MX2007014597A - Compositions and methods for treating malaria with cupredoxin and cytochrome. - Google Patents

Abstract

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. The invention provides isolated peptides that are variants, derivatives or structural equivalents of cupredoxins or cytochrome c, and compositions comprising cupredoxins and/or cytochrome c, or variants, derivatives or structural equivalents thereof, that are useful for treating or preventing malaria infection in mammals. Further, the invention provides methods to treat mammalian patients to prevent or inhibit the growth of malarial infection in mammals. The invention also provides methods to prevent the growth of malaria infection in insect vectors.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF MALARIA WITH CUPREDOXIN AND CYTOCHROME FIELD OF THE INVENTION The present invention relates to cupredoxin and cytochrome and their use, separately or in combination, to inhibit parasitaemia of the malaria parasite, and in particular to inhibit the parasitaemia of Plasmodium falciparum in red blood cells (erythrocytes). ) of mammals. The invention also relates to variants and derivatives of cupredoxin and cytochrome that maintain the ability to inhibit parasitaemia by the malaria parasite. Finally, the invention provides methods for inhibiting the spread of malaria infection in insect vectors.

BACKGROUND OF THE INVENTION About a quarter of the world population is exposed to the risk of malaria and more than one million people die of malaria each year. Of the four species of malaria parasites that infect humans, the two main species are Plasmodium falciparum and P. vivax. The merozoites of the blood stage of P. falciparum bind and parasitize erythrocytes using a variety of surface proteins (Cowman et al., FEBS Lett 476: 84-88 (2000); Baum et al. , J. Biol. Chem. 281: 5197-5208 (2006)), a major antigenic member of which is called Merozoite Superficial Protein 1 (MSP1), a 195 kDa protein. MSP1 is present in all Plasmodium invasive erythrocyte species, anchored on the surface of the merozoite by a glycosyl-phosphatidylinositol linkage. During the early stages of the erythrocyte invasion process, so soon after the release of the infected erythrocytes, the merozoite MSP1 protein undergoes a proteolytic cleavage, producing a C-terminal cutting product MSP1-42, which subsequently undergoes a second cut, producing an 11 kDa peptide MSP! -19, which remains attached to the surface of the parasite as it enters the erythrocyte. The formation of the cutting product MSP1-19 is very important for the successful invasion by the parasite since the inhibition of its proteolytic formation or its neutralization by monoclonal antibodies prevents the entry of the parasite to the erythrocytes (Blackman et al., J. Exptl., Med. 180: 389-393 (1994)). The peptide MSP1-19 is one of the most important candidates for the malaria vaccine available. The MSP1-19-specific antibodies of the human sera resistant to malaria react with the antigen and include a major inhibitory component of erythrocyte invasion (Holder &Riley, Parasitol, Today, 12: 173-174 (1996); 'Donnell et al , J. Expt. Med. 193: 1403-1412 (2001)). Serum from donors in malaria endemic regions often demonstrates a strong antibody reactivity towards Pf MSP1-19. (Nwuba et al., Infect.Immun. 70: 5328-5331 (2002)). The monoclonal antibody (mAb) G17.12 was formulated against the recombinant Pf MSP1-19 and recognizes its epitope on the surface of the parasite, demonstrating that this region of the antigen is accessible over the native MSP1 polypeptide complex (Pizarro et al. , J. Mol. Biol. 328: 1091-1103 (2003)). Interestingly, in vitro erythrocyte invasion experiments showed that infection is not inhibited in the presence of G17.12, even at a concentration of 200 μg / ml and G17.12 does not inhibit secondary processing of MSP1. . Id. The presence of antibodies that block the binding of inhibitory inhibition antibodies has also been demonstrated, thus facilitating the survival of parasites (Guevara Patino et al., J. Expt. Med. 186: 1689-1699 (1997). )), and may be responsible for the failure of G17.12 mAb to inhibit the invasion of erythrocytes by M. falciparum. Cerebral malaria, a rare but fatal infection restricted to P. falciparum invasion of brain capillaries due to sequestration of parasitized erythrocytes, is often untreatable because most drugs can not cross the blood-brain barrier to reach the capillaries of the brain. The adhesion of erythrocytes infected with P. falciparum to the capillaries of the brain is mediated by the interaction of the parasitic ligands of the Pf Emp-1 protein family expressed on the surface of erythrocytes infected with ICAM-1 and CD36 expressed on the surface of capillary endothelial cells in brain vessels. (Smith et al., Proc. Nati, Acad. Sci. USA 97: 1766-1771 (2000), Franke-Fayard et al., Proc. Nati, Acad. Sci. USA 102, 11468-11473 (2005)). Although a few drugs, such as chloroquine, which direct the heme detoxification route, are used to treat malaria, there is an increasing incidence of parasite resistance to the drugs and resistance of the mosquito vector to insecticides. Chloroquine antagonizes the polymerization of heme mediated by HRPs (proteins rich in histidine) induced by parasites, since heme monomers are highly toxic to malaria parasites. The polymerization of heme allows detoxification, which is reversed by chloroquine. Another drug, artemisinin, is effective against P. falciparum in cerebral malaria. Xa artemisinin forms adducts with heme bound to globulin in hemoglobin, which binds to HRPs to prevent polymerization of heme. There is an urgent need to find new drugs for this frightening disease that is particularly prevalent in Africa and Asia. The Current attempts at drug development are aimed at deciphering the complete parasite genome sequence, the molecular model of the malaria parasite proteins and a search for new drug targets.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to cupredoxin and cytochrome and their use, separately or together, to inhibit the propagation of parasitemia in red blood cells and other tissues infected by the malaria parasite, and in particular the parasitaemia of human red blood cells by P. falciparum. One aspect of the invention is an isolated peptide which is a variant, derivative or structural equivalent of a cupredoxin or cytochrome; and that it can inhibit the intracellular replication of a malaria parasite in the red blood cells of humans infected with malaria. Another aspect of the invention is an isolated peptide which is a variant, derivative or structural equivalent of a cupredoxin; and that it can bind a protein selected from the group consisting of PfMSPl-19 and PfMSPl-42. Another aspect of the invention is an isolated peptide which is a variant, derivative or structural equivalent of a cupredoxin or cytochrome and which can inhibit parasitaemia by malaria in infected red blood cells. with malaria. Specifically, the isolated peptide can inhibit parasitemia by malaria in the red blood cells of human infected with malaria. In some modalities, cupredoxin is an azurine, pseudoazurin, plastocyanin, rusticianin, Laz or auracyanin. Specifically, cupredoxin can be rusticianin, azurin or Laz. In some embodiments, 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 parahaemolyticus. Specifically, cupredoxin may be Thiobacillus ferrooxidans, Pseudomonas aeruginosa, Neisseria gonorrhea or Neisseria meningitidis. In other embodiments of this aspect, the cytochrome is cytochrome c or cytochrome f. In particular, cytochrome c can be from human or Pseudomonas aeruginosa. The cytochrome f can be from a cyanobacterium. In other embodiments of this aspect, the isolated peptide is a truncation of a peptide selected from the group consisting of SEQ ID NOS: 1-20 and 22. In some embodiments, SEQ ID NOS: 1-20 or 22 has at least minus 90% identity of the amino acid sequence with the isolated peptide sequence. In some embodiments of this aspect, the peptide isolated is a truncated cupredoxin or cytochrome. In some embodiments, the isolated peptide is greater than about 10 residues and not more than about 100 residues. The isolated peptide may comprise 36-89 azurine residues. Alternatively, the isolated peptide may consist of 36-89 azurine residues. Alternatively, the isolated peptide may comprise equivalent residues of a cupredoxin such as azurine 36-89. In other embodiments of this aspect, the isolated peptide is fused to an H.8 region of Laz. In another embodiment, the isolated peptide is a structural equivalent of the monoclonal antibody G17.12. Another aspect of the invention is a composition comprising at least one cupredoxin, cytochrome or isolated peptide which is a variant, derivative or structural equivalent of a cupredoxin or cytochrome that can inhibit malaria parasitaemia in red blood cells infected with malaria, in a pharmaceutical composition. Specifically, the pharmaceutical composition can be formulated for intravenous administration. The composition may comprise another anti-malaria drug or an anti-HIV drug. In some embodiments of the composition, cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisserla gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa or Vibrio parahaemolyticus.

Specifically, cupredoxin may be Thiobacillus ferrooxidans, Pseudomonas aeruginosa, Neisseria gonorrhea or Neisseria meningitidis. In some embodiments of the composition, the cytochrome is cytochrome c or cytochrome f. Specifically, cytochrome c can be from human or Pseudomonas aeruginosa. The cytochrome f can be from a cyanobacterium. In some embodiments, cupredoxin or cytochrome c is SEQ ID NOS: 1-20 or 22. Another aspect of the invention is a method for treating a patient suffering from an infection by a malaria parasite by administering to the patient an effective amount. of the composition of the invention. In specific modalities, the peptide inhibits parasitaemia by malaria in human red blood cells infected with malaria from the patient. In some modalities, the malaria parasite is Plasmodium vivax or Plasmodium falciparum. In some modalities, the patient is suffering additionally from HIV infection. In some embodiments, the composition is administered with a second composition that may contain an anti-malaria drug and / or an anti-HIV drug. In some embodiments, the composition of the invention is administered within 0 minutes to 12 hours after administration of the second composition. In some embodiments, the composition of the invention is administered to the patient orally, by inhalation, intravenously, intramuscularly or subcutaneously, and, specifically, the composition can be administered to the patient intravenously. Another aspect of the invention is a method for treating 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 for preventing malaria in mammals, which comprises administering to an insect vector in a population of insect vectors harboring a malaria parasite, an amount of the composition of the invention. In some embodiments of this method, the composition is administered to the insect vector orally. These and other aspects, advantages and characteristics of the invention will become apparent from the following figures and the detailed description of the specific modalities.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Figure 1 represents the titrations of the surface plasmon resonance junction representing the interactions of the constructions of Azurin, H.8- azurin (H.8-Az), Laz and GST-azurin (GST-Azu) with MSP1-19 and MSP1-42. (A) The binding curves demonstrating the interactions of azurine and its analogs with MSP1-19 immobilized on gold detector circuits coated with carboxymethyldextran (MSP1-19-CM5). The concentration-dependent binding of the azurine proteins to MSP1-19 to MSP1-19 was determined by means of an injection of several concentrations (0.05-300 nM) on the surface of the detector and the degree of binding was evaluated as a function of the response value of the resonance in equilibrium measured in resonance units (RU). While H.8-Az and Laz bound much more strongly than azurine, no binding was observed with GST or H.8-GST. (B) In vitro binding titers for MSP1-42 immobilized with azurine and its analogues were followed in a manner similar to that of MSP1-19 as shown in (A). The relative union affinities were determined by adjusting the data to Req = Rmax / (1+ (Kd / C)) with curve adjustments connecting the data points in the graphs. The Kd binding values MSP1-19 are: 32.2 ± 2.4 nM (azurine), 26.2 ± 2.4 nM (Laz), 11.8 ± 0.3 nM (H.8-Az), and those of the binding of MSP1-42 are: 54.3 ± 7.6 nM (azurine), 45.6 ± 2.4 nM (Laz) and 14.3 ± 1.7 nM (H.8-Az). (C) The binding titers for the interactions of the GST-Azu fusion proteins on the surface of the MSP1-19-CM5 detectors demonstrate the recognition of GST-Azu 36-128 and GST-Azu 36-89 with MSP1-19. No binding was observed with GST or GST-Azu 88-113.

