WO2006127477A2 - Compositions et methodes de traitement du paludisme au moyen de cupredoxines et de cytochromes - Google Patents

Compositions et methodes de traitement du paludisme au moyen de cupredoxines et de cytochromes Download PDF

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Publication number
WO2006127477A2
WO2006127477A2 PCT/US2006/019492 US2006019492W WO2006127477A2 WO 2006127477 A2 WO2006127477 A2 WO 2006127477A2 US 2006019492 W US2006019492 W US 2006019492W WO 2006127477 A2 WO2006127477 A2 WO 2006127477A2
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Prior art keywords
cytochrome
cupredoxin
azurin
composition
malaria
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PCT/US2006/019492
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English (en)
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WO2006127477A3 (fr
Inventor
Ananda Chakrabarty
Tapas Das Gupta
Tohru Yamada
Anita Chaudhari
Arsenio Fialho
Chang Soo Hong
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The Board Of Trustees Of The University Of Illinois
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Priority claimed from US11/244,105 external-priority patent/US7691383B2/en
Priority to JP2008512549A priority Critical patent/JP2009504567A/ja
Priority to EP06770687A priority patent/EP1882039A4/fr
Priority to CA002607739A priority patent/CA2607739A1/fr
Priority to AU2006251742A priority patent/AU2006251742A1/en
Priority to BRPI0612456-9A priority patent/BRPI0612456A2/pt
Application filed by The Board Of Trustees Of The University Of Illinois filed Critical The Board Of Trustees Of The University Of Illinois
Priority to MX2007014597A priority patent/MX2007014597A/es
Publication of WO2006127477A2 publication Critical patent/WO2006127477A2/fr
Publication of WO2006127477A3 publication Critical patent/WO2006127477A3/fr
Priority to IL187161A priority patent/IL187161A0/en
Priority to NO20076390A priority patent/NO20076390L/no
Priority to AU2010201360A priority patent/AU2010201360A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to cupredoxin and cytochrome and their use, separately or in combination, in inhibiting parasitemia of the malaria parasite, and in particular inhibiting parasitemia of Plasmodium falciparum in mammalian red blood cells.
  • the invention also relates to variants and derivatives of cupredoxin and cytochrome that retain the ability to inhibit parasitemia by the malaria parasite.
  • the invention provides methods to inhibit the spread of malaria infection in insect vectors.
  • MSPl Merozoite Surface Protein 1
  • the merozoite MSPl protein undergoes proteolytic cleavage, producing a C-terminal cleavage product MSP 1-42, which subsequently undergoes a second cleavage, producing an 11 kDa peptide MSPl -19, which remains attached to the parasite surface as it enters the erythrocyte.
  • the formation of the cleavage product MSP1-19 is very important for successful invasion by the parasite since inhibition of its proteolytic formation or its neutralization by monoclonal antibodies prevents entry of the parasite to the erythrocytes (Blackman et al, J. Exptl., Med. 180:389-393 (1994)).
  • MSP1-19 peptide is one of the most important malaria vaccine candidates available.
  • MSPl-19-specif ⁇ c antibodies from malaria-resistant human sera react with the antigen and include a major erythrocyte-invasion inhibitory component (Holder & Riley, Parasitol. Today, 12: 173-174 (1996); O'Donnell etal, J. Expt. Med. 193: 1403-1412 (2001)).
  • Serum from donors in malaria-endemic regions usually demonstrates strong antibody reactivity towards Pf MSP1-19. (Nwuba et al, Infect. Immun. 70: 5328-5331 (2002))
  • the monoclonal antibody (mAb) G 17.12 was raised against recombinant Pf MSP 1-19 and recognizes its epitope on the parasite surface, demonstrating that this region of the antigen is accessible on the native MSPl polypeptide complex (Pizarro et al, J. MoI. Biol. 328:1091-1103 (2003)).
  • erythrocyte invasion experiments in vitro showed that infection is not inhibited in the presence of G 17.12, even at 200 ⁇ g/ml concentration and G17.12 does not inhibit in vitro secondary processing of MSPl. Id.
  • the presence of antibodies that block the binding of invasion - inhibitory antibodies, thereby facilitating parasite survival has also been demonstrated (Guevara Patino et al, J. Expt. Med. 186: 1689- 1699(1997)), and may be responsible for the failure of G17.12 mAb to inhibit erythrocyte invasion by M. falciparum.
  • Cerebral malaria a rare but fatal infection restricted to P. falciparum invasion of brain capillaries because of the sequestration of parasitized erythrocytes, is often untreatable because most drugs cannot cross the blood-brain barrier to reach the brain capillaries.
  • Adhesion of P. falciparum - infected erythrocytes to brain capillaries is mediated by the interaction of parasite ligands Pf Emp-1 family of proteins expressed on the surface of infected erythrocytes with ICAM-I and CD36 expressed on the surface of capillary endothelium cells in cerebral vessels. (Smith et al., Proc. Natl. Acad. Sci. USA 97:1766- 1771 (2000); Franke-Fayard et al., Proc. Natl. Acad. Sci. USA 102, 11468-11473 (2005))
  • chloroquine that targets the heme detoxification pathway
  • Chloroquine antagonizes heme polymerization mediated by parasite-induced HRPs (histidine-rich proteins), as heme monomers are highly toxic for malaria parasites.
  • HRPs histidine-rich proteins
  • Artemisinin is effective against chloroquine-resistant P. falciparum in cerebral malaria. Artemisinin forms adducts with globin-bound heme in hemoglobin, which binds HRPs to prevent heme polymerization.
  • the present invention relates to cupredoxin and cytochrome and their use, separately or together, to inhibit the spread of parasitemia in mammalian red blood cells and other tissues infected by the malaria parasite, and in particular the parasitemia of human red blood cells by P. falciparum.
  • One aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin or cytochrome; and that can inhibit intracellular replication of a malarial parasite in malaria-infected human red blood cells.
  • Another aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin; and that can bind a protein selected from the group consisting of PfMSPl-19 and PfMSP 1-42.
  • Another aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin or cytochrome, and that can inhibit parasitemia by malaria in malaria-infected red blood cells.
  • the isolated peptide can inhibit parasitemia by malaria in P. falciparum-m ' fected human red blood cells.
  • the cupredoxin is an azurin, pseudoazurin, plastocyanin, rusticyanin, Laz or auracyanin.
  • the cupredoxin may be rusticyanin, azurin or Laz.
  • the cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluoresceins, Pseudomonas chlororaphis, Xylellafastidiosa or Vibrio par ahaemolyticus.
  • the cupredoxin may be from Thiobacillus ferrooxidans, Pseudomonas aeruginosa, Neisseria gonorrhea or Neisseria meningitidis.
  • the cytochrome is cytochrome c or cytochrome f.
  • the cytochrome c may be from human or Pseudomonas aeruginosa.
  • the cytochrome f may be from a cyanobacteria.
  • the isolated peptide is a truncation of a peptide selected from the group consisting of SEQ ID NOS: 1-20 and 22.
  • SEQ ID NOS: 1-20 or 22 has at least 90% amino acid sequence identity to the sequence of the isolated peptide.
  • the isolated peptide is a truncation of cupredoxin or cytochrome. In some embodiments, the isolated peptide is more than about 10 residues and not more than about 100 residues.
  • the isolated peptide may comprise azurin residues 36- 89. Alternatively, the isolated peptide may consist of azurin residues 36-89. Alternatively, the isolated peptide may comprise equivalent residues of a cupredoxin as azurin 36-89.
  • the isolated peptide is fused to a H.8 region of Laz.
  • the isolated peptide is a structural equivalent of monoclonal antibody G17.12.
  • compositions comprising at least one cupredoxin, cytochrome, or isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin or cytochrome that can inhibit parasitemia by malaria in malaria-infected red blood cells, in a pharmaceutical composition.
  • the pharmaceutical composition may be formulated for intravenous administration.
  • the composition may comprise another anti-malarial drug or an anti-HIV drug.
  • the cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa or Vibrio parahemolyticus.
  • the cupredoxin may be from Thiobacillus ferrooxidans, Pseudomonas aeruginosa, Neisseria gonorrhea or Neisseria meningitidis.
  • the cytochrome is cytochrome c or cytochrome f.
  • the cytochrome c may be from human or Pseudomonas aeruginosa.
  • the cytochrome f may be from a cyanobacteria.
  • the cupredoxin or cytochrome c is SEQ ID NOS: 1-20 or 22.
  • Another aspect of the invention is a method to treat a patient suffering from an infection by a malaria parasite by administering to the patient an effective amount of the composition of the invention.
  • the peptide inhibits parasitemia by malaria in the patient's malaria-infected human red blood cells.
  • the malaria parasite is Plasmodium vivax or Plasmodium falciparum.
  • the patient is additionally suffering from HIV infection.
  • the composition is administered with a second composition that may contain an anti-malarial drug and/or an anti-HIV drug.
  • the composition of the invention is administered within 0 minutes to 12 hours of the administration of second composition.
  • the composition of the invention is administered to the patient orally, by inhalation, intravenously, intramuscularly or subcutaneOusly; and, specifically, the composition may be administered to the patient intravenously.
  • Another aspect of the invention is a method to treat a patient suspected of having contact with a malaria parasite, comprising administering to the patient an effective amount of the composition of the invention.
  • Another aspect of the invention is a method to prevent malaria in mammals, comprising administering to an insect vector in a population of insect vectors harboring a malaria parasite an amount of the composition of the invention.
  • the composition is administered to the insect vector orally.
  • Figure 1 depicts surface plasmon resonance binding titrations depicting the interactions of Azurin, H.8-azurin (H.8-Az), Laz, and GST-azurin (GST-Azu) constructs with MSPl -19 and MSP 1-42.
  • A Binding curves demonstrating the interactions of azurin and its analogues with MSP1-19 immobilized on carboxymethyldextran coated gold sensor chips (MSPl -19-CM5). Concentration dependent binding of the azurin proteins to MSPl -19 was determined via injection of various concentrations (0.05-300 nM) over the sensor surface and the extent of binding was evaluated as a function of the equilibrium resonance response value measured in resonance units (RU).
  • the MSP1-19 binding Kd values are: 32.2 ⁇ 2.4 nM (azurin), 26.2 ⁇ 2.4 nM (Laz), 11.8 ⁇ 0.3 nM (H.8-Az), and those for MSP1-42 binding are: 54.3 ⁇ 7.6 nM (azurin), 45.6 ⁇ 2.4 nM (Laz) and 14.3 ⁇ 1.7 nM (H.8-Az).
  • C Binding titrations for the interactions of GST-Azu fusion proteins over the MSPl-19-CM5 sensors surface demonstrate the recognition of GST-Azu 36-128 and GST-Azu 36-89 with MSP1-19. No binding was seen with GST or GST-Azu 88- 113.
