of preneoplastic lesions is a late event. The lesions are formed after 24 days even though the glands are exposed to carcinogen much earlier during the culture period. Thus, the earlier cell signaling events, which may play a major role in development of premalignant lesions, can be monitored within the first few days of culture.
FIG. 7A depicts a comparison of p53 mRNA expression in mammary glands incubated in the presence or absence of estrogen and progesterone. After culturing the glands for four days with aldosterone plus progesterone or estradiol plus progesterone in chemically defined medium, total RNA was isolated from four glands individually in each group. RNA was converted to cDNA and real-time PCR was performed using 1 μL reverse transcription product in a MyiQ real-time PCR detection system by using iQ SYBR Green PCR Supermix. The expression of p53 was determined. Expression of two housekeeping genes 18S mitochondrial DNA and GAPDH were used for data analyses. The p53 expression was normalized to a control.
As demonstrated in FIG. 7A, mammary glands incubated with either IPAF (absence) or IPEPg (presence) did not have significant mRNA expression of p53. These control glands were normalized to 1 and relative increase or decrease caused by p28 was determined. Results showed that the glands incubated with p28 in the absence of estrogen plus progesterone exhibited 12 fold increase of p53 mRNA expression whereas there was no enhanced expression of p53 observed when the glands were incubated with p28 in the presence of estrogen and progesterone.
However, enhanced p53 expression was observed in p28 treated glands irrespective of culture conditions (presence/absence of estrogen) as determined by immunohistochemistry. FIG. 7C depicts immunohistochemical staining of p53 (photos a-f) and Ki67 (photos g-l) in mammary glands in MMOC. Mouse mammary glands were incubated with either DMBA alone (a, d, g, j) or in the presence of p28 (b, e, h, k) or Azurin (c, f, i, l) for 4 days. Paraffin embedded sections were processed for immunohistochemical staining of p53 (a-f) and Ki67 (g-l). Certain of these glands were incubated in the absence of estrogen/IPAF (a, b, c, g, h, i) and others were incubated in the presence of estrogen/IPEPg (d, e, f, j, k, l).
The cell proliferative activity determined by determining Ki67 staining by immunohistochemistry further suggested that both p28 and azurin inhibited cell proliferation. The growth promoting effect of hormone treatment resulted in intense Ki67 staining in the epithelial cells (FIG. 7C), whereas there was significant decrease in the Ki67 expression when the glands were treated with azurin. It is therefore thought that p28 stabilizes p53 protein rebuilding in the antiproliferative effects of azurin and p28.
Since recent literature indicates that estrogen interferes with p53 action in breast cancer cells, the expression of p53 in the mammary glands treated with estrogen and progesterone was examined. FIG. 7D shows the effects of azurin and p28 on the p53 and Ki67 protein expression by immunohistochemistry. Paraffin embedded sections of mouse mammary glands were incubated with IPEPg containing media, control (a, e, i); IPEPg+50 μg of p28 (b, f, j); IPEPg+10 μM Tamoxifen (c, g, k); and IPEPg+Tamoxifen+50 μg/ml p28 (d, h, l) for 4 days as indicated in the methods described in Example 2 above. After 4 days glands were fixed in buffered formalin and processed for paraffin sectioning. Immunohistochemical staining of p53 (a-d) and Ki-67 (e-h) was performed after antigen retrieval procedure. Nuclear p53 expression (i-l) in mammary epithelial cells was observed in glands incubated in the presence of Tamoxifen and Tamoxifen+p28. Similarly decreased Ki-67 was observed in Tamoxifen and Tamoxifen+p28 treated glands. H and E sections are shown.
The results showed repressed p53 expression in glands treated with estrogen and progesterone. p53 expression in glands treated with Tamoxifen, an antiestrogen, was measured to determine if estrogen mediated suppression of p53 expression can be overcome by Tamoxifen. As shown in FIG. 7B, while p28 or Tamoxifen individually did not affect the expression of p53 in mammary glands, in the presence of a combination of low concentrations of p28 and Tamoxifen there was an unexpected 125 fold increases in the p53 mRNA expression. These studies clearly showed that p28 mediated induction of p53 in mammary gland is not selective for glands incubated with glucocorticoids, however p28 can equally induce p53 in the estrogen and progesterone treated glands if the estrogen function is blocked. Immunohistochemical studies further confirmed these findings. Results showed that the estrogen and progesterone treated glands or the glands treated with the hormones and p28 or Tamoxifen individually did not express nuclear p53 protein. However when the glands are incubated with a combination of p28 and Tamoxifen, a surprising increase in nuclear expression of the p53 protein was observed (FIG. 7, l). The immunohistochemical analyses also showed that mammary glands incubated with estradiol induces upregulation of ER and PR in the glands.
There are thus significant, and previously unexpected, benefits to combining Tamoxifen and p28 at low concentrations to enhance p53 induction and thus inhibit premalignant lesion and tumor development.
Example 5
Effect of Peptide p28 in Combination With Fenretinide and Tamoxifen on DMBA-Induced Mammary Lesions
The mouse mammary gland organ culture (MMOC) assay was used to evaluate the inhibitory effects of potential chemopreventive agents on both hormone-induced structural differentiation of mammary glands and on the development of DMBA-induced preneoplastic hyperplastic alveolar nodule-like lesions in the gland. Mammary glands from young, virgin animals, when incubated for 6 days in the presence of insulin (I)+prolactin (P)+aldosterone (A), can differentiate into fully-grown glands. These glands morphologically resemble the glands obtained from pregnant mice. Aldosterone can be replaced by estrogen (E)+progesterone (Pg). Inclusion of hydrocortisone (H) to the medium stimulates the glands for functional differentiation. Mehta, R. G., and Banerjee, M. R, Acta Endocrinol., 80: 501 (1975); Mehta, R. G., and Moon, R C., Can Maturation Be Induced in Breast Cancer, In: Breast Cancer Treatment and Prognosis, Basil A. Stoll (ed.), Blackwell Press, 300, 1986. Thus, the hormone-induced structural and functional differentiation, observed in this culture system, mimics the responses to hormones observed during various physiological stages of the animal.
Two known chemopreventive compounds were selected. For MAL development fenretinide (4-HPR, N-4-Hydroxyphenyl retinamide) was selected. Fenretinide inhibits MAL development by >60% and showed clinical efficacy against breast cancer development in high-risk premenopausal women. Veronesi, U., et al, Ann. Oncol. 17: 1065-1071 (2006). The second chemopreventive agent selected for the current project was Tamoxifen for MDL. Tamoxifen has been used clinically for prevention and treatment of breast cancer patients. Since it mediates its action as an antiestrogen and is effective against MDL development in MMOC, it was evaluated in combination with p28 for prevention of MDL development in MMOC. Four experiments were conducted: two for MAL and two for MDL. For MAL, 4-HPR was used at an effective concentration either alone or in combination with increasing concentrations of p28. Similarly, for MDL experiments Tamoxifen was used at a single concentration either alone or in the presence of increasing concentrations of p28. A sample protocol is shown in Table 5.







TABLE 5







Protocol to evaluate the efficacy of peptide P28 in


combination with Fenretinide (4-HPR) on the development of


DMBA-induced mamamry alveolar lesions


in mouse mammary gland organ culture (MMOC)


Experiment No. CDG-3


Sponser: CDG











Number of




Group
Glands
Carcinogen
Agent (Conc.)










Expt 3A










1
15
DMBA (2 μg/ml)
None


2
15
DMBA (2 μg/ml)
4-HPR (1 μM)


3
15
DMBA (2 μg/ml)
P28 (12.5 μg/ml)


4
15
DMBA (2 μg/ml)
P28 (12.5 μg/ml) +





4-HPR (1 μM)


5
15
DMBA (2 μg/ml)
P28 (25 μg/ml)


6
15
DMBA (2 μg/ml)
P28 (25 μg/ml) +





4-HPR (1 μM)







Expt. 3B










7
15
DMBA (2 μg/ml)
None


8
15
DMBA (2 μg/ml)
P28 (50 μg/ml)


9
15
DMBA (2 μg/ml)
P28 (50 μg/ml) +





4-HPR (1 μM)


10
15
DMBA (2 μg/ml)
P28 (100 μg/ml)


11
15
DMBA (2 μg/ml)
P28 (100 μg/ml) +





4-HPR (1 μM)


12
15
DMBA (2 μg/ml)
Azurin (50 μg/ml)





Preparation of peptide solution: Weigh out 5 vials of 3 mg P28 and save them at −70 C. Dissolve 1 mg/2 ml filter and then add 1 ml to 4 ml medium (500 μg/5 ml to reach final conc. of 100 μg/ml) Similarly add P28 to achieve final concentrations as shown in the protocol. 4-HPR;


Culture protocol: Standard for MAL.