Figure 2. Figure 2 depicts the inhibition of parasitaemia by P. falciparum (parasite growth within RBC) by different concentrations, as shown, of Azurin, H.8-azurin (H.8-Az) and Laz. In these experiments, normal red blood cells were infected with schizonts in the absence or presence of the proteins at different concentrations, were incubated overnight and the number of intracellular parasites was recorded by thin blood smears and Gie sa staining.

Figure 3. Figure 3 represents the surface plasmon resonance binding curves for the binding of ICAMs (ICAM-1, ICAM-2, ICAM-3 and NCAM, box) with immobilized azurine. Because the large non-specific junction to the detected microcircuit Au-CM5, CM5 was added as an eluent to the run buffer (1 mg / ml of CM5 to the HBS-EP buffer). The selective recognition of azurine with ICAM-3, but not with ICAM-1 or ICAM-2, is remarkable and the concentration of the binding was 19.5 ± 5.4 nM. The Kd for the binding of NCAM with azurine, as shown in the box, was 20 ± 5.0 nM.

BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 is the azurine amino acid sequence of Pseudomonas aeruginosa. SEQ ID NO: 2 is the amino acid sequence of cytochrome C551 of Pseudomonas aeruginosa. SEQ ID NO: 3 is the Laz amino acid sequence of MC58 from Neisseria meningitidis. SEQ ID NO: 4 is the plastocyanin amino acid sequence of Phormidium laminosum. SEQ ID NO: 5 is the amino acid sequence of rusticianin from Thiobacillus ferrooxidans (Acidithiobacillus ferrooxidans). SEQ ID NO: 6 is the amino acid sequence of pseudoazurin from Achromobacter cycloclastes. SEQ ID NO: 7 is the azurine amino acid sequence of Alcaligenes faecalis. SEQ ID NO: 8 is the azurine amino acid sequence of Achromobacter xylosoxidans ssp. denitrif icans I. SEQ ID NO: 9 is the azurine amino acid sequence of Bordetella bronchiseptica. SEQ ID NO: 10 is the azurine amino acid sequence of Methylomonas sp. J. SEQ ID NO: 11 is the azurine amino acid sequence of Neisseria meningitidis Z2491. SEQ ID NO: 12 is the amino acid sequence of Pseudomonas fluorescens azurine. SEQ ID NO: 13 is the azurine amino acid sequence of Pseudomonas chlororaphis. SEQ ID NO: 14 is the azerine amino acid sequence of Xylella annoying 9a5c SEQ ID NO: 15 is the amino acid sequence of stellacyanine 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 the basic cucumber protein of Cucumis sativus. SEQ ID NO: 19 is the amino acid sequence of cytochrome c d Homo sapiens. SEQ ID NO: 20 is the amino acid sequence of cytochrome f of the cyanobacterium 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 for PCR amplification of the gene encoding Laz (laz) of Neisseria gonorrhoeae. SEQ ID NO: 24 is the reverse primer to amplify by PCR the gene encoding Laz (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 fragment of pucl8-laz. SEQ ID NO: 27 is the forward primer to PCR amplify a 0.4 kb fragment of pUC19-peace. SEQ ID NO: 28 is the reverse primer to PCR amplify a 0.4 kb fragment of pUC19-peace. 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 SEQ ID NO: 36 is an oligonucleotide for site-directed mutagenesis for the preparation of pGST-azu 88-113.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the term "cell" includes the singular or plural of the term, unless specifically described as a "simple cell." As used herein, the terms " "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical analogue of an amino acid The terms also apply to naturally occurring amino acid polymers The terms "polypeptide", "peptide" and "protein" are also included in the modifications, including, but not limited to, glycosylation, lipid binding, sulfation, gamma carboxylation of the glutamic acid residues, hydroxylation and ribosylation of ADP. It will be appreciated that the polypeptides are not always completely linear. For example, polypeptides can be branched as a result of ubiquity and can be circular (with or without branching), in general, as a result of post-translation events, which include the event of natural processing and events carried out by human manipulation that do not occur naturally. The circular, branched and branched circular polypeptides can be synthesized by the natural process without translation and also by completely synthetic methods. As used herein, the term "pathological condition" includes anatomical and physiological deviations from normal, which constitute damage to the normal state of the living animal or one of its parts, which interrupts or modifies the performance of bodily functions, and it is a response to different factors (such as malnutrition, industrial risks or weather), to specific infectious agents (such as worms, protozoan parasites, bacteria or viruses), to inherent defects of the organism (such as genetic anomalies) or combinations of these factors. As used herein, the term "condition" includes anatomical and physiological deviations from normal, which constitute damage to the normal state of the living animal or one of its parts, which interrupts or modifies the performance of bodily functions. As used herein, the term "suffering from" includes that it currently exhibits the symptoms of a condition pathological, which has a pathological condition even without observable symptoms, in the recovery of a pathological condition, or recovered from a pathological condition. As used herein, the term "parasitaemia" 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. As used herein, the term "parasitemia inhibition" refers to a decrease or reduction in the rate of increase or presence of the parasite in the blood of a mammal. Inhibition is any decrease or reduction in the rate of increase that is statistically significant compared to the control treatments. As used in this, the term "treatment" includes prevention, reduction, interruption or reversal of the progression or severity of the condition or symptoms associated with a condition being treated. As such, the term "treatment" includes medical, therapeutic and / or prophylactic administration, as appropriate. As used herein, "anti-malaria activity" includes any activity that decreases infectivity, reproduction or inhibits the progression of the life cycle of a malaria parasite. "Anti-malaria activity" includes inhibiting the growth of malaria infection by all means observed with anti-malaria drugs current As used herein, the term "antimalarial drug" refers to drugs with anti-malarial activity that can be used to decrease infectivity, reproduction or inhibit the progression of the life cycle of a malaria parasite. As used herein, the term "anti-HIV drug" refers to drugs with anti-HIV activity by which HIV infection is decreased, or the increase in the human body is prevented, by any means, including , but is not limited to, inhibition of HIV genome replication, inhibition of synthesis and / or assembly of HIV-coated proteins and inhibition of HIV entry into uninfected cells. The term "substantially pure", when used to modify the term of 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 not adulterated by, active inhibitory agents. The term "substantially pure" refers to a compound in an amount of at least about 75% dry weight, of the isolated fraction, or "75% substantially pure." More specifically, the term "substantially pure" refers to a compound of at least about 85% by weight dry, active compound, or "85% substantially pure". More specifically, the term "substantially pure" refers to a compound of at least about 95% dry weight, active compound, or "95% substantially pure". The substantially pure cupredoxin or cytochrome C551 or a variant thereof can be used in combination with one or more other substantially pure compounds, or another cupredoxin or isolated cytochrome. The phrases "isolated", "purified" or "biologically pure" refer to the material that is substantially or essentially free of the components that normally accompany the material as it is in its native state. In this way, the peptides isolated according to the invention, preferably do not contain the materials normally associated with the peptides in their in situ environment. An "isolated" region refers to a region that does not include the entire sequence of the polypeptide from which the region was derived. An "isolated" nucleic acid, protein or respective fragment thereof has been substantially removed from its environment in vivo, so that it can be manipulated by the skilled person, such as, but not limited to, nucleotide sequencing, restriction digestion, mutagenesis of directed site and subcloning in the expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in substantially pure amounts. The term "variant" as used herein with respect to a peptide, refers to variants of the amino acid sequence that may have amino acids replaced, deleted or inserted compared to the wild-type polypeptide. The variants can be truncations of the wild-type peptide. In this way, a variant peptide can be made by manipulation of the genes encoding the polypeptide. A variant can be performed by altering the basic composition or the characteristics of the polypeptide, but not at least some of its fundamental activities. For example, a "variant" of azurine can be a mutated azurine that maintains its ability to inhibit parasitaemia in red blood cells of humans infected with malaria. In some cases, a variant peptide is synthesized with non-natural amino acids, such as e- (3,5-dinitrobenzoyl) -Lys residues. (Ghadiri &Fernholz, J. Am. Chem. Soc., 112: 9633-9635 (1990)). In some embodiments, the variant has no more than 20 amino acids replaced, deleted or inserted, compared to the wild-type peptide. In some embodiments, the variant has no more than 15, 14, 13, 12 or 11 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some modalities, the variant does not have more than 10, 9, 8 or 7 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 6 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 5 or 4 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 3, 2 or 1 amino acids replaced, deleted or inserted compared to the wild-type peptide. The term "amino acid" as used herein, means an amino acid radical comprising any synthetic or naturally occurring or non-naturally occurring amino acid residue, i.e., any radical comprising at least one carboxyl and at least one amino acid residue directly linked by one, two, three or more carbon atoms, typically a carbon atom (). The term "derivative" as used herein with respect to a peptide refers to a peptide that is derived from the peptide of interest. A derivation includes the chemical modifications of the peptide, so that the peptide still maintains some of its fundamental activities. For example, a "derivative" of azurine can be a chemically modified azurine that maintains its ability to inhibit parasitemia in red blood cells infected with malaria. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, modification of polyethylene glycol (PEG), phosphorylation or glycosylation of the peptide. In addition, a derivatized peptide may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as, but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe. The term "percent (%) identity of the amino acid sequence" is defined as the percentage of amino acid residues in a polypeptide that are identical with the amino acid residues in a candidate sequence when the two sequences are aligned. To determine the% amino acid identity, the sequences are aligned and, if necessary, spaces are introduced to achieve the maximum% identity of the sequence; Conservative substitutions are not considered part of the identity of the sequence. Alignment procedures of the amino acid sequence to determine percent identity are well known to those skilled in the art. Often the publicly available computer programming elements (software), such as the BLAST, BLAST2, ALIGN2 or Megalign programming elements (DNASTAR) are used to align the peptide sequences. In a specific modality, it is used Blastp (available from the National Center for Biotechnology Information, Bethesda MD) using the missing parameters of long complexity filter, expected 10, word size 3, existence 11 and extension 1. When the amino acid sequences are aligned,% identity of the amino acid sequence of a given amino acid sequence A to, with or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain% identity of the amino acid sequence towards , with or against a given amino acid sequence B) can be calculated as:% identity of the amino acid sequence = X / Y * 100 where X is the number of amino acid residues recorded as identical matches by the alignment of the program or the sequence alignment algorithm of A and B and Y is the total number of amino acid residues in B. If the length of the amino acid sequence A does not is equal to the length of the amino acid sequence B, the% identity of the amino acid sequence from A to B will not be equal to the% identity of the amino acid sequence from B to A. When comparing larger sequences with sequences shorter, the shorter sequence will be sequence "B", unless otherwise stated. For example, when compare truncated peptides with the corresponding wild type polypeptide, the truncated peptide will be the "B" sequence. A "therapeutically effective amount" is an amount effective to prevent or diminish the development of, or to alleviate partially or totally the existing symptoms in a particular, pathological or other condition, for which the patient is being treated. The determination of a therapeutically effective amount is well within the ability of those skilled in the art.