  • Figure 2 depicts inhibition of P. falciparum parasitemia (parasite growth within the RBC) by different concentrations, as shown, of Azurin, H.8-azurin (H.8-Az) and Laz.
  • Azurin H.8-azurin
  • Laz Laz.
  • normal red blood cells were infected with schizonts in absence or in presence of the proteins at different concentrations, incubated overnight and the number of intracellular parasites was scored by thin blood smear and Giemsa staining.
  • Figure 3 depicts surface plasmon resonance binding curves for the binding of ICAMs (ICAM-I, ICAM-2, ICAM-3 and NCAM, inset) with immobilized azurin. Due to large nonspecific binding to the bare Au-CM5 chip, CM5 was added as an eluent to the running buffer (1 mg/ml CM5 to HBS-EP buffer). The selective recognition of azurin with ICAM-3, but not with ICAM-I or ICAM-2, is notable and the binding strength was 19.5 ⁇ 5.4 nM. The Kd for NCAM binding with azurin, as shown in the inset, was 20 ⁇ 5.0 nM.
  • SEQ ID NO: 1 is the amino acid sequence of azurin from Pseudomonas aeruginosa.
  • SEQ ID NO: 2 is the amino acid sequence of cytochrome Cs 51 from Pseudomonas aeruginosa.
  • SEQ ID NO: 3 is the amino acid sequence of Laz from Neisseria meningitidis MC58.
  • SEQ ID NO: 4 is the amino acid sequence of plastocyanin from Phormidium laminosum.
  • SEQ ID NO: 5 is the amino acid sequence of rusticyanin from Thiobacillus ferrooxidans ⁇ AcidithiobacUlus ferrooxidans).
  • SEQ ID NO: 6 is the amino acid sequence of pseudoazurin from Achromobacter cycloclastes.
  • SEQ ID NO: 7 is the amino acid sequence of azurin from Alcaligenes faecalis.
  • SEQ ID NO: 8 is the amino acid sequence of azurin from Achromobacter xylosox ⁇ dans ssp.denitrificans I.
  • SEQ ID NO: 9 is the amino acid sequence of azurin from Bordetella bronchiseptica.
  • SEQ ID NO: 10 is the amino acid sequence of azurin from Meihylomonas sp. J.
  • SEQ ID NO: 11 is the amino acid sequence of azurin from Neisseria meningitidis Z2491.
  • SEQ ID NO: 12 is the amino acid sequence of azurin from Pseudomonas fluorescens.
  • SEQ ID NO: 13 is the amino acid sequence of azurin from Pseudomonas chlororaphis.
  • SEQ ID NO: 14 is the amino acid sequence of azurin from Xylella fastidiosa 9a5c.
  • SEQ ID NO: 15 is the amino acid sequence of stellacyanin from Cucumis sativus
  • SEQ ID NO: 16 is the amino acid sequence of auracyanin A from Chloroflexus aurantiacus
  • SEQ ID NO: 17 is the amino acid sequence of auracyanin B from Chloroflexus aurantiacus
  • SEQ ID NO: 18 is the amino acid sequence of cucumber basic protein from Cucumis sativus
  • SEQ ID NO: 19 is the amino acid sequence of cytochrome c from Homo sapiens.
  • SEQ ID NO: 20 is the amino acid sequence of cytochrome f from cyanobacteria Phormidium laminosum.
  • SEQ ID NO: 21 is the amino acid sequence of the H.8 region of Laz from Neisseria gonorrhoeae F62.
  • SEQ ID NO: 22 is the amino acid sequence of Laz from Neisseria gonorrhoeae F62.
  • SEQ ID NO: 23 is the forward primer to PCR amplify the Laz-encoding gene ⁇ laz) of Neisseria gonorrhoeae.
  • SEQ ID NO: 24 is the reverse primer to PCR amplify the Laz-encoding gene ⁇ laz) of Neisseria gonorrhoeae.
  • SEQ ID NO: 25 is the forward primer to PCR amplify a 3.1 kb fragment of pUC18- laz .
  • SEQ ID NO: 26 is the reverse primer to PCR amplify a 3.1 kb fragment of pucl8-/ ⁇ z.
  • SEQ ID NO: 27 is the forward primer to PCR amplify a 0.4 kb fragment of pUC19- paz.
  • SEQ ID NO: 28 is the reverse primer to PCR amplify a 0.4 kb fragment of pUC19- paz.
  • SEQ ID NO: 29 is the forward primer for pGST-azu 36-128.
  • SEQ ID NO: 30 is the reverse primer for pGST-azu 36-128.
  • SEQ ID NO: 31 is the forward primer for pGST-azu 36-89.
  • SEQ ID NO: 32 is the reverse primer for pGST-azu 36-89.
  • SEQ ID NO: 33 is the forward primer for pGST-azu 88-113.
  • SEQ ID NO: 34 is the reverse primer for pGST-azu 88-113.
  • SEQ ID NO: 35 is an oligonucleotide for site directed mutagenesis for the preparation of pGST-azu 88-113.
  • SEQ ID NO: 36 is an oligonucleotide for site directed mutagenesis for the preparation of pGST-azu 88-113.
  • cell includes both the singular or the plural of the term, unless specifically described as a “single cell.”
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid. The terms also apply to naturally occurring amino acid polymers.
  • polypeptide “peptide,” and “protein” are also inclusive of modifications including, but not limited to, glycosylati ⁇ n, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated that polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination and they may be circular (with or without branching), generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods as well.
  • pathological condition includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions, and is a response to various factors (as malnutrition, industrial hazards, or climate), to specific infective agents (as worms, parasitic protozoa, bacteria, or viruses), to inherent defects of the organism (as genetic anomalies), or to combinations of these factors.
  • condition includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.
  • the term “suffering from” includes presently exhibiting the symptoms of a pathological condition, having a pathological condition even without observable symptoms, in recovery from a pathological condition, or recovered from a pathological condition.
  • the term “parasitemia” includes a condition in which parasites are present in the blood and other tissues, and in particular to indicate the presence of parasites with or without clinical symptoms.
  • the term “inhibition of parasitemia” refers to a decrease or a lessening of the rate of increase of the presence of the parasite in the blood of a mammal. Inhibition is any decrease or lessening of the rate of increase that is statistically significant as compared to control treatments.
  • treatment includes preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms associated with a condition being treated.
  • treatment includes medical, therapeutic, and/or prophylactic administration, as appropriate.
  • anti-malarial activity includes any activity that decreases the infectivity, the reproduction, or inhibits the progress of the lifecycle of a malaria parasite.
  • Anti-malarial activity includes inhibition of the growth of malaria infection by all of the means of observed with current anti-malarial drugs.
  • anti-malarial drug refers to drugs with anti-malarial activity that may be used to decrease the infectivity, the reproduction, or inhibit the progress of the lifecycle of a malaria parasite.
  • anti-HIV drug refers to drugs with anti-HIV activity HIV by which HIV infection in mammals is decreased, or prevented from increasing in the human body, by any means including, but are not limited to, inhibition of replication of the HIV genome, inhibition of synthesis and/or assembly of the HIV coat proteins, and inhibition of HIV entry into uninfected cells.
  • substantially pure when used to modify the term a polypeptide or other compound, as used herein, refers to a polypeptide or compound, for example, a polypeptide isolated from the growth medium, in a form substantially free of, or unadulterated by, active inhibitory agents.
  • substantially pure refers to a compound in an amount of at least about 75%, by dry weight, of isolated fraction, or "75% substantially pure.” More specifically, the term “substantially pure” refers to a compound of at least about 85%, by dry weight, active compound, or “85% substantially pure.” Most specifically, the term “substantially pure” refers to a compound of at least about 95%, by dry weight, active compound, or "95% substantially pure.”
  • the substantially pure cupredoxin or cytochrome C55i or a variant or derivative thereof can be used in combination with one or more other substantially pure compounds, or another isolated cupredoxin or cytochrome.
  • isolated refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • An “isolated” region refers to a region that does not include the whole sequence of the polypeptide from which the region was derived.
  • nucleic acid, protein, or respective fragment thereof has been substantially removed from its in vivo environment so that it may be manipulated by the skilled artisan, such as but not limited to nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in substantially pure quantities.
  • variant refers to amino acid sequence variants which may have amino acids replaced, deleted, or inserted as compared to the wild-type polypeptide. Variants may be truncations of the wild-type peptide.
  • a variant peptide may be made by manipulation of genes encoding the polypeptide.
  • a variant may be made by altering the basic composition or characteristics of the polypeptide, but not at least some of its fundamental activities.
  • a "variant" of azurin can be a mutated azurin that retains its ability to inhibit parasitemia in malaria-infected human red blood cells.
  • a variant peptide is synthesized with non-natural amino acids, such as ⁇ -(3,5-dinitrobenzoyl)-Lys residues. (Ghadiri & Fernholz, J. Am. Chem. Soc, 112:9633-9635 (1990))
  • the variant has not more than 20 amino acids replaced, deleted or inserted compared to wild-type peptide.
  • the variant has not more than 15, 14, 13, 12 or 11 amino acids replaced, deleted or inserted compared to wild-type peptide.
  • the variant has not more than 10, 9, 8 or 7 amino acids replaced, deleted or inserted compared to wild-type peptide.
  • the variant has not more than 6 amino acids replaced, deleted or inserted compared to wild-type peptide. In some embodiments, the variant has not more than 5 or 4 amino acids replaced, deleted or inserted compared to wild-type peptide. In some embodiments, the variant has not more than 3, 2 or 1 amino acids replaced, deleted or inserted compared to wild-type peptide.
  • amino acid as used herein, means an amino acid moiety that comprises any naturally-occurring or non-naturally occurring or synthetic amino acid residue, i.e., any moiety comprising at least one carboxyl and at least one amino residue directly linked by one, two three or more carbon atoms, typically one ( ⁇ ) carbon atom.
  • a “derivative” of azurin refers to a peptide that is derived from the subject peptide.
  • a derivation includes chemical modifications of the peptide such that the peptide still retains some of its fundamental activities.
  • a “derivative" of azurin can be a chemically modified azurin that retains its ability to inhibit parasitemia in malaria-infected red blood cells.
  • Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation or glycosylation of the peptide.
  • a derivative peptide maybe a fusion of a polypeptide or fragment thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a polypeptide that are identical with amino acid residues in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. In a specific embodiment, Blastp (available from the National Center for Biotechnology Information, Bethesda MD) is used using the default parameters of long complexity filter, expect 10, word size 3, existence 11 and extension 1.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
  • % amino acid sequence identity X/Y* 100 where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B.
  • the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • the shorter sequence will be the "B" sequence, unless stated otherwise.
  • the truncated peptide will be the "B" sequence.