The effects of p28 were evaluated at 4 different concentrations (12.5, 25, 50 and 100 μg/ml) either alone or in combination with either 4-HPR or Tamoxifen. Carcinogen treated glands without the test agent served as a measure to determine percent incidence in the absence of a chemopreventive agent. An additional control of Azurin (50 μg/ml) was included to serve as a positive control for chemoprevention. For alveolar lesions, stained glands were evaluated for the incidence of lesions (glands containing any lesions as compared to total number of glands in a given treatment group). For the ductal lesions, similar protocol was adapted; however, hormonal combination is different for alveolar and ductal lesions. A protocol for determining the effects of P28 and Tamoxifen on MDL is shown in Table 6.







TABLE 6







Protocol to evaluate the efficacy of peptide P28 in


combination with Tamoxifen on the development of


DMBA-induced mamamry Ductal lesions


in mouse mammary gland organ culture (MMOC)


Experiment No. CDG-4


Sponser: CDG











Number of




Group
Glands
Carcinogen
Agent (Conc.)










Expt. 4A










1
15
DMBA (2 μg/ml)
None


2
15
DMBA (2 μg/ml)
Tamoxifen (1 μM)


3
15
DMBA (2 μg/ml)
P28 (12.5 μg/ml)


4
15
DMBA (2 μg/ml)
P28 (12.5 μg/ml) +





Tamoxifen (1 μM)


5
15
DMBA (2 μg/ml)
P28 (25 μg/ml)


6
15
DMBA (2 μg/ml)
P28 (25 μg/ml) +





Tamoxifen (1 μM)







Expt. 4B










7
15
DMBA (2 μg/ml)
None


8
15
DMBA (2 μg/ml)
P28 (50 μg/ml)


9
15
DMBA (2 μg/ml)
P28 (50 μg/ml) +





Tamoxifen (1 μM)


10
15
DMBA (2 μg/ml)
P28 (100 μg/ml)


11
15
DMBA (2 μg/ml)
P28 (100 μg/ml) +





Tamoxifen (1 μM)


12
15
DMBA (2 μg/ml)
Azurin (50 μg/ml)





Preparation of peptide solution: Weigh out 5 vials of 3 mg P28 and save them at −70 C. Dissolve 1 mg/2 ml filter and then add 1 ml to 4 ml medium (500 μg/5 ml to reach final conc. of 100 μg/ml) Similarly add P28 to achieve final concentrations as shown in the protocol. Tamoxifen


Culture protocol: Standard for MDL.






The glands were fixed in formalin and then processed for histopathology. The sections are stained with eosin and hematoxelene and evaluated under microscope.
Organ Culture Procedure: The experimental animals used for the studies were young, virgin BALB/c female mice 3 to 4 weeks of age obtained from Charles River, Wilmington, Mass. These mice are readily available, and most of the past studies have used the BALB/c mice. The mice were treated daily by subcutaneous injections with 1 μg estradiol-17β+1 mg progesterone for 9 days. This treatment is a prerequisite inasmuch as animals not pretreated with steroids fail to respond to hormones in vitro. The entire culture procedure is described in detail in Mehta, R. G. and Banerjhee, M., Acta Endocrinol. 80: 501 (1975) and Mehta, R. G. et al., JNCI 93: 1103-1106 (2001).
Briefly, the animals were killed by cervical dislocation, and the thoracic pair of mammary glands were dissected out on silk rafts and incubated for 10 days in serum free Waymouth MB752/1 medium (5-glands/5 ml/dish). The medium was supplemented with glutamine, antibiotics (penicillin and streptomycin 100 units/ml medium) and growth-promoting hormones, 5 μg I, 5 μg P, 1 μg A and 1 μg H per ml of medium for the protocol to induce mammary alveolar lesions (MAL). For induction of ductal lesions (MDL), the medium contains 5 μg/ml I, 5 μg/ml P, 0.001 μg/ml estradiol-17β, and 1 μg/ml progesterone (Pg) (14). The carcinogen, DMBA (2 μg/ml) was added to the medium between days 3 and 4. For the present study, DMBA was dissolved in DMSO at a final concentration of 4 mg/ml, and 50 μg I was added to 100 ml medium resulting in 2 μg/ml final concentrations. The control dishes contained OMSO as vehicle. On day 4, OMBA is removed from the medium by rinsing the glands in fresh medium and transferring them to new dishes containing fresh medium without OMBA. After 10 days of incubation, the glands were maintained for another 14 days in the medium containing only I (5 μg/ml). During the entire culture period, the glands were maintained at 3° C. under 95% O2 and 5% CO2 environment. The chemopreventive agents were included in the medium during the first ten days of growth promoting phase. The test peptide p28 was evaluated at 4 concentrations ranging from 12.5 μg/ml to 100 μg/ml. Fenretinide (for MAL studies) and Tamoxifen (for MDL studies) were included (dissolved in ethanol) at 1 μM concentrations. Azurin was evaluated at 50 μg/ml in the medium. The p28 peptide was dissolved in sterile water and filtered prior to use. The medium was changed three times per week (Monday, Wednesday and Friday). At the end of the exposure, the glands were fixed in formalin. For examination of MAL, the glands were stained in alum carmine, and evaluated for the presence of the lesions. The glands were scored for the presence or absence of mammary lesions, severity of lesions per gland, and toxicity of the agent. For MDL, the glands were processed for histopathological evaluations, The glands were sectioned longitudinally into 5-micron sections and stained with eosin hematoxeline.
Chemopreventive Agents: The agents azurin and p28 were supplied by CDG Therapeutics Inc. The agents were assumed to be stable and pure.
Morphometric Analysis: The glands stored in xylene were evaluated for the presence or absence, incidence, and severity of mammary lesions for each gland under a dissecting microscope. Mammary glands were scored as positive or negative for mammary lesions, and the percent incidence was determined as a ratio of glands exhibiting lesions and the total number of glands in that group. Dilation of ducts or disintegration of mammary structure because of treatment with chemopreventive agent was considered a toxic effect. The data were subjected to statistical analysis for the incidence to determine the effectiveness of the potential chemopreventive agents.
Histopathological Evaluation of MDL: The longitudinal section of each gland was divided into several fields and each field was evaluated for ductal lesions. Briefly, the entire gland is evaluated under the scope; smaller glands will have fewer total fields as compared to larger glands. Thus, each gland will have variable number of fields. Often the number of sections through the ducts also varies greatly from gland to gland. This results in the variable number from group to group. Fields containing ductal hyperplasia or atypia were determined and were compared with total number of field evaluated for each gland. No discrimination is made between the hyperplasia or atypia and severely occluded glands. Any field containing any of these histological patterns was considered positive for the lesion. The treatment groups were compared with the controls for the severity and percent inhibition was calculated.
Statistical Analysis: If the chemopreventive agent shows 60% or greater inhibition when using 15 glands per group, this assures that the results will be statistically significant (p<0.05). Results were analyzed by Fisher's Exact Test.
Results and Discussion: The MMOC model allows evaluation of efficacy of potentially efficacious chemicals against development of mammary alveolar or ductal lesions in response to DMBA. DMBA, under appropriate incubation conditions, forms either MAL or MDL based on the hormonal milieu in the medium. Mehta, R. G. et al., JNCI 93: 1103-1106 (2001). Estrogen and progesterone treated glands in culture develop ductal lesions whereas aldosterone and hydrocortisone treated glands form estrogen progesterone independent alveolar lesions. The mammary glands not exposed to carcinogen or chemopreventive agent undergo structural regression in the absence of growth-promoting hormones, whereas treatment with DMBA for the 24-hr period between days 3 and 4 prevents the regression of structures caused by deprivation of hormones. It is assumed that this is because the glands have lost normal hormonal responsiveness and now have altered their course of development. As indicated previously, generating mammary adenocarcinoma by transplanting transformed cells into syngeneic mice has proved the preneoplastic nature of these unrepressed areas.
Effects of p28 and 4-HPR: The effects of p28 and 4-HPR alone as well as in combination on the development of alveolar lesion are summarized in Table 7 and FIG. 8.