Overview The present invention provides compositions and methods using cupredoxin and / or cytochrome to inhibit the parasitaemia of red blood cells infected with malaria and body tissues, such as brain tissue and bone tissue. Previously it was known that the different bacterial redox proteins that belong to the family of blue copper-containing proteins called cupredoxins, or iron (heme) - which contain proteins called cytochromes, enter mammalian cells, including cancer cells and induce death apoptotic cell or cause the inhibition of growth through the interruption Gl of the cell cycle. (Yamada et al., Cell Cycle 3: 752-755 (2004); Yamada et al. , Cell Cycle 3: 1182-1187 (2004)). Simple bacterial proteins, such as cupredoxin azurine or cytochrome C551, made by Pseudomonas aeruginosa, can demonstrate an activity based on their hydrophobicity. Thus, wild-type azurin induces apoptosis in murine J774 cells (Yamada et al., Infection and Immunity 70: 7054-7062 (2002)) whereas a mutant M44KM64E azurine causes cell cycle inhibition in the Gl phase in J774 cells. (Yamada et al., PNAS 101: 4770-4775 (2004)). In contrast, cytochrome C551 causes inhibition of the cell cycle in the Gl phase in J774 cells while a cytochrome C551 mutant V23DI59E induces apoptosis. (Hiraoka et al., PNAS 101: 6427-6432 (2004)). In accordance with the present invention, it is now surprisingly known that cupredoxins and cytochromes will inhibit parasitaemia in vitro in human blood red blood cells by the malaria parasite Plasmodium falciparum. In particular, azurine and Laz cupredoxins inhibit parasitaemia in P. falciparum by approximately 50% and approximately 75% respectively. See, Example 6. In addition, rusticianin and cytochromes c and f inhibited parasitemia by 20-30%. See, Example 1. In addition, it is now known that azurine 'has a discernable structural homology with the Fab fragment of the monoclonal antibody of mouse G17.12 when complexed to the Pf MSP1-19 fragment of the surface protein MSP1 of P. falciparum. See, Example 2. While the mode of inhibition is not limited to any of these means, it is thought that azurine can inhibit the parasitaemia of P. falciparum by interaction with the MSP1 protein on the surface of the parasite. Surprisingly, it is now known that azurine and Laz bind the surface proteins of P. falciparum PfMSPl-19 and PfMSPl-42 in vitro. In addition, it is now known that azurine amino acid residues 36-89 are required for binding to PfMSPl-19 and PfMSPl-42. In addition, it is now known that the H.8 domain of Laz from N. gonorrhea increases the binding of an azurine fused to PfMSPl-19 as well as the inhibition of parasitemia by P. falciparum. See, Examples 5 and 6. Due to the high structural homology between cupredoxins, it is contemplated that other cupredoxins will have the same anti-malaria activity as azurine, rusticianin or Laz. In some embodiments, cupredoxin is, but is not limited to, azurine, pseudoazurin, plastocyanin, auracyanin, Laz or rusticianin. In the specific modalities, cupredoxin is Laz, azurine or rusticianina. In other modalities, cupredoxin is a pathogenic bacterium. In a more specific modality, cupredoxin is azurine. In particularly specific modalities, azurine is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrif icans I, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis Z2491, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa 9a5 or Vibrio parahaemolyticus. In a more specific modality, azurine is from P. aeruginosa. In other specific embodiments, the cupredoxin comprises an amino acid sequence which is SEQ ID NO: 1, 2-18 or 21. In accordance with the present invention, it has been learned that cytochrome C551 from P. aeruginosa, cytochrome c human and the cytochrome f of Phormidium laminosum will inhibit the parasitaemia in the red blood cells of human infected with malaria. In a specific embodiment, the cytochrome is cytochrome C551 from P. aeruginosa, cytochrome c or human cytochrome f. In other specific embodiments, the cytochrome comprises an amino acid sequence that is SEQ ID NO: 2, 19 or 20. Due to the structural homology between cytochrome c, it is contemplated that other cytochromes will have the same anti-malaria activity as cytochrome C551 of P. aeruginosa and human cytochrome c. In some modalities, cytochrome is a pathogenic bacterium. In another specific embodiment, cytochrome inhibits parasitism in red blood cells infected with malaria, and more specifically, human blood red blood cells. In another specific modality, cytochrome inhibits the progression of the cycle cell in a mammalian cancer cell and more specifically in a J774 cell.

Compositions of the invention The invention provides peptides that are variants, derivatives or structural equivalents of cupredoxin or cytochrome. In some embodiments, the peptide is substantially pure. In other embodiments, the peptide is isolated. In some embodiments, the peptide is less than a total length of cupredoxin or cytochrome and retains some of the functional characteristics of cupredoxin or cytochrome. In some embodiments, the peptide maintains the ability to inhibit parasitaemia in red blood cells infected with malaria and more specifically, the ability to inhibit P. falciparum infection in human red blood cells. In specific embodiments, the cytochrome is cytochrome C551 from P. aeruginosa, human cytochrome c or cytochrome f cyanobacteria, and specifically SEQ ID NOS: 2, 19 and 20. In another specific embodiment, the peptide does not originate to an immune response in a mammal and more specifically a human. The invention also provides compositions comprising at least one peptide which 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 which is a cupredoxin or a variant, derivative or structural equivalent of a cupredoxin. The invention also provides compositions comprising at least one peptide that is a cytochrome, or a 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 which is a cupredoxin, cytochrome, or a variant, derivative or structural equivalent of a cupredoxin or cytochrome in a pharmaceutical composition. Due to the high structural homology between the cupredoxins, it is contemplated that other cupredoxins will have the same anti-malaria activity as the azurine of Pseudomonas aeruginosa with respect to the inhibition of parasitaemia in red blood cells infected with malaria. In some embodiments, cupredoxin is, but is not limited to, azurine, pseudoazurin, plastocyanin, rusticianin, Laz, or auracyanin. In the particularly specific modalities, cupredoxin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis Z2491, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa 9a5 or Vibrio parahaemolyticus. In a very specific modality, cupredoxin is the azure of Pseudomonas aeruginosa. In other specific embodiments, cupredoxin comprises an amino acid sequence that is SEQ ID NO: 1, 3-18 or 22. In other specific embodiments, cupredoxin is the Laz protein of Neisseria meningitidis or Neisseria gonorrhea. The invention provides variants of the amino acid sequence of a cupredoxin or cytochrome having the amino acids replaced, deleted or inserted compared to the wild-type polypeptide. The variants of the invention may be truncations of the wild-type polypeptide. In some embodiments, the composition comprises a peptide that consists of a region of a cupredoxin or cytochrome that is less than the total length of the wild-type polypeptide. In some embodiments, the composition comprises a peptide consisting of more than about 10 residues, more than about 15 residues, or more than about 20 residues of a cupredoxin or truncated cytochrome. In some embodiments, the composition comprises a peptide consisting of no more than about 100 residues, no more than about 50 residues, no more than about 40 residues, or no more than about 30 residues of a cupredoxin or truncated cytochrome. In some embodiments, the 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% identity of the amino acid sequence, at least about 95% of identity of the amino acid sequence or at least about 99% identity of the amino acid sequence. In specific embodiments, the variant of cupredoxin comprises residues 36-89 of azurine from P. aeruginosa. In other embodiments, the cupredoxin variant consists of residues 36-89 of azurine from P. aeruginosa. In other specific embodiments, the variant consists of the equivalent residues of a cupredoxin in addition to azurine. It is contemplated that other variants of cupredoxin having similar activity to azurine residues 36-89 can be designed. To do this, the amino acid sequence of the cupredoxin of interest will be aligned to the azurine sequence of Pseudomonas aeruginosa using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located in the azurine amino acid sequence of P. aeruginosa , and the equivalent residues found in the cupredoxin sequence of interest, and the equivalent residues of cupredoxin in this identified manner. Variants also include elaborate peptides with synthetic amino acids that do not occur naturally. For example, amino acids that do not occur naturally can be integrated into the variant peptide to prolong or optimize the half-life of the composition in the bloodstream. These variants include, but are not limited to, D, L-peptides (diastereomer), (Futaki et al., J. Biol. Chem. 276 (8): 5836-40 (2001); Papo et al., Cancer Res. 64 (16): 5779-86 (2004), Miller et al, Biochem Pharmacol 36 (1): 169-76, (1987)); peptides containing the usual amino acids (Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), and the incorporation of non-natural amino acids containing olefin followed by the hydrocarbon ring (Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al., Science 305: 1466-1470 (2004)) and peptides comprising e- (3, 5) residues. dinitrobenzoyl) -Lys. In other embodiments, the peptide of the invention is a derivative of a cupredoxin or cytochrome. The cupredoxin or cytochrome derivatives are chemical modifications of the peptide, so that the peptide still maintains some of its fundamental activities. For example, a "derivative" of azurine can be a chemically modified azurine that maintains its ability to inhibit malaria parasitaemia in mammalian cells. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, modification of polyethylene glycol (PEG), phosphorylation and glycosylation of the peptide. In addition, a derivatized peptide may be a fusion of a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof to a chemical compound, such as, but not limited to, another peptide, drug molecule or other therapeutic agent or pharmaceutical or a detectable probe. Derivatives of interest include chemical modifications by which the half-life in the blood stream of the peptides and compositions of the invention can be extended or optimized, such as by the various methods well known to those skilled in the art, including, but they are 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)), N- and C-terminal modifications (Labrie et al., Clin. Invest. Med. 13 (5): 275-8, (1990)), and the incorporation of an unnatural amino acid containing olefin followed by the hydrocarbon ring (Schaf eister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al., Science 305: 1466-1470 (2004)). In one embodiment, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, is fused to an H.8 region of Laz of Neisseria meningitidis or Neisseria gonorrhea. An example of such a peptide is the H.8-Peace fusion protein described in Example 4. In a modality specific, H.8 is fused to the C-terminus of the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof. In another embodiment, region H.8 is SEQ ID NO: 21, or a variant, derivative or structural equivalent thereof. It is contemplated that the peptide of the composition of the invention may be more than one variant, derivative and structural equivalent of a cupredoxin or cytochrome. For example, the peptide may be a truncation of the azurine that has been PEGylated, thereby making it a variant and a derivative. In one embodiment, the peptides of the invention are synthesized with non-natural, α-disubstituted amino acids containing olefin-bearing linkages, followed by a ruthenium-catalyzed "ring" of hydrocarbon by metathesis of olefin. (Scharmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000); Walensky et al. , Science 305: 1466-1470 (2004)). In addition, peptides that are structural equivalents of azurine can be fused to other peptides, thereby making a peptide that is an equivalent and a structural derivative. These examples are exclusively to illustrate and not to limit the invention. The variants, derivatives or structural equivalents of cupredoxin or cytochrome may or may not bind copper. In another embodiment, the peptide may be a structural equivalent of a cupredoxin or cytochrome. Examples of Studies that determine significant structural homology between cupredoxins and cytochromes and other proteins include Toth et al. (Developmental Cell 1: 82-92 (2001)). Specifically, the significant structural homology between a cupredoxin or cytochrome and its structural equivalents is determined using the VAST algorithm (Gibrat et al., Curr Opin Struct Biol 6: 377-385 (1996), Madej et al., Proteins 23: 356- 3690 (1995)). In specific embodiments, the VAST p-value of 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. In other embodiments, the homology Significant structural significance between a cupredoxin or cytochrome and its structural equivalents is determined using the DALÍ algorithm (Holm &Sander, J. Mol.Biol. 233: 123-138 (1993).) In the specific modalities, the DALY Z record for a structural comparison of pairs is at least about 3.5, at least about 7.0 or at least about 10.0 In another embodiment, the variant or derivative of cupredoxin has significant structural homology to the Fab fragment of mouse monoclonal antibody G17. 12. An example of how this structural similarity can be determined can be found in Example 3. Specifically, the Significant structural homology between a cupredoxin and the Fab fragment of the mouse monoclonal antibody G17.12 can be determined using the VAST algorithm (Gibrat et al., id .: Madej et al., id.). In specific embodiments, the VAST p-value of a structural comparison of a cupredoxin to the Fab fragment of the mouse monoclonal antibody G17.12 may be less than about 10 ~ 4, less than about 10"5, less than about 10" 6. or less than about 10-7. In other specific embodiments, the VAST registry of a structural comparison of a cupredoxin to the Fab fragment of the mouse monoclonal antibody G17.12 can be greater than about 9, greater than about 10, greater than about 11 or greater than about 12. In some modalities, the variant, derivative or structural equivalent of it has some of the functional characteristics of the azurine of P. aeruginosa, cytochrome c55? of P. aeruginosa, cytochrome c of human or cytochrome f cyanobacteria. In a specific embodiment, the peptide of the invention inhibits parasitaemia by malaria in red blood cells infected with malaria, and more specifically parasitaemia by P. falciparum in the red blood cells of human infected with P. falciparum. The invention also provides the variants, derivatives and structural equivalents of cupredoxin and cytochrome C551 that maintain the ability to inhibit parasitemia in red blood cells infected with malaria, and more specifically parasitaemia by red blood cells of human infected with P. falciparum. The inhibition of parasitaemia by red blood cells of human infected with P. falciparum can be determined by the method described in Example 6. Because it is now known that cupredoxins and cytochrome can inhibit parasitaemia in infected red blood cells. With malaria, it is now possible to design variants, derivatives and structural equivalents of cupredoxins and cytochrome that maintain this antimalarial activity. These variants and derivatives can be made, for example, by creating a "library" of different variants and derivatives of cupredoxins and cytochromes, and then testing each for anti-malarial activity using one of the many methods known in the art, such as the representative method in Example 6. It is contemplated that the different variants, derivatives and structural equivalents of the cupredoxins and cytochromes with anti-malaria activity may be used in the methods of the invention, instead of, or in addition to the cupredoxins and cytochromes mentioned in the present. This method for selecting variants and derivatives can be adapted for any of the activities of P. aeruginosa azurine, cytochrome C551 of P. aeruginosa, human cytochrome c or cytochrome f cyanobacteria described herein. In other embodiments, the peptide of the invention inhibits the intracellular replication of the malaria parasite in human blood cells. Methods for determining intracellular replication of the malaria parasite are well known in the art, and one such method is described in Example 2. In some embodiments, the peptide of the invention binds to the surface proteins of P. falciparum PfMSPl-19 and / or PfMSPl-42 with a relative binding affinity that is statistically greater than a non-binding control protein. A peptide can be tested for this activity using surface plasmon resonance analysis as described in Example 5. Other methods for determining whether a protein binds to another are well known in the art and can also be used. In another embodiment, the peptide of the invention binds to ICAM-3 or NCAM with a relative binding affinity that is statistically greater than a non-binding control protein. A peptide can be tested for this activity using surface plasmon resonance analysis as described in Examples 7 and 5. Other methods for determining whether a protein binds to each other are well known in the art and can also be used.

