(12) Patent Application Publication (10) Pub. No.: US 2009/0286719 A1 Das Gupta Et Al

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US 20090286719A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0286719 A1 Das Gupta et al. (43) Pub. Date: Nov. 19, 2009

(54) MODIFICATION OF CUPREDOXIN DERVED Publication Classification PEPTDES AND METHODS OF USE (51) Int. Cl. THEREOF A638/08 (2006.01) C07K 7/06 (2006.01) (76) Inventors: Tapas K. Das Gupta, River Forest, C07K 7/08 (2006.01) IL (US); Craig W. Beattie, C07K 7/50 (2006.01) Chicago, IL (US) C07K I4/2 (2006.01) A638/10 (2006.01) Correspondence Address: A638/12 (2006.01) DON.J. PELTO A638/16 (2006.01) Sheppard, Mullin, Richter & Hampton LLP CI2P 2L/02 (2006.01) 1300 ISTREET, NW, 11TH FLOOR EAST C07K L/107 (2006.01) WASHINGTON, DC 20005 (US) (52) U.S. Cl...... 514/11; 530/324; 530/326; 530/325: 530/327: 530/317; 514/12: 514/15: 514/14; (21) Appl. No.: 12/389,120 514/13:435/68.1:530/345 (22) Filed: Feb. 19, 2009 (57) ABSTRACT The present invention provides modified cupredoxin derived Related U.S. Application Data peptides with pharmacologic activity that have improved (63) Continuation-in-part of application No. 12/314.703, pharmacokinetic properties, and methods to use them to treat filed on Dec. 15, 2008, which is a continuation-in-part mammals suffering from various conditions related to the of application No. 1 1/853,497, filed on Sep. 11, 2007, pharmacologic activities. Modifications of the cupredoxin which is a continuation-in-part of application No. derived peptides include amino acid sequence variants and 1 1/244,105, filed on Oct. 6, 2005. structural derivations that increase the plasma half-life of the peptide, increase the specific activity of the pharmacologic (60) Provisional application No. 61/013,709, filed on Dec. activity, decrease immunogenicity, and decrease the biotrans 14, 2007, provisional application No. 60/843,388, formation of the peptides. The modified cupredoxin derived filed on Sep. 11, 2006, provisional application No. peptides can be used in methods to treat mammals for cancer, 60/616,782, filed on Oct. 7, 2004, provisional applica conditions related to inappropriate angiogenesis, viral and tion No. 60/680,500, filed on May 13, 2005, provi bacterial infections, and specifically HIV and malaria, con sional application No. 60/700,297, filed on Jul. 19, ditions related to ephrin signaling, and to deliver cargo com 2005. pounds, including diagnostic compounds, to cancer cell

Patent Application Publication Nov. 19, 2009 Sheet 1 of 65 US 2009/0286719 A1

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Structure : AZurin truncation with alpha-helical structure

Structure 2: Result of 70 ns Simulation.

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Measurement of thioether bridge positions based on distances between Co. atoms in a simulated structure.

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MODIFICATION OF CUPREDOXIN DERVED midium laminosum and pseudoaZurin form Achronobacter PEPTIDES AND METHODS OF USE cycloclastes also are cytotoxic towards macrophages. U.S. THEREOF Pat. Pub. No. 20060040269, published Feb. 23, 2006. Detailed studies of various domains of the azurin molecule CROSS REFERENCE TO RELATED suggested that amino acids 50-77 (p.28) (SEQ ID NO: 13) APPLICATIONS represented a putative protein transduction domain (PTD) 0001. This application claims priority under 35 U.S.C. SS critical for internalization and Subsequent apoptotic activity. 119 and 120 to, and is a continuation in part of U.S. patent Yamada et al., Cell. Microbial. 7:1418-1431 (2005), although application Ser. No. 12/314.703, filed on Dec. 15, 2008, possible routes of cellular entry were not identified which claims priority to U.S. Patent Application Ser. No. 0004 AZurin is now also known to have other pharmaco 61/013,709, filed on Dec. 14, 2007; and is a continuation in logic activities of therapeutic importance. It is known to part of U.S. patent application Ser. No. 1 1/853,497, filed Sep. inhibit angiogenesis in human umbilical vascular endothe 11, 2007, which claims priority to Provisional U.S. Applica lium cells (HUVECs). U.S. patent application Ser. No. tion Ser. No. 60/843,388, filed Sep. 11, 2006; and is a con 1 1/488,693, filed Jul. 19, 2006. AZurin from Paeruginosa is tinuation in part of U.S. patent application Ser. No. 1 1/244. also known for its ability to inhibit the growth of HIV-1 105, filed Oct. 6, 2005, which claims priority to U.S. infection in peripheral blood mononuclear cells and to inhibit Provisional Patent Application No. 60/616,782, filed Oct. 7, parasitemia of malaria-infected mammalian red blood cells. 2004, U.S. Provisional Patent Application No. 60/680,500, Chaudhari et al., Cell Cycle. 5: 1642-1648 (2006). AZurin filed May 13, 2005, and U.S. Provisional Patent Application from P. aeruginosa is also known to interfere with the ephrin No. 60/700,297, filed Jul. 19, 2005. The entire content of signaling system in various mammalian cells and tissues. those applications are fully incorporated herein by reference. U.S. patent application Ser. No. 11/436,592, filed May 19, 2006. FIELD OF THE INVENTION 0005. AZurin, and in particular, two peptides derived from aZurin, an 18-mer and a 28-mer, have therefore been found to 0002 The present invention relates to modified cupre be useful therapeutically and diagnostically. However, the doxin derived peptides with pharmacologic activity that have efficacy of atherapeutic agent in body of the patient is depen improved pharmacokinetic properties, and methods to use dent on several factors. In addition to the activity of the them to treat mammals Suffering from various conditions therapeutic drug itself, there are also the pharmacokinetic related to the pharmacologic activities. Modifications of the properties of the therapeutic drug, and how it relates to the cupredoxin derived peptides include amino acid sequence various processes that take place after the drug is adminis variants and structural derivations that may increase the tered, i.e., absorption, distribution, metabolism and excretion. plasma half-life of the peptide, increase the specific activity These pharmacokinetic properties of the drug describe how of the pharmacologic activity, decrease immunogenicity, and/ and to what extent these biological processes influence the or decrease the biotransformation of the peptides. The modi efficacy of the administered drug, and these properties fied cupredoxin derived peptides can be used in methods to include the drug half-life in the blood stream, the hepatic treat mammals for cancer, conditions related to inappropriate first-pass metabolism of the drug, the volume distribution of angiogenesis, viral and bacterial infections, and specifically the drug, the degree of albumin binding of the drug, etc. Each HIV and malaria, conditions related to ephrin signaling and of these pharmacokinetic properties can have a profound deliver cargo compounds, including diagnostic compounds, effect on the efficacy of the drug. to cancer cells. 0006. The site of absorption of the drug into the blood stream of the patient depends on the route of administration. BACKGROUND For example, orally administered drugs may be absorbed 0003. The cupredoxin azurin from Pseudomonas aerugi more at one site of the alimentary tract than another site due nosa is a promising new therapeutic and diagnostic molecule. to the chemical and physical nature of the drug. Absorption by Two redox proteins elaborated by P. aeruginosa, the cupre parenteral administration, on the other hand, is not only faster doxin aZurin and cytochrome css (Cyticss), both enter J774 than oral administration, but the blood levels of the drug are cells and show significant cytotoxic activity towards the far more predictable because much less of the drug is lost, human cancer cells as compared to normal cells. Zaborina et particularly in intravenous administration. The bioavailabil al., Microbiology 146: 2521-2530 (2000). AZurin can also ity is the fraction of the administered drug that reaches the enter human melanoma UISO-Mel-2 or human breast cancer systemic circulation. MCF-7 cells. Yamada et al., PNAS 99: 14098-14103 (2002); 0007. The distribution of the drug from the bloodstream Punjet al., Oncogene 23:2367-2378 (2004): Yamada et al., into the extracelluar fluid (interstitium) and/or cells of the Cell. Biol. 7:1418-1431 (2005). In addition, aZurin from P tissues may be altered by various aspects of the drug. The aeruginosa preferentially enters J774 murine reticulum cell distribution of the drug in the body may be expressed as the sarcoma cells, forms a complex with and stabilizes the tumor “volume distribution of the drug, which is a hypothetical Suppressor protein p53, enhances the intracellular concentra volume of liquid into which the drug is disseminated. The tion of p53, and induces apoptosis. Yamada et al., Infection structure of the drug may influence the drug distribution in and Immunity, 70:7054-7062 (2002). AZurin also caused a that hydrophobic drugs more readily move across most bio significant increase of apoptosis in human osteosarcoma cells logical membranes, and thus may be distributed within cells as compared to non-cancerous cells. Ye et al., Ai Zheng of the tissues. A drug may also be bound to blood proteins and 24:298-304 (2003). Rusticyanin from Thiobacillus ferrooxi its passage into Surrounding tissues thus delayed. For dans can also enter macrophages and induce apoptosis. example, when in the blood stream, naproxen is 99% bound to Yamada et al., Cell Cycle 3:1182-1187 (2004): Yamada et al., plasma proteins, penicillin G is 60% bound, amoxicillin only Cell. Micro. 7:1418-1431 (2005). Plastocyanin from Phor 20% bound and minoxidil is unbound. Howard C. Ansel et al., US 2009/0286719 A1 Nov. 19, 2009

Pharmaceutical Dosage Forms and Delivery Systems 129 macologic activities of interest include (1) entering a mam (Lippincott, Williams and Wilkins 1999). A bound drug is malian cancer cell, (2) not entering non-cancerous mamma neither exposed to the body's detoxification processes, nor is lian cells, (3) entering pre-malignant mammalian cells, (4) it removed from the bloodstream by filtration through the killing mammalian cancer cells, (5) killing pre-malignant renal glomeruli. The bound drug is referred to as the inactive mammalian cells, (6) inhibiting the growth of a mammalian portion while the unbound portion is considered the active cancer cell, (7) inhibiting HIV-1 infection, (8) inhibiting para portion. The bound portion of the drug serves as a reservoir of sitemia of malaria-infected red blood cells, (9) interfering the drug that is then released into the bloodstream in an with ephrin signaling system and (10) inhibiting angiogen unbound active form when the level of free drug is no longer Sufficient to ensure protein Saturation. Therefore, a drug that CS1S. is bound in the bloodstream will remain in the body for longer 0013 The modified cupredoxin derived peptide may be periods of time and will require a less frequent dosage. derived from a cupredoxin from Pseudomonas aeruginosa, 0008. The metabolism of the drug in the patient will also Phormidium laminosum, Ulva pertussis, Thiobacillus fer affect its efficacy. Many drugs undergo biotransformation rooxidans, Achromobacter cycloclastes, Pseudomonas Syrin before being excreted from the body. The biotransformation gae, Neisseria meningitidis, Vibrio parahaemolyticus, Bor of a drug may result in a form of the drug that is more water detella bronchiseptica, Bordetella pertussis, Chloroflexus soluble, ore ionized, less capable of binding proteins in the aurantiacus and Neisseria gonorrhoeae. The cupredoxin plasma and tissues, less able to penetrate cell membranes, and may beaZurin, plastocyanin, rusticyanin, pseudoaZurin, aura other aspects that make the drugs less pharmacologically cyanin Stellacyanin, cucumber basic protein or aZurin-like active. The biotransformed drug may therefore be rendered protein. In specific embodiments, the cupredoxin may be one less toxic and more readily excreted. There are four major of SEQID NOS: 1-12. ways by which drugs are biotransformed: oxidation, reduc tion, hydrolysis, and conjugation. Oxidation reactions are 0014. The isolated modified cupredoxin derived peptide primarily catalyzed by oxidases bound to the endoplasmic may be a truncation of the cupredoxin. In specific embodi reticulum within the liver cells. Reduction reactions are cata ments, the peptide may be one of SEQ ID NOS: 13-47. On lyzed by reductases primarily in the gut and liver. Hydrolytic other specific embodiments, SEQID NOS: 1-12 are at least breakdown is catalyzed by esterases primarily in the liver. about 90% identical to the isolated peptide. Glucuronide conjugation, the most common pathway of 0015. In some embodiments, the isolated modified cupre biotransformation of a drug, occurs by a combination of the doxin derived peptide may be less susceptible to hydrolysis drug with glucuronic acid, forming an ionic form of the drug than the corresponding unmodified cupredoxin. Specifically, that is easily eliminated from the body. Christensen et al., J. the isolated peptide may have one or more asparagine or Pharm. Pharmacol. 37:91-95 (1985). Other biotransforma serine residues in the sequence of the cupredoxin derived tive processes that increase elimination include methylation peptide that are replaced with another amino acid residue, and acylation. specifically a glutamic acid or threonine residue. 0009 Excretion of the drug from the body may occur by 0016. In some embodiments, the isolated modified cupre various routes. The kidney plays the dominant role of elimi doxin derived peptide is less Susceptible to deamidation. In nating the drug in the urine. However, the drug can also be specific embodiments, one or more glycine residues of the eliminated from the plasma through the liver. With drugs that cupredoxin derived peptide are replaced with another amino are orally administered in particular, the liver may play an acid residue, specifically a threonine or alanine residue. In important role in determining the plasma half-life of the drug. Some embodiments, one or more of the glycine residues in the cupredoxin derived peptide that are equivalent to residues 58 SUMMARY OF THE INVENTION or 63 of Pseudomonas aeruginosa azurin (SEQ ID NO: 1) 0010. One aspect of the invention provides an isolated may be replaced. In another specific embodiment, the isolated modified cupredoxin derived peptide that is a variant, trunca peptide may comprise SEQID NO:30. tion or derivative of a cupredoxin derived peptide. In some 0017. In some embodiments, the isolated modified cupre embodiments, the modification includes cyclization. In one doxin derived peptide is less susceptible to oxidation. Spe embodiment the cyclization is enzymatic cyclization. In cifically, the isolated peptide may have one or more methion another embodiment the cyclization creates a thioether ine or cysteine residues of the cupredoxin derived peptide cyclization. In another embodiment the thioether cyclization replaced with another amino acid residue, specifically a leu is formed by the addition of cysteine residues to dehydroala cine or valine residue. In a specific embodiment, one or more nine and dehydrobutrine residues. In another embodiment the methionine residues of the cupredoxin derived peptide that dehydroalanine and dehydrobutrine residues originate from are equivalent to residues 56 or 64 of Pseudomonas aerugi dehydration of serine and threonine respectively. In another nosa azurin (SEQID NO: 1) is replaced. In another specific embodiment Nisb dehydrates the serine and threonine. embodiment, the isolated peptide may comprise SEQID NO: 0011. In one embodiment, the isolated modified cupre 31 or SEQID NO:32. doxin derived peptide has improved pharmacokinetic prop 0018. In some embodiments, the isolated modified cupre erties as compared to the unmodified cupredoxin derived doxin derived peptide may be less susceptible to diketopip peptide. The improved pharmacokinetic property may be one erazine and pyroglutamic acid formation. Specifically, the or more of the peptide (1) is less susceptible to biotransfor isolated peptide may have a glycine residue in positions 1, 2 mation in the patient, (2) is excreted from the body of the or 3 from the N-terminus of the cupredoxin derived peptide patient at a slower rate, (3) has increased stability of its replaced with another amino acid residue. Further, the iso tertiary structure and (4) has longer plasma half-life. lated peptide may have a proline residue in position3 from the 0012. Additionally, the isolated peptide may have at least N-terminus of the cupredoxin derived peptide that is replaced one pharmacologic activity of a cupredoxin. Specific phar with another amino acid residue. Further, the isolated peptide US 2009/0286719 A1 Nov. 19, 2009