TABLE 7







Effects of combination of P28 and 4-HPR on DMBA


Induced Mammary Alveolar Lesions in MMOC


Chemopre P28 and 4-HPR


Experiment No.: CDG-3 and 4













Treatment
No. Glands
Glands with
Percent
Percent


Gp. No.
DMBA + X
per group
lesions
Incidence
Inhibition










Exp. 1












1
None
15
9
60.0



2
4-HPR (1 μM)
15
3
20.0
67%


3
P28 (12.5 μg/ml))
15
4
26.7
*56% 


4
P28 (12.5 μg/ml) + 4-HPR (1 μM)
15
3
20.0
67%


5
P28 (25 μg/ml)
15
3
20.0
67%


6
P28 (25 μg/ml) + 4-HPR (1 μM)
15
2
13.3
78%







Exp. 2












7
None
15
10
66.7



8
P28 (50 μg/ml)
15
3
20
70%


9
P28 (50 μg/ml) + 4-HPR (1 μM)
15
2
13.3
80%


10 
P28 (100 μg/ml)
15
2
13.3
80%


11 
P28 (100 μg/ml) + 4-HPR (1 μM)
15
3
20
70%


12 
Azurin (50 μg/ml)
15
3
20
70   





Fisher's Exact Test


*= Gp1 vs 3 = NS p = 0.1394


All Others VS Gp 1: p < 0.05






All glands were photographed and the pictures are shown in FIG. 10. The peptide p28 inhibited MAL formation by 67% at 25 μg/ml concentration. Increasing the concentration of p28 further up to 100 μg/ml resulted in inhibition to 80%. Combination chemoprevention studies indicated that 4-HPR inhibited MAL development as expected by 67%. Azurin suppressed the MAL development by about 70%. Statistical analyses indicated that the effects of p28 (>25 μg/ml), 4-HPR and all combinations were statistically significant compared to DMBA control (p<0.01, Fisher's ExactTest).
Effects of p28 and Tamoxifen—Table 8 and FIG. 9 summarize results for the chemoprevention of MDL by P28 and in combination with Tamoxifen.







TABLE 8







EFFECT OF POTENTIAL CHEMOPREVENTIVE AGENT ON


DMBA-INDUCED MAMMARY DUCTAL LESIONS IN


ORGAN CULTURE: BALB/C SCREENING


Chemopreventive agents: P28 and Tamoxifen


Experiment No.: CDG-4














No. of fields
Fields with

Percent


Group No.
Treatment
examined
ductal lesions
Multiplicity %
Inhibition










Expt. 4 A












1
DMBA
84
69
82.1



2
DMBA + Tamoxifen (1 μM)
71
30
42.3
49%


3
DMBA + P28 (12.5 μg/ml)
79
44
55.7
32%


4
DMBA + P28 (12.5 μg/ml) +
88
21
23.9
71%



Tamoxifen (1 μM)


5
DMBA + P28 (25 μg/ml)
73
18
24.7
70%


6
DMBA + P28 (25 μg/ml) +
68
12
17.6
79%



Tamoxifen (1 μM)







Expt. 4 B












7
DMBA
87
62
71.3



8
DMBA + P28 (50 μg/ml)
63
15
23.8
67%


9
DMBA + P28 (50 μg/ml) +
78
15
20
72%



Tamoxifen (1 μM)


10 
DMBA + P28 (100 μg/ml)
82
12
14.8
80%


11 
DMBA + P28 (100 μg/ml) +
73
14
19.2
73%



Tamoxifen (1 μM)