In some specific embodiments, 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 can be observed by the ApoAlert ™ confocal microscope using a MITOSENSOR ™ APOLERT ™ Mitochondrial Membrane Kit (Clontech Laboratories, Inc., Palo Alto, California, USA), measuring the activity of caspase-8, caspase-9 and caspase-3 using the method described in Zou et al. (J. Biol. Chem. 274: 11549-11556 (1999)), and detecting fragmentation of nuclear DNA that induces apoptosis using, for example, the APOLERT ™ DNA fragmentation kit (Clontech Laboratories, Inc., Palo Alto , California, USA). In another specific embodiment, the peptide of the invention induces the disruption of cell growth in a mammalian cancer cell, more specifically a Jll cell. The disruption of cell growth can be determined by measuring the degree of progression of the cell cycle, such as the method found in Yamada et al. (PNAS 101: 4770-4775 (2004)). In another specific embodiment, the peptide of the invention inhibits the progression of the cell cycle in a mammalian cancer cell, more specifically a J774 cell.

Cupredoxins These small, blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or are of unknown function. The copper ion is bound exclusively by the protein matrix. A distorted trigonal plane array, special to two ligands of histidine and one of cysteine around copper, gives rise to electronic properties very peculiar to the metallic site and an intense blue color. A number of cupredoxins have been characterized in a crystallographic manner at medium to high resolution. In general, cupredoxins have a low sequence homology, but a high structural homology. (Gough &Clothia, Structure 12: 917-925 (2004); De Rienzo et al., Protein Science 9: 1439-1454 (2000)). For example, the amino acid sequence of azurine is 31% identical to that of auracyanin B, 16.3% to that of rusticianin, 20.3% to that of plastocyanin and 17.3% to that of pseudoazurin. See Table 1. However, the structural similarity of these proteins is more pronounced. The p value of VLAST for the comparison of the structure of azurine to auracyanin B is 10 ~ 7'4, azurine to rusticianin is 10 ~ 5, azurine to plastocyanin is 10 ~ 5'6 and azurine to the pseudoazurin is 10"4-1.

All cupredoxins have a beta sandwich or beta barrel of eight-strand Greek key and have a highly conserved site architecture. (De Rienzo et al., Protein Science 9: 1439-1454 (2000)). A prominent hydrophobic connection, due to the presence of many long-chain aliphatic residues such as methionines and leucines, is present around the copper site in azurines, amycinins, cyanobacterial plastocyanins, cucumber basic protein and to a lesser extent, pseudoazurin and the eukaryotic plastocyanins. Id. Hydrophobic connections are also found to a lesser degree in stellacyanine and rusticianine copper sites, but have different characteristics. Id.

Table 1. Sequence and structural alignment of azurine (1JZG) from P. aeruginosa to other proteins using the VAST algorithm.

PDB Length% of value Register RMSD4 Description of identity P2 alignment1 aa 1AOZ A 2 82 18. 3 10 12.2 1. 9 Ascorbate oxidase 1QHQ A 113 31 10 -7. 12 eleven . 9 AuracyaninB PDB Length% of value Register RMSD4 Description of identity P2 alignment1 aa 1V54 B 1 79 20.3 10"11.2 2.1 Cytochrome c oxidase 1GY2 A 92 16.3 10 -5.0 11.1 1.8 Rusticianina 3MSP A 74 8.1 10 -6.7 10.9 2.5 Main motile sperm protein5 1IUZ 74 20.3 10 -5.6 10.3 2.3 Plastocyanin 1KGY E 90 5.6 10 ~ 4 * 6 10.1 3.4 Efrinab2 1PMY 75 17.3 10"4 * 1 9.8 2.3 Pseudoazurin • "• Lined length: The number of equivalent pairs of alpha C atoms superimposed between the two structures, that is, how many residues have been used to calculate the 3D overlay 2P-VAL: The p-value of VAST is a measure of the meaning of the comparison, expressed as a probability For example, if the p-value is 0.001, then the probabilities are 1000 to 1 against the observation of a pair of this quality by a pure probability. p of 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 corresponds, in this way, to the value of p for the comparison of pairs of each domain pair, divided by 500. 3Registro: Record of similarity of structure VAST. This number is related to the number of superimposed secondary structural elements and the quality of such superposition. Higher VAST records correlate with a greater similarity. 4RMSD: The residual of the superposition of the root of the square mean in Angstroms. This number is calculated after the optimal superposition of two structures, such as the square root of the distances of the square average between the equivalent alpha C atoms. Note that the scales of the RMSD value with the degree of structural alignments and that this size should be taken into consideration when using RMSD as a descriptor of global structural similarity. 5The main sperm protein of C. elegans was shown to be an erythrin antagonist in the maturation of oocytes (Kuwabara, 2003"The multifaceted C. elegans major sperm protein: an ephrin signaling antagonist in oocyte maturation" Genes and Development, 17 : 155-161 Azurine Azurines are proteins that contain copper of 128 amino acid residues that belong to the family of cupredoxins involved in the transfer of electrons in plants and some bacteria. The azurines include those of P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans (SEQ ID NO: 8). (Murphy et al., J. Mol. Biol. 315: 859-871 (2002)). The identity of the amino acid sequence between the azurines varies between 60-90%, these proteins showed a strong structural homology. All azurines have a ß sandwich radical with a Greek key and a single copper atom is always placed in the same region of the protein. In addition, azurines have an essentially neutral hydrophobic connection that surrounds the copper site. Id.