may have an asparagine residue at the N-terminus of the 0025. In some embodiments, the isolated modified cupre cupredoxin derived peptide replaced with another amino acid doxin derived peptide may have one or more PEG (polyeth residue. ylene glycol) molecules covalently bonded to the cupredoxin 0019. In some embodiments, the isolated modified cupre derived peptide. Specifically, the isolated peptide may have doxin derived peptide may be less Susceptible to racemiza one or more PEG molecules is covalently bonded to one or tion. Specifically, the isolated peptide may have one or more more cysteine residues of the cupredoxin derived peptide. In amino acid residues of the cupredoxin derived peptide specific embodiments, the isolated peptide may have one or replaced with the D-isomer of the amino acid residue. In one more PEG molecules are covalently bonded to one or more specific embodiment, all of the amino acid residues of the cysteine residues equivalent to one or more of residues 3, 6, cupredoxin derived peptide are replaced with the D-isomers and 112 of Pseudomonas aeruginosaaZurin (SEQID NO: 1). of the amino acid residues. In another specific embodiment, In another embodiment, a cysteine residue may be substituted the isolated peptide comprises SEQID NO: 45. into the cupredoxin derived peptide and may be covalently 0020. In some embodiments, the isolated modified cupre bonded to a PEG molecule. doxin derived peptide may be less Susceptible to degradation. 0026. In another embodiment, the isolated peptide may Specifically, the N-terminus of the cupredoxin derived pep have one or more PEG molecules covalently bonded to the tide may be acetylated. Further, the C-terminus of the cupre cupredoxin derived peptide at a lysine, cysteine, histidine, doxin derived peptide may be amidated. In one specific arginine, aspartic acid, glutamic acid, serine, threonine, embodiment, the isolated peptide is SEQID NO:33. tyrosine, N-terminal amino group, or C-terminal carboxylic 0021. In some embodiments, the isolated modified cupre acid. In specific embodiments, the isolated peptide has one or doxin derived peptide is modified to increase the stability of more lysine residues or C-terminal carboxylic acids its tertiary structure. Specifically, the isolated peptide may be covalently bonded to a PEG molecule. In another embodi modified to increase the stability of a least one C.-helix. In ment, one or more lysine, cysteine, histidine, arginine, aspar Some embodiments, at least one glycine, proline, serine, tic acid, glutamic acid, serine, threonine and tyrosine residues aspartic acid, alanine, threonine, Valine, glutamine, aspar may be substituted into the cupredoxin derived peptide and agine, cysteine, histidine, lysine, and arginine amino acid may be covalently bonded to a PEG molecule. residue of the cupredoxin derived peptide is replaced with 0027. In other embodiments, one or more PEG molecules leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, may be covalently bonded to one or more amino groups of the tryptophan or methionine. In a specific embodiment, the cupredoxin derived peptide, or randomly covalently bonded replaced residue of the cupredoxin derived peptide may be to the cupredoxin derived peptide. within equivalent residues to residues 53-56,58-64 and 68-70 0028. The average molecular weight of the PEG mol of P aeruginosa aZurin. In other specific embodiments, the ecules per cupredoxin derived peptide may be about 200 to glutamine at a residue equivalent to residue 57 of P aerugi about 100,000 daltons. The cupredoxin derived peptide may nosa aZurin may be replaced with a tryptophan residue, the be covalently bonded to one or more branched PEG mol threonine at a residue equivalent to residue 52 of P. aerugi ecules, specifically where the branched PEG molecule is nosa aZurin may be replaced with a tryptophan residue, the about 50 kDa. The cupredoxin derived peptide may be threonine at a residue equivalent to residue 61 of P aerugi covalently bonded to one or more linear PEG molecules, nosa aZurin may be replaced with a tryptophan residue, and/ specifically where the linear PEG molecule is about 5 kDa. or the glycine at a residue equivalent to residue 63 of P 0029. Another aspect of the invention is a pharmaceutical aeruginosa aZurin is replaced with a tryptophan residue. In composition which may comprise a modified cupredoxin other specific embodiments, the isolated peptide comprises derived peptide and a pharmaceutically acceptable carrier. one of SEQID NOS: 34-44. 0030. Another aspect of the invention is a method to treat 0022. In other embodiments, the isolated peptide may conditions Suffered by mammals which may comprise have two or more lysine residues of the cupredoxin derived administering to the mammal a therapeutically effective peptide substituted with e-(3,5-dinitrobenzoyl)-lysine resi amount of the modified cupredoxin derived peptides. In spe dues in an i(i+4) spacing. Specifically, the replaced residues cific embodiments, the mammal is human. of the cupredoxin derived peptide may be within residues 0031. Another aspect of the invention is an isolated pep equivalent to residues 53-56, 58-64 and 68-70 of P. aerugi tide which comprises, or alternatively consists of the amino nosa aZurin. acid Sequence X 0023. In other embodiments, the isolated peptide may SXAADXXXVVXDXXs ASGLDKDYLKPDX, have histidine-cysteine or histidine-histidine residue pairs (SEQ ID NO:48); wherein X is selected from the group substituted into the cupredoxin derived peptide at an i(i+4) consisting of L and acetylated-L: X is selected from the spacing, and at least one of Cu, Zn, Cd and Ru. In a specific group consisting of T and W: X is selected from the group embodiment, the isolated peptide may have the replaced resi consisting of M. L and V: X is selected from the group dues of the cupredoxin derived peptide within residues consisting of Q and W: Xs is selected from the group consist equivalent to residues 53-56, 58-64 and 68-70 of P. aerugi ing of G and A: X is selected from the group consisting of T nosa aZurin. and W, X, is selected from the group consisting of G, T and 0024. In another embodiment, the isolated peptide may W: X is selected from the group consisting of M, Land V; and have one or more pairs of native amino acid residues in the X is selected from the group consisting of Dandamidated-L. cupredoxin derived peptide substituted with O.C.-disubsti 0032. Another aspect of the invention is an isolated pep tuted non-natural amino acids with olefin-bearing tethers that tide comprising, or alternatively consisting of the amino acid correspond to the native amino acids. The isolated peptide Sequence may have the replaced residues of the cupredoxin derived XDPKLYDKDLGSAXXDXVVXXXDAAX.SX peptide within residues equivalent to residues 53-56, 58-64 (SEQ ID NO:49); wherein X is selected from the group and 68-70 of P aeruginosa azurin. consisting of D and acetylated-D: X is selected from the US 2009/0286719 A1 Nov. 19, 2009

group consisting of M. L and V: X is selected from the group 0054 SEQ ID NO: 21 is the amino acid sequence of the consisting of G, T and W: X is selected from the group 57-89 amino acid fragment of auracyanin B of Chloroflexus consisting of T and W: Xs is selected from the group consist aurantiacus. ing of G and A: X is selected from the group consisting of Q 0055 SEQ ID NO: 22 is the amino acid sequence of the and W, X, is selected from the group consisting of M. L and 50-77 amino acid fragment of azurin from Bordetella pertus V:Xs is selected from the group consisting of Tand W. and X SS. is selected from the group consisting of L and amidated-L. 0056 SEQ ID NO. 23 is the amino acid sequence of the 0033. Another aspect of the invention is an isolated pep 89-115 amino acid fragment of the Laz protein from Neis tide comprising, or consisting of the sequences of SEQ ID seria meningitidis. NOS: 50-3504, which are cupredoxin derived peptides modi 0057 SEQ ID NO: 24 is the amino acid sequence of the fied using one or more of the techniques and methods dis 53-70 amino acid fragment of azurin from Pseudomonas closed herein. aeruginosa. 0058 SEQ ID NO: 25 is the amino acid sequence of the BRIEF DESCRIPTION OF THE SEQUENCES 53-64 amino acid fragment of aZurin from Pseudomonas aeruginosa. 0034 SEQ ID NO: 1 is the amino acid sequence of wt 0059 SEQ ID NO: 26 is the amino acid sequence of the aZurin from Pseudomonas aeruginosa. 51-77 amino acid fragment from azurin from Pseudomonas 0035) SEQID NO: 2 is the amino acid sequence of plas aeruginosa. tocyanin from Phormidium laminosum. 0060 SEQ ID NO: 27 is the amino acid sequence of the 0036 SEQID NO: 3 is the amino acid sequence of rusti 51-77 amino acid fragment from azurin from Pseudomonas cyanin from Thiobacillus ferrooxidans. Syringae. 0037 SEQ ID NO. 4 is the amino acid sequence of 0061 SEQID NO: 28 is the amino acid sequence of the is pseudoaZurin from Achromobacter cycloclastes. the 52-78 amino acid fragment from azurin from Vibrio para 0038 SEQID NO. 5 is the amino acid sequence of azurin haemolyticus. from Pseudomonas Syringae. 0062 SEQ ID NO: 29 is the amino acid sequence of the 0039 SEQ ID NO: 6 is the amino acid sequence of Laz 51-77 amino acid fragment from azurin from Bordetella from Neisseria gonorrhoeae. bronchiseptica. 0040 SEQID NO: 7 is the amino acid sequence of the Laz 0063 SEQID NO:30 is an artificial sequence for a variant from Neisseria meningitides. form of the 50-77 amino acid region of Pseudomonas aerugi 0041 SEQ ID NO: 8 is the amino acid sequence of the nosa aZurin. aZurin from Vibrio parahaemolyticus. 0064 SEQID NO:31 is an artificial sequence for a variant 0042 SEQ ID NO: 9 is the amino acid sequence of the form of the 50-77 amino acid region of Pseudomonas aerugi aZurin from Bordetella bronchiseptica. nosa aZurin. 0043 SEQ ID NO: 10 is the amino acid sequence of the 0065. SEQID NO:32 is an artificial sequence for a variant auracyanin. A from Chloroflexus aurantiacus form of the 50-77 amino acid region of Pseudomonas aerugi 0044 SEQID NO: 11 is the amino acid sequence of the nosa aZurin. auracyanin B from Chloroflexus aurantiacus. 0066 SEQID NO:33 is an artificial sequence for a variant 0045 SEQ ID NO: 12 is the amino acid sequence of the form of the 50-77 amino acid region of Pseudomonas aerugi aZurin from Bordetella pertussis. nosa aZurin. 0046 SEQ ID NO: 13 is the amino acid sequence of the 0067 SEQID NO:34 is an artificial sequence for a variant 50-77 amino acid fragment of wt-aZurin (p28) from form of the 50-77 amino acid region of Pseudomonas aerugi Pseudomonas aeruginosa. nosa aZurin. 0047 SEQ ID NO: 14 is the amino acid sequence of the 0068 SEQID NO:35 is an artificial sequence for a variant 50-67 amino acid fragment of wt-aZurin (p18) from form of the 50-77 amino acid region of Pseudomonas aerugi Pseudomonas aeruginosa. nosa aZurin. 0048 SEQ ID NO: 15 is the amino acid sequence of the 0069 SEQID NO:36 is an artificial sequence for a variant 36-128 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. 0049 SEQ ID NO: 16 is the amino acid sequence of the (0070 SEQID NO:37 is an artificial sequence for a variant 36-89 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. 0050 SEQ ID NO: 17 is the amino acid sequence of the (0071 SEQID NO:38 is an artificial sequence for a variant 36-77 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. 0051 SEQ ID NO: 18 is the amino acid sequence of the (0072 SEQID NO:39 is an artificial sequence for a variant 36-50 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. 0052 SEQ ID NO: 19 is the amino acid sequence of the (0073 SEQID NO: 40 is an artificial sequence for a variant 50-66 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. 0053 SEQ ID NO: 20 is the amino acid sequence of the 0074 SEQID NO: 41 is an artificial sequence for a variant 67-77 amino acid fragment of wt-aZurin from Pseudomonas form of the 50-77 amino acid region of Pseudomonas aerugi aeruginosa. nosa aZurin. US 2009/0286719 A1 Nov. 19, 2009

0075) SEQID NO: 42 is an artificial sequence for a variant O097 SEQID NO:3518 is a sequence for a variantform of form of the 50-77 amino acid region of Pseudomonas aerugi the 50-77 amino acid region of Pseudomonas aeruginosa nosa aZurin. aZurin. 0076 SEQID NO: 43 is an artificial sequence for a variant 0098 SEQID NO:3519 is a sequence for a variantform of form of the 50-77 amino acid region of Pseudomonas aerugi the 50-77 amino acid region of Pseudomonas aeruginosa nosa aZurin. aZurin. 0.077 SEQID NO:44 is an artificial sequence for a variant 0099 SEQID NO:3520 is a sequence for a variantform of form of the 50-77 amino acid region of Pseudomonas aerugi the 50-77 amino acid region of Pseudomonas aeruginosa nosa aZurin. aZurin. 0078 SEQID NO:45 is an artificial sequence for a variant 01.00 SEQID NO:3521 is a sequence for a variant form of form of the 50-77 amino acid region of Pseudomonas aerugi the 50-77 amino acid region of Pseudomonas aeruginosa nosa aZurin. aZurin. 0079 SEQID NO: 46 is a conserved amino acid sequence 0101 SEQID NO:3522 is a sequence for a variant form of from aZurins where D is aspartic acid, G is glycine, Y is the 50-77 amino acid region of Pseudomonas aeruginosa tyrosine, K is lysine and X is any amino acid. aZurin. 0080 SEQID NO: 47 is a conserved amino acid sequence 01.02 SEQID NO:3523 is a sequence for a variantform of from aZurins where D is aspartic acid, G is glycine, Y is the 50-77 amino acid region of Pseudomonas aeruginosa tyrosine, K is lysine and X is any amino acid. aZurin. 0081 SEQID NO: 48 is an artificial sequence of modifi (0103) SEQID NO:3524 is a sequence for a variant form of cations to aZurin 50-77 of Pseudomonas aeruginosa. the 50-77 amino acid region of Pseudomonas aeruginosa 0082 SEQID NO: 49 is an artificial sequence of modifi aZurin. cations to the D-isomer of azurin 50-77 of Pseudomonas 0104 SEQID NO:3525 is a sequence for a variantform of aeruginosa. the 50-77 amino acid region of Pseudomonas aeruginosa 0083 SEQ ID NOS: 50-3504 are sequences for variant aZurin. forms of the 50-77 amino acid region of Pseudomonas 0105 SEQID NO:3526 is the amino acid sequence of the aeruginosa aZurin. 60-77 amino acid fragment (p18b) of wt-aZurin from 0084 SEQ ID NO: 3505 is the amino acid sequence of Pseudomonas aeruginosa. Pep42, a cyclic 13-mer oligopeptide. 0106 SEQ ID NO: 3527 is the amino acid sequence of 0085 SEQID NO:3506 is the amino acid sequence of the Arg-8. 66-77 amino acid fragment (p12) of wt-aZurin from Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE FIGURES 0086 SEQID NO:3507 is a sequence for a variantform of 0107 FIG. 1 depicts the images of whole mouse scans of the 50-77 amino acid region of Pseudomonas aeruginosa mice that have been injected with labeled p18 (SEQID NO: aZurin. 14). IRDyeR) labeled p18 (SEQ ID NO: 14) (125 ug) was 0087 SEQID NO:3508 is a sequence for a variantform of injected intravenously and athymic mice were scanned at the 50-77 amino acid region of Pseudomonas aeruginosa indicated time periods for detection of labeled dye in tumors aZurin. and organs using the Odyssey R. Infrared Imaging System. 0088 SEQID NO:3509 is a sequence for a variantform of 0.108 FIG. 2 depicts the images from whole mouse and the 50-77 amino acid region of Pseudomonas aeruginosa organs scans of mice that have been injected with labeled p18 aZurin. (SEQID NO: 14). 125 ug IRDye R labeled p18 (SEQID NO: 0089 SEQID NO:3510 is a sequence for a variantform of 14) 120 h post-injection i.v. (immediately before sacrifice). the 50-77 amino acid region of Pseudomonas aeruginosa Excised organs scanned and shown on right. p18 (SEQ ID aZurin. NO: 14) signal was seen from kidneys and Mel-2 tumors. 0090 SEQID NO:3511 is a sequence for a variant form of 0109 FIG. 3. MeI-2 subcutaneous tumor with 125 ug the 50-77 amino acid region of Pseudomonas aeruginosa IRDye(R) labeled p18 (SEQ ID NO: 14) administered i.v. 3 aZurin. weeks after Mel-2 cells were injected, and Odyssey(R) infra 0091 SEQID NO:3512 is a sequence for a variant form of red scan was performed 48 hours later. Images recorded with the 50-77 amino acid region of Pseudomonas aeruginosa the 800 nm channel represent specific p18 (SEQID NO: 14) azin. signal from IRDyeR, and those with the 700 nm channel 0092 SEQID NO:3513 is a sequence for a variantform of represent background. p18 (SEQID NO: 14) signal was seen the 50-77 amino acid region of Pseudomonas aeruginosa from kidneys and Mel-2 tumors. aZurin. 0110 FIG. 4. AZurin truncation with alpha-helical struc 0093 SEQID NO:3514 is a sequence for a variant form of ture and the results of a 70 ns simulation. the 50-77 amino acid region of Pseudomonas aeruginosa 0111 FIG. 5. Measurement of thioether bridge positions aZurin. in p28 (SEQ ID NO: 13) based on distances between Ca 0094) SEQID NO:3515 is a sequence for a variantform of atoms in a simulated Structure. the 50-77 amino acid region of Pseudomonas aeruginosa 0112 FIG. 6 (A), (B) and (C). Depicts photographs show aZurin. ing penetration of azurin derived peptides, p 18 (SEQID NO: 0095 SEQID NO:3516 is a sequence for a variant form of 14) and p28 (SEQID NO: 13), into cancer cell lines of diverse the 50-77 amino acid region of Pseudomonas aeruginosa histogenesis and their normal counterparts. (A) Photos show aZurin. ing penetration of Alexafluor 568 labeled p28 (SEQ ID NO: 0.096 SEQID NO:3517 is a sequence for a variantform of 13) or p18 (SEQID NO: 14) after 2 hrs at 37°C. The cationic the 50-77 amino acid region of Pseudomonas aeruginosa Arg-8 (SEQID NO: 3527) was used as a control. (B) Graphs aZurin. depicting flow eytometric analysis of the penetration of Alex US 2009/0286719 A1 Nov. 19, 2009