p28 (12.5 μg/ml) suppressed 32% of the MDL formation, whereas Tamoxifen alone inhibited nearly 50% of the lesions. The combination of low P28 and Tamoxifen increased inhibition to 71%. All chemopreventive agents at all concentrations were statistically significant compared to DMBA alone. At the same time, p28 (12.5 μg/ml) or Tamoxifen alone were also significantly different as compared to combination treatment. Results show that p28 can suppress both MAL and MDL development. p28 (12.5 μg/ml) in combination with Tamoxifen results in significantly enhanced chemopreventive activity compared to either agent alone. Azurin and p28 inhibited both estrogen progesterone dependent ductal lesions as well as independent alveolar lesions.
Mechanism of Preferential Entry into Cells
Example 6
Entry of p18 and p28 into Human Cell Lines
Cell Culture and Cell Lines: Human cancer and non-cancer (immortalized and non-immortalized) cell lines were obtained from ATCC [lung cancer (A549 and NCI-H23 adenocarcinoma), normal lung (CCD-13Lu), prostate cancers (DU145 and LN-CAP), normal prostate (CRL11611), breast cancer (MCF-7), normal breast (MCF-10A), colon cancer (HCT116), normal colon (CCD33Co), fibrosarcoma (HT1080), and ovarian cancer (SK-OV3 adenocarcinoma)]. Normal fibroblasts isolated from skin were established. Normal ovarian cells (HOSE6-3) were donated by Dr. S. W. Tsao (University of Hong Kong). Melanoma lines (UISO-Mel-2, 23, 29) were established and characterized. All cells except UISO-Mel-2 were cultured in MEM-E (Invitrogen, Carlsbad, Calif.) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biological Inc., Lawrenceville, Ga.), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 C in 5% CO2 or air.
Proliferation assays/Cell growth: Melanoma cells were seeded (four replicates) in flat bottom 24 well plates (Becton Dickinson, Franklin Lakes, N.J.) at a density of 12×103 cells/well. After 24 hrs media was changed and fresh p18, p28, azurin or a similar volume of media without peptide (eight replicates) added daily for 72 hr. Cells were then counted in a Beckman Coulter (Z 1 coulter particle counter). Values represent the mean±SD of 4 replicates.
MITT Assay: Melanoma cells were seeded at a density of 2000 cells/well in flat-bottomed 96 well plates (Becton Dickinson, Franklin Lakes, N.J.) and allowed to attach for 24 hrs. Freshly prepared peptide (10 μl) or culture medium was then added to each well. After 24 hrs, medium was changed and p18, p28 or azurin added daily. After 72 hr incubation, 10 μl of MTT reagent (Trevigen, Gaithersburg, Md.) was added to each well, the samples incubated for 3 hr, RT/sig 100 μl of detergent added to each well, and the samples incubated for an additional 3 hr at 37° C. Absorbance was measured with a SpectraMax 340 plate reader (Molecular Devices Corporation, Sunnyvale, Calif.) and percent change in the absorbance at 570 nm in treated cells relative to untreated controls determined. Values represent the mean±SD. Significance between control and treated groups was determined by Student's t-test.
Peptide synthesis: All azurin derived peptides including p18, Leu50-Gly67 LSTAADMQGVVTDGMASG (SEQ ID NO. 25), p28 Leu50-Asp77 LSTAADMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO. 2), p18b Val60-Asp77 VTDGMASGLDKDYLKPDD (SEQ ID NO. 38), MAP, Mastoparan-7, and poly arginine (Arg8, SEQ ID NO: 82) were synthesized by C S Bio, Inc. (Melo Park, Calif.). Peptides were received as lyophilized powder aliquoted and stored at −20° C. in air-tight desiccators. All peptides were subsequently analyzed by mass spectrometry and reverse phase HPLC as >95% purity and mass balance.
Predictive modeling for azurin peptides: GENETYX software (ver. 6.1) was used to generate Robson structure models for azurin derived peptides. Garnier, J., Osguthorpe, D. J., and Robson, B., J Mol Biol, 120: 97-120 (1978). The MAPAS Software was used to predict a given protein structure for strong membrane contacts and define regions of the protein surface that most likely form such contacts. Sharikov, Y. et al., Nat Methods, 5: 119 (2008). If a protein, i.e., azurin, has a membranephilic residue score (MRS)>3, membranephilic area score (MAS)>60%, and coefficient of membranephilic asymmetry (Kmpha)>2.5, there is a high probability that the protein has a true membrane-contacting region.
Peptide/Protein labeling: Peptides were dissolved in 1 ml PBS mixed with Alexafluor 568 dye (Molecular Probes, Eugene, Oreg.) at a 1:2 protein:dye ratio, 100 μl sodium bicarbonate added, and the mixture incubated overnight at 4° C. with continuous stirring. Labeled peptide was separated from free dye by dialyzing against cold-PBS using Slide-A-Lyzerg Dialysis Cassettes 1000 MWCO for p12 and 2000 MWCO for others (Pierce Biotechnology, Rockford, Ill.).
Cell penetrationfconfocal analysis: Cells were seeded on glass coverslips and allowed to attach overnight at 37° C. under 5% CO2. Cells were rinsed with fresh media and incubated at 37° C. for 2 hrs in pre-warmed media containing Alexafluor 568 labeled azurin peptides (20 μM) or Arg8 (SEQ ID NO: 82) (5 μM), or media alone. Following incubation, coverslips were rinsed 3× with PBS, cells fixed in 2.5% formalin for 5 min, and washed 2× in PBS, once in d.i. H2O, and coverslips mounted in media containing 1.5 μg/ml DAPI for nuclear counter staining (VECTASHIELD® Vector Laboratories, Burlingame Calif.). Cellular uptake and distribution were photographed under an inverted confocal laser scanning microscope (‘Model LC510, Carl Zeiss Inc., Gottingen, Germany).
Peptide co-localization with lysosomes or mitochondria was determined by incubating cells growing on a glass coverslip for 2 hrs at 37° with Alexafluor 568 labeled azurin or peptides. Mitrotracker (MitroTracker® Green FM Invitrogen Corporation, Carlsbad, Calif.) or lysotracker (LysoTracker® Green DND-26 Invitrogen Corporation, Carlsbad, Calif.) was added (final concentration 1 μM) for the last 30 mins of incubation. Cells were rinsed 3× with PBS, fixed in 2.5% formalin for 5 mins, washed 2× with PBS and incubated in 0.1% Triton-X100 in PBS for 15 min. Cells were then incubated with 1 μg/ml rabbit anti-human golgin 97 or anti-human caveolin I (Abeam, Cambridge Mass.) in PBS with 1% BSA. After 1 hr incubation at 4° C., coverslips were washed once with PBS, incubated 10 min in PBS containing Alexafluor 468 conjugated goat anti-rabbit antibody, washed 2× in PBS and once in d.i.H20. Coverslips were then mounted in media containing 1.5 μg/ml DAPI for nuclear counter staining. Colocalization (yellow) of Alexafluor 568 (red) and Alexafluor 468 (green) was analyzed and photographed.
UISO-Mel-2 cells on coverslips were preincubated in MEM-E containing 100 μg/ml heparin sulfate (Sigma-Aldrich, St. Louis, Mo.) for 30 min and p18, p28 or Arg8 (SEQ ID NO: 82) added to bring the final concentration to 20 μM. After 1 hr, coverslips were washed, fixed, and analyzed as described above.
Cell penetration by FFACS: Cells (1.0×106/500 μl PBS) were incubated for 2 hrs at 37° C. with Alexafluor 568 labeled p18 or p28 (20 μM), Arg8 (SEQ ID NO: 82) (5 μM), or media alone washed 3× in PBS, fixed in 2.5% formalin for 5 min, washed twice in PBS, resuspended in 200 μl PBS, and passed through a screen to obtain a single cell suspension. Samples were analyzed with a MoFlo Cell Sorter (Dako, Glostrup, Denmark) λex 568 nm and λem 603 nm and the fold increase of the mean fluorescence intensity over background levels calculated. Results represent mean fluorescence of three separate experiments.
Entry inhibitors: UISO-Mel-2 cells (3×105 per 300 μl), maintained in phenol red-, serum-free MEM-E at 37° C., were pretreated with inhibitors, including: Chloropromazine (inhibitor of clathrin-mediated endocytosis, 10 μg/ml, 60 min); Amiloride (macropinocytosis inhibitor, 50 μM, 30 min); Nystatin (50 μg/ml, 30 min); Methyl-β-cyclodextrin (MβCD, 5 mM, 60 min); Filipin (inhibitor of caveolae-mediated endocytosis, 3 μg/ml, 60 min); Taxol (microtubule stabilizer, 20 μM, 30 min); Staurosporine (cell cycle inhibitor, 250 nM, 10 min); Sodium azide (metabolic inhibitor, 1 mM, 60 min); Oauabain (ATPase-dependent Na+/K+ pump inhibitor, 50 mM, 60 min); Brefeldin A (BFA; Golgi apparatus disruptor, 100 μM, 60 min); Wortmannin (early endosome inhibitor, 100 nM, 30 min); Monensin (inhibits at late endosome/lysosome, 10 μM, 60 min); Nocodazole (inhibits caveosome formation, 10 μM, 60 min); Cytochalasin D (actin filament and microtubule disrupter, 5 μM, 30 min); Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BnGalNac; O-linked glycosylation inhibitor, 3 mM, 48 hrs); Tunicamycin (N-linked glycosylation inhibitor, 20 μg/ml, 48 hrs); and Neuraminidase (cleave sialic acid residues from proteins, IU/ml, 30 min). Final concentrations were derived from the dose response curves of individual inhibitors. Alexafluor 568 labeled p18 or p28 (20 μM) were then added, incubated for 1 hr, and the cells washed, fixed and prepared for flow cytometric analysis as described above.
Cell Membrane Toxicity Assays/LDH Leakage Assay: An LDH leakage assay was performed according to the manufacturer's instructions (CytoTox-One, Promega, Wis.) with 100 μl of UISO-Mel-2 cells (5×103). Cells without peptides/proteins were used as a negative control. Experiments were carried out in triplicate (data represent mean±SEM).