  • a “therapeutically effective amount” is an amount effective to prevent or slow the development of, or to partially or totally alleviate the existing symptoms in a particular condition, pathological or otherwise, for which the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the present invention provides compositions and methods that use cupredoxin and/or cytochrome to inhibit parasitemia of malaria-infected mammalian red blood cells and bodily tissues, such as brain tissue and bone tissue.
  • cupredoxins the blue copper-containing proteins
  • iron (haem) - containing proteins the iron (haem) - containing proteins called cytochromes
  • wild type (wt) azurin induces apoptosis in the murine J774 cells (Yamada et al, Infection and Immunity 70:7054-7062 (2002)) while a mutant M44KM64E azurin causes cell cycle inhibition at the Gl phase in J774 cells.
  • wt cytochrome C 55 causes cell cycle inhibition at the Gl phase in J774 cells while a mutant V23DI59E cytochrome c 55 i induces apoptosis.
  • cupredoxins and cytochromes will inhibit in vitro parasitemia in human red blood cells by the malaria parasite Plasmodium falciparum.
  • the cupredoxins azurin and Laz inhibit parasitemia in P. falciparum by about 50% and about 75% respectively.
  • rusticyanin and cytochromes c and f inhibited parasitemia by 20-30 %. See, Example 1.
  • azurin has a discernable structural homology to the Fab fragment of G17.12 mouse monoclonal antibody when complexed to the Pf MSP1-19 fragment of the MSPl surface protein of P. falciparum. See, Example 2. While not limiting the mode of inhibition to any one means, it is thought that azurin may inhibit parasitemia of P. falciparum by interaction with the MSPl protein on the parasite's surface.
  • cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, auracyanin, Laz or rusticyanin.
  • the cupredoxin is Laz, azurin or rusticyanin.
  • the cupredoxin is from a pathogenic bacteria.
  • the cupredoxin is azurin.
  • the azurin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp.denitrificans I, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis Z2491, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa 9a5 or Vibrio par ⁇ haemolyticus.
  • the azurin is from P. aeruginosa.
  • the cupredoxin comprises an amino acid sequence that is SEQ ID NO:1, 2-18 or 21.
  • the cytochrome is cytochrome C5 51 from P. aeruginosa, human cytochrome c or cytochrome f.
  • the cytochrome comprises an amino acid sequence that is SEQ ID NO: 2, 19 or 20.
  • the cytochrome is from a pathogenic bacterium.
  • the cytochrome inhibits parasitism in malaria- infected red blood cells, and more specifically, human red blood cells.
  • the cytochrome inhibits cell cycle progression in a mammalian cancer cell, and more specifically in a J774 cell.
  • the invention provides for peptides that are variants, derivatives or structural equivalents of cupredoxin or cytochrome.
  • the peptide is substantially pure. In other embodiments, the peptide is isolated. In some embodiments, the peptide is less that a full length cupredoxin or cytochrome, and retains some of the functional characteristics of the cupredoxin or cytochrome. In some embodiments, the peptide retains the ability to inhibit parasitemia in malaria-infected red blood cells, and more specifically the ability to inhibit P. falciparum infection in human red blood cells. In specific embodiments, the cytochrome is P.
  • the peptide does not raise an immune response in a mammal, and more specifically a human.
  • the invention also provides compositions comprising at least one peptide that is a cupredoxin, cytochrome, or variant, derivative or structural equivalent of a cupredoxin or cytochrome.
  • the invention also provides compositions comprising at least one peptide that is a cupredoxin or variant, derivative or structural equivalent of a cupredoxin.
  • the invention also provides compositions comprising at least one peptide that is a cytochrome, or variant, derivative or structural equivalent of a cytochrome. In other embodiments, the composition consists essentially of the peptide.
  • the invention also provides compositions comprising at least one peptide that is a cupredoxin, cytochrome, or variant, derivative or structural equivalent of a cupredoxin or cytochrome in a pharmaceutical composition.
  • cupredoxins Because of the high structural homology between the cupredoxins, it is contemplated that other cupredoxins will have the same anti-malarial activity as Pseudotnonas aeruginosa azurin with regards to inhibition of parasitemia in malaria- infected red blood cells.
  • the cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin, Laz or auracyanin.
  • the cupredoxin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp.denitrificans I, Bordetella br ⁇ nchiseptica, Methylomonas sp., Neisseria meningitidis Z2491, Neisseria gonorrhea, Pseudomonas fiuorescens, Pseudomonas chlororaphis, Xylella fastidiosa 9a5 or Vibrio parahaemolyticus.
  • the cupredoxin is azurin from Pseudomonas aeruginosa. In other specific embodiments, the cupredoxin comprises an amino acid sequence that is SEQ ID NO: 1, 3-18, or 22. In other specific embodiments, the cupredoxin is the Laz protein from Neisseria meningitidis or Neisseria gonorrhea.
  • the invention provides for anlino acid sequence variants of a cupredoxin or cytochrome which have amino acids replaced, deleted, or inserted as compared to the wild- type polypeptide. Variants of the invention may be truncations of the wild-type polypeptide.
  • the composition comprises a peptide that consists of a region of a cupredoxin or cytochrome that is less that the full length wild-type polypeptide. In some embodiments, the composition comprises a peptide that consists of more than about 10 residues, more than about 15 residues or more than about 20 residues of a truncated cupredoxin or cytochrome.
  • the composition comprises a peptide that consists of not more than about 100 residues, not more than about 50 residues, not more than about 40 residues or not more than about 30 residues of a truncated cupredoxin or cytochrome.
  • composition comprises a peptide to which a cupredoxin or cytochrome, and more specifically to SEQ ID NOS: 1-20 or 22 has at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity or at least about 99% amino acid sequence identity.
  • the variant of cupredoxin comprises P. aeruginosa azurin residues 36-89. In other embodiments, the variant of cupredoxin consists of P. aeruginosa azurin residues 36-89. In other specific embodiments, the variant consists of the equivalent residues of a cupredoxin other that azurin.
  • cupredoxin variants can be designed that have a similar activity to azurin residues 36-89.
  • the subject cupredoxin amino acid sequence will be aligned to the Pseudomonas aeruginosa azurin sequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located on the P. aeruginosa azurin amino acid sequence, and the equivalent residues found on the subject cupredoxin sequence, and the equivalent residues of the cupredoxin thus identified.
  • the variants also include peptides made with synthetic amino acids not naturally occurring.
  • non-naturally occurring amino acids may be integrated into the variant peptide to extend or optimize the half-life of the composition in the bloodstream.
  • Such variants include, but are not limited to, D,L-peptides (diastereomer), (Futaki et ah, J. Biol. Chem. 276(8):5836-40 (2001); Papo et al, Cancer Res. 64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol. 36(1): 169-76, (1987)); peptides containing unusual amino acids (Lee et ah, J. Pept. Res.
  • the peptide of the invention is a derivative of a cupredoxin or cytochrome.
  • the derivatives of cupredoxin or cytochrome are chemical modifications of the peptide such that the peptide still retains some of its fundamental activities.
  • a "derivative" of azurin can be a chemically modified azurin that retains its ability to inhibit the malaria parasitemia in mammalian cells.
  • Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation and glycosylation of the peptide.
  • a derivative peptide maybe a fusion of a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.
  • a chemical compound such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.
  • Derivatives of interest include chemical modifications by which the half-life in the bloodstream of the peptides and compositions of the invention can be extended or optimized, such as by several methods well known to those in the art, including but not limited to, circularized peptides (Monk et al, BioDrugs 19(4):261-78, (2005); DeFreest et al, J. Pept. Res.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is fused to a H.8 region of Laz from Neisseria meningitidis or Neisseria gonorrhea.
  • H.8-Paz fusion protein described in Example 4.
  • the H.8 is fused to the C-terminus of the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof.
  • the H.8 region is SEQ ID NO: 21, or a variant, derivative or structural equivalent thereof.
  • the peptide of the composition of invention may be more than one of a variant, derivative and structural equivalent of a cupredoxin or cytochrome.
  • the peptide may be a truncation of azurin that has been PEGylated, thus making it both a variant and a derivative.
  • the peptides of the invention are synthesized with ⁇ , ⁇ -disubstituted non-natural amino acids containing olefin-bearing tethers, followed by an all-hydrocarbon "staple" by ruthenium catalyzed olefin metathesis. (Scharmeister et al, J. Am. Chem. Soc.
  • peptides that are structural equivalents of azurin may be fused to other peptides, thus making a peptide that is both a structural equivalent and a derivative.
  • peptides that are structural equivalents of azurin may be fused to other peptides, thus making a peptide that is both a structural equivalent and a derivative.
  • the peptide may be a structural equivalent of a cupredoxin or cytochrome.
  • studies that determine significant structural homology between cupredoxins and cytochromes and other proteins include Toth et al. ⁇ Developmental Cell 1 :82-92 (2001)). Specifically, significant structural homology between a cupredoxin or cytochrome and its structural equivalents are determined by using the VAST algorithm (Gibrat et al, Curr Opin Struct Biol 6:377-385 (1996); .Madej et al, Proteins 23:356-3690 (1995)).
  • the VAST p value from a structural comparison of a cupredoxin or cytochrome to the structural equivalent is less than about 10 '3 , less than about 10 '5 , or less than about 10 "7 .
  • significant structural homology between a cupredoxin or cytochrome and its structural equivalents are determined by using the DALI algorithm (Holm & Sander, J. MoI. Biol. 233:123-138 (1993)).
  • the DALI Z score for a pairwise structural comparison is at least about 3.5, at least about 7.0, or at least about 10.0.
  • the variant or derivative of cupredoxin has a significant structural homology to the Fab fragment of Gl 7.12 mouse monoclonal antibody.
  • An example of how this structural similarity can be determined can be found in Example 3.
  • significant structural homology between a cupredoxin and the Fab fragment of G 17.12 mouse monoclonal antibody can be determined by using the VAST algorithm (Gibrat et at, id.; Madej et ah, id.).
  • the VAST p-value from a structural comparison of a cupredoxin to the Fab fragment of G17.12 mouse monoclonal antibody can be less than about 10 "4 , less than about 10 '5 , less than about 10 "6 , or less than about 10 "7 .
  • the VAST score from a structural comparison of a cupredoxin to the Fab fragment of Gl 7.12 mouse monoclonal antibody can be greater than about 9, greater than about 10, greater than about 11 or greater than about 12.
  • the variant, derivative or structural equivalent thereof has some of the functional characteristics of the P. aeruginosa azurin, P. aeruginosa cytochrome C 551 , human cytochrome c or cyanobacterial cytochrome f.
  • the peptide of the invention inhibits parasitemia by malaria in malaria-infected red blood cells, and more specifically parasitemia by P. falciparum in P. falciparum-mfected human red blood cells.