afluor 568 labeled p28 (SEQID NO: 13) or p 18(SEQID NO: incubation, mitotracker/lysotracker probes were added and 14) into the same cell lines after 2 hrs at 37° C. (C) Graphs cells incubated for 30 min. Cells were counterstained with depicting fold increase over fluorescence from normal cells. DAPI (blue). Colocalization of azurin, p28 (SEQID NO: 13) Similar observations of p28 (SEQID NO: 13) or p18 (SEQID or p 18 (SEQID NO: 14) appears as a yellow florescence. NO: 14) entry into 4 melanoma cell lines show a several fold 0117 FIG. 11 (A) and (B). Graphs depicting UISO-Mel-2 increase over fluorescence from normal cells. cells that were incubated with increasing concentrations of 0113 FIG. 7 (A) and (B). Depicts photographs showing aZurin, p28 (SEQID NO: 13), or p 18(SEQID NO: 14) at 37° entry of azu 60-77 (p18b) (SEQID NO:3526) and azu 66-77 (p12) (SEQID NO:3506) into cancer and normal cells. Cells C. for 72 hrs. MTT (A): Direct cell count (B). Cell viability were incubated with alexafluor 568 labeled p18b (SEQ II) (MTT) or cell number in control wells were considered as NO:3526) (A) or p 12 (SEQID NO:3506) (B) at 37° C. for 2 100%. Data represent mean+SEM. hrs and images recorded by confocal microscopy. 0118 FIG. 12. (A) through (H). Depict photographs show 0114 FIG. 8. Graphs depicting cellular membrane toxic ing uptake of compounds by cells, taken using a confocal ity of aZurin and its peptides. (A) LDH leakage assay of microscope after treatment of cells with proteins and/or UISOMel-2 cells exposure for 10 minto different concentra buffer. (A) Human brain tumor LN-229 cells were pretreated tions of p28 (SEQ ID NO: 13), p.18 (SEQ ID NO: 14) and with 20 uMofunlabeled proteins or PBS buffer for 2 hours, aZurin at 37°C. A standard lysis buffer (cytotox-one reagent) then washed three times using PBS buffer. All buffer was was included as a positive control. Changes in fluorescence discarded and then 20 uM of Alex568-Paz was added for 30 following exposure were measured at k.X 560 nm and kem minutes at 37°C. (B) The IN-229 cells were then treated with 590 nm. Lysis buffer was defined as 100% LDH release. Data 20 uM of unlabeled proteins or PBS buffer and 20 uM of represent % of positive fluorescence of control. Data are Alex568-Paz for 30 minutes at 37° C. (C) Another group of shown as mean+SEM. (B) Hemoglobin leakage from human human brain tumor LN-229 cells were pretreated with 10 uM erythrocytes incubated with p28 (SEQID NO: 13), p 18(SEQ unlabeled proteins or PBS buffer for 2 hours, then washed ID NO: 14) and azurin. Human erythrocytes were incubated three times using PBS buffer. All buffer was discarded and with peptide for 30 min at 37° C. and absorbance at 540 nm then 10 uM of Alex568-Paz was added for 30 minutes at 37° determined. Hemoglobin release following 0.1% Triton C. (D) The LN-229 cells were then treated with 10 Munla X-100 was defined as 100% hemoglobin release. Data repre beled proteins or PBS buffer and 10 uM of Alex568-Paz for sent meant-SEM of triplicate determinations. 30 minutes at 37° C. (E) Human brain tumor LN-229 cells 0115 FIG. 9 (A), (B), (C) and (D). Depicts photographs were treated with 20 uMofunlabeled proteins or PBS buffer showing temperature dependent and competitive internaliza and 20 uM of Alex568-Paz for 30 minutes at 37° C. (F) tion of p28 (SEQID NO: 13) and p18 (SEQID NO: 14) into Human brain tumor LN-229 cells were treated with 20 uM of UISO-Mel-2 cells. Penetration of Alexafluor 568 labeled p28 Alex568-H.8 for 30 minutes at 37°C. (G) Human brain tumor (SEQID NO: 13) (A) or p 18(SEQID NO: 14)(B) at 2011 M LN-229 cells were treated with 20 uMAlex568-proteins for was evaluated by confocal microscopy at different tempera 30 minutes at 37° C. (H) Human breast adenocarcinoma tures. (C) and (D) Confocal analysis of entry of Alexafluor MCF-7 cells were treated with 20 uM of Alex568-proteins for 568 labeled p28 (SEQID NO: 13) (C) or p 18 (SEQID NO: 30 minutes at 37° C. 14) (D) at 5uM into UISO-Mel-2 cells after 30 minat 37° C. 0119 FIG. 13. (A) through (C). Graphs and charts depict in the presence/absence of unlabeled peptide (200 fold ing peptide binding and entry into cells. (A) UISO-Mel-2 or excess). fibroblast cells (3x10 cells) were suspended in MEME 0116 FIG. 10 (A), (B), (C) and (D). (A) Depicts photo media without phenol red. Reactions were started by adding graphs showing confocal analysis of 28, p 18 (SEQ ID NO: Alexafluor 568-conjugated p28 (SEQID NO: 13) at 10, 50, 14) (20 uM) and Arg-8 (SEQID NO:3527) (10 uM) entry into 100, 150,250, 300 and 400 uM for 30, 60,90 and 120 secon UISO-Mel-2 cells after 1 hr at 37° C. in the presence/absence ice. Cells were analyzed by flow cytometry. (B) The K and of heparin sulfate (100 g/ml). (B) Graphs showing flow V, were calculated by plotting peptide concentration (LM) cytometricanalysis of p28 (SEQID NO: 13) or p18 (SEQID vs. velocity (MFI/sec). (C) Peptide binding and entry was NO: 14) entry in the presence of inhibitors. Cell fluorescence determined using whole Mel2 cells (50,000 cells/ml), were intensity in the absence of inhibitor (control) was considered incubated for 30 minat37°C. with increasing concentrations as 100%. (C) Graphs depicting FRCS analysis of p28 (SEQ (0-175 nM) of radiolabeled azurin in the presence/absence of IDNO: 13) and p18 (SEQID NO:14) entry into fibroblasts in 1000 fold excess of unlabeled p28 (SEQ ID NO: 13), or presence of inhibitors. (D) Depicts photographs showing aZurin, and radioactivity remaining in the cell pellet counted colocalization of p 18(SEQID NO: 14) and p28 (SEQID NO: using a gamma counter. Radioactivity in cells incubated with 13) with caveolin I (Panel 1). UISO-MeI-2 cells were incu 'I azurin alone was considered total binding; radioactivity bated with Alexafluor 568 labeled p18 (SEQ ID NO: 14) or in the presence of unlabeled azurin or p28 (SEQID NO: 13) p28 (SEQ ID NO: 13) (20 uM) or media for 2 hrs at 37° C. was considered nonspecific binding. Specific binding was Cells were fixed and processed for anti-caveolin 1 immun determined by Subtracting nonspecific binding from total ostaining. Confocal analysis of entry of Alexafluor 568 binding and Scatchard plots generated. labeled p18 (SEQID NO: 14) or p28 (SEQ ID NO: 13) (20 I0120 FIG. 14. (A) through (C). Depict side and back uM) into UISO-Mel-2 cells after 2 hrs at 37° C. followed by photographs of mice with melanoma MEL-23 tumors taken antigolgin 97 antibodies (Panel 2). Colocalization of Alex after injection with p28 (SEQID NO: 13) dye complex at 60 afluor 568 labeled azurin, p28 (SEQ ID NO: 13) and p18 umolar concentration in 250LL scans and after injection with (SEQID NO: 14) (red) with mitotracker (green) (Panel 3) and control PBS at (A) 24 hours and (B) 48 hours. (C) depicts side Lysotracker (green) (Panel 4) dyes in UISO-Mel-2 cells. and back photographs of mice with melanoma MEL-23 Cells were incubated at 37°C. with 20 uMaZurin, p28 (SEQ tumors taken after injection with p28 (SEQID NO: 13) at 200 IDNO: 13), p.18 (SEQID NO: 14) or media only. After 90 min LM concentration at 24 and 48 hours. US 2009/0286719 A1 Nov. 19, 2009

0121 FIG. 15. (A) through (C). Depict side and back p18 (SEQID NO:14), p28 (SEQIDNO: 13), and Arg-8 (SEQ photographs of mice with melanoma MEL-23 tumors taken IDNO:3527) into the tail veins of mice with HCT-116tumors after injection with p18 (SEQID NO: 14) at 60 umolar con and organs. centration at (A) 17 hours, (B) 24 hours, and (C) 46 hours. (C) 0.133 FIG. 27. (A) and (B). (A) Depicts photographs of also depicts photographs of mouse organs, including the mouse organs taken 24 hours after injection of 120 LM con heart, lung, liver, kidney, spleen, and brain, taken 46 hours centrations of p18 (SEQID NO: 14), p28 (SEQID NO: 13), after injection of p18 (SEQID NO: 14). and Arg-8 (SEQID NO:3527) into the tail veins of mice with 0122 FIG. 16. (A) and (B). (A) Depicts side and back HCT-116 tumors and organs. (B) Depicts side photographs of photographs of mice with tumors taken 12 hours after injec mice with HCT-116 tumors taken 21 hours after injection of tion with p18 (SEQID NO: 14), p28 (SEQID NO: 13), and 120 uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ Arg-8 (SEQ ID NO:3527) at 60 umolar concentration. (B) ID NO: 13), and Arg-8 (SEQ ID NO: 3527) into their tail Depicts photographs of mouse organs, including mouse veins. brains, taken 12 hours after injection with p18 (SEQID NO: 0.134 FIG. 28, (A) and (B). (A) Depicts side and back 14), p28 (SEQ ID NO: 13), and Arg-8 (SEQID NO:3527). photographs of mice with HCT-116 24 hours after injection (0123 FIG. 17, (A) and (B). (A) Depicts side and back with 120M concentrations of p28 (SEQID NO: 13), 47 days photographs of mice with melanoma MEL-6 tumors taken 40 after injection of 1 million cells into tail veins. (B) Depicts hours after injections of 600 uM concentrations of p18 (SEQ photographs of mouse organs taken from mice with HCT-116 ID NO: 14) and Arg-8 (SEQ ID NO: 3527) into tail veins. 4 hours after injection with 120 uM concentrations of p28 Animals treated with p18 (SEQ ID NO: 14) received 0.5 (SEQ ID NO: 13), 47 days after injection of 1 million cells million cells, and animals treated with Arg-8 (SEQ ID NO: into tail veins. 3527) received 1 million cells. (B) Depicts photographs of 0.135 FIG. 29. Depicts photographs of organs from mouse organs taken 40 hours after injections of 600 PM MEL-6 mice taken 24 hours after treatment with 120 uM concentrations of p18 (SEQID NO: 14) and Arg-8 (SEQ ID concentrations of p18 (SEQID NO: 14), p28 (SEQID NO: NO:3527). 13), and Arg-8 (SEQID NO:3527). 0.124 FIG. 18, (A) and (B). (A) Depicts side and back (0.136 FIG. 30, (A) and (B). (A) Depicts side and back photographs of mice with melanoma MEL-23 tumors taken photographs of MEL-6 mice taken 22 hours after injection of 16 hours after injections of 60 uM concentrations of p28 120 uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ (SEQID NO: 13), p 18(SEQID NO: 14), and Arg-8 (SEQID ID NO: 13), and Arg-8 (SEQ ID NO:3527), and 6060 uM NO: 3527). (B) Depicts side and back photographs of mice concentration of Arg-8 (SEQ ID NO: 3527). (B) Depicts with melanoma MEL-23 tumors taken 24 hours after injec photographs of MEL-6 mouse organs after treatment with tions of 60 uM concentrations of p28 (SEQID NO: 13), p 18 120 uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ (SEQ ID NO: 14), and Arg-8 (SEQID NO:3527). ID NO: 13), and Arg-8 (SEQ ID NO: 3527), and 60 uM 0.125 FIG. 19. Depicts photographs of mouse organs concentration of Arg-8 (SEQID NO:3527). taken 48 hours after injection of 60LM concentrations of p28 0.137 FIG. 31. (A) and (B). (A) Depicts photographs of (SEQ ID NO: 13) and p18 (SEQ ID NO: 14) dye peptide organs from HT-1080 mice taken 22 hours after treatment complex into mice with melanoma MEL-23. with 60 and 120 uM concentrations of p18 (SEQID NO: 14), 0126 FIG. 20. Depicts photographs of mouse organs p28 (SEQ ID NO: 13), and Arg-8 (SEQ ID NO:3527). (B) taken 24 hours after injection of 60LM concentrations of p28 Depicts side-by-side photographs of brains from HT-1080 (SEQII) NO: 13) into mice with MEL-23 tumors and organs. mice taken 22 hours after treatment with 60 and 120 uM 0127 FIG. 21. Depicts side and back photographs of mice concentrations of p18 (SEQID NO: 14), p28 (SEQID NO: with melanoma MEL-23 tumors taken 16 hours after injec 13), and Arg-8 (SEQ ID NO: 3527), demonstrating the dif tions of 60 uM concentrations of p28 (SEQ ID NO: 13) and ferences between uptake of p18 (SEQ ID NO: 14) and p28 Arg-8 (SEQID NO:3527). (SEQ ID NO: 13) into the brain. 0128 FIG.22. Depicts side and back photographs of mice 0.138 FIG. 32. Depicts side and back photographs of with melanoma MEL-23 tumors taken 16 hours after injec HT-1080 mice during Doxorubicin vs. p28 (SEQID NO: 13) tions of 60 uM concentrations of p18 (SEQID NO: 14). study taken 16 hours after treatment with 60 and 120 uM 0129 FIG. 23. Depicts side photographs of mice with concentrations of p18 (SEQID NO: 14), p28 (SEQID NO: tumors taken 10 and 24 hours after high dose treatment with 13), and Arg-8 (SEQID NO:3527). 240 uM concentrations of p18 (SEQID NO: 14), p28 (SEQ I0139 FIG. 33. (A) and (B). (A) Depicts photographs of ID NO: 13), and Arg-8 (SEQID NO:3527). organs from HT-1080 mice taken 22 hours after treatment 0130 FIG. 24. Depicts side and back photographs of mice with 60 and 120 uM concentrations of p28 (SEQID NO: 13) with MCF-7 tumors and organs taken 28 hours after high dose and Arg-8 (SEQ ID NO: 3527). (B) Depicts side-by-side treatment with 240 uM concentrations of p18 (SEQID NO: photographs of brains from HT-1080 mice taken 22 hours 14), p28 (SEQID NO: 13), and Arg-8 (SEQ ID NO:3527). after treatment with 60 and 120 uM concentrations of p28 Also depicts photographs of mouse organs with MCF-7 taken (SEQID NO: 13) and Arg-8 (SEQID NO:3527). 28 hours after high dose treatment with 240 uM concentra 0140 FIG. 34. (A) and (B). (A) Depicts photographs of tions of p18 (SEQ ID NO: 14), p28 (SEQ ID NO: 13), and organs from HT-1080 mice taken 22 hours after treatment Arg-8 (SEQID NO:3527). with 60 and 120 uM concentrations of p18 (SEQID NO: 14) 0131 FIG. 25. Depicts side and back photographs of mice and Arg-8 (SEQ ID NO: 3527). (B) Depicts side-by-side with tumors taken 50 hours after high dose treatment with 240 photographs of brains from HT-1080 mice taken 22 hours uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ ID after treatment with 60 and 120 uM concentrations of p 18 NO: 13), and Arg-8 (SEQID NO:3527). (SEQID NO: 14) and Arg-8 (SEQID NO:3527). 0132 FIG. 26. Depicts photographs of mouse organs 0141 FIG. 35. (A) through (E). Depicts photographs of taken 24 hours after injection of 120 uM concentrations of HT-1080 mice with lung metastases treated via their tail veins US 2009/0286719 A1 Nov. 19, 2009