Plastocyanins 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 the white poplar plastocyanin in 1978, the structure of the algae plastocyanins (Scenedesmus, Enteromorpha, Chlamydomonas) and plants (French bean) has been determined by NMR or crystallographic methods, and the white poplar structure has been refined at a resolution of 1.33 Á. SEQ ID NO: 4 shows the amino acid sequence of plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium. Despite the sequence divergence between the algal plastocyanins and vascular plants (eg, 62% sequence identity between the Chlamydomonas and the white poplar proteins), the three-dimensional structures are preserved (e.g., 0.76 A deviation). rms in the positions of C alpha between Chlamydomonas and Poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end and a pronounced negative connection, eight-barrel beta-antiparallel barrel, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and negative and hydrophobic connections are proposed that are involved in the recognition of physiological reaction partners. The experiments of chemical modification, cross-linking and site-directed mutagenesis have confirmed the importance of negative and hydrophobic connections in the binding interactions with cytochrome f, and have validated the model of two electron transfer connections' functionally significant involving plastocyanin . A The putative electron transfer pathway is relatively short (approximately 4A) and involves the copper ligand exposed to solvent His-87 in the hydrophobic connection, while the other is longer (approximately 12-15A) and involves the residue almost conserved Tyr-83 in the negative connection, Redinbo et al. , J. Bioenerg. Biomembr. 26: 49-66 (1994).

Rusticianins Rusticianins are single chain polypeptides containing blue copper obtained from a Thiobacillus (now called Acidithiobacillus). The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidative cupredoxin rusticianin of Thiobacillus ferrooxidans (SEQ ID NO: 5) has been determined by anomalous multi-wavelength diffraction and refined at a resolution of 1.9A . The rusticianins are composed of a central beta sandwich fold, composed of sheets b of six and seven strands. Like other cupredoxins, the copper ion is coordinated by a group of four conserved residues (His 85, Cysl38, Hisl43, Metl48) arranged in a distorted tetrahedron. Walter, R.L. et al. , J. Mol. Biol., Vol. 263, pp-730-51 (1996).

Pseudoazurins Pseudoazurins are a family of the single chain polypeptide containing blue copper. The amino acid sequence of the pseudoazurin obtained from Achromobacter cycloclastes is shown in SEQ ID NO: 6. The analysis of the X-ray structure of the pseudoazurin shows that it has a structure similar to the azurines, although there is a low sequence homology between these proteins. There are two main differences between the global structure of pseudoazurines and azurines. There is an extension of the term carboxy in the pseudoazurines, in relation to the azurines, which consists of two alpha helices. In the middle peptide region the azurines contain a prolonged, shortened turn in the pseudoazurins, which forms a fold containing a short helix. The only major differences in the site of the copper atom are the conformation of the MET side chain and the length of the Met-S copper bond, which is significantly shorter in pseudoazurin than in azurine.

Phytocyanins Proteins identified as phytocyanins include, but are not limited to, cucumber basic protein, stellacyanin, mavicianin, umecianin, a cupredoxin from the cucumber skin, a putative blue copper protein in the pods of peas, and a blue copper protein from Arabidopsis thaliana. In all, except the basic protein of cucumber and the pea pod protein, the axial methionine ligand normally found in the blue copper sites is replaced by glutamine.

Auracyanin Three small blue copper proteins designated as auracyanin A, auracyanin B-1 and auracyanin B-2 have been isolated from the green, thermophilic, photosynthetic Chloroflexus aurantiacus bacterium. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from form A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates apparent molecular masses of the monomer of 14 (A), 18 (B-2 ) and 22 (Bl) kDa. The amino acid sequence of auracyanin A has been determined and showed that auracyanin A is a 139 residue polypeptide. (Van Dreissche et al., Protein Science 8: 947-957 (1999)). His58, Cysl23, Hisl28 and Metl32 are spaced in a manner that is expected if the metal ligands are evolutionary conserved as in the known small copper proteins, plastocyanin and azurine. The prediction of the secondary structure also indicates that auracyanin has a general beta-barrel structure similar to that of the Pseudomonas aeruginosa azurine and the plastocyanin of the white poplar leaves. However, auracyanine appears to have sequence characteristics of small copper protein sequence classes. The overall similarity with a consensus sequence of azurine is approximately the same as that of a plastocyanin consensus sequence, that is, 30.5%. The region of the N-terminal sequence 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids. Id. See the example of the amino acid sequence SEQ ID NO: 16 for the A chain of the auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Access No. AAM12874). The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of the auracyanin B of Chloroflexus aurantiacus has been studied. (Bond et al., J. Mol. Biol. 306: 47-67 (2001)). With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurine. As in the other cupredoxins, one of the Cu ligands is located in strand 4 of the polypeptide, and the other three are placed along a long loop between strands 7 and 8. The geometry of the Cu site is described with reference to amino acid spacing between the last three ligands. The Cu-binding domain characterized by the crystallographic form of auracyanin B is probably bound to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits a significant sequence identity with the known junctions in several other electron-transfer proteins associated with membranes. The amino acid sequences of the B forms are presented in McManus et al. (J. Biol Chem. 267: 6531-6540 (1992)). See the exemplary amino acid sequence SEQ ID NO: 17 for the B chain of the auracyanin from Chloroflexus aurantiacus (NCBI Protein Data Bank Access No. 1QHQA).

Stellacyanin Stellacyanines are a subclass of phytocyanines, a ubiquitous family of plant cupredoxins. An example of a stellacyanine sequence is included herein as SEQ ID NO: 15. The crystal structure of umecianin, a stellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc. 127: 158-166 (2005)) and cucumber stellacyanine (Hart et al., Protein Science 5: 2175-2183 (1996)). The protein has a global fold similar to the other phytocyanines. The tertiary structure of the ectodomain of the ephrin B2 protein bears a significant similarity to stellacyanin (Toth et al., Developmental Cell 1: 83-92 (2001)). An example of an amino acid sequence of a stellacyanin is found in the National Center for Biotechnology Information Protein Data Bank as Accession No. 1JER, SEQ ID NO: 15.

Cucumber Basic Protein An example of an amino acid sequence of a cucumber basic protein is included herein as SEQ ID NO: 18. The crystal structure of the cucumber basic protein (CBP), a blue copper protein type 1, has been refined at the resolution of 1.8 Á. The molecule resembles other blue copper proteins because they have a Greek key beta keg structure, except that the keg is open on one side and is best described as a "beta sandwich" or "beta taco". (Guss et al., J. Mol. Biol. 262: 686-705 (1996)). The tertiary structure of the ectodomain of the ephrin protein B2 carries a high similarity (rms deviation from 1.5 Á for the 50 carbons a) to the basic cucumber protein. (Toth et al., Developmental Cell 1: 83-92 (2001)). The Cu atom has the coordination of NNSS 'from normal blue copper with Cu-N bond lengths (His39) = 1.93A, Cu-S (Cys79) = 2.16A, Cu-N (His84) = 1.95A, Cu -S (Met89) = 2.61 Á. A disulfide bond, (Cys52) -S-S- (Cys85), seems to play an important role in the stabilization of the molecular structure. The polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanines) as well as a non-metalloprotein, Ra3 ragweed allergen, with which CBP has a high degree of sequence identity. The proteins currently identifiable as phytocyanins are CBP, stellacyanine, mavicianin, umecianin, a cucumber shell cupredoxin, a putative blue copper protein in pea pods and a blue copper protein from Arabidopsis thaliana. In all but CBP and the pea pod protein, the axial methionine ligand normally found in the blue copper sites is replaced by glutamine. An example of a sequence for the basic cucumber protein is found in NCBI Protein Data Bank Access No. 2CBP, SEQ ID NO: 18.

Cytochrome Cytochrome C55? The cytochrome C551 of P. aeruginosa (Pa-C551) is a monomeric redox protein of 82 amino acid residues (SEQ ID NO: 2), involved in dissimilar denitrification as the physiological electron donor of nitrite reductase. The functional properties of Pa-C551 have been investigated extensively. Reactions with small non-physiological inorganic redox reagents and with other macromolecules, such as blue copper, eukaryotic cytochrome c and nitrite reductase proteins from the physiological partner have provided at least one test for protein-protein electron transfer. The three-dimensional structure of Pa-C551, which is a member of cytochromes of bacterial class I, shows a heme low simple spin with His-Met ligation and the typical polypeptide fold that, however, leaves the borders of pyrrole rings II and III of 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 additional exposure of the heme border to the propionate level 13. The distribution of the charged residues on the surface of Pa-C551 is very anisotropic : one side is richer in acid residues while the other exhibits a ring of positive side chains, mainly lysines, located at the edge of a hydrophobic connection that surrounds the heme crack. This connection comprises Glyll, Vall3, Ala1, Met22, Val23, Pro58, Ile59, Pro60, Pro62, Pro63 and Ala65 residues. The distribution of the anisotropic charge leads to a large dipole moment that is important for the formation of electron transfer complexes. The charge distribution described above for Pa-C551 has been reported for other electron transfer proteins and their electron acceptors. In addition, the modification by site-directed mutagenesis of the residues within the hydrophobic or charged connection has shown for different proteins the importance of surface complementarity for binding and electron transfer. As an example, the evidence for the relevance of the hydrophobic connection for the electron transfer properties of the azurine of P. aeruginosa comes from the studies carried out on mutants of the Met44 and Met64 residues changed to the negatively and positively charged amino acids. Id. The cytochrome c domain has a fold consisting of a series of alpha helices and inverse changes which serve to wind the heme covalently bound within a hydrophobic pocket. This domain can be found in cytochrome c monodomain proteins, such as cytochrome c6, cytochrome Cs52, cytochrome c59 and mitochondrial cytochrome c. The cytochrome c-type domain is present in a number of other proteins, such as in cytochrome cdl nitrite reductase as the C-domain of N-terminal heme, in quinoprotein alcohol dehydrogenase as the C-terminal domain, in the amine dehydrogenase A chain of quinohemoprotein as domains 1 and 2, and in the cytochrome bci complex as the bci domain of cytochrome. The structural analysis with the VAST algorithm (cytochrome C551 of Pseudomonas aeruginosa as an investigation) showed significant structural neighbors (P values between 10"10 * 3 to 10 ~ 4 * 5) only for cytochromes.