  • the invention also provides for the variants, derivatives and structural equivalents of cupredoxin and cytochrome C 551 that retain the ability to inhibit parasitemia in malaria-infected red blood cells, and more specifically parasitemia by P. falciparum in P. falciparum-infected human red blood cells.
  • the inhibition of parasitemia by P. falciparum in P. falciparum-m ' fQcted human red blood cells may be determined by the method described in Example 6.
  • cupredoxins and cytochrome can inhibit parasitemia in malaria-infected red blood cells
  • variants, derivatives and structural equivalents of cupredoxins and cytochrome that retain this anti-malarial activity.
  • Such variants and derivatives can be made by, for example, creating a "library" of various variants and derivatives of cupredoxins and cytochromes, and then testing each for antimalarial activity using one of many methods known in the art, such the exemplary method in Example 6.
  • cupredoxins and cytochromes with anti-malarial activity can be used in the methods of the invention, in place of or in addition to the cupredoxins and cytochromes mentioned herein.
  • This method of selecting variants and derivatives may be adapted for any of the activities of P. aeruginosa azurin, P. aeruginosa cytochrome C551, human cytochrome c or cyanobacterial cytochrome f disclosed herein.
  • the peptide of the invention inhibits intracellular replication of the malaria parasite in human red blood cells.
  • Methods to determine the intracellular replication of the malaria parasite are well known in the art, and one such method is described in Example 2.
  • the peptide of the invention binds to the PfMSPl -19 and/or PfMSP 1-42 P. falciparum surface proteins with a relative binding affinity that is statistically greater a non-binding control protein.
  • a peptide can be tested for this activity by using surface plasmon resonance analysis as described in Example 5. Other methods to determine whether one protein binds to another are well known in the art and may be used as well.
  • the peptide of the invention binds to ICAM-3 or NCAM with a relative binding affinity that is statistically greater a non-binding control protein.
  • a peptide can be tested for this activity by using surface plasmon resonance analysis as described in Examples 7 and 5. Other methods to determine whether one protein binds to another are well known in the art and may be used as well.
  • the peptides of the invention induce apoptosis in a mammalian cancer cell, more specifically a J774 cell.
  • the ability of a peptide to induce apoptosis may be observed by mitosensor ApoAlertTM confocal microscopy using a MITOSENSORTM APOLERTTM Mitochondrial Membrane Sensor kit (Clontech Laboratories, Inc., Palo Alto, California, U.S.A.), by measuring caspase-8, caspase-9 and caspase-3 activity using the method described in Zou et al. (J. Biol. Chem.
  • the peptide of the invention induces cellular growth arrest in a mammalian cancer cell, more specifically a J774 cell.
  • Cellular growth arrest can be determined by measuring the extent of inhibition of cell cycle progression, such as by the method found in Yamada et al. (PNAS 101:4770-4775 (2004)).
  • the peptide of the invention inhibits cell cycle progression in a mammalian cancer cell, more specifically a J774 cell. Cupredoxins
  • cupredoxins small blue copper proteins
  • electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or are of unknown function.
  • the copper ion is solely bound by the protein matrix.
  • a special distorted trigonal planar , arrangement to two histidine and one cysteine ligands around the copper gives rise to very peculiar electronic properties of the metal site and an intense blue color.
  • a number of cupredoxins have been crystallographically characterized at medium to high resolution.
  • cupredoxins in general have a low sequence homology but high structural homology.
  • Gough & Clothia Structure 12:917-925 (2004); De Rienzo et al, Protein Science 9:1439-1454 (2000).
  • amino acid sequence of azurin is 31% identical to that of auracyanin B, 16.3% to that of rusticyanin, 20.3 % to that of plastocyanin, and 17.3% to that of pseudoazurin. See Table 1.
  • the structural similarity of these proteins is more pronounced.
  • VAST p value for the comparison of the structure of azurin to auracyanin B is 10 "74 , azurin to rusticyanin is 10 "5 , azurin to plastocyanin is 10 "5 6 , and azurin to psuedoazurin is 10 "4 1 .
  • AU of the cupredoxins possess an eight-stranded Greek key beta-barrel or beta- sandwich fold and have a highly conserved site architecture.
  • a prominent hydrophobic patch due to the presence of many long chain aliphatic residues such as methionines and leucines, is present around the copper site in azurins, amicyanins, cyanobacterial plastocyanins, cucumber basic protein and to a lesser extent, pseudoazurin and eukaryotic plastocyanins.
  • Hydrophobic patches are also found to a lesser extent in stellacyanin and rusticyanin copper sites, but have different features. Id.
  • VAST p value is a measure of the significance of the comparison, expressed as a probability. For example, if the p value is 0.001, then the odds are 1000 to 1 against seeing a match of this quality by pure chance.
  • the p value from VAST is adjusted for the effects of multiple comparisons using the assumption that there are 500 independent and unrelated types of domains in the MMDB database. The p value shown thus corresponds to the p value for the pairwise comparison of each domain pair, divided by 500.
  • VAST structure-similarity score The VAST structure-similarity score. This number is related to the number of secondary structure elements superimposed and the quality of that superposition. Higher VAST scores correlate with higher similarity.
  • RMSD The root mean square superposition residual in Angstroms. This number is calculated after optimal superposition of two structures, as the square root of the mean square distances between equivalent C-alpha atoms. Note that the RMSD value scales with the extent of the structural alignments and that this size must be taken into consideration when using RMSD as a descriptor of overall structural similarity. 5 C. elegans major sperm protein proved to be an ephrin antagonist in oocyte maturation (Kuwabara, 2003 "The multifaceted C. elegans major sperm protein: an ephrin signalling antagonist in oocyte maturation" Genes and Development, 17:155-161.
  • the azurins are copper containing proteins of 128 amino acid residues which belong to the family of cupredoxins involved in electron transfer in plants and certain bacteria.
  • the azurins include those from P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitriflcans (SEQ ID NO: 8).
  • PA P. aeruginosa
  • A. xylosoxidans A. denitriflcans
  • SEQ ID NO: 8 A. denitriflcans
  • azurins have a characteristic ⁇ -sandwich with Greek key motif and the single copper atom is always placed at the same region of the protein.
  • azurins possess an essentially neutral hydrophobic patch surrounding the copper site. Id.
  • the plastocyanins are soluble proteins of cyanobacteria, algae and plants that contain one molecule of copper per molecule and are blue in their oxidized form. They occur in the chloroplast, where they function as electron carriers. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 A resolution. SEQ ID NO: 4 shows the amino acid sequence of plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium.
  • Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface.
  • the copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners.
  • Rusticyanins are blue-copper containing single-chain polypeptides obtained from a Thiobacillus (now called Acidithiobacillus).
  • the X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 5) has been determined by multiwavelength anomalous diffraction and refined to 1.9A resolution.
  • the rusticyanins are composed of a core beta- sandwich fold composed of a six- and a seven-stranded b-sheet.
  • the copper ion is coordinated by a cluster of four conserved residues (His 85, Cysl38, Hisl43, Metl48) arranged in a distorted tetrahedron. Walter, R.L. et ah, J. MoI. Biol., vol. 263, pp- 730-51 (1996).
  • the pseudoazurins are a family of blue-copper containing single-chain polypeptide.
  • the amino acid sequence of pseudoazurin obtained from Achromobacter cycloclastes is shown in SEQ ID NO: 6.
  • the X-ray structure analysis of pseudoazurin shows that it has a similar structure to the azurins although there is low sequence homology between these proteins.
  • azurins In the mid-peptide region azurins contain an extended loop, shortened in the pseudoazurins, which forms a flap containing a short ⁇ -helix.
  • the only major differences at the copper atom site are the conformation of the MET side-chain and the Met-S copper bond length, which is significantly shorter in pseudoazurin than in azurin.
  • the proteins identifiable as phytocyanins include, but are not limited to, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana.
  • cucumber basic protein and the pea-pod protein the axial methionine ligand normally found at blue copper sites is replaced by glutamine.
  • auracyanin A Three small blue copper proteins designated auracyanin A, auracyanin B-I, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus.
  • the two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form.
  • the sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates apparent monomer molecular masses as 14 (A), 18 (B-2), and 22 (B-I) kDa.
  • auracyanin A The amino acid sequence of auracyanin A has been determined and showed auracyanin A to be a polypeptide of 139 residues. (Van Dreissche et ah, Protein Science 8:947-957 (1999).) His58, Cysl23, Hisl28, and Metl32 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general beta-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes.
  • the overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%.
  • the N-terminal sequence region 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids. Id. See exemplary amino acid sequence SEQ ID NO: 16 for chain A of auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Accession No. AAM12874).
  • the auracyanin B molecule has a standard cupredoxin fold.
  • the crystal structure of auracyanin B from Chloroflexus aurantiacus has been studied. (Bond et al, J. MoI. Biol. 306:47-67 (2001).) With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin.
  • one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8.
  • the Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands.
  • the crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.
  • the amino acid sequences of the B forms are presented in McManus et al. (J. Biol Chem. 267:6531-6540 (1992).). See exemplary amino acid sequence SEQ ID NO: 17 for chain B of auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Accession No. IQHQA).
  • Stellacyanins are a subclass of phytocyanins, a ubiquitous family of plant cupredoxins.
  • An exemplary sequence of a stellacyanin is included herein as SEQ ID NO: 15.
  • the crystal structure of umecyanin, a stellacyanin from horseradish root (Koch et al, J. Am. Chem. Soc. 127:158-166 (2005)) and cucumber stellacyanin (Hart el al, Protein Science 5:2175-2183 (1996).).
  • the protein has an overall fold similar to the other phytocyanins.
  • the ephrin B2 protein ectodomain tertiary structure bears a significant similarity to stellacyanin. (Toth et al, Developmental Cell 1:83-92 (2001).)
  • An exemplary amino acid sequence of a stellacyanin is found in the National Center for Biotechnology Information Protein Data Bank as Accession No. IJER, SEQ ID NO: 15.
  • An exemplary amino acid sequence from a cucumber basic protein is included herein as SEQ ID NO: 18.
  • the crystal structure of the cucumber basic protein (CBP), a type 1 blue copper protein, has been refined at 1.8 A resolution.
  • the molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich” or "beta-taco”.
  • the ephrinB2 protein ectodomian tertiary structure bears a high similarity (rms deviation 1.5 A for the 50 ⁇ carbons) to the cucumber basic protein.
  • a disulphide link, (Cys52)-S-S-(Cys85) appears to play an important role in stabilizing the molecular structure.
  • the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identity.
  • the proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana.
  • CBP CBP
  • stellacyanin mavicyanin
  • umecyanin a cucumber peeling cupredoxin
  • a putative blue copper protein in pea pods a putative blue copper protein in pea pods
  • a blue copper protein from Arabidopsis thaliana In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine.
  • An exemplary sequence for cucumber basic protein is found in NCBI Protein Data Bank Accession No.2CBP, SEQ ID NO: 18.