with (A) 3 mg/kg Doxorubicin IP3 treatments; (B) 5 mg/kg 0153 FIG. 47. Depicts side and back photographs of IP p28 (SEQIDNO: 13) daily; (C) PBS control, PBSIP daily; MEL-6 mice taken 72 hrs after injection of Arg-8 (SEQ ID (D) 10 mg/kg IP p28 (SEQID NO: 13) daily; (E) 20 mg/kg IP NO:3527) and p18 (SEQID NO: 14), 41 days post injection daily. of cells. 0142 FIG. 36. (A) and (B). (A) Depicts photographs of 0154 FIG. 48. Depicts back photographs of mice taken organs from HT-1080 mice in an animal study, whereby after injection of Arg-8 (SEQID NO:3527) and p18 (SEQID 1x10 cells are injected into tail veins (43 days) and all treated NO: 14). mice have lung metastases, taken 24 and 26 hours after 60LM 0155 FIG. 49. Depicts side and front photographs of mice concentrations of p28 (SEQ ID NO: 13) injected into tail taken 3, 24, and 48 hours after injection of Arg-8 (SEQ ID veins. Animal 6982 was dead when photographed. (B) NO: 3527) and p18 (SEQ ID NO: 14). Depicts side and back photographs of HT-1080 mice in an animal study, whereby 1x10° cells are injected into tail veins DETAILED DESCRIPTION OF THE (43 days), taken 22 hours after 60 LM concentrations of p28 EMBODIMENTS (SEQ ID NO: 13) injected into tail veins. Animal 6982 was dead when photographed. Definitions 0143 FIG. 37. Depicts side and back photographs of 0156. As used herein, the term “cell includes both the HT-1080 mice in an animal study, whereby 1x10° cells are singular or the plural of the term, unless specifically described injected into tail veins (43 days), taken 26 hours after 60 LM as a “single cell.” concentrations of p28 (SEQ ID NO: 13) injected into tail 0157. As used herein, the terms “polypeptide.” “peptide.” veins. and “protein’ are used interchangeably to refer to a polymer 014.4 FIG. 38, (A) and (B). Depicts photographs of (A) of amino acid residues. The terms apply to amino acid poly organs from mice and (B) back views of mice in Balb-C mers in which one or more amino acid residues are an artifi peptide study taken 12 hours after treatment with 60 and 120 cial chemical analogue of a corresponding naturally occur uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ ID ring amino acid. The terms also apply to naturally occurring NO: 13), and Arg-8 (SEQID NO:3527). amino acid polymers. The terms “polypeptide.” “peptide.” 0145 FIG. 39. (A) and (B). Depicts photographs of (A) and “protein’ are also inclusive of modifications including, organs from mice and (B) side views of mice in Balb-C but not limited to, glycosylation, lipid attachment, Sulfation, peptide study taken 24 hours after treatment with 60 and 120 gamma-carboxylation of glutamic acid residues, hydroxyla uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ ID tion and ADP-ribosylation. It will be appreciated that polypeptides are not always entirely linear. For instance, NO: 13), and Arg-8 (SEQID NO:3527). polypeptides may be branched as a result of ubiquitination 0146 FIG. 40. Depicts side and back photographs of and they may be circular (with or without branching), gener MEL-6 mice (0.5 million cells injected via tail vein) 16 hours ally as a result of post-translation events, including natural after injection into tail veins of 60LM concentrations of p 18 processing event and events brought about by human manipu (SEQ ID NO: 14) and Arg-8 (SEQID NO:3527). lation which do not occur naturally. Circular, branched and 0147 FIG. 41, (A) through (D). Depicts photographs of branched circular polypeptides may be synthesized by non mouse organs, and specifically mouse brains, after treatment translation natural process and by entirely synthetic methods with p18 (SEQID NO: 14) and p28 (SEQID NO: 13). as well. A synthetic peptide is one made without the aid of 0148 FIG. 42. Depicts photographs of organs from cellular components. Synthetic methods to make peptides are MEL-6 mice taken 24 hours after treatment with p28 (SEQID well known in the art and are commercially available. Further, NO: 13), p.18 (SEQ ID NO: 14), and Arg-8 (SEQ ID NO: this invention contemplates the use of both the methionine 3527). containing and the methionine-less amino terminal variants 014.9 FIG. 43. (A) through (C). (A) Depicts side and back of the protein of the invention. photographs of MEL-6 mice 3 hours after injection with 60 0158. As used herein, the term “condition' includes ana uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ ID tomic and physiological deviations from the normal that con NO: 13), and Arg-8 (SEQID NO:3527). (B) Depicts side and stitute an impairment of the normal state of the living animal back photographs of MEL-6 mice, and photographs of organs or one of its parts, that interrupts or modifies the performance from MEL-6 mice, taken 22 hours after injection with 60 LM of the bodily functions. concentrations of p18 (SEQID NO: 14), p28 (SEQID NO: 0159. As used herein, the term “inhibit cell growth' means 13), and Arg-8 (SEQID NO:3527). (C) Depicts photographs the slowing or ceasing of cell division and/or cell expansion. of organs from MEL-6 mice 24 hours after injection with 60 This term also includes the inhibition of cell development or uM concentrations of p18 (SEQ ID NO: 14), p28 (SEQ ID increases in cell death. NO: 13), and Arg-8 (SEQID NO:3527). 0160. As used herein, the term “suffering from includes 0150 FIG. 44, (A) and (B). Depict uptake of p18 (SEQID presently exhibiting the symptoms of a condition, having a NO: 14) and p28 (SEQID NO: 13) into (A)mouse brains and condition even without observable symptoms, in recovery (B) mouse organs). from a condition, and recovered from a condition. 0151 FIG. 45. Depicts side and back photographs of 0.161. A used herein, the term “treatment includes pre MEL-6 mice in study whereby 0.5 million cells injected I.V. venting, lowering, stopping, or reversing the progression or into tail vein (44 days post), taken 120 hours after injection severity of the condition or symptoms associated with a con into tail vein of 24 uM concentrations of p18 (SEQ ID NO: dition being treated. As such, the term “treatment includes 14) and Arg-8 (SEQID NO:3527). medical, therapeutic, and/or prophylactic administration, as 0152 FIG. 46. Depicts photographs of organs from appropriate. MEL-6 mice taken 168 hours after treatment with p18 (SEQ 0162. A “therapeutically effective amount” is an amount ID NO: 14). effective to prevent, lower, stop or reverse the development US 2009/0286719 A1 Nov. 19, 2009 of or to partially or totally alleviate a particular condition, or substantially pure.” The term “substantially pure' may also the existing symptoms of a particular condition for which the be used to modify a synthetically made protein or compound, subject being treated. Determination of a therapeutically where, for example, the synthetic protein is isolated from the effective amount is well within the capability of those skilled reagents and by-products of the synthesis reaction(s). in the art. 0172. The term “pharmaceutical grade.” as used herein, 0163 As used herein, the term “pharmacologic activity” when referring to a peptide or compound of the invention, is means the effect of a drug or other chemical on a biological a peptide or compound that is isolated Substantially or essen system. The effect of chemical may be beneficial (therapeu tially from components which normally accompany the mate tic) or harmful (toxic). The pure chemicals or mixtures may rial as it is found in its natural state, including synthesis be of natural origin (plant, animal, or mineral) or may be reagents and by-products, and Substantially or essentially synthetic compounds. isolated from components that would impair its use as a 0164. As used herein, the term “premalignant’ means pre pharmaceutical. For example, a “pharmaceutical grade' pep cancerous, or before abnormal cells divide without control. tide may be a isolated away from any carcinogen. In some 0.165. As used herein, the term “pharmacokinetic prop instances, “pharmaceutical grade' may be modified by the erty” refers to a parameter that describes the disposition of an intended method of administration, Such as “intravenous active agent or drug in an organism or host. Representative pharmaceutical grade.” in order to specify a peptide or com pharmacokinetic properties include: plasma half-life, hepatic pound that is substantially or essentially isolated from any first-pass metabolism, volume of distribution, degree of blood substance that would render the composition unsuitable for serum protein, e.g. albumin, binding, etc. intravenous administration to a patient. For example, an 0166 As used herein, the term "plasma half-life” refers to “intravenous pharmaceutical grade peptide may be isolated the time for one-half of an administered drug to be eliminated from detergents, such as SDS, and anti-bacterial agents. Such from the plasma of the patient through biological processes, as azide. e.g., biometabolism, excretion, etc. (0173 The phrases "isolated,” “purified” or “biologically 0167 As used herein, the term “volume of distribution” pure” refer to material which is substantially or essentially refers to the distribution and degree of retention of a drug free from components which normally accompany the mate throughout the various compartments of an organisms, e.g. rial as it is found in its native state. Thus, isolated peptides in intracellular and extracellular spaces, tissues and organs, etc. accordance with the invention preferably do not contain This factor is expressed as the “apparent volume of distribu materials normally associated with the peptides in their in situ tion, or V, which is the estimated Volume of the body into environment. An "isolated region refers to a region that does which the drug has distributed. A large Vd Suggests that the not include the whole sequence of the polypeptide from drug has distributed more broadly throughout the body and which the region was derived. An "isolated nucleic acid, may be associated with the longer half-life because a lesser protein, or respective fragment thereofhas been Substantially portion of the drug will be in the plasma and thus delivered to removed from its in vivo environment so that it may be the elimination points, the kidney and the liver. manipulated by the skilled artisan, such as but not limited to 0168 As used herein, the term “degree of blood serum nucleotide sequencing, restriction digestion, site-directed binding refers to the propensity of a drug to be bound by a mutagenesis, and Subcloning into expression vectors for a blood serum protein, such as albumin. nucleic acid fragment as well as obtaining the protein or (0169. As used herein, the term “efficacy” refers to the protein fragment in Substantially pure quantities. effectiveness of a particular active agent for its intended pur 0.174. The term “wild-type.” as used herein to refer to a pose, i.e. the ability of a given active agent to cause its desired peptide, mean that the peptide has the same sequence as one pharmacologic effect. naturally occurring. 0170 As used herein, the term “specific activity” refers to 0.175. The term “variant' as used herein with respect to a the amount of product formed by an enzyme in a given peptide, refers to amino acid sequence variants that may have amount of time under given conditions per milligram of amino acids replaced, deleted, or inserted as compared to the enzyme. Specific activity is equal to the rate of reaction mul wild-type polypeptide. Variants may be truncations of the tiplied by the volume of reaction divided by the mass of wild-type peptide. A “deletion' is the removal of one or more enzyme. In the case of a transport peptide, the specific activity amino acids from within the wild-type protein, while a “trun will be the amount of transport peptide or transport peptide cation' is the removal of one or more amino acids from one or cargo complex internalized into a cell in a given amount of more ends of the wild-type protein. Thus, a variant peptide time under given conditions per milligram of transport pep may be made by manipulation of genes encoding the tide or transport peptide-cargo complex. polypeptide. A variant may be made by altering the basic 0171 The term “substantially pure,” as used herein, when composition or characteristics of the polypeptide, but not at used to modify a protein or other cellular product of the least Some of its fundamental pharmacologic activities. For invention, refers to, for example, a protein isolated from the example, a “variant' of the Pseudomonas aeruginosa transit growth medium or cellular contents, in a form Substantially peptide may be a mutated Pseudomonas aeruginosa transit free of, or unadulterated by, other proteins and/or active peptide that retains its ability to enter cancer cells. In some inhibitory compounds. The term “substantially pure’ refers to cases, a variant peptide is synthesized with non-natural amino a factor in an amount of at least about 75%, by dry weight, of acids, such as e-(3,5-dinitrobenzoyl)-Lys residues. (Ghadiri isolated fraction, or at least “75% substantially pure.” More & Fernholz, J. Am. Chem. Soc., 112:9633-9635 (1990)). In specifically, the term “substantially pure' refers to a com some embodiments, the variant has not more than 20, 19, 18, pound of at least about 85%, by dry weight, active compound, 17 or 16 amino acids replaced, deleted or inserted compared or at least “85% substantially pure.” Most specifically, the to wild-type peptide or a portion thereof. In some embodi term “substantially pure” refers to a compound of at least ments, the variant has not more than 15, 14, 13, 12 or 11 about 95%, by dry weight, active compound, or at least "95% amino acids replaced, deleted or inserted compared to wild US 2009/0286719 A1 Nov. 19, 2009 type peptide or a portion thereof. In some embodiments, the tity to, with, or against a given amino acid sequence B) can be variant has not more than 10, 9, 8 or 7 amino acids replaced, calculated as: deleted or inserted compared to wild-type peptide or a portion % amino acid sequence identity=X/Y*100 thereof. In some embodiments, the variant has not more than 0181 where 6 amino acids replaced, deleted or inserted compared to wild 0182 X is the number of amino acid residues scored as type peptide or a portion thereof. In some embodiments, the identical matches by the sequence alignment program's variant has not more than 5 or 4 amino acids replaced, deleted or algorithm's alignment of A and B and or inserted compared to wild-type peptide or a portion 0183 Y is the total number of amino acid residues in B. 0184. If the length of amino acid sequence A is not equal to thereof. In some embodiments, the variant has not more than the length of amino acid sequence B, the '% amino acid 3, 2 or 1 amino acids replaced, deleted or inserted compared sequence identity of A to B will not equal the 96 amino acid to wild-type peptide or a portion thereof. sequence identity of B to A. When comparing longer 0176 The term “amino acid,” as used herein, means an sequences to shorter sequences, the shorter sequence will be amino acid moiety that comprises any naturally-occurring or the “B” sequence. For example, when comparing truncated non-naturally occurring or synthetic amino acid residue, i.e., peptides to the corresponding wild-type polypeptide, the any moiety comprising at least one carboxyl and at least one truncated peptide will be the “B” sequence. amino residue directly linked by one, two, three or more General carbon atoms, typically one (a) carbon atom. An amino acid 0185. The present invention relates to cupredoxin derived peptides that maintain one or more pharmacologic activities may be an L-isomer or a D-isomer of an amino acid. of the cupredoxin and which may have improved pharmaco 0177. The term “modified residue' as used herein refers to kinetic properties, such as improved stability, specific activ an amino acid that has been modified using a method or ity, half-life in the bloodstream, and/or decreased immuno technique which may include, but is not limited to, one or genicity, among others. Additionally, the present invention more of the methods and techniques disclosed herein. relates to compounds derived from the modified cupredoxin derived peptides, which in turn also maintain one or more (0178. The term “derivative” as used herein with respect to pharmacologic activities of the cupredoxin and which have a peptide refers to a peptide that is derived from the subject improved pharmacokinetic properties. Finally, the invention peptide. A derivation includes chemical modifications of the relates to methods to use the modified cupredoxin derived peptide such that the peptide still retains some of its funda peptides and compound made from them to treat and/or diag mental pharmacologic activities. For example, a "derivative' nose various conditions Suffered by mammalian patients, and of a Pseudomonas aeruginosa transport peptide can be a to research various conditions suffered by mammalian patients. The Sequence Listing Submitted herewith is incor chemically modified Pseudomonas aeruginosa transport porated by reference in its entirety herein. peptide that retains its ability to enter cancer cells. Chemical modifications of interest include, but are not limited to, ami Compositions dation, acetylation, sulfation, polyethylene glycol (PEG) 0186 The invention relates to peptides that are modifica tions of cupredoxin derived peptides. In some embodiments modification, phosphorylation or glycosylation of the pep the modified peptides have improved pharmacokinetic prop tide. In addition, a derivative peptide maybe a fusion of a erties. In some embodiments, these modified cupredoxin polypeptide to a chemical compound. Such as, but not limited derived peptides retain at least one pharmacologic activity of to, another peptide, drug molecule or other therapeutic or the cupredoxin. In some embodiments, the modified cupre pharmaceutical agent or a detectable probe. doxin derived peptides are isolated, Substantially pure, or pharmaceutical grade. In specific embodiments, the modified 0179 The term “percent (%) amino acid sequence iden cupredoxin derived peptides are intravenous pharmaceutical tity” is defined as the percentage of amino acid residues in a grade. polypeptide that are identical with amino acid residues in a 0187 Cupredoxins, and specifically aZurin from candidate sequence when the two sequences are aligned. To Pseudomonas aeruginosa, are known to have several useful determine% amino acid identity, sequences are aligned and if pharmacologic activities that are useful for treating and/or necessary, gaps are introduced to achieve the maximum '% diagnosing mammalian patients, and for conducting research on conditions suffered by mammalian patients. For example, sequence identity; conservative Substitutions are not consid many cupredoxin proteins, such as Pseudomonas aeruginosa ered as part of the sequence identity. Amino acid sequence aZurin, have the ability to specifically enter and kill many alignment procedures to determine percent identity are well types of mammalian cancer cells. Yamada et al., Cell. Biol. known to those of skill in the art. Often publicly available 7:1418-1431 (2005); Hiraoka et al., PNAS 101:6427-6432 computer software such as BLAST, BLAST2, ALIGN2 or (2004); Hiraoka et al., Biochem. Biophys. Res. Comm. 338: Megalign (DNASTAR) software is used to align peptide 1284-1290 (2005); U.S. patent application Ser. No. 1 1/244, sequences. In a specific embodiment, Blastp (available from 105, filed Oct. 6, 2005: U.S. patent application Ser. No. the National Centerfor Biotechnology Information, Bethesda 10/720,603, filed Nov. 24, 2003: U.S. patent application Ser. Md.) is used using the default parameters of long complexity No. 10/047,710, filed Jan. 15, 2002; U.S. patent application Ser. No. 1 1/485,252, filed Jul. 13, 2006, all of which are filter, expect 10, word size 3, existence 11 and extension 1. expressly incorporated herein by reference in their entirety. 0180. When amino acid sequences are aligned, the % AZurin from Paeruginosa is also known to inhibit the growth amino acid sequence identity of a given amino acid sequence of viral or bacterial infection, and more specifically HIV-1 A to, with, or against a given amino acid sequence B (which infection in peripheral blood mononuclear cells and also to can alternatively be phrased as a given amino acid sequence A inhibit parasitemia of malaria-infected mammalian red blood that has or comprises a certain% amino acid sequence iden cells. Chaudhariet al., Cell Cycle. 5:1642-1648 (2006); U.S. US 2009/0286719 A1 Nov. 19, 2009