Methods of use The invention provides methods for treating patients with a malaria infection or in danger of acquiring it, or inhibiting the spread of the malaria parasite. These methods comprise administering to a patient or an insect vector a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, which inhibits parasitaemia or mammalian cells infected with malaria. The inhibition of parasitaemia can be determined by many methods well known in the art. One method is described in Example 6, and determines the inhibition of parasitaemia in the red blood cells of human infected with malaria. In other embodiments, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, inhibits the intracellular replication of the malaria parasite in the red blood cells and is administered to the patient or insect vector. Methods for determining intracellular replication of the malaria parasite are well known in the art, and one such method is described in Example 2. The mode of the invention is not limited to any particular mechanism, and the inhibition of parasitaemia It can result from many factors, including, but not limited to, inhibiting the replication of the parasite in infected blood cells, inhibiting the parasite that infects uninfected blood cells, inhibiting the parasite's growth cycle, and inhibition of the entrance of the parasite in mammalian cells. The invention provides methods for treating patients suffering from infection by a malaria parasite by administering an effective amount of at least one protein which is a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof. Patients who can be treated by this method are any mammal that may be infected by a malaria parasite and specifically are human patients. Known malaria parasites that 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 ( specific to monkey). It has also been learned that cupredoxins and cytochrome C551 are also effective against HIV-1 infections, as described in the co-filed application. "Compositions And Methods For Treating HIV Infection With Cupredoxin And Cytochrome c," Provisional US Patent Application No., the disclosure of which is expressly incorporated herein by reference. In addition, co-infections with HIV and malaria are very common in many areas of the world, and particularly in sub-Saharan Africa. In some modalities, the patient suffering from infection by a malaria parasite is also suffering from HIV infection. In some modalities, the The method of treatment of the invention also comprises administering anti-HIV drugs. In some modalities, anti-HIV drugs are coadministered. The invention also provides methods for treating a patient suspected of having contact with a malaria parasite, administering an effective amount of at least one peptide which is a cupredoxin and / or a cytochrome, or a variant, derivative or structural equivalent of a cupredoxin or a cytochrome. A patient suspected of having contact with a malaria parasite, for example, if such a patient lives or has traveled to a region of the world where malaria infection of other of the patient's species is common. Treatment by this method can be started when the patient is near, or has already come in contact with, the malaria parasite. Contact with malaria parasites is often presented with an insect vector such as mosquitoes, so that the abundant areas in these insects and the malaria parasite are considered to be among the areas where a patient would have a high probability of coming in contact with a malaria parasite. Such areas of the world include, but are not limited to, parts of Africa, Asia and Latin America. In addition, a patient may be suspected of having contact with the malaria parasite if it has come into contact with blood infected with a malaria parasite, is intentionally exposed to malaria parasite or accidentally injected with blood or drugs contaminated with the parasite. The cupredoxin or cytochrome, or a 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 skilled in the art. Specific embodiments, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome, are administered orally, topically, by inhalation, by injection, more specifically, intravenously, intramuscularly or subcutaneously. In one embodiment, the methods may comprise co-administering to a patient a unit dose of the compositions comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, and a unit dose of the compositions comprising an anti-malaria drug. and / or an anti-HIV drug in any order. These compositions may be administered at almost the same time, or within about a given time after administration of the other, for example, about one minute to about 60 minutes, or about 1 hour to about 12 hours of the other. The invention also provides methods for inhibiting the spread of the malaria parasite in a population of insect vector harboring a malaria parasite, administering to an insect vector in the population, at least one of a cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome, at an amount that is effective to reduce the infectivity of the parasite in a coexisting mammal population. In specific embodiments, the insect vector is a mosquito, and more specifically a mosquito of the species Anopheles gambiae. In this method, the administration of the cupredoxin or cytochrome, or a variant, derivative or structural equivalent of the cupredoxin or cytochrome can be performed by placing the peptides in the compositions that will be consumed by the insect vector, however, any form that is contemplated is contemplated. put the peptide in contact with the malaria parasite in the intestine of the insect vector. Many methods for administering chemicals to insect populations that produce such consumption are known in the art. In another embodiment, a transmissible genetic element passing from one mosquito to another will be operably linked to the cupredoxin coding sequence, operably linked to a constitutive promoter, cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome will be produced within the Anopheles gambiae infected with P. falciparum and will interfere with its replication / survival in the mosquito. Other forms of administration of the peptides to the insect vector include, but are not limited to, fusing the cupredoxin or cytochrome, or a variant, derivative or structural equivalent of the cupredoxin or cytochrome genes to genes of other proteins normally consumed by the insects. The amount of the 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 modalities, 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 promoter categories are available for use: the inducible and constitutive promoters. Inducible promoters include, but are not limited to, heat shock promoters. Preferably, the heat shock promoter is an insect heat shock promoter, for example, the hsp70 promoter from Drosophila melanogaster, which is capable of triggering the expression of genes in heterologous organisms, including the fruit fly of the Mediterranean. The invention also encompasses the use of the hsp70 promoter from the fruit fly of the Mediterranean (Papadimitriou et al., Insect Mol Biol 7: 279-90 (1998)). Alternative systems can be based on induction with antibiotic tetracycline. (Heinrich and Scott, PNAS 97: 8229-8232, (2000)). The heat shock promoters are inducible by raising the temperature of the conditions under which the fruit fly of the Mediterranean is being cultivated. For example, at 23-25 ° C, the hsp70 promoter is active at low levels or not at all. This allows the larva of the insect to develop without effort induced by the production of a heterologous protein. However, at higher temperatures, such as 37-42 ° C, the hsp70 promoter is induced and expresses the heterologous protein at a high level. The inducible promoters can be constructed based on the known inducible gene control elements. For example, inducible promoters can be constructed by combining a drug or hormone-sensitive element that can be administered in the diet. In a preferred embodiment, a human estrogen responsive element (ERE) can be used to regulate the expression of the protein of interest, provided that the vector is transformed with a second coding sequence expressing the human estrogen receptor. Constitutive promoters can also be used to Express the protein and / or other proteins required in the insect larva. For example, the constitutive promoter may be a cytoplasmic actin promoter. The cytoplasmic actin promoter from D. melanogaster has been cloned (Act5C) and is highly active in mosquitoes (Heinrich and Scott, PNAS 97: 8229-8232, (2000)). The cytoplasmic actin genes and their promoters can also be isolated from other insects, including the fruit fly of the Mediterranean. Other examples include the cytoplasmic tubulin promoter, for example, the cytoplasmic tubulin promoter of the Mediterranean fruit fly. The promoters that control the secreted polypeptides can be used, optionally together with the appropriate signal sequences, to direct the secretion of the protein to the hemolymph. For example, the larval serum protein promoter can be used (Benes et al., Mol.Gennet Gene 249 (5): 545-556 (1995)). The technology of mass culture for mosquitoes is highly developed. For protein production, cultures * of larvae that have been started at different times can be synchronized by appropriate temperature regimes. This is possible because the growth rates depend on the temperature of the environment; At 18 ° C the growth rates of the larvae decrease in approximately 50%.

Pharmaceutical compositions comprising cupredoxin and / or cytochrome and the variants and derivatives thereof. Pharmaceutical compositions comprising cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be manufactured in any conventional manner, for example, by the processes of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entraining or lyophilization. The cupredoxin and / or substantially pure cytochrome and the variants, derivatives and structural equivalents thereof can be easily combined with a pharmaceutically acceptable carrier well known in the art. These vehicles allow the preparation to be formulated as a tablet, pill, lozenge, capsule, liquid, gel, syrup, paste, suspension and the like. Suitable vehicles or excipients may also include, for example, fillers and cellulose preparations. Other excipients may include, for example, flavoring agents, coloring agents, anti-adherents, thickeners and other acceptable additives, adjuvants or binders. In some embodiments, the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation may contain insect vector harboring a malaria parasite, administering to an insect vector in the population, at least one of a cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome, at an amount that is effective to reduce the infectivity of the parasite in a coexisting mammal population. In specific embodiments, the insect vector is a mosquito, and more specifically a mosquito of the species Anopheles gambiae. In this method, the administration of the cupredoxin or cytochrome, or a variant, derivative or structural equivalent of the cupredoxin or cytochrome can be performed by placing the peptides in the compositions that will be consumed by the insect vector, however, any form that is contemplated is contemplated. put the peptide in contact with the malaria parasite in the intestine of the insect vector. Many methods for administering chemicals to insect populations that produce such consumption are known in the art. In another embodiment, a transmissible genetic element passing from one mosquito to another will be operably linked to the cupredoxin coding sequence, operably linked to a constitutive promoter, cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome will be produced within the Anopheles gambiae infected with P. falciparum and will interfere with its replication / survival in the mosquito. Other forms of administration of the peptides to the insect vector include, but are not limited to, fusing the cupredoxin or cytochrome, or a variant, derivative or structural equivalent of the cupredoxin or cytochrome genes to genes of other proteins normally consumed by the insects. The amount of the 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 modalities, 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 promoter categories are available for use: the inducible and constitutive promoters. Inducible promoters include, but are not limited to, heat shock promoters. Preferably, the heat shock promoter is an insect heat shock promoter, for example, the hsp70 promoter from Drosophila melanogaster, which is capable of triggering the expression of genes in heterologous organisms, including the fruit fly of the Mediterranean. The invention also encompasses the use of the hsp70 promoter from the fruit fly of the Mediterranean (Papadimitriou et al., Insect Mol Biol 7: 279-90 (1998)). Alternative systems can be based on induction with antibiotic tetracycline. (Heinrich and Scott, PNAS 97: 8229-8232, (2000)). The heat shock promoters are inducible by raising the temperature of the conditions under which the fruit fly of the Mediterranean is being cultivated. For example, at 23-25 ° C, the hsp70 promoter is active at low levels or not at all. This allows the larva of the insect to develop without effort induced by the production of a heterologous protein. However, at higher temperatures, such as 37-42 ° C, the hsp70 promoter is induced and expresses the heterologous protein at a high level. The inducible promoters can be constructed based on the known inducible gene control elements. For example, inducible promoters can be constructed by combining a drug or hormone-sensitive element that can be administered in the diet. In a preferred embodiment, a human estrogen responsive element (ERE) can be used to regulate the expression of the protein of interest, provided that the vector is transformed with a second coding sequence expressing the human estrogen receptor. Constitutive promoters can also be used to Express the protein and / or other proteins required in the insect larva. For example, the constitutive promoter may be a cytoplasmic actin promoter. The cytoplasmic actin promoter from D. melanogaster has been cloned (Act5C) and is highly active in mosquitoes (Heinrich and Scott, PNAS 97: 8229-8232, (2000)). The cytoplasmic actin genes and their promoters can also be isolated from other insects, including the fruit fly of the Mediterranean. Other examples include the cytoplasmic tubulin promoter, for example, the cytoplasmic tubulin promoter of the Mediterranean fruit fly. The promoters that control the secreted polypeptides can be used, optionally together with the appropriate signal sequences, to direct the secretion of the protein to the hemolymph. For example, the larval serum protein promoter can be used (Benes et al., Mol.Gennet Gene 249 (5): 545-556 (1995)). The technology of mass culture for mosquitoes is highly developed. For protein production, larval cultures that have been started at different times can be synchronized by appropriate temperature regimes. This is possible because the growth rates depend on the temperature of the environment; At 18 ° C the growth rates of the larvae decrease in approximately 50%.