  • Cytochrome C 551 from P. aeruginosa is a monomeric redox protein of 82 amino-acid residues (SEQ ID NO: 2), involved in dissimilative denitrification as the physiological electron donor of nitrite reductase.
  • SEQ ID NO: 2 The functional properties of Pa-C551 have been extensively investigated. The reactions with non-physiological small inorganic redox reactants and with other macromolecules, like blue copper proteins, eukaryotic cytochrome c and the physiological partner nitrite reductase have provided a test for protein-protein electron transfer.
  • Pa-C551 which is a member of bacterial class I cytochromes, shows a single low-spin heme with His-Met ligation and the typical polypeptide fold which however leaves the edges of pyrrole rings II and III of the heme exposed (Cutruzzola et al, J. Inorgan. Chem. 88:353-61 (2002)).
  • the lack of a 20-residue omega loop, present in the mammalian class I cytochromes, causes further exposure of the heme edge at the level of propionate 13.
  • the distribution of charged residues on the surface of Pa-C551 is very anisotropic: one side is richer in acidic residues whereas the other displays a ring of positive side chains, mainly lysines, located at the border of a hydrophobic patch which surrounds the heme crevice.
  • This patch comprises residues Glyll, VaI 13, AIaW, Met22, Val23, Pro58, Ile59, Pro ⁇ O, Pro62, Pro63 and Ala65.
  • the anisotropic charge distribution leads to a large dipolar moment which is important for electron transfer complex formation.
  • the charge distribution described above for Pa-C551 has been reported for other electron transfer proteins and their electron acceptors. Moreover, modification by site- directed mutagenesis of residues within the hydrophobic or charged patch has shown for different proteins the importance of surface complementarity for binding and electron transfer. As an example, evidence for the relevance of the hydrophobic patch for the electron transfer properties of azurin from P. aeruginosa came from the studies carried out on mutants of residues Met44 and Met64 changed to positively and negatively charged amino acids. Id. The cytochrome c-type domain has a fold consisting of a series of alpha helices and reverse turns that serve to envelop the covalently bound haem within a hydrophobic pocket.
  • This domain can be found in monodomain cytochrome c proteins, such as cytochrome c6, cytochrome C 552 , cytochrome c 45 g and mitochondrial cytochrome c.
  • the cytochrome c-type domain occurs in a number of other proteins, such as in cytochrome cdl -nitrite reductase as the N-terminal haem c domain, in quinoprotein alcohol dehydrogenase as the C-terminal domain, in Quinohemoprotein amine dehydrogenase A chain as domains 1 and 2, and in the cytochrome bci complex as the cytochrome bo ⁇ domain.
  • Structural analysis with VAST algorithm cytochrome C 551 from Pseudomonas aeruginosa as a query
  • showed significant structural neighbors P values between 10 ⁇ i ⁇ 3 to 10 "45 ) only for cytochromes.
  • the invention provides methods to treat patients with a malarial infection or at danger of acquiring one, or inhibit the spread of the malaria parasite. These methods comprise administering to a patient or an insect vector a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof which inhibits parasitemia of malaria-infected mammalian cells.
  • the inhibition of parasitemia can be determined by many methods well known in the art. One method is described in Example 6, and determines the inhibition of parasitemia in malaria-infected human red blood cells.
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof inhibits intracellular replication of the malaria parasite in human red blood cells and is administered to the patient or insect vector.
  • the mode of the invention is not limited to any particular mechanism, and inhibition of parasitemia may result from many factors, including but not limited to, inhibition of replication of the parasite in infected blood cells, inhibition of parasite infecting uninfected blood cells, inhibition in the growth cycle of the parasite and inhibition of parasite entry into the mammalian cell.
  • the invention provides methods to treat patients suffering from infection by a malaria parasite by administering an effective amount of at least one protein that is a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof.
  • the patients that may be treated by this method are any mammal that can be infected by a malaria parasite, and specifically are human patients.
  • Malaria parasites known to infect mammals include, but are not limited to, Plasmodium falciparum, P. vivax, P. berghei (rodent-specific), P. yoelli (murine-specific), P. cynomolgi and P. knowlesi (monkey-specific).
  • cupredoxins and cytochrome C 551 are also effective against HIV-I infections, as disclosed in a co-filed application. "Compositions And Methods For Treating HIV Infection With Cupredoxin And Cytochrome c," U. S Provisional Patent
  • the patient suffering from infection by a malaria parasite is also suffering from infection by HIV.
  • the method of treatment of the invention also comprises administering anti- HIV drugs.
  • the anti-HIV drugs are co-administered.
  • the invention also provides methods to treat a patient suspected of having contact with a malaria parasite by administering an effective amount of at least one peptide that is a cupredoxin and/or a cytochrome, or a variant, derivative or structural equivalent of a cupredoxin or a cytochrome.
  • a patient can be suspected of having contact with a malaria parasite, for example, if that patient lives or has traveled in a region of the world where malaria infection of others of the patient's species is common. Treatment by this method may be commenced when the patient is about to, or has already, come into contact with the malaria parasite.
  • a patient can be suspected of having contact with the malaria parasite if they have come into contact with blood infected with a malaria parasite, are intentionally exposed to the malaria parasite, or accidentally injected with blood or drugs contaminated with the parasite.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome can be administered to the patient by many routes and in many regimens that will be well known to those in the art.
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome is administered orally, topically, by inhalation, by injection, more specifically, intravenously, intramuscularly or subcutaneously.
  • the methods may comprise co-administering to a patient one unit dose of compositions comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, and one unit dose of compositions comprising an anti-malarial drug and/or an anti-HIV drug, in either order.
  • compositions may be administered at about the same time, or within about a given time following the administration of the other, for example, about one minute to about 6o minutes, or about 1 hour to about 12 hours of the other.
  • the invention also provides methods to inhibit the spread of the malaria parasite in an insect vector population harboring a malaria parasite by administering to an insect vector in the population at least one of a cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome, at an amount that is effective to reduce the infectivity of the parasite in a co-existant mammalian population.
  • the insect vector is a mosquito, and more specifically a mosquito from the species Anopheles gambiae.
  • cupredoxin or cytochrome or variant, derivative or structural equivalent of cupredoxin or cytochrome
  • administration of the cupredoxin or cytochrome can be accomplished by placing the peptides in compositions that will be consumed by the insect vector, however any manner that brings the peptide into contact with the malaria parasite in the insect vector's gut is contemplated. Many methods to administer chemicals to insect populations which produce such consumption are known in the art.
  • a transmissible genetic element that passes from one mosquito to another will be operably connected to the cupredoxin coding sequence operably connected to a constitutive promoter, the cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome will be produced inside the Anopheles gambiae infected with P. falciparum and will interfere with its replication/survival in the mosquito.
  • Other manners of administration of the peptides to the insect vector include, but are not limited to, fusing the cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome genes to genes from other proteins normally consumed by the insects.
  • the amount of peptides administered to the insect vector should be an amount effective to reduce the infectivity of the malaria parasite in a mammal when the insect vector comes into contact with a mammal. In specific embodiments, the amount administered should be effective to reduce the infectivity of the malaria parasite when the insect vector comes into contact with a human.
  • Mosquito larvae are suitable for use in the present invention and preferably, the promoter used is a strong promoter.
  • Two alternative categories of promoter are available for use: inducible and constitutive promoters.
  • Inducible promoters include, for example, heat shock promoters.
  • the heat shock promoter is an insect heat shock promoter, for example the Drosophila melanogaster hsp70 promoter, which is capable of driving the expression of genes in heterologous organisms, including medfly.
  • the invention also encompasses the use of the medfly hsp70 promoter (Papadimitriou et ah, Insect MoI Biol 7:279-90(1998) ).
  • Alternative systems may be based on induction with the antibiotic tetracycline. (Heinrich and Scott, PNAS 97:8229-8232, (2000))
  • Heat shock promoters are inducible by raising the temperature of the conditions under which the medfly are being cultured. For example, at 23-25° C, the hsp70 promoter is active at low levels or not at all. This allows the insect larva to develop without stress induced by the production of a heterologous protein. At higher temperatures, however, such as 37-42° C, the hsp70 promoter is induced and expresses the heterologous protein at a high level.
  • Inducible promoters may be constructed based on known inducible gene control elements. For example, inducible promoters may be constructed by combining an element responsive to a drug or hormone which may be administered in the diet. In a preferred embodiment, a human oestrogen responsive element (ERE) may be used to regulate expression of the protein of interest, as long as the insect is transformed with a second coding sequence which expresses the human oestrogen receptor.
  • EEE human oestrogen responsive element
  • constitutive promoters may also be used to express the protein and/or other proteins required in the insect larva.
  • the constitutive promoter may be a cytoplasmic actin promoter.
  • the D. melanogaster cytoplasmic actin promoter has been cloned (Act5C) and is highly active in mosquitoes (Huynh and Zieler, J. MoI. Biol. 288:13-20(1999) ).
  • Cytoplasmic actin genes and their promoters may also be isolated from other insects, including medfly.
  • Other examples include the cytoplasmic tubulin promoter, for instance the medfly cytoplasmic tubulin promoter.
  • Promoters which control secreted polypeptides may be used, optionally together with appropriate signal sequences, to direct secretion of the protein to the haemolymph.
  • the larval serum protein promoter may be employed (Benes et ah, MoI. Gen. Genet 249(5):545-556 (1995)).
  • compositions comprising Cupredoxin and/or Cytochrome and Variants and Derivatives Thereof
  • compositions comprising cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof can be manufactured in any conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the substantially pure cupredoxin and/or cytochrome, and variants, derivatives and structural equivalents thereof can be readily combined with a pharmaceutically acceptable carrier well-known in the art.
  • Such carriers enable the preparation to be formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, and the like.
  • Suitable carriers or excipients can also include, for example, fillers and cellulose preparations.
  • excipients can include, for example, flavoring agents, coloring agents, detackifiers, thickeners, and other acceptable additives, adjuvants, or binders.
  • the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation may contain at least one preservative.
  • General methodology on pharmaceutical dosage forms is found in Ansel et ah, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore MD (1999)).
  • composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof used in the invention may be administered in a variety of ways, including by injection (e.g., intradermal, subcutaneous, intramuscular, intraperitoneal and the like), by inhalation, by topical administration, by suppository, by using a transdermal patch or by mouth.
  • injection e.g., intradermal, subcutaneous, intramuscular, intraperitoneal and the like
  • topical administration e.g., by topical administration, by suppository, by using a transdermal patch or by mouth.
  • General information on drug delivery systems can be found in Ansel et ah, Id..
  • the composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof can be formulated and used directly as injectibles, for subcutaneous and intravenous injection, among others.