patent application Ser. No. 1 1/436,591, filed May 19, 2006: (1996); Cruciani, R. A., et al., Proc Natl Acad Sci USA 88: U.S. patent application Ser. No. 11/436,590, filed May 19, 3792-3796 (1991); Papo, N. and Shai, Y., Biochemistry, 42: 2006, both of which are expressly incorporated herein by 9346-9354 (2003). Peptides generated throughphage-display reference in their entirety. AZurin from P. aeruginosa is also technology, though rather specific for lymphatic and tumor known to interfere with the ephrin signaling system in various blood vessels, also do not induce cytotoxicity by direct pen mammaliancells and tissues. U.S. patent application Ser. No. etration of cancer cells. 11/436,592, filed May 19, 2006, which is expressly incorpo 0190. In contrast, aZurin and the two peptides derived from rated herein by reference in its entirety. Further, peptides it (p.28, SEQ. ID 13, and p18, SEQ. ID 14) possess the unique derived from PaeruginosaaZurinare known to inhibit angio property of preferentially entering cancer cells and inhibiting genesis in mammalian cells, and specifically human umbili their proliferation through cytostatic and cytotoxic mecha cal vascular endothelium cells (HUVECs). U.S. patent appli nisms. It has been shown by confocal microscopy and FACS cation Ser. No. 1 1/488,693, filed Jul. 19, 2006, which is that p18 (SEQ. ID 14) is the minimal motif responsible for expressly incorporated herein by reference in its entirety. In aZurin's preferential entry into human cancer cells. Some embodiments, the modified cupredoxin derived pep 0191 In addition to entering cancer cells, p 18 (SEQ ID tides of the invention retain at least one pharmacologic activ NO: 14) and p28 (SEQ ID NO: 13) are able to enter tumors ity of the cupredoxin from which they are derived. The phar and mammalian organs, as is shown in FIGS. 14 through 49. macologic activity of cupredoxin may be any useful activity which were obtained using the methods disclosed in Example of a cupredoxin. Pharmacologic activities of particular inter 9. Surprisingly, p 18 (SEQID NO: 14) and p28 (SEQID NO: est include, but are not limited to, the ability to specifically 13) are also able to penetrate the blood-brain barrier and enter enter mammalian cancer cells, the inability to enter non mammalian brains, as demonstrated by, for example, FIGS. cancerous mammalian cells, the ability to enter pre-malig 16A, 16B, 17B. 19, 20, 24, 26, 27A, 28B, 29, 30B, 31 A-B, nant mammalian cells, the ability to kill mammalian cancer 33A-C, 34A-C, 36A, 38A, 39A, 41A-D, 42,43B, 44A-B, and cells, the ability to kill pre-malignant mammalian cells, the 46. ability to inhibit the growth of viral or bacterial infection, the 0.192 Redox proteins, such as azurin, are not normally ability to inhibit the HIV-1 infection in peripheral blood classified as CPPs, or anti-proliferative agents. The amphip mononuclear cells, the ability to inhibit parasitemia by athic, azurin fragments p18 (SEQID NO: 14) and p28 (SEQ malaria in malaria-infected red blood cells, and the ability to ID NO: 13) contain the 54-67 amino acid C.-helical structure inhibit angiogenesis in mammalian cells, and specifically of aZurin as well as a partial C-sheet structure and describe a HUVECs. Methods to measure the amount of pharmacologic minimal sequence for cancer cell entry and cell cycle inhibi activity of the peptide are provided in the above referenced tory activity, respectively. The entry of azurin, p28 (SEQ ID applications and publications. NO: 13), and p18 (SEQ ID NO: 14) is distinct from that of 0188 AZurin, a cupredoxin from Pseudomonas aerugi cationic CPPs. Aberrant N-glycosylation on several cell sur nosa, utilizes a protein transduction domain (PTD) to selec face receptors, including integrins and cadherins, is associ tively enter human cancer cells. The minimal motif PTD used ated with changes in progression and metastasis of cancers of by these cupredoxins had not been previously identified. The diverse histogenesis, suggesting a role for as yet unknown mechanisms of entry by azurin, p 18 (SEQ ID 14) and p28 N-glycoslyated cell surface protein(s) in the initial steps of (SEQ ID 13) are detailed here and in the examples provided aZurin, p18 (SEQ ID NO: 14) and p28 (SEQ ID NO: 13) below. Generally, PTDs cluster into two groups based on their penetration. Partridge, E. A., et al. Science 306: 120-124 structural characteristics, cationic residues or amphipathic (2004); Seales, E. C., et al., Cancer Res 65:4645-4652 (2005). C.-helix, although several fall into both classes. In general, 0193 The temperature dependent entry of cationic CPPs, cationic peptides initially interact with the cell membranes of which Supports an endocytotic component to cell penetration, prokaryotic and eukaryotic species by binding to negatively is reflected in the entry of aZurin and amino acid fragment charged Surface glycoproteins, facilitating efficient entry into 50-77 (p28 SEQ. ID 13). Yamada, T., et al., Cell Microbiol 7: a broad range of normal and malignant cell lines. Kondejew 1418-1431 (2005). The entry of amino acids 50-67 of azurin ski, L. H., etal, J Biol Chem 277: 67-74 (2002); Fuchs, S. M. (p18 SEQ. ID 14) into normal and malignant cells appears and Raines, R. T., Biochemistry, 43: 2438-2444 (2004). The accelerated relative to p28 (SEQID NO: 13). The lower K, binding of cationic peptides to HS is consistent with their high and higher V of p18 (SEQID NO: 14) suggest that amino affinity for HS (Kd-109 nM), a value well in excess of that acids 50-67 define an amphipathic structure when associated reported for azurin. p18 (SEQ ID 14) and p28 (SEQ ID 13). with phospholipid membranes that more closely represents Tran, D. etal, Proc Natl AcadSci USA84: 7957-7961 (1987). the actual PTD of azurin. However, an energy dependent 0189 However, the cytotoxic effects exerted by synthetic, endocytotic or pore related process does not appear to be the cationic, amphipathic C-helical diastereomeric peptides are only entry mechanism available to these peptides. For not generally specific to cancer cells. Amphipathic cell pen example, the metabolic and membrane potential inhibitors etrating peptides (CPPs) that are cytolytic to cancer cells sodium azide and ouabain (Na+K+ ATPase inhibitor), which either disrupt the cancer cell membrane, alter mitochondrial inhibit the entry-of cationic peptides, did not impair the entry permeability, or act through a specific receptor mediated of either p18 (SEQID NO: 14) or p28 (SEQID NO: 13) into mechanism. Leuschner, C. and Hansel, W., Curr Pharm Des, UISO-Mel-2 cells or fibroblasts (FIG. 10 B.C), suggesting 10: 2299-2310 (2004). Synthetic magainins, a linear, helical, that either peptide may penetrate the cell membrane directly. channel-forming or ionophore class of peptides including (0194 p18 (SEQ ID NO: 14), p28 (SEQ ID NO: 13) and those exclusively comprised of Lys, Ala, and Leu residues aZurin penetrate the plasma membrane and reach late endos rapidly and irreversibly lyse hematopoietic and solid tumor mes, lysosomes and the golgi associated with caveolae in target cells at doses below those cytotoxic to normal cells, but what is thought to be a dynamin-independent clathrinin do not have the property of preferentially penetrating cancer dependent carrier mediated manner. Kirkham, M. and Parton, cells. Javapour, M. M., et al., J Med Chem, 39: 3107-3113 R. G., Biochem Biophys Acta 1746: 349-363 (2005). US 2009/0286719 A1 Nov. 19, 2009

Nocodazole, which disrupts caveolae transport and caveolae (0197) p18 (amino acids 50-67 of azurin, SEQ. ID 14) and mediated endocytosis inhibited penetration by 50-65%. The p28 (amino acids 50-77 of azurin, SEQ. ID 13) are not bound striking inhibition of penetration by nocodazole and relative by cell membrane glycosaminoglycans and preferentially lack of inhibition by cytochalasin-D, which disrupts actin penetrate cancer cells via endocytotic, caveosome directed filaments, Supports caveolae mediated entry. Notably, the and caveosome independent pathways. The cellular penetra lack of effect of staurosporine demonstrates that dynamin tion of p18 (SEQ ID NO: 14) and p28 (SEQ ID NO: 13) is does not play a large role in the penetration of either peptide. unique relative to all current CPPs in its preference for cancer Id. This route of entry has been described for integral cell cells. Surprisingly, the C-terminal 10-12 amino acids of p28 Surface components and seemingly disparate molecules, i.e., (SEQ ID NO: 13) are demonstrated to comprise the domain dextran, and abroad range of pathogens or their products that responsible for cell cycle inhibition and apoptotic activity/ also utilize caveolae to bypass classic endocytotic pathways. cytotoxicity. Furthermore, this same domain is most likely to Depletion of cholesterol from the plasma membrane with contact specific residues on a cell membrane and thus facili B-methylcylodextran, filipin or nystatin to disrupt lipid rafts, tate entry; amino acids 69, 70, 75, 76, and 85 of azurin in plasma membrane domains that provide fluid platforms to particular provide contact to the cell membrane. Once inter segregate membrane components and compartmentalize nalized, p28 (SEQID NO: 13) inhibits cancer cell prolifera membranes, significantly inhibited the penetration of p 18 tion initially through a cytostatic mechanism. Thus, it is now (SEQ ID NO: 14) (50%) and p28 (SEQID NO: 13) (-60%) known that p18 (SEQ ID NO: 14) and p28 (SEQID NO: 13) into UISO-Mel-2 cells and fibroblasts (35% and 42%, respec account for the preferential entry of aZurin into human cancer tively) demonstrating that a significant percentage (~60%) of cells and a significant amount of the anti-proliferative activity p18 (SEQID NO: 14) and p28 (SEQ ID NO: 13) penetrates of aZurin on human cancer cells, respectively. the plasma membrane via caveolae. Caveolae are a 50- to 0198 The cupredoxin derived peptides may be any cupre 100-nm omega-shaped Subset of lipidraft invaginations of the doxin, or variant, derivative or structural equivalent of cupre plasma membrane defined by the presence of caveolin spe doxin, or a truncation thereof. In some embodiments, the cific proteins (caveolin-1, -2, or -3) that function as regulators cupredoxin derived peptide retains at least one pharmaco of signal transduction. logic activity of the cupredoxin. In some embodiments, the 0.195 Brefeldin A disrupts the Golgi apparatus and inhib cupredoxin may be, but is not limited to, aZurin, plastocyanin, ited p18 (SEQID NO: 14) accumulation, so it follows that this rusticyanin, pseudoaZurin, auracyanin oraZurin-like protein. pathway is also utilized in p18 (SEQ ID NO: 14) and p28 The cupredoxin derived peptides may be from any organism, (SEQID NO: 13) entry and intracellular transport. Cell pen including but not limited to Pseudomonas aeruginosa, Phor etration of p18 (SEQID NO: 14) and p28 (SEQID NO: 13) midium laminosum, Thiobacillus ferrooxidans, Achromo via caveolae comports with the evidence that inhibitors of bacter cycloclastes, Pseudomonas syringa, Neisseria menin N-glycosylation reduce cell entry by ~60% in UISO-Mel-2 gitidis, Vibrio parahaemolyticus, Bordetella bronchiseptica, cells and 25% and 35% respectively in fibroblasts. The per Bordetella pertussis, Chloroflexus aurantiacus and Neisseria centile differences between p18 (SEQ ID NO: 14) and p28 gonorrhoeae. In some embodiments, the cupredoxin may be (SEQ ID NO: 13) entry relate to the numbers of N-glycosy aZurin, specifically from an organism including but not lim lation membrane structures in cancer vs. normal cells and the ited to Pseudomonas aeruginosa, Pseudomonas syringae. relative route of entry of p28 (SEQID NO: 13) and p18 (SEQ Neisseria gonorrhoeae, Vibrio parahaemolyticus, or Borde ID NO: 14) via this mechanism. tella bronchiseptica. (0196. AZurin, p28 (SEQ ID NO: 13), and p18 (SEQ ID 0199 The cupredoxin derived peptides may be any vari NO: 14) all bind to cancer cells with high affinity and high ant, derivative or structural equivalent of a cupredoxin. In capacity relative to many other potential anti-cancerpeptides. Some embodiments, the cupredoxin derived peptides may be After binding, this protein/receptor complex localizes in a truncation of a cupredoxin. The cupredoxin derived pep caveolae and is internalized, eventually moving (via caveo tides may also be any cupredoxin peptide that is known in the Somes) to the golgi, ER, and nucleus. In addition to caveolar art and/or described in previous applications, such as U.S. mediated entry, kinetic analysis also demonstrates that p28 patent application Ser. No. 1 1/244,105, filed Oct. 6, 2005; (SEQ ID NO: 13) and p18 (SEQ ID NO: 14) penetrate the U.S. patent application Ser. No. 10/720,603, filed Nov. 24, plasma membrane via a non-clathrin caveolae mediated pro 2003: U.S. patent application Ser. No. 10/047,710, filed Jan. cess. A clathrin- and caveolin-independent pathway can exist 15, 2002, now U.S. Pat. No. 7,084,105: U.S. patent applica as a constitutive internalization mechanism, Such as for the tion Ser. No. 1 1/485,252, filed Jul. 13, 2006: U.S. patent interleukin 2 receptor and for certain glycosyl-phosphatidyli application Ser. No. 11/436,591, filed May 19, 2006: U.S. nositol (GPI)-anchored proteins. Lamaze, C., et al., Mol Cell patent application Ser. No. 1 1/436,590, filed May 19, 2006: 7: 661-671 (2001); Sabharanjak, S., et al., Dev Cell, 2: 411 U.S. patent application Ser. No. 11/436,592, filed May 19, 423 (2002). Clathrin- and caveolin-independent endocytosis 2006; and U.S. patent application Ser. No. 1 1/488,693, filed is also used by pathogens to invade cells, either exclusively, as Jul.19, 2006. All of these applications are expressly incorpo for the murine polyoma virus, or in combination with a con rated by reference herein in their entirety. In some embodi ventional pathway, as is the case for the influenza virus. ments, the peptide is isolated. In some embodiments, the Ewers, H., et al, Proc Natl Acad Sci USA 102: 15110-15115 peptide is Substantially pure orpharmaceutical grade. In other (2005); Sieczkarski, S. B. and Whittaker, G. R., J. Virol, embodiments, the peptide is in a composition that comprises, 76:10455-10464 (2002). An increase in caveolin-1 expres or consists essentially of the peptide. In another specific sion in cancer cells over normal cells is not likely to be the embodiment, the peptide does not raise an immune response sole basis for the preferential entry of azurin, p28 (SEQ ID in a mammal, and more specifically a human. NO: 13) and p18 (SEQID NO: 14) into cancer cells. Fibro 0200. The cupredoxin derived peptides may beamino acid blasts and a number of other normal cells also have significant sequence variants which have amino acids replaced, deleted, numbers of caveolae on their surface. or inserted as compared to the wild-type cupredoxin. These US 2009/0286719 A1 Nov. 19, 2009