Hemolysis assay: Human whole blood samples (2-3 ml) were centrifuged for 10 min at 1000×g, and the pellets washed once with PBS and once with HKR buffer pH7.4 (18). Cell pellets were then resuspended in HKR buffer to 4% erythrocytes, 50 μl transferred to a 1.5 ml tube with 950 μl of peptides, azurin (5, 50 and 100 μM) or 0.1% Triton X-100 in HRK buffer to completely disrupt the RBC membrane. MAP and Mastoparan7 (Bachem California, Inc., Torrance, Calif.) were used as positive controls. After 30 min incubation at 37° C. with rotation, tubes were centrifuged for 2 min at 1000×g, 300 μl of supernatants transferred to a 96-well plate and absorbance recorded at 540 nm.
Kinetics of Entry: UISO-Mel-2 cells (5×105 cells) in 1.5 ml tubes were suspended in MEME media without phenol red. Reactions were started by adding either Alexa fluor 568-conjugated p18 at 0, 10, 20, 50, 100, 150 and 200 μM for 5, 10, 15 and 20 sec., or Alexafluor 568-conjugated p28 at 1, 10, 25, 50, 100, 150 and 200 μM for 30, 60, 90 and 120 sec on ice. After incubation, 1 ml of cold-PBS was added to the 250 μl reaction in mixture. Cells were centrifuged twice at 600×g for 2 min at 4° C. At least 10,000 fixed cells were analyzed by flow cytometry in each reaction and their background and relative fluorescence calculated.
I125 Labeling of Azurin and Competition Assays: Peptide binding and entry was determined using a whole cell assay with UISO-Mel-2 cells in HEPES solution (50,000 cells/ml), were incubated for 30 min at 37° C. with increasing concentrations (0-175 nM) of radiolabeled azurin in the presence/absence of 1000 fold excess of unlabeled p18, p28, or azurin, then washed 3 times with ice cold PBS, and radioactively remaining in the cell pellet counted using a gamma counter. Radioactivity in cells incubated with I125 azurin alone was considered total binding; radioactivity in the presence of unlabeled azurin, p18, or p28 was considered nonspecific binding. Specific binding was determined by subtracting nonspecific binding from total binding and Scatchard plots generated.
Example 7
Domain of p28 Responsible for Preferential Entry into Cancer Cells
Initial data from peptide-GST constructs defined aa 50-77 of azurin as a putative PTD for cell penetration, which fits well with structural evidence for an α-helical region encompassing residues 54-67 of azurin stabilizing the azurin molecule. Confocal analyses initially suggested that p28 and p18 of p28/azurin (FIG. 11 A) penetrated human melanoma, prostate, lung, breast and ovarian cancer cells with relatively similar efficiency, but did not penetrate histologically matched normal cell lines to the same degree (FIG. 11 A). A singular exception was CCD13-Lu, a cell line derived from lung fibroblasts. The cationic Arg8 (SEQ ID NO: 82) was rapidly and efficiently taken up into fibroblasts (FIG. 11 A) and all other normal cell lines tested (data not shown).
These observations were essentially confirmed by a more sensitive FACs analyses (FIG. 11B) where p28 fluorescence was about 0.5-6 and p18 about 0.5-3 fold higher than the corresponding normal cell line, with the exception of lung cancer. A similar pattern in intracellular fluorescence intensity was observed within a histopathologic subtype, melanoma, where the relative intensity of p18 was about 50% of that observed with p28 (FIG. 11C). Fluorescence intensity over background was also consistently lower in normal and cancer cell pairs exposed to p18 than p28 (data not shown), again suggesting less p 18 entered individual cells. In all cases, the degree of entry of p18 and p28 into either cancer or normal cells was significantly less than that observed with Arg8 (SEQ ID NO: 82), where no preference for entry was observed (FIG. 11A). The predicted Robson structure (data not shown) of p18 suggests that the C-terminal amino acids form a partial β-sheet. This and the shorter length of p18, which lacks the hydrophilic C-terminal 10 amino acids (aa 68-77) (SEQ ID NO: 40) of p28, suggests that p18, as a putative PTD for azurin, may have a more rapid entry into cancer and normal cells via a non-endocytotic over an endocytotic or membrane receptor mediated process. MAPAS data (MRS 3.74, MAS 87.1, Kmpha 2.37) predict that aa's 69, 70, 75, 76, 85 of azurin provide the best opportunity for membrane contact, suggesting the C-terminal region of p28, not present on p18 (aa 50-67) is most likely to contact specific residues on the cell membrane, irrespective of a cell's status.
The preferential penetration of p18 and p28 was confirmed by exposing the same cell lines to azurin 60-77 (p18b), or aa 66-77 (SEQ ID NO: 39), the C-terminal 12 aa of p28 (FIG. 12 A, B). Here, the preferential penetration observed with p18 and p28 was completely abolished. p18b (theoretical p 4.13) has a short α-helix and partial β-sheet, and is extremely hydrophilic which together may negate preferential entry. p12 (theoretical pI 4.33) lacks a secondary α-helical structure, but is also hydrophilic suggesting overall hydrophilicity may be a major contributor to the decrease in selectivity of cell penetration.
Example 8
Cell Penetration is not a Result of Membrane Disruption
Cell penetration by azurin, p28, and p18 does not result from membrane disruption. An LDH leakage assay using UISO-Mel-2 cells in the presence of 5-100 μM p28, p18 or azurin (FIG. 13 A) suggested that neither peptide nor azurin entered cells by altering plasma membrane integrity. The lack of membrane disruption was confirmed by determining the hemolytic activity of azurin, p28, and p18 on human erythrocytes against the receptor mimetic MAP and mast cell degranulating peptide mastoparan 7, which translocates cell membranes as an amphipathic alpha-helix, and activates heterotrimeric G proteins. Mastoparan 7 caused complete cell lysis at 25 μM, while azurin, p28, and p18 had no hemolytic effect when compared to control (no peptide) (FIG. 13 B).
Example 9
p18/p28 Penetration is Energy Dependent and Saturable
The penetration of p28 (FIG. 14 A) and p18 (FIG. 14 B) into UISO-Mel-2 cells is temperature dependent. Cell penetration and intracellular transport occurs relatively slowly over 3 hr at 4° C., while entry and intracellular transport through various compartments is rapid at 22 and 37° C. as p18 and p28 were present in the nucleus of UISO-Mel-2 cells within 2 hrs post exposure. The penetration of 5 μM p28 (FIG. 14 C) or p18 (FIG. 14 D) into UISO-Mel-2 cells after 30 min in the presence of a 200 fold excess of unlabeled peptide was severely curtailed, suggesting that entry was a saturable process and specific receptors or cell surface proteins or specific residues were, at least in part, responsible for initial entry.
Example 10
Kinetics of p28 and p18
The kinetics of p28 and p18 entry into UISO-Mel-2 cells relative to human fibroblasts was calculated after incubation, when cells were fixed and mean fluorescence intensity (MFI) determined. The Km and Vmax of each peptide were calculated by plotting peptide concentration (μM) vs velocity (MFI/sec) or by Scatchard analysis. Although the penetration of azurin fragments 50-67 (p18: Vmax 2.46, Km 101.6) and 50-77 (p28: Vmax 1.87, Km 159.1) into cancer and normal cells (Vmax 2.88, Km 102.1 and Vmax 1.89, Km 166.0, respectively) differs significantly from each other, with p18 entering −42% faster, the rate of the entry of each peptide into normal and cancer cells is virtually identical. The increase in amount of fluorescence following exposure of cancer cells to p28 relative to p18 is likely due to the increase in the amount of p28 entering malignant cells. 125I azurin and p18 bound to UISO-Mel-2 cells with a similar affinity. In contrast, significantly more p28 (Kd 2.5 μm, Bmax 3.0 pm) bound to UISO-Mel-2 cells with a higher affinity when exposed for a longer period of time (20 min vs 2 min) at a higher temperature (37° C. vs 4° C.) than either p18 (Kd 18 min, Bmax 0.51 pm) or azurin (Kd 10 μm and 0.48 pm). These results suggest that azurin, p28, and p18 all bind with relatively high affinity and capacity to a site on the cancer and normal cell surface prior to entry, but may enter via more than one mechanism.
Example 11
p18/p28 Penetration Involves Caveolae and the Golgi Complex
Peptides called cell-penetrating peptides (CPPs) or cell-delivery vectors (CDVs), such as penetratin, transportan, Tat (amino acids 47-57 or 48-60), and the model amphipathic peptide MAP, are short, amphipathic and cationic peptides and peptide derivatives, usually containing multiple lysine and arginine residues. Fischer, P. M., Med Res Rev, 27: 755-795 (2007). They form a class of small molecules receiving significant attention as potential transport agents or delivery vehicles for a variety of cargoes, including cytotoxic drugs, anti-sense oligo-nucleotides, proteins, and peptides, in gene therapy, and as decoy peptides. Hallbrink, M. et al Biochim. Biophys. Acta 1515: 101-109 (2001); Lindgren, M., et al Trends Pharmacol. Sci. 21: 99-103 (2000); Gusarova, et al, J Clin Invest, 117: 99-111 (2007); Melnick, A., Biochem Soc Trans, 35: 802-806 (2007); Astriab-Fisher et al., Pharm Res, 19: 744-754 (2002); El-Andaloussi et al, J Gene Med, 8:1262-1273 (2006); Cashman et al., Mol Ther, 6: 813-823 (2002).