  • the injectable formulation in particular, can advantageously be used to treat patients that are at risk of an malaria-infection, likely to have an malaria-infection or have an malaria-infection.
  • the composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof can also be taken orally after mixing with protective agents such as polypropylene glycols or similar coating agents.
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof may be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide.
  • the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide.
  • the intravenous fluids for use administering the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof may be composed of crystalloids or colloids.
  • Crystalloids as used herein are aqueous solutions of mineral salts or other water-soluble molecules.
  • Colloids as used herein contain larger insoluble molecules, such as gelatin.
  • Intravenous fluids may be sterile.
  • Crystalloid fluids that may be used for intravenous administration include but are not limited to, normal saline (a solution of sodium chloride at 0.9% concentration), Ringer's lactate or Ringer's solution, and a solution of 5% dextrose in water sometimes called D5W, as described in Table 2.
  • Ringer's lactate also has 28 mmol/L lactate, 4 mmol/L K " ⁇ " a .-..n * .d J 3 o * m»- TM mo ⁇ il//L ⁇
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof may be delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the proteins and a suitable powder base such as lactose or starch.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof may be formulated as solutions, gels, ointments, creams, suspensions, and the like, as are well known in the art.
  • administration is by means of a transdermal patch.
  • cupredoxin and/or cytochrome c and variants and derivatives thereof compositions may also be formulated in compositions containing conventional suppository bases.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof can be readily formulated by combining the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof with pharmaceutically acceptable carriers well known in the art.
  • a solid carrier such as mannitol, lactose, magnesium stearate, and the like may be employed; such carriers enable the cupredoxin and/or cytochrome and variants and derivatives thereof to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • suitable excipients include fillers such as sugars, cellulose preparation, granulating agents, and binding agents.
  • sustained-release formulations that include a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof allow for the release of cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof over extended periods of time, such that without the sustained release formulation, the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof would be cleared from a subject's system, and/or degraded by, for example, proteases and simple hydrolysis before eliciting or enhancing a therapeutic effect.
  • compositions of the invention can be extended or optimized by several methods well known to those in the art, including but not limited to, circularized peptides (Monk et al, BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), D,L-peptides (diastereomer), (Futaki et al, J. Biol. Chem. Feb 23;276(8):5836-40 (2001); Papo et al, Cancer Res. 64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol.
  • the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like.
  • suitable carrier known to those of ordinary skill in the art may be employed to administer the compositions of this invention, the type of carrier will vary depending on the mode of administration.
  • Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.
  • compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof can be administered formulated as pharmaceutical compositions and administered by any suitable route, for example, by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical, percutaneous, i.e., transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary) administration.
  • the pharmaceutical formulations thereof can be administered in any amount effective to achieve its intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In specific embodiments, the therapeutically effective amount is generally from about 0.01-20 mg/day/kg of body weight.
  • the compounds comprising cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof are useful for the treatment and/or prophylaxis of malaria infection, alone or in combination with other active agents.
  • the appropriate dosage will, of course, vary depending upon, for example, the compound of cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof employed, the host, the mode of administration and the nature and severity of the conditions being treated. However, in general, satisfactory results in humans are indicated to be obtained at daily dosages from about 0.01-20 mg/kg of body weight.
  • An indicated daily dosage in humans is in the range from about 0.7 mg to about 1400 mg of a compound of cupredoxin or cytochrome C 551 , or variant, derivative or structural equivalent thereof conveniently administered, for example, in daily doses, weekly doses, monthly doses, and/or continuous dosing.
  • Daily doses can be in discrete dosages from 1 to 12 times per day.
  • doses can be administered every other day, every third day, every fourth day, every fifth day, every sixth day, every week, and similarly in day increments up to 31 days.
  • dosing can be continuous using patches, i.v. administration and the like.
  • cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is, in some embodiments, through the coadministration of cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof with other drugs used for malaria therapy.
  • the cupredoxin and/or cytochrome c are part of an cocktail or co- dosing containing or with other malaria therapeutics.
  • Malaria therapeutics of interest include, but are not limited to, proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, mefloquine, l,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide, and combinations thereof.
  • cupredoxin or cytochrome Cs 51 , or variant, derivative or structural equivalent thereof is, in some embodiments, the same as currently used to introduce anti-HIV drugs, such as the protease-inhibitor-containing cocktails. Such methods are well-known in the art.
  • the cupredoxin or cytochrome C 5 51, or variant, derivative or structural equivalent thereof are part of an cocktail or co-dosing with anti-HIV therapeutics.
  • Anti-HIV drugs include, but are not limited to, reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine [ZerifJ), and 3TC (lamivudine [Epivir]), nonnucleoside reverse transcriptase inhibitors (NNRTIS): delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), a lopinavir and ritonavir combination (Kaletra), saquinavir (Invirase), indinavir sulphate (Crixivan), amprenavir (Agenerase), and nelfinavir (Viracept).
  • AZT zidovudine [Retrovir]
  • ddC zalcitabine [Hivid]
  • HAART highly active antiretroviral therapy
  • the exact formulation, route of administration, and dosage is determined by the attending physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide plasma levels of the active cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof which are sufficient to maintain therapeutic effect.
  • the desired cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is administered in an admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is delivered as DNA such that the polypeptide is generated in situ.
  • the DNA is "naked," as described, for example, in Ulmer et ah, (Science 259:1745-1749 (1993)) and reviewed by Cohen (Science 259:1691-1692 (1993)).
  • the uptake of naked DNA may be increased by coating the DNA onto a carrier, e.g., biodegradable beads, which are then efficiently transported into the cells.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems.
  • Vectors used to shuttle genetic material from organism to organism, can be divided into two general classes: cloning vectors are replicating plasmid or phage with regions that are essential for propagation in an appropriate host cell and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector.
  • An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA, such as the DNA of a cupredoxin and/or a cytochrome.
  • the introduced DNA is operably-linked to elements such as promoters that signal to the host cell to highly transcribe the inserted DNA.
  • Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. Operably-linking a cupredoxin or cytochrome and variants and derivatives thereof polynucleotide to an inducible promoter can control the expression of the cupredoxin or cytochrome and variants and derivatives thereof in response to specific factors.
  • Examples of classic inducible promoters include those that are responsive to ⁇ -interferon, heat shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511 (1990)) and tetracycline.
  • desirable inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but, however, are responsive in those cells when the induction agent is exogenously supplied.
  • useful expression vectors are often plasmids.
  • other forms of expression vectors such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) are contemplated.
  • Vector choice is dictated by the organism or cells being used and the desired fate of the vector.
  • vectors comprise signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters, and transcription termination sequences.
  • one may clone a cupredoxin or a cytochrome gene into a vector transmissible in the malaria parasite-harboring mosquitoes to prevent the parasite from replicating inside the mosquitoes.
  • the transmissibility of the vector will allow the spread of the cupredoxin/cytochrome to neighboring mosquitoes that are infected with the malaria parasites as well.
  • the exact formulation, route of administration, and dosage is determined by the attending physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide plasma levels of the active cupredoxin and/or cytochrome and variants and derivatives thereof which are sufficient to treat the patient and/or maintain therapeutic effect. Generally, the desired cupredoxin and/or cytochrome and variants and derivatives thereof can be administered in an admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions used in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the cupredoxin and/or cytochrome and variants and derivatives thereof, active agents, for inhibiting or stimulating the secretion of cupredoxin and/or cytochrome and variants and derivatives thereof, or a mixture thereof into preparations which can be used therapeutically.
  • Kits Comprising Cupredoxin And/Or Cytochrome C And Variants And Derivatives Thereof
  • kits containing one or more of the following in a package or container: (1) a biologically active composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof; (2) a pharmaceutically acceptable adjuvant or excipient; (3) a vehicle for administration, such as a syringe; (4) instructions for administration.
  • a biologically active composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof
  • a pharmaceutically acceptable adjuvant or excipient such as a syringe
  • a vehicle for administration such as a syringe
  • instructions for administration such as a syringe
  • kits containing one or more of the following in a package or container (1) a biologically active composition comprising a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof; (2) an malaria therapeutic, including, but not limited to, proguanil, chlofproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin, artefiene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, meflo
  • the kit also comprises an anti-HIV therapeutic in a package or container.
  • Anti-HIV therapeutics of interest include, but are not limited to, reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine [Zerit]), and 3TC (lamivudine [Epivir]), nonnucleoside reverse transcriptase inhibitors (NNRTIS): delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), a lopinavir and ritonavir combination (Kaletra), saquinavir (Invirase), indinavir sulphate (Crixivan), amprenavir (Agenerase), and nelfinavir (Viracept).
  • AZT zidovudine [Retrovir]
  • the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.
  • the reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain the lyophilized polypeptide or polynucleotide of cupredoxin and/or cytochrome c and variants and derivatives thereof, or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold similar reagents.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may comprise foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to be mixed. Removable membranes may be glass, plastic, rubber, etc.
  • Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, flash memory device, etc.. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
  • Cupredoxin or cytochrome may be chemically modified or genetically altered to produce variants and derivatives as explained above. Such variants and derivatives may be synthesized by standard techniques.
  • cupredoxin and cytochrome changes can be introduced by mutation into cupredoxin or cytochrome coding sequence that incur alterations in the amino acid sequences of the encoded cupredoxin or cytochrome that do not significantly alter the ability of cupredoxin or cytochrome to inhibit parasitemia in malaria-infected red blood cells.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the cupredoxin without altering biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the cupredoxins are predicted to be particularly non-amenable to alteration, and thus "essential.”
  • amino acids for which conservative substitutions that do not change the activity of the polypeptide can be made are well known in the art. Useful conservative substitutions are shown in Table 3, "Preferred substitutions.” Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the invention so long as the substitution does not materially alter the biological activity of the compound.
  • Residues are divided into groups based on common side-chain properties as denoted in Table 4.
  • Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more specifically into non-conserved sites.
  • the variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis Carter, Biochem J. 2Y1 ⁇ -1 (1986); Zoller and Smith, Methods Enzymol. 154:329-350 (1987)
  • cassette mutagenesis cassette mutagenesis
  • restriction selection mutagenesis Wells et ah, Gene 34:315-323 (1985)
  • other known techniques can be performed on the cloned DNA to produce the cupredoxin or cytochrome Cs 51 variant DNA.
  • cupredoxins and cytochrome CsS 1 can also be used to create variant cupredoxin and cytochrome Cs 51 to be used in the methods of the invention.
  • the Cl 12D and M44KM64E mutants of azurin are known to have cytotoxic and growth arresting activity that is different from the native azurin, and such altered activity can be useful in the treatment methods of the present invention.
  • One embodiment of the methods of the invention utilize cupredoxin and/or cytochrome and variants and derivatives thereof retaining the ability inhibit the growth of malaria infection in mammalian cells.