Pharmaceutical compositions comprising cupredoxin and / or cytochrome and the variants and derivatives thereof. Pharmaceutical compositions comprising cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be manufactured in any conventional manner, for example, by the processes of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entraining or lyophilization. The cupredoxin and / or substantially pure cytochrome and the variants, derivatives and structural equivalents thereof can be easily combined with a pharmaceutically acceptable carrier well known in the art. These vehicles allow the preparation to be formulated as a tablet, pill, lozenge, capsule, liquid, gel, syrup, paste, suspension and the like. Suitable vehicles or excipients may also include, for example, fillers and cellulose preparations. Other excipients may include, for example, flavoring agents, coloring agents, anti-adherents, thickeners and other acceptable additives, adjuvants or binders. In some embodiments, the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation may contain at least one conservator. The general methodology regarding pharmaceutical dosage forms is found in Ansel et al. , Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams &Wiikins, Baltimore MD (1999)). The composition comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof used in the invention, can 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 suppositories, using a transdermal patch or by mouth. General information on drug delivery systems can be found in Ansel et al., Id. In some embodiments, the composition comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be formulated and used directly as injectables, for subcutaneous or intravenous injection, among others. The injectable formulation, in particular, can be used advantageously to treat patients who are at risk of malaria infection, who are likely to be infected by malaria or have a malaria infection. The composition comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be taken orally after mixing with the protective agents, such as polypropylene glycols or similar coating agents. When administration is by injection, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be formulated in aqueous solutions, specifically in physiologically compatible buffers, such as Hank's solution, Ringer's solution or physiological saline buffer. The solution may contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof may be in powder form for constitution with an appropriate vehicle, eg, sterile, pyrogen-free water, before use. In some embodiments, the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide. When administration is by intravenous fluids, the intravenous fluids for use, the administration of the cupredoxin or cytochrome, or a 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. The colloids used herein contain large insoluble molecules, such as gelatin. Intravenous fluids can be sterile. Crystalloid fluids that can be used for intravenous administration include, but are not limited to, normal saline (a sodium chloride solution at a concentration of 0.9%), Ringer's lactate or Ringer's solution, and a 5% solution. of dextrose in water, often called D5W, as described in Table 2.

Table 2. Composition of common crystalloid solutions Solution Other name [Na +] [Cl ~] [Glucose] D5W 5% dextrose 252 2/3 & 1/3 3.3% of 51 51 168 dextrose / 0.3% saline Saline 0.45% NaCl 77 77 average normal Solution Other name [Na +] [Cl ~] [Glucose] Saline 0.9% NaCl 154 154 0 normal Lactate of Ringer's Solution 130 109 0 Ringer * * Ringer's lactate also has 28 mmol / L of lactate, 4 mmol / L of K + and 3 mmol / L of Ca2 +. When the administration is by inhalation, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be released in the form of an aerosol spray from pressurized containers or a nebulizer with the use of an appropriate propellant., for example, dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to release a measured quantity. Capsules or cartridges of, for example, gelatin, for use in an inhaler or insufflator can be formulated to contain a mixture of proteins and an appropriate powder base, such as lactose or starch. When administration is by topical administration, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be formulated as solutions, gels, ointments, creams, suspensions and the like, as are well known in the art. In some modalities, the administration is by means of a transdermal patch. When the administration is by means of a suppository (eg, rectal or vaginal), the cupredoxin and / or cytochrome and the variants and derivatives of the same compositions can also be formulated in compositions containing the conventional suppository bases. When the administration is oral, a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof can be formulated easily by combining the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof with the pharmaceutically acceptable carriers well known in the art. The technique. A solid carrier, such as mannitol, lactose, magnesium stearate and the like; such vehicles allow the cupredoxin and / or cytochrome and the variants and derivatives thereof to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, pastes, suspensions and the like, for oral ingestion to a patient what is going to be For oral solid formulations, such as, for example, powders, capsules and tablets, suitable excipients include fillers, such as sugars, cellulose preparation, granulating agents and binding agents.

Other conventional carriers, as are well known in the art, also include multivalent vehicles, such as bacterial capsular polysaccharides, a dextran or a genetically engineered vector. In addition, sustained-release formulations that include a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof allow the release of the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof for extended periods of time. so that without the prolonged release formulation, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof would be clarified from a patient's system and / or degraded, eg, by proteases and simple hydrolysis, before provoke or improve a therapeutic effect. The half-life in the bloodstream of the compositions of the invention can be prolonged or optimized by several methods well known 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. 36 (1) : 169-76, (1987)); peptides containing the usual amino acids (Lee et al., J. Pept. Res. 63 (2): 69-84 (2004)), N- and C-terminal modifications (Labrie et al., Clin. Invest. Med. 13 (5): 275-8, (1990)), and hydrocarbon ring (Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al., Science 305: 1466-1470 (2004)). Of particular interest are the d-isomerization (substitution) and modification of the stability of the peptide by means of the substitution D or the substitution of the amino acid L and the hydrocarbon ring. In the various embodiments, the pharmaceutical composition includes carriers and excipients (including, but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, agents thickening 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, agents humectants and the like. It will be recognized that, while any vehicle known to those skilled in the art can be employed to administer the compositions of this invention, the type of vehicle will vary depending on the mode of administration. The compounds can also be encapsulated within liposomes using the well-known technology. The biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are described, for example, in U.S. Patent No. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. The pharmaceutical compositions can be sterilized by well-known, conventional sterilization techniques or can 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 before administration.

Administration of cupredoxin and / or cytochrome and variants and derivatives thereof Cupredoxin or cytochrome or a variant, derivative or structural equivalent thereof can be administered formulated as the pharmaceutical compositions and administered by any appropriate route, for example, by oral administration , buccal, inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical, percutaneous, that is, transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary). The pharmaceutical formulations thereof can administered in any effective amount 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 0.01-20 mg / day / kg body weight. The compounds comprising cupredoxin or cytochrome or a 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. Of course, the appropriate dosage will vary depending on, for example, the compound used of cupredoxin or cytochrome or a variant, derivative or structural equivalent thereof, 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 in daily dosages of approximately 0.01-20 mg / kg body weight. As indicated, the daily dosage in humans is in the range of about 0.7 mg to about 1400 mg of a cupredoxin or cytochrome c55? Compound, or a variant, derivative or structural equivalent thereof, administered conveniently, for example , in daily doses, weekly doses, monthly doses and / or continuous dosing. Daily doses can be discrete dosages from 1 to 12 times per day.

Alternatively, doses can be administered every day, every third day, every fourth day, every fifth day, every sixth day, every week and similarly in daily increments up to 31 days. Alternatively, the dosage can be continuous using patches, i.v. administration. and similar. The method for introducing cupredoxin or cytochrome or a variant, derivative or structural equivalent thereof to patients is, in some embodiments, through the co-administration of cupredoxin or cytochrome or a variant, derivative or structural equivalent thereof with other drugs used for malaria therapy. These methods are well known in the art. In a specific embodiment, cupredoxin and / or cytochrome c are part of a cocktail or contain a co-dosing or 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, amopirochin, sulfonamides, artemisinin, artephlene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, mefloquine, 1,1-hexadecamethylenebis (N-methylpyrrolidinium) dibromide, and combinations thereof. The method for introducing cupredoxin or cytochrome C551, or a variant, derivative or structural equivalent thereof to patients is, in some embodiments, the same as that currently used to introduce anti-HIV drugs, such as cocktails containing the inhibitor. of protease. Such methods are well known in the art. In a specific embodiment, cupredoxin or cytochrome c55 ?, or a variant, derivative or structural equivalent thereof are part of a 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 [Zerit]) and 3TC (lamivudine [Epivir] ), non-nucleoside reverse transcriptase inhibitors (NNRTIS): delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), a combination of lopinavir and ritonavir (Kaletra), saquinavir (Invirase), indinavir sulfate (Crixivan), amprenavir (Agenerase) and nelfinavir (Viracept). In some modalities, a combination of several drugs called highly active antiretroviral therapy (HAART) is used to treat patients. The exact formulation, the route of administration and the dosage are determined by the attending physician from the point of view of the patient's condition. The dosage amount and range can be individually adjusted to provide the plasma levels of the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof, which are sufficient to maintain a therapeutic effect. In general, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof are administered in a mixture with a selected pharmaceutical carrier with respect to the intended route of administration and standard pharmaceutical practice. In one aspect, the cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof are deliberated as DNA, so that the polypeptide is generated in itself. In one embodiment, the DNA is "naked" as described, for example in Ulmer et al. , (Science 259: 1745-1749 (1993)) and reviewed by Cohen (Science 259: 1691-1692 (1993)). The uptake of naked DNA can be increased by coating the DNA on a vehicle, for example, biodegradable beads, which are then transported efficiently in the cells. In such methods, DNA can be present within any variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. The techniques for incorporating DNA into such expression systems are well known to those skilled in the art. See, for example, WO90 / 11092, WO93 / 24640, WO 93/17706 and U.S. Patent No. 5,736,524. The vectors, used to launch the genetic material from organism to organism, can be divided into two general classes: the cloning vectors are a plasmid or phage of replication with regions that are essential for propagation in an appropriate host cell and in which the Strange DNA can be inserted, the foreign DNA replicates and propagates 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 the foreign genetic material into a host cell or tissue to transcribe and translate the foreign DNA, such as the DNA of a cupredoxin and / or cytochrome. . In the expression vectors, the introduced DNA is operably linked to elements, such as promoters that signal the host cell to highly transcribe the inserted DNA. Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. The operably linking of a cupredoxin or cytochrome and the variants and polynucleotide derivatives thereof to an inducible promoter can control the expression of the cupredoxin or cytochrome and the variants and derivatives thereof in response to specific factors. Examples of classical inducible promoters include those which are sensitive to interferon, heat shock, heavy metal ions and steroids, such as glucocorticoids (Kaufman, Methods Enzymol 185: 487-511 (1990)) and tetracycline. Other desired inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but, however, are sensitive to these cells when the induction agent is delivered exogenously. In general, useful expression vectors are often plasmids. However, other forms of expression vectors are contemplated, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses). The choice of vector is dictated by the organism or cells that are used and the desired destination of the vector. In general, the vectors comprise signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters and transcription termination sequences. As an example, a cupredoxin or cytochrome gene can be cloned into a transmissible vector in mosquitoes carrying the malaria parasite to prevent parasite replication within mosquitoes. The transmission capacity of the vector will allow the propagation of cupredoxin / cytochrome to neighboring mosquitoes that are also affected with parasites of malaria. The exact formulation, the route of administration and the dosage is determined by the attending physician from the point of view of the patient's condition. The dosage amount and range can be adjusted individually to provide plasma levels of active cupredoxin and / or cytochrome and variants and derivatives thereof, which are sufficient to treat the patient and / or maintain the therapeutic effect. In general, cupredoxin and / or cytochrome and the desired variants and derivatives thereof can be administered in a mixture with a selected pharmaceutical carrier with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions used in accordance with the present invention can be formulated in the conventional manner, using one or more physiologically acceptable carriers, comprising excipients and auxiliaries that facilitate the processing of cupredoxin and / or cytochrome and variants and derivatives thereof, active agents, for inhibiting or stimulating the secretion of cupredoxin and / or cytochrome and the variants and derivatives thereof, or a mixture thereof in the preparations that can be used therapeutically.