As a class, cationic CPPs such as pTat and Arg8 (SEQ ID NO: 82) enter cells by initially binding to anionic, sulfated proteoglycans prior to endocytosis. Incubation of p28 and p18 and Arg8 (SEQ ID NO: 82) with UISO-Mel-2 cells under serum free conditions in the presence/absence of 100 μg/ml heparin sulfite (HS) significantly reduced the amount of intracellular Arg8 (SEQ ID NO: 82), but did not alter the entry of either p28 or p18 (FIG. 15 A). The penetration of p18 and p28 into UISO-Mel-2 cells in the presence or absence of a specific inhibitor of O-linked glycosylation, BnGalNac, and neruaminidase, which cleaves sialic acid residues, was further characterized (FIG. 15 B), and no inhibition of penetration was observed. However, tunicamycin, an inhibitor of N-linked glycosylation, significantly reduced the penetration of p18 and p28 across the cell membrane.
The entry of p18 and p28 into UISO-Mel-2 cells was also analyzed using inhibitors of energy dependent transport mechanisms, i.e., ATP. Sodium azide (FIG. 15 B) and ouabain (Na+ K+ ATPase pump) did not significantly inhibit the penetration of either peptide suggesting non endocytosic pathways might also be involved in the penetration of these peptides. Chlorpromazine (CPZ), a specific inhibitor of clathrin mediated endocytosis, also had no effect on penetration, nor did the macropinocytosis inhibitor amiloride. (FIG. 15 B). Stabilization of microtubules with taxol had no effect on penetration, but disruption of actin filaments and macropinocytosis with Cytochalasin D produced a small (−20%), reproducible inhibition of the penetration of p18 and p28. The lack of effect of amiloride suggests that the inhibitory activity of Cytochalasin D is probably through its effect on actin filaments.
Inhibition of the cell cycle with staurosporine did not block penetration, suggesting that penetration was not cell cycle specific. The lack of effect of staurosporine on p18 and p28 penetration of the cancer cell plasma membrane also suggests that a Src kinase/tyrosine kinase dependent pathway was not involved in penetration, was dynamin independent, and hence independent of caveolac budding. Neither p18 nor p28 co-localized with flotillin-1 (data not shown) a protein that resides within the plasma membrane and in a specific population of endocytic intermediates, again arguing against a role for flotillin and dynamin in internalization. In contrast, nocodazole, which disrupts caveolae transport and inhibitors of cholesterol mobilization and hence, caveolae-mediated endocytosis, inhibited penetration 50-65%.
The intracellular disposition of p18 and p28 was then analyzed using wortmannin, an inhibitor of early endosome formation, monensin, which inhibits late endosome/lysosome, and brefeldin A (BFA), a disrupter of the Golgi apparatus. Wortmannin did not block the intracellular accumulation of either p18 or p28 suggesting that, unlike cholera toxin, a caveolae to early endosome pathway is not involved in the intracellular trafficking of p18 and p28. The lack of early endosome involvement in the intracellular trafficking of p18 and p28 also suggests that clathrin mediated endocytosis is not involved in internalization of these peptides.
However, monensin (FIG. 15 B) and BFA reduced the intracellular accumulation of both peptides with a greater inhibitory effect on p28 (30%) than p18 ( ̃10%) (FIG. 15 B). The penetration of p28 and p18 into fibroblasts was also inhibited by MβCD, nocodazole, monensin and tunicamycin, but not by amiloride, sodium azide, and CPZ (FIG. 15 C). This suggests that at least one mechanism of entry into cancer and normal cells may be similar, but additional preferential accumulation into cancer cells may be a function of the number of common membrane receptors or structures, ie., caveolae (FIG. 15 D, panels 1, 2). Alexafluor 568 labeled p18 and p28 co-localized with caveolin-1 and golgin 97 antibodies (FIG. 15 D panels 1,2). This confirms that these organelles are involved in the intracellular trafficking of p18 and p28. Interestingly, azurin, but neither p18 nor p28 colocalized with mitochondrial specific fluorescence (FIG. 15 D panel 3). In contrast, p28 and azurin, but not p18, co-localized with lysosomes (FIG. 15 D panel 4).
Example 12
Functional Analysis of p28 and p18
Azurin inhibits the growth of several human cancer cell lines in vitro and in vivo. FIGS. 16 A and B illustrate the effect of p18 and p28 relative to azurin and dacarbazine (DTIC) on UISO-Mel-2 cells as determined by MTT and cell count. After 72 hrs exposure, azurin decreased (p<0.05) cell survival at 100 and 200 μM −15% (FIG. 15 A). p28 had inhibited cell survival 14 and 22% (p<0.05) at 100 and 200 μM, respectively. In contrast, p18 had no effect, while dacarbazine (DTIC) produced a significant dose-related decrease on UISO-Mel-2 survival. Azurin and p28 (200 μM) also significantly decreased the survival of UISO-Mel-23 and 29 cells. p18 had no effect on UISO-Mel-2 cell proliferation.
The apparent increase ( ̃30-35%; UISO-Mel-2) in p28 and azurin inhibition of melanoma cell proliferation, as measured by direct cell counting, suggests that the inhibitory effect may reside primarily at the level of cell cycle with apoptosis subsequent to any delay. Although p18 penetrated cancer cells preferentially, unlike p28, it had virtually no inhibitory activity on cell proliferation. This result indicates that the cytostatic and cytotoxic activity of p28 likely lies in the C-terminal 10-12 aa of the sequence.
Example 13
Inhibition of Cancer Growth Through p53 Using Azurin-Derived Peptides: Materials and Methods
Cell culture. Human breast cancer cell lines, MCF-7 (p53 wt), obtained from ATCC (Manassas, Va.) and MDD2 (p53 dominant negative) from Dr. Andrei V. Gudkov (Lerner Research Institute, Cleveland, Ohio) were cultured in MEM-E (Invitrogen, Carlsbad, Calif.) containing 2 mM L-glutamine, 0.1 mM essential amino acids supplemented with 10% heat inactivated fetal bovine serum, 100 Units/ml penicillin and 100 μg/ml streptomycin.
Bacterial culture and isolation of azurin Escherichia coli JM109 was used as the host strain for production of wild type azurin. Culture conditions and protein purification steps were as described in Yamada, et al., Infect Immun, 70:7054-7062 (2002) and Goto, et al., Mol Microbiol, 47:549-449 (2003).
Peptide synthesis. All azurin-derived peptides including p18, Leu50-Gly67 LSTAADMQGVVTDGMASG (SEQ ID NO: 25), p28 Leu50-Asp77 LSTAADMQGVVTDGMASGLDKDYLKPDD, (SEQ ID NO: 2) p18b Val60-Asp77 VTDGMASGLDKDYLKPDD (SEQ ID NO: 38), p12 Ser66-Asp77 SGLDKDYLKPDD (SEQ ID NO: 39), and poly arginine (Arg8) (SEQ ID NO: 82) were synthesized by CS Bio, Inc. (Menlo Park, Calif.) as >95% purity and mass balance.
Proliferation assays. Cells were seeded in MEM-E in quadruplicate into 24-well plates (Becton Dickinson, Franklin Lakes, N.J.) at a density of 12×103 cells/well and incubated in the presence of 5, 50, 100 and 200 μM p28 for 24, 48 and 72 hr. Media was changed daily. Control wells received MEM-E without p28 (8 replicates). Doxorubicin (10 μM) was used as positive control (Z1 coulter; Beckman Coulter Inc., Fullerton, Calif.). Values represent (%) of control. Significance between control and treated groups was determined by Student's t-test.
MTT Assay: MCF-7 cells were seeded at a density of 2000 cells/well (quadruplicate) allowed to attach for 24 hrs, and freshly prepared peptide (10 μL) or MEM-E added to each well. After 24 hrs, medium and p18, p28, azurin or doxorubicin were added daily. After incubation, 101 of MTT reagent (Trevigen, Gaithersburg, Md.) was added to each well, the samples incubated for 3 hr at RT, 100 μl of detergent added to each well, and incubated for an additional 3 hr at 37° C. Absorbance (570 μm) was measured (SpectraMax 340 plate reader, Molecular Devices Corporation, Sunnyvale, Calif.) and percent change in treated cells determined. Significance (p<0.05) between control and treated groups was determined by Student's t-test.
Xenograft model. Estradiol pre-treated (0.72 mg/pellet, 60-day release; Innovative Research, Sarasota, Fla.) female athymic mice (Harlan; 4-5 weeks old) received 3×106 MCF-7 cells s.c. in the right flank and randomized into control and experimental groups prior to treatment. Control animals received PBS/castor oil i.p. Paclitaxel, 15 μmol/kg in PBS/castor oil was injected i.p. on days 10, 14, 21 and 25 post-tumor cell inoculation, or p28, 5 or 10 mg/kg in sterile PBS i.p. daily was injected for 30 days. Tumor volume was determined 3×/week. Body weights were measured twice weekly. Mice were necropsied on day 31 and all tumors collected for histopathology and immunocytochemistry. Significance (p<0.05) between control and treated groups was determined by Student's t-test.
Immunocytochemistry. BrdU, 50 mg/kg body wt, was injected i.p., 2 hrs prior to necropsy. Tumor cell nuclei labeled with BrdU were identified with an anti-BrdU monoclonal antibody (Beckon Dickinson, Franklin Lakes, N.J.). p53 expression was quantified in formalin fixed, 5μ paraffin sections treated with 10 mM citrate buffer in a pressure cooker for 6 min. Cooled slides were treated with 3% H2O2 for 10 min to block endogenous peroxidase, covered with blocking serum for 10 min, and exposed to p53 antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, Calif.) for 2 hrs at room temperature. Rat anti-mouse IgG2a was used as the second antibody. Cells expressing p53 were identified using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif.) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma Aldrich, St. Louis, Mo.). Slides were counterstained with hematoxylin. Ten non-overlapping fields (250 cells/field) from each tumor periphery were screened (40×) for p53 labeled cells.