variants may be truncations of the wild-type cupredoxin. In Soc. 122:5891-5892 (2000); Walenski et al., Science 305: Some embodiments, amino acids may be replaced with 1466-1470 (2004)), and peptides comprising e-(3,5-dini unnatural or modified amino acids. An unnatural amino acid trobenzoyl)-Lys residues. is one other than the 20 commonamino acids. The cupredoxin 0204. In other embodiments, the cupredoxin derived pep derived peptides comprise a region of a cupredoxin that is less tide is a derivative of a cupredoxin. The derivatives of cupre that the full length wild-type polypeptide. In some embodi doxin are chemical modifications of the peptide such that the ments, the cupredoxin derived peptides comprise more than peptide still retains some of its fundamental pharmacologic about 10 residues, more than about 15 residues or more than activities. For example, a "derivative' of azurin can be a about 20 residues of a truncated cupredoxin. In some embodi chemically modified aZurin that retains its ability to inhibit ments, the cupredoxin derived peptides comprise not more the growth of mammalian cancer cells. Chemical modifica tions of interest include, but are not limited to, hydrocarbon than about 100 residues, not more than about 50 residues, not stabling, amidation, acetylation, Sulfation, polyethylene gly more than about 40 residues, not more than about 30 residues col (PEG) modification, phosphorylation and glycosylation or not more than about 20 residues of a truncated cupredoxin. of the peptide. In addition, a derivative peptide maybe a In some embodiments, a cupredoxin has to the cupredoxin fusion of a cupredoxin, or variant, derivative or structural derived peptide, and more specifically SEQID NOS: 1-12 at equivalent thereof to a chemical compound. Such as but not least about 70% amino acid sequence identity, at least about limited to, another peptide, drug molecule or other therapeu 80% amino acid sequence identity, at least about 90% amino tic or pharmaceutical agent or a detectable probe. Derivatives acid sequence identity, at least about 95% amino acid of interest include chemical modifications by which the half sequence identity or at least about 99% amino acid sequence life in the bloodstream of the peptides and compositions of the identity. invention can be extended or optimized, such as by several 0201 In specific embodiments, the cupredoxin derived methods well known to those in the art, including but not peptide comprises P. aeruginosa azurin residues 50-77 (SEQ limited to, circularized peptides (see, for example Monket al., IDNO:13), azurin residues 50-67 (SEQIDNO: 14), oraZurin BioDrugs 19(4):261-78, (2005): DeFreest et al., J. Pept. Res. residues 36-89 (SEQID NO: 16). In other embodiments, the 63(5):409-19 (2004)), N- and C-terminal modifications (see, variant of cupredoxin consists of P aeruginosa aZurin resi for example Labrie et al., Clin. Invest. Med. 13(5):275-8, dues 50-77 (SEQID NO:13), azurin residues 50-67 (SEQID (1990)), and olefin-containing non-natural amino acid fol NO:14), oraZurin residues 36-89 (SEQID NO:16). In other lowed by hydrocarbon Stapling (see, for example Schafmeis specific embodiments, the variant consists of the equivalent teret al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski residues of a cupredoxin other that aZurin. To determine the et al., Science 305:1466-1470 (2004)). equivalent residues of another cupredoxin, the Subject cupre 0205. In another embodiment, the peptide is a structural doxin amino acid sequence will be aligned to the Pseudomo equivalent of a cupredoxin or a truncation of a cupredoxin. nas aeruginosa aZurin sequence using BLAST, BLAST2, Examples of studies that determine significant structural ALIGN2 or Megalign (DNASTAR), the relevant residues homology between cupredoxins and other proteins include located on the Paeruginosa aZurinamino acid sequence, and Tothet al. (Developmental Cell 1:82-92 (2001)). Specifically, the equivalent residues found on the Subject cupredoxin significant structural homology between a cupredoxin and the sequence, and the equivalent peptide thus designed. structural equivalent is determined by using the VAST algo 0202 In one embodiment of the invention, the cupredoxin rithm. Gibrat et al., Curr Opin Struct Biol 6:377-385 (1996): derived peptide comprises at least amino acids 57 to 89 of Made et al., Proteins 23:356-3690 (1995). In specific auracyanin B of Chloroflexus aurantiacus (SEQID NO: 21). embodiments, the VAST p value from a structural comparison In another embodiment of the invention, the cupredoxin of a cupredoxin to the structural equivalent is less than about derived peptide comprises at least amino acids 51-77 of 10, less than about 10, or less than about 107. In other Pseudomonas syringae azurin (SEQ ID NO: 27). In another embodiments, significant structural homology between a embodiment of the invention, the cupredoxin derived peptide cupredoxin and the structural equivalent is determined by comprises at least amino acids 89-115 of Neisseria menin using the DALI algorithm. Holm & Sander, J. Mol. Biol. gitidis Laz (SEQID NO. 23). In another embodiment of the 233:123-138 (1993). In specific embodiments, the DALI Z. invention, the cupredoxin derived peptide comprises at least score for a pairwise structural comparison is at least about 3.5. amino acids 52-78 of Vibrioparahaemolyticus azurin (SEQ at least about 7.0, or at least about 10.0. ID NO: 28). In another embodiment of the invention, the 0206. One specific cupredoxin derived peptide of interest cupredoxin derived peptide comprises at least amino acids is a fusion of the entry domain of cupredoxin with a cargo 51-77 of Bordetella bronchiseptica azurin (SEQID NO: 29). compound. In some embodiments, cupredoxin derived pep 0203 The cupredoxin derived peptides also include pep tides may specifically enterinto a mammalian cancer cell, and tides made with synthetic amino acids that are not naturally thus may be used to deliver a cargo compound into a cell, and occurring. For example, non-naturally occurring amino acids specifically into a cancer cell. A cupredoxin transport peptide may be integrated into the variant peptide to extend or opti comprises a cupredoxin entry domain. The term “cupredoxin mize the half-life of the composition in the bloodstream. Such entry domain refers to a fragment of a cupredoxin that variants include, but are not limited to, D.L-peptides (diaste includes the amino sequence that is required for the entry of reomer), (see, for example Futaki et al., J. Biol. Chem. 276 cupredoxin into a mammalian cancer cell. In specific embodi (8):5836-40 (2001); Papo et al., Cancer Res. 64(16):5779-86 ments, the cupredoxin transport peptide is SEQ ID NOS: (2004); Miller et al, Biochem. Pharmacol. 36(1):169-76, 13-17, or equivalent residues from another cupredoxin. The (1987); peptides containing unusual amino acids (see, for present invention encompasses cupredoxin transport peptides example Lee et al., J. Pept. Res. 63(2):69-84 (2004)), olefin complexed with cargo compounds that have been modified to containing non-natural amino acid followed by hydrocarbon improve their pharmacokinetic properties. The cargo com Stapling (see, for example Schafmeister et al., J. Am. Chem. pound as well as the cupredoxin transport peptide may be US 2009/0286719 A1 Nov. 19, 2009

modified by the methods described herein to improve phar carbamate functionality. In such embodiments, the enzymatic macokinetic properties. These complexes can then be used in cleavage within the cancer cell delivers an unmodified cupre the methods of the invention to deliver the cargo compound doxin derived protein or drug inside the cell. into mammalian cancer cells to treat patients Suffering from 0210. In some embodiments, the cupredoxin-derived pep cancer. Cargo compounds delivered by the materials and tide is conjugated with a nanoparticle, for example a noble methods of the present invention include, but are not limited metal Such as gold or platinum, to create a hybrid system that to, proteins, lipoproteins, polypeptides, peptides, polysaccha can be used in therapeutic applications, diagnostics, and rides, nucleic acids, including anti-sense nucleic acids, dyes, imaging. fluorescent and radioactive tags, microparticles or nanopar 0211. In some embodiments, amino acids residues in the ticles, toxins, inorganic and organic molecules, metals, Small cupredoxin derived peptides that are conserved among cupre molecules, and drugs. In some embodiments, the drugs and doxins with the desired pharmacologic activity are conserved toxins kill tumor cells. Such cupredoxin transport peptides in modified cupredoxin derived peptides with improved phar and complexes made with them are provided in U.S. patent macokinetic properties. For example, it is known that within application Ser. No. 1 1/244,105, filed Oct. 6, 2005, which is the cupredoxin entry domain of Pseudomonas aeruginosa expressly incorporated herein by reference in its entirety. aZurin, several residues are conserved among aZurins and 0207. In some embodiments, the fusion between the cargo aZurin-like proteins from several species, Pseudomonas compound and the cupredoxin derived peptide may be by a aeruginosa, Pseudomonas Syringae. Neisseria gonorrhoeae, linkage that is selectively cleaved after entry of the fused Vibrio parahaemolyticus, and Bordetella bronchiseptica. peptide into the cell. In some embodiments, such a linkage Yamada et al., Cell. Microbiol. 7:1418-1431 (2005). In some improves the pharmacokinetic properties of the cupredoxin embodiments, the cupredoxin derived peptide retains one or transport peptide-cargo compound complex. In some more amino acid residues corresponding to residues 62, 63. embodiments, the stability of the cupredoxin transport pep 69, 72, 74 and 77 P aeruginosa azurin (SEQ ID NO: 1). In tide-cargo compound complex in plasma is enhanced. In another embodiment, the cupredoxin peptide comprises a Some embodiments, the linkage may be one that is selectively conserved amino acid sequence DGXXXXXDXX cleaved in the lysosomes of a mammalian cell. In other YXKXXD (SEQID NO: 46) or DGXXXXDXXYXKXXD embodiments, the linkage may be one that is cleaved by (SEQID NO: 47) where D is aspartic acid, G is glycine, Y is cathespin B, a cysteine protease located in the lysosomes or tyrosine, K is lysine and X is any amino acid. extracellularly in proximity to cancerous or arthritic sites. In some such embodiments, the linkage is a Val-Cit linkage. Modifications 0208. In another embodiment, the peptide is a cupredoxin, 0212. The present invention relates to modifications of or variant, structural equivalent, or derivative thereofthat is a cupredoxin derived peptides that are variants orderivatives or conjugate of Pep42, a cyclic 13-mer oligopeptide, truncations, and in specific embodiments, maintain one or CTVALPGGYRVRVC (SEQID. NO:3505) that specifically more pharmacologic activities, and/or that improve the phar binds to glucose-regulated protein 78 (GRP78) and is inter macokinetic properties of the peptide. These modifications nalized into cancer cells. The cupredoxin or variant, structural include, but are not limited to, variants and derivatives of the equivalent, or derivative of cupredoxin may be conjugated peptides that may increase their stability, specific activity, with Pep42 (SEQ ID. NO: 3505) pursuant to the synthesis plasma half life, and/or decrease immunogenicity of the methods disclosed in Yoneda et al., “A cell-penetrating pep cupredoxin derived peptide, while retaining the ability of the tidic GRP78 ligand for tumor cell-specific prodrug therapy.” cupredoxin to enter mammal cancer cells and/or inhibit the Bioorganic & Medicinal Chemistry Letters 18: 1632-1636 growth of mammalian cancer cells. Such variants include, but (2008), the disclosure of which is incorporated in its entirety are not limited to, those which decrease the hydrolysis of the herein. peptide, decrease the deamidation of the peptide, decrease the 0209. In some embodiments the cupredoxin derived pep oxidation, decrease the immunogenicity and/or increase the tide may be attached to Pep42 (SEQ ID. NO:3505). In some structural stability of the peptide. It is contemplated that two embodiments, Pep42 (SEQ ID. NO: 3505) may be further or more of the modifications described herein may be com fused with a drug. It is believed that Pep42 (SEQ ID. NO: bined in one modified cupredoxin derived peptide, as well as 3505) specifically binds to glucose regulated protein 78 combinations of one or more modifications described herein (GRP78), which is overexpressed in cancer cells, and is spe with other modification to improve pharmacokinetic proper cifically present on the cancer cell Surface. Yoneda, et al., ties that are well know to those in the art. Many methods to Bioorganic & Medicinal Chemistry Letters 18 1632-1636 design such variants and derivatives are well know in the art. (2008). Pep42 (SEQID. NO:3505) is efficiently internalized 0213. One method of chemically modifying a cupredoxin through the GRP78 receptor. Thus, in some embodiments, a or cytochrome c551 or variant, derivative, truncation, or cupredoxin derived peptide-Pepa2 fusion peptide may have structural equivalent thereof may be to follow the steps taken enhanced specificity to cancer cells. In some such embodi to design an anti-HIV small protein, CCL-5 (RANTES) with ments, the fusion between Pep42 (SEQID. NO:3505) and the improved pharmaceutical properties by, for example, hydro cupredoxin derived protein may be through a Val-Cit linkage, phobic N-terminal modification, total protein-polymer con a cathespin B. In some embodiments, the Pep42-drug conju jugate chemicals synthesis, coded and noncoded amino acid gate may be via a Val-Cit linkage. Pep42-drug conjugates mutagenesis, peptide backbone engineering, and site-specific containing cathepsin B-cleavable linkers are likely to be polymer attachment. Anti-HIV proteins can be designed by stable in the plasma and selectively release their drug specifi incorporating natural and unnatural amino acid residues into cally in the targeted tissue. In some embodiments of the CCL-5 analogues baring polymer Substituents at varying invention, Pep42 (SEQ ID. NO: 3505) is linked to p-ami attachment positions. Studies indicate that in vitro anti-HIV nobenzylalcohol via an amidic bond, which is then attached activity of polymer-modified CCL-5 derivatives correlates to the drug or cupredoxin derived peptide via a carbonate or with CCR-5 signaling, so changes to the peptide should not US 2009/0286719 A1 Nov. 19, 2009

disrupt CCR-5 activity. See Miranda, et al., J. Am. Chem. B-t-butyl-alanine, B-cyclopentyl alanine, photo-Leu, thiazo Soc. 129: 13153-13159 (2007), the disclosure of which is lidine-2-carboxylic acid, thiazolidine-4-carboxylic acid, 3,4- incorporated in its entirety herein. dehydro proline, L-aZetidine-2-carboxylic acid, 1-amino 0214 Biotransformation cyclopentanoic acid, 1-aminocyclohexanoic acid, B-hydroxy 0215 One approach to improving the pharmacokinetic acids, N-methyl His, N-methyl Asp, C.-hydroxy acids, and properties of the peptides is to create variants and derivatives O-hydroxy methionine. The Hartmann and Miranda refer of the cupredoxin derived peptides that are less susceptible to ences, and all unnatural building blocks and amino acids biotransformation. Biotransformation may decrease the phar described and disclosed by these references, are hereby incor macologic activity of the peptide as well as increase the rate porated by reference in their entirety herein. In some embodi at which it is eliminated from the patient’s body. One way of ments, such amino acids may be incorporated in cupredoxin achieving this is to determine the amino acids and/or amino derived peptides. acid sequences that are most likely to be biotransformed and 0218. Other modifications may include the use of optically to replace these amino acids with ones that are not susceptible active C-amino acids. The use of optically active C-amino to that particular transformative process. acids and their derivatives is being expanded for their use in 0216. In some embodiments, the cupredoxin derived pep pharmaceuticals, agrochemicals and as chiral ligands. In par tides may include unnatural amino acids or modified amino ticular, chiral glycine and alanine equivalents plan an impor acids. In some embodiments, the introduction of certain tant role. At least one stereoselective strategy for constructing unnatural amino acids enhances the pharmacokinetic proper C.-amino acids has been proposed, allowing for enantiopure ties of the cupredoxin derived peptide. Such introduction may C.-amino acids in predetermined stereochemistry. Lu, et al. be site-specific and may be done to avoid certain biochemical Asymmetric Synthesis of C.-amino acids: Preparation and modifications in vivo. Exemplary unnatural amino acids alkylation of monocyclic iminolactones derived from C.-Me include 3-amino acids (e.g. b3 and b2), homo-amino acids, thyltrans-cinnamaldehyde' published on Internet on Sep. 11, cyclic amino acids, aromatic amino acids, Pro and Pyr deriva 2008 (to be published in J. Org. Chem.). The modified cupre tives, 3-substituted Alanine derivatives, Glycine derivatives, doxin derived peptides may be synthesized using the optically Ring-substituted Phe and Tyr Derivatives, C.C. disubstituted active C-amino acids to produce enantiomerically enriched amino acids, Linear Core Amino Acids and Diamino Acids. iterations. Such unnatural amino acids may be incorporated into pep 0219) Hydrolysis is generally a problem in peptides con tides by site directed modification, ribosomal translation, or taining aspartate. Aspartate is Susceptible to dehydration to by chemical synthesis of the peptide. Each of these methods form a cyclic imide intermediate, causing the aspartate to be may be applied in synthesizing cupredoxin derived peptides. converted to the potentially inactive iso-aspartate analog, and 0217 For example, modified cupredoxin derived peptides ultimately cleaving the peptide chain. For example, in the may be synthesized by the use of wild-type Aminoacyl-tRNA presence of aspartic acid proline in the peptide sequence, synthetases (AARSs) with unnatural amino acids building for the acid catalyzed formation of cyclic imide intermediate can the production of unnatural cupredoxin variants. See Hart result to cleavage of the peptide chain. Similarly, in the pres man, et al., PLoS One, 2(10): e972 (2007); Miranda, et al., J. ence of aspartic acid glycine in the peptide sequence, the Am. Chem. Soc. 129: 13153-13159 (2007). The specificity of cyclic intermediate can be hydrolyzed either into the original the ribosomal translation apparatus limits the diversity of aspartate form (harmless) or into the iso-aspartate analog. unnatural amino acids that may be incorporated into peptides Eventually, all of the aspartate form can be completely con using ribosomal translation. Over ninety unnatural building Verted into the iso-aspartate analog. Similarly sequences with blocks that are AARS substrates have been uncovered includ serine can also be dehydrated to form a cyclic imide interme ing side chain and backbone analogs. Hartman, et al., PLoS diate that can cleave the peptide chain. Cleavage of the pep One, 2(10): e972 (2007). Over fifty unnatural amino acids tide may result in reduced plasma half-life as well as reduced may be incorporated into peptides with high efficiency using specific pharmacologic activity of the peptide. an all-enzymatic translation system, with peptides containing 0220. It is contemplated that substituting other amino up to thirteen different unnatural amino acids. Hartman, et al., acids for asparagine and/or serine in the sequence of the PLoS One, 2(10): e972 (2007). Unnatural amino acids cupredoxin derived peptide may result in a peptide with include, but are not limited to, 4-fluoro-glutamates, 4-methyl improved pharmacokinetic properties Such as a longer analogs, L-threo-B aspartic acid, S-2-aminoethyl cysteine, plasma half-life and increased specific activity of a pharma trans-dehydrolysine, aza-leucine, L-canavanine, L-N'-me cologic activity of the peptide. In one contemplated variant, at thyl arginine, N hydroxy arginine, vinyl-L-NIO, DL-f-hy one or more asparagine residues of the cupredoxin derived droxy norvaline, L-Gluy-methyl ester, L-Asp B-methyl ester, peptide may be replaced with anotheramino acid residue, and L-glutamic acid y-hydrazide, L-albizzine, L-theanine, B-2- specifically a glutamic acid residue. In another contemplated thiazolyl-alanine, B-(1,2,4-triazol-3-yl-alanine), 3-fluoro variant, one or more serine residues of the cupredoxin derived tyrosine, 3-fluoro valine, 3-nitro tyrosine, 2-fluoro Phe, peptide may be replaced with anotheramino acid residue, and 2-thienyl Ala, B-methyl Phe, B-thienyl Ser, p-nitrophenylala specifically a threonine residue. In some variants of cupre nine, 3-(thianaphthen-3-yl)-L-alanine, L-quisqualic acid, doxin derived peptide, one or more asparagine residues and ibotenic acid, DL-C.-(2-thienyl)glycine, L-phenylglycine, one or more serine residues are Substituted. In some embodi 2-amino hex-5-ynoic acid, crotylglycine, L-norleucine, ments, conservative Substitutions are made. In other embodi L-norvaline, L-ethionine, L-B-azidohomoalanine, 6,6,6,-trif ments, non-conservative Substitutions are made. luoronorleucine, 2-amino-4,4,4-trifluorobutyric acid, L-C- 0221 Deamidation of amino acid residues is a particular propargyl glycine, L-allyl glycine, B-Cyclopropyl alanine, problem in biotransformation. This base-catalyzed reaction photo-Met, 3-fluoro-valine, t-butyl-glycine, O-methyl-L- frequently occurs in sequences containing asparagine gly threonine, (2S,3S)-2-amino-3-methoxybutanoic acid, 4-thia cine or glutamine glycine and follows a mechanism analo isoleucine, L-cyclohexylglycine, 5'5",5'-trifluoro leucine, gous to the aspartic acid glycine sequence above. The de US 2009/0286719 A1 Nov. 19, 2009