  • the methods of the present invention utilize cupredoxin variants such as the M44KM64E mutant, having the ability to cause cellular growth arrest.
  • Example 1 In Vitro Inhibition of P. falciparum Parasitemia by Cupredoxin and
  • cupredoxins bacterial wt azurin, M44KM64E azurin, rusticyanin and cyanobacterial plastocyanin, as well as the cytochromes Pseudomonas aeruginosa cytochrome C 551 , human cytochrome c and Phormidium laminosum cytochrome f were tested in a normal red blood cell (RBC) assay at 200 ⁇ g/ml concentrations at 30 hours post inoculation.
  • RBC red blood cell
  • Hct RBCs 200 ⁇ l of 10% Hct RBCs were added to each of 24 wells (final 2% Hct at ImI) in addition to 30 ⁇ l complete RPMI containing recombinant cupredoxin or cytochrome proteins at 666 ⁇ M for a final concentration of 200 ⁇ M.
  • the control showed 9.5% parasitemia (standard error 1.3%), wt azurin 6.9% (s.e. 1.4%), M44KM64E azurin 9.1% (s.e. 1.0%), rusticyanin 7.2% (s.e. 0.7%), cytochrome c 55 i 7.5% (s.e. 1.5%), human cytochrome c 8.4% (s.e. 0.4%), plastocyanin 8.1% (s.e. 1.3%) and cytochrome f 6.6% (s.e. 1.0%), suggesting that cupredoxins such as wt azurin and rusticyanin and cytochromes such as cytochrome for cytochrome c S5 i demonstrated 20 to 30% inhibition of parasitemia.
  • red blood cells were loaded to an intracellular recombinant protein concentration of 200 ⁇ g/ml using a hypotonic ghost preparation. Cells where then washed, resuspended and infected with schizont-stage parasites (P. falciparum) as described in Example 1. The red blood cell ghosts were incubated for 19 hours and 40 hours and giemsa smears were made.
  • Wt azurin, M44KM64E mutant azurin, plastocyanin, cytochrome C 5 5 1 , human cytochrome c and cynobacterial cytochrome f proteins showed parasitemia varying from 4.2 to 5.4% .
  • Table 5 Cupredoxin and cytochrome inhibition of P. falciparum infection of red blood cell ghosts.
  • Example 3 Structural Homology between Azurin and Fab Fragment of G17.12 Monoclonal Antibody Complexed with Pf MSP1-19
  • cupredoxins show structural similarity to the variable domains of the immunoglobulin superfamily members.
  • the DALI algorithm Holm & Park, Bioinformatics 16:566-567 (2000)
  • Azurin exhibits structural similarity to the Fab fragment of G17.12 monoclonal antibody in complexation with Pf MSP1-19 fragment of the MSPl merozoite surface protein of P. falciparum.
  • cupredoxins including azurin demonstrate structural similarities in having two anti-parallel ⁇ sheets packed face to face and linked by a disulfide bridge to the variable domains of the immunoglobulin superfamily members as well as extracellular domains of the intercellular adhesion molecules (ICAM) and their ligands.
  • IAM intercellular adhesion molecules
  • ITOP B The complex Structure of Binding Domains 22 2.5 ofICAM-3 and Alphabeta2
  • the laz gene from Neisseria gonorrhoeae was cloned based on its known sequence (SEQ ID NO: 22).
  • the P. aeruginosa azurin gene (SEQ ID NO: 1), termed paz , and the sequence of the H.8 epitope of laz from N. gonnerrhoeae (SEQ ID NO: 21), were used to clone in frame the H.8 epitope gene in the 5 '-end of paz to produce H.8-p ⁇ z or in the 3 '-end of paz to generate paz-H.8.
  • aeruginosa PAO 1 cloned into HindIII Acad. Sci. USA 99:14098- and Pstl digested pUCl9, Ap r 14103 (2002); Yamada, et al, Proc. ⁇ atl. Acad. Sci. USA 101:4770-4775 (2004) pUC18-H.8- Fusion plasmid encoding H.8 from ⁇ .
  • Km r ⁇ ovagen pET29a-gst pET29a derivative containing the gst Herein gene Km r pGEX-5X-3- pGEX-5X-3 derivative containing H.8- Herein H.8 encoding region, Ap r pET29a-#rt- pET29a derivative containing gst-H.8 Herein H.8 gene, Km r
  • the forward and reverse primers used were 5'-CCGGAATTCCGGCAGGGATGTTGTAAATATCCG-S' (SEQ ID NO: 23) and S'-GGG ⁇ TAC ⁇ GCCGTGGCAGGCATACAGCATTTCAATCGG-S 1 (SEQ ID NO: 24) where the additionally introduced restriction sites of EcoRI and Kpnl sites are underlined respectively.
  • the plasmids expressing fusion H.8 of N. gonorrhoeae Laz and azurin of P. aeruginosa were constructed by PCR with pUC19-p ⁇ z and pUC18-/ ⁇ z as templates.
  • a 3.1 kb fragment was amplified with pUC18-/ ⁇ z as a template and primers, 5'-(phosphorylated)GGCAGCAGGGGCTTCGGCAGCATCTGC-3' (SEQ ID NO: 25) and 5'-CTGCAGGTCGACTCTAGAGGATCCCG-S' (SEQ ID NO: 26) where a Sail site is underlined.
  • a PCR amplified a 0.4 kb fragment was obtained from p ⁇ JCl9-paz as a template and primers, 5'-(phosphorylated)GCCGAGTGCTCGGTGGACATCCAGG-3' (SEQ ID NO: 27) and S'-TACTCGAGTCACTTCAGGGTCAGGGTG-S' (SEQ ID NO: 28) where a Xhol site is underlined.
  • a Sail digested PCR fragment from pUC 18-/ ⁇ z and Xhol digested PCR fragment from p ⁇ JC19-paz were cloned to yield an expression plasmid pUC18-H.8-j9oz (Table 7).
  • E. coli JMl 09 was used as a host strain for expression of azurin and its derivative genes.
  • Recombinant E. coli strains were cultivated in 2 X YT medium containing 100 ⁇ g/ml ampicillin, 0.1 mM IPTG and 0.5 mM CuSO 4 for 16 h at 37°C to produce the azurin proteins.
  • Plasmid Construction for Fusion GST Proteins Plasmids expressing fusion glutathione S-transferase (GST)-truncated wt-azurin (azu) derivatives were constructed by a polymerase chain reaction using proofreading DNA polymerase. For pGST-azu 36-128, an amplified PCR fragment was introduced into the BamHl and EcoRI sites of the commercial GST expression vector pGEX-5X (Amersham Biosciences, Piscataway, NJ).
  • GST fusion glutathione S-transferase
  • azu truncated wt-azurin
  • the fragment was amplified with pUC19-azu as a template and primers, 5'-CGGGATCC CCG GCA ACC TGC CGA AGA ACG TCA TGG GC-3'(SEQ ID NO: 29) and 5'-CGGAATTC GCA TCA CTT CAG GGT CAG GG-3' (SEQ ID NO: 30), where the additionally introduced BamHl and EcoRI sites are underlined respectively.
  • Carboxyl-terminus truncation of azu gene was cumulatively performed by introducing a stop codon using QuickChange site-direct mutagenesis kit (Stratagene, La Jolla, CA).
  • pGST-azu 36-89 For pGST-azu 36-89, a stop codon were introduced into Gly90.
  • the plasmid carrying pGST-azu 36-128 was used as template DNA.
  • Three sets of oligonuclotides for site-direct mutagenesis are shown as follows.
  • pGST-azu 88-113 carboxyl terminus truncation of azu gene was cumulatively performed by introducing stop codon using QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA).
  • QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA).
  • pGST-azu 88-113 a stop codon was introduced into Phel 14.
  • the plasmid carrying pGST-azu 88-128 was used as the template.
  • an amplified PCR fragment was introduced into the BamHl and EcoRI sites of the commercial GST expression vector pGEX-5X (Amersham Biosciences).
  • the fragment was amplified with pUC19-azu as the template and primers, 5'-CGGGGATCC CCG GCT CGG GCG AGA AGG AC-3' (SEQ ID NO: 33) and 5'-CGGGAATTC TCC ACT TCA GGG TCA GGG TG- 3' (SEQ ID NO: 34) where the additionally introduced BamHl and EcoRI sites are underlined respectively.
  • oligonucleotides for site directed mutagenesis are shown as follows for the preparation of pGST-azu 88-113: 5'-GTT CTT CTG CAC CTA GCC GGG CCA CTC CG- 3' (SEQ ID NO: 35) and 5'-CGG AGT GGC CCG GCT AGG TGC AGA AGA AC-3' (SEQ ID NO: 36).
  • pGST-azu 88-113 was used to transform E. coli XL-I Blue strains. Plasmid extraction was performed using a commercial kit (Qiagen, Venlo, The Netherlands) and PCR sequencing were performed to assess plasmid insertion and transfection.
  • E. coli BL21 (DE3) was used as a host strain for expression of the gst and its fusions derivatives.
  • E. coli strain XLl -Blue transformed with pGST-azu plasmids was grown in LB media with ampicillin for three hours at 37 0 C upon which IPTG induction (0.4 mM) was performed an subsequent incubation for 2-4 h at 37°C to maximize the expression levels.
  • Cells were isolated by centrifugation, resuspended in 25 mL of IX PBS buffer. Subsequent cell lysis involved two sequential treatments of the cell suspension via sonication (20 min on ice) and heat-cold shock in acetone-dry ice bath (using the appropriate protease inhibitors).
  • Binding studies were performed by injecting protein eluents (50 ⁇ l) over the protein-CM5 surface at flow rates of 30 ⁇ l/min with a 120 sec time delay at the end of the injections.
  • Protein eluents included GST-azurin fusion proteins (GST, GST-Azu 36- 128, GST-Azu 36-89, and GST-Azu 88-113, as described in Example 4).
  • Sensor chip surfaces were regenerated between protein injections using 100 mM NaOH (10 ⁇ l injection pulse). All binding studies were run in parallel against a negative flow channel with bare Au- CM5 sensor surface to correct for nonspecific binding to the chips.
  • both H.8-azurin and Laz demonstrated a higher affinity of binding with the merozoite surface protein MSPl cleavage products, with characteristic Kd values of 32.2 nM between azurin and MSP1-19 and 54.3 nM between azurin and MSPl-42.
  • the Kd values between H.8-azurin and MSP1-19 and MSPl-42 were 11.8 nM and 14.3 nM while such values between Laz and MSP1-19 and MSPl-42 ranged from 26.2 nM and 45.6 nM respectively.
  • H.8 epitope might facilitate binding of the H.8-azurin or Laz to the PfMSPl-19 or PfMSPl-42 moieties
  • glutathione S-transferase GST
  • H.8-GST glutathione S-transferase
  • a fusion derivative H.8-GST where the H.8 epitope was fused in the N-terminal of GST was tested.