Confocal microscopy: Cells were seeded overnight on glass cover slips at 37° C. under 5% CO2, rinsed with fresh media, and incubated at 37° C. for 2 hr in pre-warmed media containing Alexa Fluor 568 labeled peptides (20 μM), Arg8 (SEQ ID NO: 82) (5 μM), or media alone. After incubation, cover slips were rinsed 3× with PBS, fixed in 2.5% formalin for 5 min, washed 2× in PBS, once in d.i.H2O, and mounted in media containing 1.5 μg/ml DAPI to counter stain nuclei (VECTASHIELD®, Vector Laboratories). Cyclin B1 and p21 staining were determined in fixed cells, permiabilized by methanol and acetone, washed with PBS and incubated with anti-p21 or cyclin B at a 1:200 dilution (Santa Cruz Biotechnology). Secondary antibody conjugated Alexa Fluor 568 was used at 1:100 dilution. Cellular uptake and intracellular distribution were determined using an inverted confocal laser scanning microscope (Model LC510, Carl Zeiss Inc., Gottingen, Germany).
Kinetics: MCF-7 and MDD2 cells (3×105 cells) were suspended in MEM-E without phenol red. Reactions were started by adding Alexa Fluor 568-conjugated p28 at 1, 10, 25, 50, 100, 150 and 200 μM for 30, 60, 90 and 120 sec on ice. After incubation, 1 ml of cold-PBS was added to the reaction mixture and cells centrifuged 2× at 600×g for 2 min at 4° C. At least 10,000 fixed cells were analyzed for each time point and concentration by flow cytometry and their background and relative fluorescence calculated.
Cell cycle analysis. MCF-7 and MDD2 cells were incubated with 50 μM of p28 for 48 and 72 hr at 37° C., washed twice with phosphate-buffered saline (PBS) and fixed with 70% ethanol at −20° C. Fixed cells were washed twice with PBS and stained by 50 μg/ml of propidium iodide (PI) in PBS containing 20 μg/ml of RNase A. Flow cytometry (EPICS Elite ESP, Beckman Coulter, Fullerton, Calif.) was used to determine DNA content. A minimum of ten thousand cells were collected in each experiment.
Immunoblotting. MCF-7 and MDD2 cells were cultured with 50 μM p28 for 0, 24, 48 and 72 hr. and whole cell lysates prepared according to the methods described earlier (3). Cell lysates for phosphorylated cdc2 (p-cdc2) was prepared in 10 mM NaF, 137 mM NaCl, 1 mM NaVO4, 10 mM EDTA, 1% NP-40, 1 mM DTT and proteinase inhibitors (Sigma Aldrich). Antibodies against p53, p27, CDKs, cyclins (Santa Cruz Biotechnology), p21 (Invitrogen) were used according to the suppliers' instructions. Actin expression was determined with a monoclonal actin antibody (Santa Cruz Biotechnology) and protein bands visualized using ECL reagent (Santa Cruz Biotechnology).
Anti-p28 antibody. A cysteine was introduced at the N-terminus of p28 (CS Bio Inc., Menlo Park, Calif.), and then the peptide was conjugated with Keyhole limpet hemocyanin through the thiol groups of the cysteine residue, the complex was inoculated intradermally and subcutaneously, and a polyclonal antibody specific for 11-28aa of p28 (60-77aa of azurin) in rabbits (New Zealand White, Covance, Mich.) was generated. Antibody titer was determined by direct ELISA using p28 (0-3 μg/well). An antibody dilution of 1:140,000 was sufficient to give a reproducible change in absorbance of 0.5 at 450 nm after 15 min incubation with substrate (1-Step PNPP, Pierce, Rockford, Ill.) at 25° C., when 96 well-plates (Nunc, Rochester, N.Y.) were coated with 1 μg/well p28.
GST pull-down assay. p28 binding to p53 was assayed using a GST pull down assay essentially as described in Punj, et al, Oncogene 23:2367-2378 (2004). Purified GST-p28 (10 and 20 μg/reaction), GST-MDM2 (20 μg/reaction) and GST alone (20 μg/reaction) were bound to Glutathione Sepharose 4B beads (GE Healthcare, NJ) and unbound peptide removed by washing 2× with PBS. Whole cell lysates of MCF-7 cells were generated with PBS/0.1% Triton X-100 containing proteinase inhibitor cocktail (Sigma-Aldrich) on ice for 15 min, and centrifuged at 14000 r.p.m. for 30 min at 4° C. Resultant supernatants were mixed with beads, incubated for 2 hr at 4° C., washed 2× with PBS to remove unbound cell lysate and then boiled in SDS-sample buffer prior to loading on 10% SDS-PAGE. Membranes were incubated with skim milk (5%) in TBST (Tris/0.05% Tween20) and polyclonal p53 antibody (FL-393, Santa Cruz Biotechnology) in 5% skim milk at 4° C., washed 3× with TBST, secondary rabbit IgG-HRP antibody (Sigma-Aldrich) added, incubated for 1 hr at room temperature (r/t), and washed 3× with TBST.
Potential binding sites on p53 were identified as follows. Interaction at the MDM2 binding site (18-23aa) of p53 was analyzed using a GST-pull down assay in the presence of p28 (10-50 molar excess) and p53 bands detected by immunoblotting (IB). Three different anti-p53 antibodies, Pab 1801 (32-79 aa; Santa Cruz Biotechnology), ab 2433 (277-296aa; Abeam Inc., Cambridge, Mass.) and Pab1802 (306-393aa; Santa Cruz Biotechnology), that represent the broadest coverage of the p53 protein available, were each reacted with GST-p53 immobilized beads in the presence of p28. After incubation, samples were washed 2× with PBS to remove unbound p28, boiled in native PAGE sample buffer (Tri/glycerol/BPB) and loaded on 5% Native-PAGE. Samples were transferred to PVDF membrane by electroblotting (0.2 Amp for 1 hr), membranes blocked with skim milk (5%) in TBST and incubated with a polyclonal antibody to p28 (1:5000 dilution) in 5% skim milk at 4° C. After washing with TBST, HRP-conjugated rabbit anti-IgG antibody (1:7000 dilution, Santa Cruz Biotechnology) was applied. p28 bands were visualized using ECL reagent. Binding domains on p28 were identified using a competition assay between p28 and the p28 fragments p12, p18 and p18b for GST-p53 (20 μg/reaction) immobilized on Glutathione Sepharose 4B beads. Reactions were incubated for 2 hr at 4° C., washed 2× with PBS to remove unbound p28, then boiled in native PAGE sample buffer (Tri/glycerol/BPB) and loaded on 5% Native-PAGE. Proteins were transferred to a PVDF membrane by electro blotting (0.2 Amp for 1 hr), blocked, and incubated with the polyclonal antibody to p28 at 4° C. for 16 hr. p28 bands were visualized with ECL reagent. Band intensity was determined using Gel & Graph Digitizing Software, UN-SCAN-ITTM (Silk Scientific Inc., Orem, Utah) and the ratio of specific protein/actin calculated. Numbers displayed below each protein band are relative percentage of the protein babd intensity immediately prior to treatment (0 hr expressed as 100%).
p53 DNA-binding activity. Nuclear fractions (Nuclear Extraction kit, Active Motif, Carlsbad, Calif.) were isolated from MCF-7 cells after incubation with either 50 μM p28 or azurin at for 24 h according to the manufacturers' instructions. Nuclear extract supernatants were collected by centrifugation at 14,000 rpm for 10 min at 4° C. Protein concentrations were determined using the Bradford method. DNA-binding activity of p53 was measured using a TransAM p53 kit (Active Motif). Briefly, 40 μl of binding buffer containing DTT and poly[d(I-C)] was introduced to each well to prevent non-specific binding to the p53 consensus oligonucleotide. Nuclear extracts were applied to each well, with H2O2-treated or buffer only as positive and negative controls, respectively, and incubated 1 hr r/t. Wells were washed 3× and 100 μl of p53 antibody (1:1000 dilution) applied and incubated at r/t for 1 hr. After washing, secondary antibody conjugated with HRP was added, samples incubated for 1 hr and developed for 3 min in the dark. p53 binding to DNA was determined by absorbance at 450 and 655 mm.
Example 14
Effect of p28 Treatment on the Growth of Human Cancer Cells In Vitro and In Vivo
It is believed that azurin exerts its anti-cancer activity through induction of a p53-mediated apoptosis. FIGS. 18 A and B show the effect of p28 and doxorubicin on wt p53 (positive) MCF-7 cells as determined by direct cell count and MTT assay. p28 initially inhibited the proliferation of MCF-7 cells in vitro (FIG. 17 A) in a dose and time related manner producing a significant decrease (p<0.05) in cell number  ̃23% at 5 μM and  ̃36% at 50-200 μM after 24 hr exposure. Doxorubicin (DNA intercalating agent) also significantly inhibited cell growth in time-dependent manner. Cell survival determined by MTT assay was not significantly altered by p28, while doxorubicin exhibited a significant time related decrease in MCF-7 cell survival (FIG. 17 B). p28 also produced a significant dose related decrease in the volume of xenografted MCF-7 cells in athymic mice over a daily, 30-day i.p. exposure (FIG. 17 C), decreasing tumor volume (p<0.05) to that observed with Paclitaxel®, without inducing either a loss in body weight or behavioral change. By day 30, 10 mg/kg p28 daily i.p. inhibited MCF-7 growth to a greater extent ( ̃20%) than 15 mmol/kg Paclitaxel® on days 10, 14, 21 and 25 post-tumor cell inoculation. The reduction in BrdU labeling associated with the p28-induced decrease in tumor volume suggested cell cycle was inhibited (Table 9). In contrast, the reduction in BrdU labeling and tumor volume was accompanied by a slight increase in nuclear p53-staining in p28 and a significant increase in the Paclitaxel® treated group compared to control (Table 9).