amidation of the asparagine glycine sequence forms a is replaced with a leucine or valine residue. In other specific cyclic imide intermediate that is Subsequently hydrolyzed to embodiments, one or more of the methionines at residues 56 form the aspartate or iso-aspartate analog of asparagine. In and 64 of Paeruginosa azurin (SEQID NO: 1), or equivalent addition, the cyclic imide intermediate can lead to racemiza methionine residues in other cupredoxin derived peptides, are tion into D-aspartic acid or D-iso-aspartic acid analogs of replaced with leucine or valine. In some embodiments, con asparagine, all of which can potentially lead to inactive forms servative Substitutions are made. In other embodiments, non of the peptide. conservative Substitutions are made. In specific embodi 0222. It is contemplated that deamidation in the cupre ments, the cupredoxin peptides of the invention comprise one doxin peptides may be prevented by replacing a glycine, of the following sequences, wherein the underlined amino asparagine and/or glutamine of the asparagine glycine or acid is Substituted into the wild type Pseudomonas aerugi glutamine glycine sequences of the cupredoxin with nosa aZurin sequence: anotheramino 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 LSTAADLQGVVTDGLASGLDKDYLKPDD (SEQ ID NO: 31) the peptide. In some embodiments, the one or more glycine o residues of the cupredoxin derived peptide are replaced by another amino acid residue. In specific embodiments, one or LSTAADVOGVVTDGVASGLDKDYLKPDD. (SEQ ID NO: 32) more glycine residues of the cupredoxin derived peptide are 0225. Another biotransformative process that may affect replaced with a threonine or an alanine residue. In some the pharmacologic activity, plasma half-life and/or immuno embodiments, the one or more asparagine or glutamine resi genicity of the cupredoxin derived peptides is diketopipera dues of the cupredoxin derived peptide are replaced by Zine and pyroglutamic acid formation. Diketopiperazine for another amino acid residue. In specific embodiments, one or mation usually occurs when glycine is in the third position more asparagine or glutamine residues of the cupredoxin from the N-terminus, and more especially if proline or gly derived peptide are replaced with analanine residue. In other cine is in position 1 or 2. The reaction involves nucleophilic specific embodiments, the glycine at residues 58 and/or 63 of attack of the N-terminal nitrogen on the amide carbonyl P. aeruginosa azurin (SEQID NO: 1), or equivalent glycines between the second and third amino acid, which leads to the of other cupredoxins, are replaced with an alanine or a threo cleavage of the first two amino acids in the form of a dike nine. In other specific embodiments, the methionine at resi topiperazine. On the other hand, pyroglutamic acid formation due 59 of PaeruginosaaZurin (SEQID NO: 1), or an equiva may be almost inevitable if glutamine is in the N-terminus. lent methionine residue of another cupredoxin derived This is an analogous reaction where the N-terminal nitrogen peptide, is replaced by an alanine residue. In other specific attacks the side chain carbonyl carbon of glutamine to form a embodiments, the glycine at residue 63 of P aeruginosa deaminated pyroglutamayl peptide analog. This conversion aZurin (SEQ ID NO: 1), or an equivalent glycine residue of also occurs in peptide containing asparagine in the N-termi another cupredoxin derived peptide, is replaced by an threo nus, but to a much lesser extent. nine residue. In some embodiments, conservative Substitu 0226. It is contemplated that diketopiperazine and pyro tions are made. In other embodiments, non-conservative Sub glutamic acid formation may be decreased in cupredoxin stitutions are made. In specific embodiments, the modified derived peptides by replacing glycine in position 1, 2, or 3 cupredoxin derived peptide of the invention comprises the from the N-terminus, proline in position 3 from the N-termi following sequence, wherein the underlined amino acids are nus, or asparagine at the N-terminus of the peptide with Substituted into the wild type Pseudomonas aeruginosa another amino acid residue. In some embodiments, a glycine aZurin sequence: 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 LSTAADMQAVVTDTMASGLDKDYLKPDD. (SEQ ID NO: 30) threonine or alanine residue. In another embodiment, a pro 0223 Reversible and irreversible oxidation of amino acids line at position 3 from the N-terminus of the cupredoxin are other biotransformative processes that may also pose a derived peptide is replaced with another amino acid residue. problem that may reduce the pharmacologic activity, and/or In specific embodiments, the proline is replaced by analanine plasma half-life of cupredoxin derived peptides. The cysteine residue. In another embodiment, an asparagine at the N-ter and methionine residues are the predominant residues that minus is replaced with anotheramino acid residue. In specific undergo reversible oxidation. Oxidation of cysteine is accel embodiments, the asparagine residue is replaced by a erated at higher pH, where the thiol is more easily deproto glutamine residue. In some embodiments, conservative Sub nated and readily forms intra-chain or inter-chain disulfide stitutions are made. In other embodiments, non-conservative bonds. These disulfide bonds can be readily reversed in vitro Substitutions are made. by treatment with dithiothreitol (DTT) or tris(2-carboxyeth 0227. Another biotransformative process that may affect ylphosphine) hydrochloride (TCEP). Methionine oxidizes by the pharmacologic activity, plasma half-life and/or immuno both chemical and photochemical pathways to form methion genicity of the cupredoxin derived peptide is racemization. ine sufoxide and further into methionine sulfone, both of This term is loosely used to refer to the overall loss of chiral which are almost impossible to reverse. integrity of the amino acid or peptide. Racemization involves 0224. It is contemplated that oxidation in the cupredoxin the base-catalyzed conversion of one enantiomer (usually the derived peptides may be prevented by replacing methionine L-form) of an amino acid into a 1:1 mixture of L- and D-enan and/or cysteine residues with other residues. In some embodi tiomers. One way to improve stability of the peptide in gen ments, one or more methionine and/or cysteine residues of the eral is by making a retro-inverso (D-isomer) peptide. The cupredoxin derived peptide are replaced by another amino double inversion of peptide structure often leaves the surface acid residue. In specific embodiments, the methionine residue topology of the side-chain intact and has been used exten US 2009/0286719 A1 Nov. 19, 2009 sively to stabilize biologically active peptides. Snyder et al., growth of gram-positive bacteria. In lantibiotics, dehydroala PLoS Biol. 2:0186-0193 (2004). A D-amino acid substituted nine and dehydrobutyrine are created by enzyme mediated Tat is internalized into cells as well as the L-amino acid dehydration of serine and threonine residues. Cysteines are peptide. Futaki et al., J. Biol. Chem. 276:5836-5840 (2001): then enzymatically coupled to the dehydrated serine and Huq et al., Biochemistry 38:5172-5177 (1999). In some threonine residues to form thioether cyclizations. Naturally embodiments, one or more amino acid residues of the cupre occurring lantibiotics show Such couplings via thioether doxin derived peptide are replaced by the D-isomer of that bonds between residues that are up to 19 residues apart. amino acid residue. In other embodiments, all of the amino Thioether ring formation depends upon the leader peptide. acid residues of the cupredoxin derived peptide are replaced The location of the cyclization depends upon the cyclase with D-isomers of those residues. In one embodiment, the mediated regio- and stereospecific ring closure and the posi modified cupredoxin derived peptide is a retro-inverso tions of the dehydratable serine and threonine residues. (D-isomer) version of the cupredoxin derived peptide. In a 0232. The best characterized of the lantibiotics is nisin—a specific embodiment, the modified cupredoxin derived pep pentacyclic peptide antiobiotic produced by Lactococcus lac tide is tis. Nisin is composed of four methyllanthionines, one lanthionine, two dehydroalanines, one dehydrobutyrine, and twenty-six unmodified amino acids. Nisin's five thioether DDPKLYDKDLGSAMGDTVVGOMDAATSL. (SEO ID NO : 45) cross-links are formed by the addition of cysteine residues to 0228. In other specific embodiments, the modified cupre dehydroalanine and dehydrobutyrine residues that originate doxin derived peptides are retro-inverso versions of cupre from serine and threonine. Nisin contains thioether-contain doxin derived peptides, including SEQID NOS: 1777-3504. ing amino acids that are posttranslationally introduced by a 0229. Other methods to protect a cupredoxin derived pep membrane-associated enzyme complex. This enzyme com tide from biotransformative degradation are N-acetylation plex includes: transporter NisT serine and threonine dehy and C-amidation. These derivatives may protect the peptide dratase Nisb, and cyclase NisC. Nisb dehydrates serine and from degradation and may make the cupredoxin derived pep threonine residues, converting them into dehydroalanine and tide more closely mimic the charge state of the alpha amino dehydrobutyrine, respectively. This is followed by NisC cata and carboxyl groups in the native protein. Peptides with the lyzed enantioselective coupling of cysteines to the formed N-acetylation and/or C-amidation can be provided by com dehydroresidues. NisT facilitates the export of the modified mercial Suppliers. In one embodiment of the invention, the premisin. Another enzyme, Nisp cleaves the nisin leader pep N-terminus of the cupredoxin derived peptide may be acety tide from premisin. lated. In another embodiment of the invention, the C-terminus 0233. The cyclase NisC has been well characterized. Liet of the cupredoxin derived peptides may be amidated. In one al, “Structure and Mechanism of the Lantibiotic Cyclase specific embodiment, the modified cupredoxin derived pep Involved in Nisin Biosynthesis' 311 Science, 1464-67 (2006) tide is (hereby incorporated by reference in its entirety). 0234. An analysis of cyclization in lantibiotics has led to the identification of amino acid sequences and characteristics Acetylation-LSTAADMQGVVTDGMASGLDK (SEQ ID NO: 33) in peptides that favor cyclization. It has been shown that the DYLKPDD-amidation. Nisb enzyme dehydrates more often where certain amino acids flank the serine and threonine residues. It has been 0230 Cyclization is an additional manner of biotransfor shown that cyclization occurs more often in lantibiotic mation that may be beneficial to therapeutic peptides includ propeptides where hydrophobic, nonaromatic residues are in ing the cupredoxins as described herein. Cyclization may proximity to the serine and threonine residues. The flanking stabilize therapeutic peptides, allowing them to be stored residues of the modified cysteines are typically less hydro longer, be administered at lower doses and be administered phobic than the flanking residues of the modified threonines less frequently. Cyclization has been shown to protect pep and serines. Exceptions have been found, including hexapep tides against peptidase and protease degradation. Cyclization tides VSPPAR,YTPPAL and FSFFAF. The hexapeptides sug can be done chemically or enzymatically. Enzymatic cycliza gest that the presence of a proline at position 3 or 4 or having tion is generally less problematic than chemical cyclization, phenylalanine flanking both sides may prohibit dehydration. as chemical cyclization can lack in regio- and Stereospecific The rings are typically formed by coupling a dehydrated ity, can lead to multimerization in lieu of cyclization and can residue to a C-terminally located cysteine. However, rings require complicated multistep processes. Indeed, it has been may be formed by coupling a dehydrate residue to a N-ter shown that thioether cyclization is more protective and stable minally located cysteine. than a disulfide bond against proteolytic enzymes. 0235. It has also been shown that the nisin dehydrating and 0231. Enzymatic cyclization has been shown in lantibiot transport enzymes are not specific to nisin and may, in fact, be ics—(methyl)lanthionine-containing bacterial peptides. E.g., used to modify non-nisin peptides (and non-lantibiotic pep R. Rink, et al., “Lantibiotic Structures as Guidelines for the tides). Nisb has been shown to dehydrate serine and threo Design of Peptides That Can Be Modified by Lantibioitic nine residues in peptides such as human peptide hormones Enzymes' 44 Biochem. 8873-82 (2005); R. Rink, et al., when such peptides are N-terminally fused to the lantibiotic “Production of Dehydroamino Acid-Containing Peptides by leader peptide. On non-lantibiotic peptides, similar ring for Lactococcus lactis '73:6 Applied and Environmental Micro mation characteristics apply; namely, the extent of dehydra biology, 1792-96 (2007): R. Rink, et al., “NisC, the Cylcase tion can be controlled by the amino acid context of the flank of the Lantibiotic Nisin, Can Catalyze Cyclization of ing region of the dehydratable serine and threonine residues. Designed Nonlantibiotic Peptides' 46 Biochem., 13179-89 The presence of hydrophobic flanking residues (e.g., alanine (2007) (each of which is hereby incorporated by reference in and valine) around the serines and threonines allowed full its entirety). Lantibiotics are produced by and inhibit the dehydration and therefore enhanced thioether ring formation. US 2009/0286719 A1 Nov. 19, 2009