  • GST nor the H.8-GST bound PfMSPl-19 (Fig. IA) or MSPl-42 (Fig. IB), although H.8-GST showed a weak binding with MSPl-42.
  • Glutathione S transferase and some of the fusion proteins where parts of azurin were fused to GST (Yamada et al, Cell. Microbiol. 7:1418-1431 (2005), and Example 4) were tested for their ability to bind to MSP1-19.
  • the extent of parasitemia was determined using schizont stage parasites and normal red blood cells (RBC).
  • Normal red blood cells (RBCs) were washed twice in serum-free medium and resuspended to 10% hematocrit in complete RPMI. 200 ⁇ l of 10% hematocrit RBCs were added to each of 24 wells in addition to 300 ⁇ l complete RPMI without or with azurin, H.8-azurin or Laz at various concentrations.
  • Schizont stage P. falciparum parasites were prepared by centrifuging a late-stage culture through a Percoll cushion at 3200 rpm for 10 min. For infection, 4xlO 6 parasites per well in 500 ⁇ l volume were added at time zero. The plate was incubated overnight (about 16 h) and then scored by thin blood smear and Giemsa stain at that time.
  • azurin does not enter normal cells such as macrophages, mast cells, etc, and the effect of azurin, H.8-azurin or Laz is at the entry level rather than the intracellular replication of the parasite.
  • the data in Fig. 2 demonstrate the potential antimalarial action of azurin, H.8-azurin and Laz through interference in the invasion of the RBC by the parasites.
  • Example 7 Azurin binds ICAMs
  • a pharmaceutical comprising one or more cupredoxin and/or a cytochrome is administered to a patient.
  • the sterile pharmaceutical preparation is in the form of 0.5 ml single-dose ampules of sterile Pseudomonas aerugninosa azurin in a pharmaceutical preparation designed for intraveneous administration, as will be well known to those in the art.
  • the pharmaceutical preparation is stored at 4° C. and protected from light before administration.
  • azurin is prepared at five different concentrations: 10 ⁇ g, 30 ⁇ g, 100 ⁇ g, 300 ⁇ g and 800 ⁇ g azurin per 0.5 ml dose.
  • the pharmaceutical preparation is given intraveneously to thirteen volunteers for each 10 doses. Volunteers receive primary treatment at day 0 and subsequent doses identical doses at every other day for three weeks. Volunteers are observed for immediate toxic effects for twenty minutes after injection. Twenty-four and forty-eight hours later, they are examined for evidence of fever, local tenderness, erythema, warmth, induration and lymphadenopathy, and are asked about complaints of headache, fever, chills, malaise, local pain, nausea and joint pain. Before each dose, blood and urine samples are taken for full laboratory examination. Complete blood count and serum chemistry profiles are rechecked two days after each dose.
  • the presence of the malaria parasite are determined by light microscopic examination (ME) of the stained blood smears, or the ICT Malaria P.f.lP.v. test kits ( Binax, Inc., Portland, ME) . The results demonstrate the effectiveness of the therapy.
  • a transmissible genetic element that passes from one mosquito to another will be operably connected to the cupredoxin coding sequence operably connected to a constitutive promoter.
  • the P. aeruginosa azurin will therefore be produced inside the Anopheles gambiae infected with P. falciparum and will interfere with its replication/survival in the mosquito.
  • This mosquito will then be introduced to an endemic area so that the azurin- harboring genetic element will spread to other P. falciparum-infected A. gambiae mosquitoes, inhibiting P. falciparum growth or survival.
  • a malaria therapy comprising one or more cupredoxin and/or a cytochrome, for treatment of malaria infection in humans.
  • the sterile pharmaceutical preparation is in the form of 0.5 ml single-dose ampules of sterile P. aeruginosa azurin in a pharmaceutical preparation designed for intraveneous administration, as will be well known to those in the art.
  • the pharmaceutical preparation is stored at 4° C. and protected from light before administration.
  • P. aeruginosa azurin is prepared at five different concentrations: 10 ⁇ g, 30 ⁇ g, 100 ⁇ g, 300 ⁇ g and 800 ⁇ g azurin/cytochrome C 551 (1:1 on molecule basis) per 0.5 ml dose.
  • the pharmaceutical preparation is given intraveneously to thirteen volunteers for each 10 doses. Volunteers receive primary treatment at day 0 and subsequent doses identical doses at every other day for three weeks. Volunteers are observed for immediate toxic effects for twenty minutes after injection. Twenty-four and forty-eight hours later, they are examined for evidence of fever, local tenderness, erythema, warmth, induration and lymphadenopathy, and are asked about complaints of headache, fever, chills, malaise, local pain, nausea and joint pain. Before each dose, blood and urine samples are taken for full laboratory examination. Complete blood count and serum chemistry profiles are rechecked two days after each dose.
  • the presence of the malaria parasite are determined by light microscopic examination (ME) of the stained blood smears, or the ICT Malaria P.f./P.v. test kits ( Binax, Inc., Portland, ME) . The results demonstrate the effectiveness of the therapy.

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Abstract

L'invention concerne des cuprédoxines et des cytochromes et leur utilisation, conjointement ou séparément, pour inhiber la propagation d'une parasitémie dans les globules rouges d'un mammifère et dans d'autres tissus infectés par le parasite du paludisme, et en particulier la parasitémie des globules rouges humains par P. falciparum. L'invention concerne notamment des peptides isolés qui sont des variants, des dérivés ou des équivalents structurels de cuprédoxines ou du cytochrome c, ainsi que des compositions comprenant des cuprédoxines et/ou le cytochrome c, ou des variants, des dérivés ou des équivalents structurels de ceux-ci, ces peptides et ces compositions présentant une utilité pour traiter ou prévenir une infection paludéenne chez des mammifères. L'invention concerne en outre des méthodes de traitement de patients mammifères destinées à prévenir ou inhiber la progression d'une infection paludéenne chez des mammifères. L'invention concerne enfin des méthodes pour prévenir la progression d'une infection paludéenne chez des insectes vecteurs.
PCT/US2006/019492 2005-05-20 2006-05-19 Compositions et methodes de traitement du paludisme au moyen de cupredoxines et de cytochromes WO2006127477A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
MX2007014597A MX2007014597A (es) 2005-05-20 2006-05-19 Composiciones y metodos para el tratamiento de malaria con cupredoxina y citocromo.
EP06770687A EP1882039A4 (fr) 2005-05-20 2006-05-19 Compositions et methodes de traitement du paludisme au moyen de cupredoxines et de cytochromes
CA002607739A CA2607739A1 (fr) 2005-05-20 2006-05-19 Compositions et methodes de traitement du paludisme au moyen de cupredoxines et de cytochromes
AU2006251742A AU2006251742A1 (en) 2005-05-20 2006-05-19 Compositions and methods for treating malaria with cupredoxin and cytochrome
BRPI0612456-9A BRPI0612456A2 (pt) 2005-05-20 2006-05-19 composiÇÕes e mÉtodos para o tratamento da malÁria com cupredoxina e citocromo
JP2008512549A JP2009504567A (ja) 2005-05-20 2006-05-19 クプレドキシンおよびシトクロムでマラリアを治療するための組成物および方法
IL187161A IL187161A0 (en) 2005-05-20 2007-11-05 Compositions and methods for treating malaria with cupredoxin and cytochrome
NO20076390A NO20076390L (no) 2005-05-20 2007-12-11 Sammensetninger og medtoder for behandling av malaria med cupredoxin og cytokrom
AU2010201360A AU2010201360A1 (en) 2005-05-20 2010-04-06 Compositions and methods for treating malaria with cupredoxin and cytochrome

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US68281305P 2005-05-20 2005-05-20
US60/682,813 2005-05-20
US11/244,105 US7691383B2 (en) 2004-10-07 2005-10-06 Cupredoxin derived transport agents and methods of use thereof
US11/244,105 2005-10-06
US78086806P 2006-03-10 2006-03-10
US60/780,868 2006-03-10

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1883650A2 (fr) * 2005-05-20 2008-02-06 The Board Of Trustees Of The University Of Illinois Compositions et méthodes pour traiter une infection vih à l'aide de cuprédoxine et de cytochrome c
JP2009502797A (ja) * 2005-07-19 2009-01-29 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ 血管形成をキュプレドキシンで制御するための組成物および方法
US7491394B2 (en) 2001-02-15 2009-02-17 The Board Of Trustees Of The University Of Illinois Cytotoxic factors for modulating cell death
US8188251B2 (en) 2005-07-19 2012-05-29 The Board Of Trustees Of The University Of Illinois Transport agents for crossing the blood-brain barrier and into brain cancer cells, and methods of use thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7491394B2 (en) * 2001-02-15 2009-02-17 The Board Of Trustees Of The University Of Illinois Cytotoxic factors for modulating cell death
AU2002255649A1 (en) * 2001-03-02 2002-09-19 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Band 3 antigenic peptides, malaria polypeptides and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1882039A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7491394B2 (en) 2001-02-15 2009-02-17 The Board Of Trustees Of The University Of Illinois Cytotoxic factors for modulating cell death
US8545812B2 (en) 2004-10-07 2013-10-01 The Board Of Trustees Of The University Of Illinois Transport agents for crossing the blood-brain barrier and into brain cancer cells and methods of use thereof
US9309292B2 (en) 2004-10-07 2016-04-12 The Board Of Trustees Of The University Of Illinois Transport agents for crossing the blood-brain barrier and into brain cancer cells, and methods of use thereof
EP1883650A2 (fr) * 2005-05-20 2008-02-06 The Board Of Trustees Of The University Of Illinois Compositions et méthodes pour traiter une infection vih à l'aide de cuprédoxine et de cytochrome c
EP1883650A4 (fr) * 2005-05-20 2009-06-10 Univ Illinois Compositions et méthodes pour traiter une infection vih à l'aide de cuprédoxine et de cytochrome c
JP2009502797A (ja) * 2005-07-19 2009-01-29 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ 血管形成をキュプレドキシンで制御するための組成物および方法
US8188251B2 (en) 2005-07-19 2012-05-29 The Board Of Trustees Of The University Of Illinois Transport agents for crossing the blood-brain barrier and into brain cancer cells, and methods of use thereof

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AU2010201360A1 (en) 2010-04-29
CA2607739A1 (fr) 2006-11-30
IL187161A0 (en) 2008-02-09
NO20076390L (no) 2008-02-15
KR20080019615A (ko) 2008-03-04
MX2007014597A (es) 2008-01-21
AU2006251742A1 (en) 2006-11-30
EP1882039A4 (fr) 2008-08-20
JP2009504567A (ja) 2009-02-05
BRPI0612456A2 (pt) 2009-02-17
WO2006127477A3 (fr) 2007-03-08

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