TABLE 9







BrdU and p53 in MCF-7 xenograft tumors













N
BrdU (%)
p53 (%)







Control
7
21.0 ± 2.7
15.6 ± 0.82



p28 (5 mg/kg)
4
17.6 ± 0.75*
15.8 ± 0.51



p28 (10 mg/kg)
3
16.1 ± 1.4*
17.7 ± 0.92



Paclitaxel
6
 9.0 ± 1.8**
25.4 ± 0.65**







All tumors were collected on day 31 post treatment. Values represent Mean ± SEM.



*p < 0.025;



**p < 0.01 from respective control;



student's T-test.






Example 15
Inhibition of Cell Cycle Progression by p28
Cell cycle analysis of the two isogenic breast cancer cell lines, MCF-7 (p53 wt) and MDD2 (p53 dominant negative), revealed an increased cell population at the G2/M phase after exposure to p28 for 48-72 hrs and subsequent induction of apoptosis at 72 hrs in MCF-7 cells (FIG. 19 A). There was essentially no inhibition of cell cycle progression or apoptosis in p28-treated MDD2 cells (FIG. 18 B). The lack of cell cycle inhibition and apoptosis in p28-treated MDD2 cells (FIG. 18 B) was not due to a difference in p28 entry into MDD2 cells (FIG. 18 C) or difference in Vmax (MCF-7: 1.83 MFI/sec, MDD2: 2.21 MFI/sec) or Km (MCF-7: 144.3 μM, MDD2: 147.9 μM).
Example 16
p53 Levels are Elevated by p28
Azurin forms a complex with p53 and elevates intracellular p53 levels in MCF-7 cells. The intracellular level of p53 in MCF-7 cells also significantly increased with time post exposure to p28 (FIG. 19 A). A GST pull-down assay suggested p28 binds to p53 (FIG. 19 B). Here, GST-p28 and GST-MDM2 successfully pulled down p53 from MCF-7 cell lysates, but GST alone did not. Molar increases of p28 did not compete for binding with GST-MDM2 (FIG. 19 C) suggesting that aa 18-23 of p53 were not a preferred binding site for p28. An additional GST-pull down assay in the presence or absence of p53 antibodies, which recognize different motifs of the p53 protein (aa 32-79, 277-296 and 306-393), did not block p28 binding to p53, suggesting that p28 binds to a region of p53 outside these recognition sites (FIG. 19 C).
When Sepharose 4B-glutathione beads immobilized with GST-p53 protein were incubated with p28 and either aa 66-77, aa 50-67, or aa 60-77 of azurin, (p28 fragments p12, p18 and p18b) respectively, significant amounts of p28 were displaced by p18 and p18b, but only weakly when p12 was used as the competitor (FIG. 19 D). These results suggest that maximal binding to p53 occurs within aa 11-28 of p28 (aa 60-77 of azurin).
As p28 enhances intracellular levels of p53, the DNA-binding activity of p53 obtained from MCF-7 cell nuclear extracts treated with p28 or azurin was also examined. p53 DNA-binding activity in the nuclear fraction of MCF-7 cells treated by p28 and azurin was 1.8 and 2.3 fold higher than control (p>0.1, p28 vs azurin). The p53 wt consensus, but not the mutated oligonucleotide sequence, completely blocked the p28 induced increase in p53, confirming that the p53 in nuclear extracts of MCF-7 cells binds specifically to the consensus oligonucleotide sequence for wt p53 (FIG. 19 E).
Example 17
Modulation of Cell Cycle Related Proteins by p28
Upregulation of the CDK inhibitors (CDKIs), p21 and p27, blocks cell cycle progression. p28 increased intracellular levels of p21, p27, CDK6 and cyclin B1 over control in MCF-7 cells with time post-exposure (FIG. 20 A). The levels of CDK2 and cyclin A, essential proteins in the mitotic process, subsequently decreased with time post-exposure in p28 treated MCF-7 cells (FIG. 20 A). In contrast, p53, cdc2, CDK2, CDK4 and CDK6 essentially remained constant in MDD2 cells (FIG. 20 B), while cyclin A and cyclin B1 (48 hrs) increased slightly. Since p21 can be expressed by a p53-independent pathway in MDD2 cells, p21 remained detectable. p28 did not alter the level of p21, however (FIG. 20 B). In contrast, p27 was not detectable in untreated or p28 exposed MDD2 cells. The increased levels of p21 and cyclin 131 in MCF-7 cells detected by immunoblotting in response to p28 are reflected by their increase in nuclear and cytosolic compartments, respectively (FIGS. 20 C and D). Exposure of MCF-7 cells to p28 also induced the accumulation of phosphorylated cdc2 (p-cdc2), the inactive form of cdc2. The level of p-cdc2 did not increase following exposure of MDD2 cells to p28 (FIG. 20 E).

## Claims

1. A pharmaceutical composition comprising:
a first chemopreventive agent that is a truncation of azurin, wherein the truncation consists of an amino acid sequence selected from one or more of the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34; and
a second chemopreventive agent.

2. The pharmaceutical composition of claim 1, wherein the second chemopreventive agent is an antiestrogen.

3. The pharmaceutical composition of claim 2, wherein the second chemopreventive agent is selected from the group consisting of Tamoxifen, fenretinide, and aromatase inhibitors.

4. The pharmaceutical composition of claim 1, wherein the truncation binds to the N-terminal binding domain of p53.

5. The pharmaceutical composition of claim 1, wherein the truncation binds to the DNA binding domain of p53.

6. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

8. A kit comprising the composition of claim 6 in a vial.