The presence of an N-terminal aspartate and C-terminally the Cys there is a slight preference for charged hydrophilic flanked arginine prevented dehydration. It also shown that the residues, especially glutamate/aspartate. Depending on the presence of proline residues and phenylalanine residues is size of the thioether ring, the bulkiness of the amino acids that disfavorable for dehydration. Generally, the presence of participate in the ring matters. hydrophilic flanking residues prevented dehydration of the serine and threonine residues. Hydrophobic flanking favors 0242. In one embodiment, the truncated aZurin sequence dehydration; hydrophilic flanking disfavors dehydration. is SEQ ID NO. 3507, LSTAADMQGVVTDGMASGLD Studies have shown that where dehydration does occur, the KDYLTPGC. A thioether bridge is formed between positions average hydrophobicity of the flanking residues of serines 25 and 28, and will be fully protected against carboxyeti and threonine is positive 0.40 on the N-terminal side and dases. Positions 2,3 and 25 will be dehydrated, but neither the 0.13 on the C-terminal side. Also, the average hydrophobicity import sequence, nor the sequence thought to be relevant for of the residues flanking serines and threonines that are not interaction with p53, is altered by thioether ring introduction. dehydrated is negative -0.36 on the N-terminal side and— As such, peptide activity should not be altered. The threonine 1.03 on the C-terminal side. Dehydration is not restricted by is between two hydrophobic amino acids and hence is the presence of a series of flanking threonine residues and is expected to be fully dehydrated by dehydratase, Nisb. not restricted by the distance between the nisin leader peptide according to specific guidelines. See Rink et al., Biochemis and the residue to be dehydrated. try 2005. The same guidelines also predict cyclization involv 0236 NisC has been shown to catalyze the regiospecific ing positions 25 and 28 by cyclase NisC, especially because formation of thioether rings in peptides unrelated to naturally of the aspartate located before the cysteine. occurring lantibiotics. Generally, Such peptides must be fused 0243 In another embodiment, the truncated azurin to the nisin leader peptide. In some cases, thioether rings may sequence is LSTAADCQGVVTDGMASGLDKDYLKPDD form spontaneously, for example where a dehydroalanine is (SEQ ID NO. 3508) and the thioether bridge is formed spaced by two amino acids from a cysteine. Unlike sponta between positions 3 and 7. The ring between position 3 and 7 neous cyclization, NisC catalyzed cyclization is stereospe mimics ring A ofnisin and makes use of the existing threonine cific for dehydrated pre-nisin. Consequently, the methyl at position 2. The aspartate at position 6 will favorcyclization. lanthionines and lanthionine in nisin are in the DL configuration. It is thought that cyclization in nonlantibiotic 0244. In another embodiment, the truncated azurin peptides will also be stereospecific sequence is LSTAACMQGVVTDGMASGLDKDYLK 0237. These principles can be applied to the compounds PDD, (SEQ ID NO. 3509) and the threonine in position 2 is described herein, including cupredoxins and variants and utilized to form a thioether bridge. truncations thereof. 0245. In another embodiment, two or more of the thioether 0238. Thioether Bridges rings in the truncated aZurins described in the paragraphs 0239. In nature, lantibiotic-enzyme-induced thioether above are combined into one peptide. bridges occur with up to 19 amino acids under the bridge. 0246. In another embodiment, many truncated azurin Thioether bridges with 2 to 4 amino acids under the bridge are sequences can be created and screened for threonine rings by abundant. analyzing the peptides with a ring of one lanthionine and two 0240. In some embodiments, the cupredoxin may be to three additional amino acids under the sulfur bridge. As modified by introducing thioether bridges into the structure. examples, this might involve one or combinations of the The azurin truncation p28 (SEQ ID NO: 13), for example, sequences below, where the bolded mutations facilitate com may be modified using this method. Extended molecular plete modification: dynamics simulations (70 ns) using Software package GRO MACS (www.gromacs.org) Suggest that, at 37°C., the region LSTACDMOGVVTDGMASGLDKDYLKPDD (SEQ ID NO. 3510) of the p28 (SEQID NO: 13) alpha helix from position 6 to 16 is unstable, and that the peptide tends to adopt a beta sheet LSTAATMOCVVTDGMASGLDKDYLKPDD (SEQ ID NO. 3511) conformation. (See FIG. 4). This, together with the fact that the part of the molecule presumed to be responsible for inter LSTAATMOGCVTDGMASGLDKDYLKPDD (SEQ ID NO. 3512) action with p53 remains solvent exposed, suggests that intro LSTAANTOGCVTDGMASGLDKDYLKPDD (SEQ ID NO. 3513) duction of a thioether bridge in this region of the p28 (SEQID NO: 13) peptide may not affect its functionality. LSTAANTOGVCTDGMASGLDKDYLKPDD (SEQ ID NO. 3514) 0241. The amino acid sequence of p28 is SEQID NO: 13 STAADMAWCTDGMASGLDKDYLKPDD (SEO ID NO. 3515) (LSTAADMQGVVTDGMASGLDKDYLKPDD). The amino acid sequence known as p18 is SEQ ID NO: 14 STAADMAWWCDGMASGLDKDYLKPDD (SEQ ID NO. 3516) (LSTAADMQGVVTDGMASG). Within p28 (SEQID NO: 13), the sequence SGLDKD may interact with p53. Thioether LSTAADMOTWVCDGMASGLDKDYLKPDD (SEO ID NO. 3517) bridges can be formed between Ser/Thr on the N-side to Cys LSTAADMOTWWTCGMASGLDKDYLKPDD (SEQ ID NO. 3518) on the C-side. The serine/threonine may be dehydrated and Subsequently coupled to the cysteine. Threonines are pre LSTAADMOATWTCGMASGLDKDYLKPDD (SEO ID NO. 3519) ferred since they are more easily dehydrated than serines. LSTAADMOATVTDCMASGLDKDYLKPDD (SEQ ID NO. 352O) Generally, hydrophobic flanking residues (at least one) to the threonine are preferred since they enhance the extent of dehy LSTAADMOGVTADCMASGLDKDYLKPDD (SEQ ID NO. 3521) dration. Negatively charged amino acids, glutamate and aspartate, that are flanking residues have a strong negative LSTAADMQGWTADGCASGLDKDYLKPDD (SEQ ID NO. 3522) effect on dehydration. Generally, hydrophilic flanking resi LSTAADMOGVVTNGCASGLDKDYLKPDD (SEQ ID NO. 3523) dues, especially glycine, do not favor dehydration. Preceding US 2009/0286719 A1 Nov. 19, 2009

0247. A practical approach would be to genetically make a doxin derived peptide may be substituted. In still more spe large number of Such sequences and select a group for puri cific embodiments, the serine and/or glutamine residues in fication on the basis of extent of modification and level of residues 53-56, 58-64 and 68-70 of P aeruginosa azurin production. (SEQ ID NO: 1), or equivalent residues of other cupredoxin 0248. In another embodiment, a thioether bridge is formed derived peptides, may be replaced. In another specific between a threonine at position 12 in p28 (SEQ ID NO: 13) embodiment, the glutamine residue at amino acid residue 57 and the c-terminus of the peptide. The distance between the of P aeruginosa azurin (SEQ ID NO: 1), or an equivalent Co. of position 13 and the aspartate at position 28 might be residue of another cupredoxin derived peptide, may be 17.52 angstroms, larger than 1.5 nanometers, implying sig replaced, more specifically replaced with tryptophan. In nificant alteration of the structure of the peptide. (See FIG.5.) another specific embodiment, the threonine residue at amino 0249. In another embodiment, the peptide sequence is acid residue 52 of Paeruginosa azurin (SEQID NO: 1), oran LSTAADMQGVVTATMGSGLCKDYLKPDD, (SEQ ID equivalent residue of another cupredoxin derived peptide, NO. 3524) with a thioether bridge from position 14 to posi may be replaced, more specifically replaced with tryptophan. tion 2 at a distance of 4.38 angstroms. The mutation of aspar In another specific embodiment, the threonine residue at tate at position 13 to alanine favors dehydration of threonine amino acid residue 61 of P. aeruginosaaZurin (SEQID NO: at position 14. Mutation of alanine at position 16 to glycine 1), or an equivalent residue of another cupredoxin derived completely prevents dehydration of serine at position 17 and peptide, may be replaced, more specifically replaced with enhances cyclization. tryptophan. In another specific embodiment, the glycine resi 0250 In another embodiment, the peptide sequence is due atamino acid residue 63 of Paeruginosa azurin (SEQID LSTAADMQGVVTDLTASGLCKDYLKPDD, (SEQ ID NO: 1), or an equivalent residue of another cupredoxin NO. 3525) with the thioether bridge from position 15 to derived peptide, may be replaced, more specifically replaced position 20 at a distance of 5.83 angstroms. In this situation, with tryptophan. In another specific embodiment, one or mutation of glycine at position 14 to leucine favors dehydra more threonine, glutamine or glycine residues at amino acid tion of threonine at position 15. residues 52, 57, 61 or 63 of PaeruginosaaZurin (SEQID NO: 0251 Tertiary Structure Stabilization 1), or an equivalent residue of another cupredoxin derived 0252. The stability of the tertiary structure of the cupre peptide, may be replaced, more specifically replaced with doxin derived peptide will affect most aspects of the pharma tryptophan. In specific embodiments, the cupredoxin peptide cokinetics, including the pharmacologic activity, plasma comprises one of the following sequences wherein the under half-life, and/or immunogenicity among others. See lined amino acid is substituted into the wild type Kanovsky et al., Cancer Chemother. Pharmacol. 52:202-208 (2003); Kanovsky et al., PNAS 23:12438-12443 (2001). Pep tide helices often fall apart into random coils, becoming more LSWAADMQGVVTDGMASGLDKDYLKPDD; (SEQ ID NO. 34) Susceptible to protease attack and may not penetrate cell LSTAADMWGVVTDGMASGLDKDYLKPDD; (SEQ ID NO: 35) membrane well. Schafineister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000). Therefore, one way to stabilize the overall LSTAADMQGVWWDGMASGLDKDYLKPDD; (SEQ ID NO: 36) structure of the peptide is to stabilize the C-helix structure of LSTAADMOGVVTDWMASGLDKDYLKPDD (SEO ID NO : 37) the peptide. The intra-molecular hydrogen bonding associ ated with helix formation reduces the exposure of the polar LSWAADMWGVVTDGMASGLDKDYLKPDD; (SEQ ID NO: 38) amide backbone, thereby reducing the barrier to membrane penetration in a transport peptide, and thus increasing related LSWAADMQGVWWDGMASGLDKDYLKPDD; (SEQ ID NO: 39) pharmacologic activities and increasing the resistance of the LSWAADMQGVVTDWMASGLDKDYLKPDD; (SEQ ID NO: 40) peptide to protease cleavage. Id. Pseudomonas aeruginosa aZurin (SEQID NO: 1) has C-helices at residues 53-56,58-64 LSTAADMWGVWWDGMASGLDKDYLKPDD; (SEQ ID NO: 41) and 68-70. LSTAADMWGVVTDWMASGLDKDYLKPDD; (SEQ ID NO: 42) 0253) One method to stabilize an O.-helix is to replace in the C-helix helix breaking amino acid residues such as gly LSTAADMQGVWWDWMASGLDKDYLKPDD; (SEQ ID NO: 43) cine, proline, serine and aspartic acid, or helix neutral amino o acid residues such as alanine, threonine, Valine, glutamine, LSWAADMWGVWWDWMASGLDKDYLKPDD. (SEQ ID NO: 44) asparagine, cysteine, histidine, lysine or arginine, with helix forming residues, such as leucine, isoleucine, phenylalanine, In other embodiments, equivalent amino acids in other cupre glutamic acid, tyrosine, tryptophan and methionine or helix doxin derived peptides are substituted with tryptophan. favoring amino acid residue Substitutions, for example 0254 Another method to stabilize an O-helix tertiary C.-amino-isobutyric acid (Aib). See Miranda et al., J. Med. structure involves using unnatural amino acid residues Chem. 51, 2758-2765 (2008). It is contemplated that the capable of t-stacking. For example, in Andrews and Tabor C.-helix of cupredoxin derived peptides may be stabilized by (Tetrahedron 55:11711-11743 (1999)), pairs of e-(3,5-dini replacing one or more glycine, proline, serine and/or aspartic trobenzoyl)-Lys residues were substituted into the C-helix acid residues with other amino acids. In specific embodi region of a peptide at different spacings. The overall results ments, the glycine, proline, serine, aspartic acid, alanine, showed that the i,(i+4) spacing was the most effective stabi threonine, Valine, glutamine, asparagine, cysteine, histidine, lizing arrangement. Increasing the percentage of water, up to lysine and/or arginine residues are replaced by leucine, iso 90%, increased the helical content of the peptide. Pairs of leucine, phenylalanine, glutamic acid, tyrosine, tryptophan, e-acyl-Lys residues in the same i,(i+4) spacing had no stabi Aib and/or methionine residues. See Lee et al., Cancer Cell lizing effect, indicating that the majority of the stabilization Intl. 11:21 (2005). In other specific embodiments, one or arises from U-7L interactions. In one embodiment, the cupre more serine or glutamine residues in the C-helices of a cupre doxin derived peptide may be modified so that the lysine US 2009/0286719 A1 Nov. 19, 2009 20 residues are substituted by e-(3,5-dinitrobenzoyl)-Lys resi carbon cross-link method is proven to increase the stabiliza dues. In a specific embodiment, the lysine residues may be tion of helical structure, protease resistant and cell-perme Substituted by e-(3,5-dinitrobenzoyl)-Lys inai,(i+4) spacing. ability. Walensky et al., Science, 305, 1466-1470 (2004). 0255 Another method to stabilize an O-helix tertiary C.C.-disubstituted non-natural amino acids containing olefin structure uses the electrostatic interactions between side bearing tethers are incorporated into peptides. Ruthenium chains in the C-helix. When His-Cys or His-His residue pairs catalyzed olefin metathesis generates an all-hydrocarbon were substituted in into peptides inani,(i+4) arrangement, the “staple' to cross-link the helix. Schafineister et al., J. Am. peptides changed from about 50% helical to about 90% heli Chem. Soc., 122, 5891-5892 (2000); Walensky et al., id. cal on the addition of Cu, Zn or Cd ions. When ruthenium Non-natural amino acids containing olefin-bearing tethers (Ru) salts were added to the His-His peptides, an exchange may be synthesized according to methodology provided in inert complex was formed, a macrocyclic cis-Ru—(NH) Schafineister et al. (id.) and Williams and Im (J. Am. Chem. Lal" complex where L2 are the side chains of two histidines, Soc., 113:9276-9286 (1991)). In some embodiments, the which improved the helix stability. Ghadiri and Fernholz, J. cupredoxin derived peptides are stabilized by all-hydrocar Am. Chem. Soc. 112, 9633-9635 (1990). In some embodi bon Staples. In specific embodiments, one or more pairs of ments, the cupredoxin derived peptides may comprise mac C.C.-disubstituted non-natural amino acids containing olefin rocyclic cis-Ru—(NH), Lal" complexes where L is the bearing tethers corresponding to the native amino acids are side chains of two histidines. In some embodiments, one or substituted into the C-helices of the cupredoxin derived pep more histidine-cysteine or histidine-histidine residue pairs tide. In other embodiments, one or more pairs of O.C.-disub may be substituted an i,(i+4) arrangement into the C-helices stituted non-natural amino acids containing olefin-bearing of the cupredoxin derived peptide. In other embodiments, one tethers corresponded to the native amino acids are substituted or more histidine-cysteine or histidine-histidine residue pairs into residues 53-56, 58-64 and 68-70 of Paeruginosa azurin may be substituted an i,(i+4) arrangement in residues 53-56, (SEQ ID NO: 1), or equivalent residues of other cupredoxin 58-64 and 68-70 of PaeruginosaaZurin (SEQID NO: 1), or derived peptides. equivalent residues of other cupredoxin derived peptides. In 0259. In some embodiments, the modified cupredoxin Some embodiments, the cupredoxin derived peptide may fur derived peptide may comprise ther comprise Cu, Zn, Cd and/or Ru ions. XSXAADXXXVVXDXXASGLDKDYLKPDX 0256 Another method to stabilize an O-helix tertiary (SEQIDNO:48), where X is Loracetylated-L, X is Tor W. structure involves disulfide bond formation between side X is M. L or V. X is Q or W, Xs is G or A, X is T or W, X, chains of the C-helix. It is also possible to stabilize helical is G, T or W, X is M. L or V, and X is D or amidated-D. In structures by means of formal covalent bonds between resi other embodiments, the modified cupredoxin derived peptide dues separated in the peptide sequence. The commonly may consist of employed natural method is to use disulfide bonds. Pierret et XSXAADXXXVVXDXXASGLDKDYLKPDX al., Intl. J. Pept. Prot. Res., 46:471-479 (1995). In some (SEQIDNO:48), where X is Loracetylated-L, X is Tor W. embodiments, one or more cysteine residue pairs are Substi X is M. L or V. X is Q or W, X is G or A, X is T or We X, tuted into the C-helices of the cupredoxin derived peptide. In is G, T or W, X is M. L or V, and Xg is D or amidated-D. In other embodiments, one or more cysteine residue pairs are other embodiments, the modified cupredoxin derived peptide substituted at residues 53-56, 58-64 and 68-70 of P aerugi may comprise nosa azurin (SEQID NO: 1), or equivalent residues of other XDPKLYDKDLGSAXXDXVVXXXDAAX.SX cupredoxin derived peptides. (SEQID NO:49), where X is D or acetylated-D, X is M. L. 0257 Another method to stabilize an O.-helical tertiary or V. X is G, Tor W, X is T or W, Xs is G or A, X is Q or W. structure involves the use of side chain lactam bridges. A X, is M. L or V. X is T or W. and X is L or amidated.-L. In lactam is a cyclic amide which can form from the cyclization other embodiments, the modified cupredoxin derived peptide of amino acids. Side chain to side chain bridges have been may consist of Successfully used as constraints in a variety of peptides and XDPKLYDKDLGSAXXDXVVXXXDAAXSX peptide analogues, such as amphipathic or model C.-helical (SEQ ID NO:49), where X is D or acetylated-D, X is M, L peptides, oxytocin antagonists, melanoptropin analogues, or V. X is G, Tor W, X is Tor W, X is G or A, X is Q or W. glucagon, and SDF-1 peptide analogues. For example, the X, is M. L or V. X is T or W. and X is L or amidated-L. Glucagon-like Peptide-1 (GLP-1) gradually assumes a heli Specific peptides of interest are listed in Table 3. cal conformation under certain helix-favoring conditions and can be stabilized using lactam bridging. Miranda et al., J. TABLE 3 Med. Chem, 51, 2758-2765 (2008). These lactam bridges Modified Curedoxin-Derived Peptides may be varied in size, effecting stability and binding affinity. Id. Such modifications improved the stability of the com *LSTAADMOGVVTDGMASGLDKDYLKPDD (SEO ID NO: 5O) pounds in plasma. Id. Depending on the space between the cyclization sites and choice of residues, lactambridges can be LSWAADMOGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 51) used to induce and stabilize turn or helical conformations. In *LSWAADMOGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 52) Some embodiments, one or more cupredoxin or variant ana logues are prepared with lactam bridging between nearby LSTAADLQGWVTDGMASGLDKDYLKPDD (SEO ID NO: 53) amino acids (such as i to i+4 glutamic acid-lysine con *LSTAADLOGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 54) straints). In some embodiments, the cupredoxin derived pep tide may comprise Such modifications to enhance C.-helix LSWAADLQGWVTDGMASGLDKDYLKPDD (SEO ID NO: 55) COntent. *LSWAADLOGVVTDGMASGLDKDYLKPDD (SEO ID NO. 56) 0258 Another method to stabilize an O-helix tertiary structure is the all-carbon cross-link method. The all-hydro