Kits (equipment) comprising cupredoxin and / or cytochrome C and the variants and derivatives thereof In one aspect, the invention provides kits containing one or more of the following, in a container or container: (1) a biologically active composition comprising a cupredoxin or cytochrome, or a 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. Also contemplated are the modalities in which two or more of the components (1) - (4) are in the same container. In another aspect, the invention provides kits containing one or more of the following in a container or container: (1) a biologically active composition comprising a cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof; (2) a malaria therapeutician, which includes, but is not limited to, proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine, amopiroquin, sulfonamides , artemisinin, arteflene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, mefloquine, dibromide 1,16-hexadecamethylenebis (N-methylpyrrolidinium); (3) a pharmaceutically acceptable adjuvant or excipient; (4) a vehicle for administration, such as a syringe; (5) instructions for administration. Also contemplated are the modalities in which two or more of the components (1) - (5) are in the same container or container. In some embodiments, the kit also comprises an anti-HIV therapeutic in a container or container. The anti-HIV therapeutics of interest include, but are not limited to, AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine [Zerit]) and 3TC (lamivudine [Epivir]), inhibitors of non-nucleoside reverse transcriptase (NNRTIS): delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), a combination of lopinavir and ritonavir (Kaletra), saquinavir (Invirase), indinavir sulfate (Crixivan), amprenavir (Agenerase) and nelfinavir (Viracept). In some modalities, a combination of several drugs called highly active antiretroviral therapy (HAART) is included in the kit. When a kit is supplied, the different components of the composition can be packaged in separate containers and mixed immediately before use. Such packaging of the components separately can allow long-term storage without losing the functions of the components assets. The reagents included in the kits can be supplied of any kind, so that the life of the different components is conserved and not adsorbed or altered by the materials of the container. For example, sealed glass ampoules may contain the lyophilized polypeptide or cupredoxin and / or cytochrome c polynucleotide and variants and derivatives thereof, or buffers that have been packaged under a neutral, non-reactive gas, such as nitrogen. The vials can consist of any material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramics, metal or any other material usually employed to hold similar reagents. Other examples of suitable containers include simple bottles that can be made of similar substances, such as ampoules and sachets, which may comprise sheet-coated interiors, such as aluminum or an alloy. Other containers include test tubes, ampules, flasks, bottles, syringes or the like. The containers may have a sterile access port, such as a bottle having a stopper that can be punctured by a hypodermic injection needle. Other containers may have two compartments that are separated by an easily removable membrane that, once removed, allows the components to mix. The removable membranes can be Glass, plastic, rubber, etc. The kits can also be supplied with instructional materials. The instructions can be printed on paper or other substrate, and / or can be supplied as an electronic readable medium, such as a disk drive, CD-ROM, DVD-ROM, Zip disk, video cassette, audiocassette, instant memory device, etc. Detailed instructions can not be physically associated with the kit; in fact, a user can go to a site on the internet network specified by the manufacturer or distributor of the kit, or be provided as an email.

Modification of cupredoxin and / or cytochrome Cupredoxin or cytochrome can be chemically modified or genetically altered to produce variants and derivatives as explained above.

These variants and derivatives can be synthesized by standard techniques. In addition to allelic variants of cupredoxin and cytochrome that occur naturally, changes can be introduced by mutation in the coding sequence of cupredoxin or cytochrome that incur alterations in the amino acid sequences of the cupredoxin or encoded cytochrome that do not alter significantly the ability of cupredoxin to inhibit parasitemia in blood cells blood reds infected with malaria. A "non-essential" amino acid residue is a residue that can be altered from the wild type sequences of cupredoxin without altering the biological activity, while an "essential" amino acid residue is required for such biological activity. For example, the amino acid residues that are conserved among the cupredoxins are predicted to be particularly not sensitive to alteration, and thus, "essential". The amino acids for which conservative substitutions can be made that do not change the activity of the polypeptide are well known in the art. Useful conservative substitutions are shown in Table 3, "Preferred substitutions". Conservative substitutions by which an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the invention, provided that the substitution does not materially alter the biological activity of the compound.

Table 3. Preferred substitutions Substitutions Substitutions Original residue preferred specimens Ala (A) Val, Leu, He Val Arg l: R) Lys, Gln, Asn Lys Asn l: N) Gln, His, Lys, Arg Gln Asp 1 ID) Glu Glu Cys l; c) Be Ser Gln 1 IQ) Asn Asn Glu 1: E) Asp Asp Gly l; G) Pro, Ala Ala His 1: H) Asn, Gln, Lys, Arg Arg Leu, Val, Met, Ala, He I) Leu Phe, Norleucine Norleucine, He, Val, Leu L) He Met, Ala, Phe Lys K) Arg, Gln, Asn Arg Met M) Leu, Phe, He Leu Leu, Val, He, Ala, Phe F) Leu Tyr Pro 1: P) Ala Ala Ser i [S) Thr Thr Thr l (T) Be Ser Trp i (W) Tyr, Phe Tyr Tyr i [Y) Trp, Phe, Thr, Ser Phe He, Leu, Met, Phe, Val V) Leu Ala, Norleucine Non-conservative substitutions that affect (1) the structure of the polypeptide skeleton, such as a conformation of β-film or helix a, (2) the charge, (3) hydrophobicity or (4) the volume of the side chain of the target site can modify the function of the cytotoxic factor. The residues are divided into groups based on the properties of the common side chain as represented in Table 4. Non-conservative substitutions cause the exchange of a member of one of these classes by another class. Substitutions can be introduced at the sites of the conservative substitution or more specifically at the non-conserved sites.

Table 4. Classes of amino acids Class Amino Acids Norleucine, Met, Ala, Val, Hydrophobic Leu, He Hydrophilic Neutral Cys, Ser, Thr Acid Asp, Glu Basic Asn, Gln, His, Lys, Arg Interruption of the Gly, Pro conformation of the aromatic chain Trp, Tyr, Phe Variant polypeptides can be made using methods known in the art, such as oligonucleotide-mediated mutagenesis (targeted site), alanine scanning and PCR mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237: 1-7 (1986), Zoller and Smith, Methods Enzymol 154: 329-350 (1987)), cassette mutagenesis, restriction selection mutagenesis (Wells et al. ., Gene 34: 315-323 (1985)) or other known techniques can be performed on the cloned DNA to produce variant cupredoxin or cytochrome C551 DNA. Known mutations of cupredoxins and cytochrome C551 can also be used to create a variant of cupredoxin and cytochrome C551 for use in the methods of the invention. For example, the mutants of azurine C112D and M44KM64E are known to have cytotoxic and growth arrest activity that is different from native azurine, and such altered activity may be useful in the methods of treatment of the present invention. One embodiment of the methods of the invention uses cupredoxin and / or cytochrome and variants and derivatives thereof which maintain the ability to inhibit the growth of malaria infection in mammalian cells. In another embodiment, the methods of the present invention use the cupredoxin variants, such as the mutant M44KM64E, which has the ability to cause growth arrest cell phone. A more complete understanding of the present invention can be obtained with reference to the following specific Examples. The Examples are described exclusively for the purposes of illustration and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated as circumstances, may be suggested or made records. Although the specific terms have been used herein, such terms are intended in a descriptive sense and not for the purpose of limitations. Modifications and variations of the invention as set forth above, may be made without departing from the spirit and scope thereof, and therefore, only such limitations should be imposed as indicated by the appended claims. EXAMPLES Example 1: In vitro inhibition of Parasitemia of P. falcípsuram by cupredoxin and cytochrome The cupredoxins azurine wt bacterial, M44KM64E azurine, rusticianin and cyanobacterial plastocyanin, as well as the cytochrome cytochrome c55? of Pseudomonas aeruginosa, human cytochrome c and cytochrome f of Phormidium laminosum were tested in a normal red blood cell (RBC) test at concentrations of 200 μg / ml at 30 hours post-inoculation. In In these experiments, the normal RBCs were washed twice in serum-free medium and resuspended in 10% hematocrit in complete RPMI. 200 μl of 10% Het RBCs were added to each of the 24 wells (2% final Het to 1 ml) in addition to 30 μl of complete RPMI containing the recombinant cupredoxin or cytochrome proteins at 666 μM for a concentration end of 200 μM. The parasites of the schizont stage were prepared by centrifuging a culture in the late stage through a Percoll pad at 3200 rpm for 10 minutes. For infection, 4xl06 parasites / well in a volume of 500 μl were added at t = 0 h. The plate was incubated for 30 hours and recorded by thin blood smears and Giemsa staining at that time. The control showed 9.5% parasitemia (standard error 1.3%), wt azurine 6.9% (1.4%), 9.1% azurine M44KM64E (1.0%), 7.2% rusticianin (0.7%), 7.5% cytochrome C551 (1.5%), 8.4% human cytochrome c (0.4%), 8.1% plastocyanin (1.3% sc) and 6.6% cytochrome f (1.0%), suggesting that cupredoxins, such as aztine wt and rusticianin and cytochromes, such as cytochrome or cytochrome C551 demonstrated 20 to 30% inhibition of parasitaemia. When cupredoxins were tested for their effects at different stages of the parasite's life cycle (0 - 24 hours, ring formation, 24-36 hours, trophozoite, 36-48 hours, schizont), the control showed 0.1% average ring formation and 9.4% trophozoite formation, while wt azurine showed no ring formation, but a 6.9% formation of trophozoite; cytochrome f showed 0.2% ring formation, but had a significantly low trophozoite formation (6.3%). Remarkably, rusticianin exhibited a very high ring formation (2.0%) and significantly reduced (5.2%) the formation of trophozoite. The others did not have a significant effect. The parasites in the rusticianina - the treated samples appeared diseased and colored compared to the rest of the samples, showing a significant inhibitory and toxic effect of rusticianin in the development of parasites.

Example 2: In vitro inhibition of intracellular replication of P. falcipaxvan by rusticianin To determine whether bacterial redox proteins can inhibit intracellular replication of malaria parasites, red blood cells were loaded at an intracellular recombinant protein concentration of 200 μg / ml using a hypotonic phantom preparation. The cells were then washed, resuspended and infected with the parasites of the schizont stage (P. falciparum) as described in Example 1. The ghosts of the Red blood cells were incubated for 19 hours and 40 hours and Giemsa smears were made. Compared with normal red blood cell infections in Example 1, only rusticianin decreased total parasitaemia in loaded cellular phantom cultures. At 19 hours, there was no significant difference in the invasion and formation of the ring, with empty ghosts at 5.0 ± 0.4% and phantoms loaded with rusticianin at 4.5 ± 1.0%. However, at 40 hours, the ghosts loaded with rusticianina had a lower level of infection. No major effects were observed at 19 hours with any of the bacterial proteins. However, at 40 hours, untreated control phantoms showed 4.6 ± 0.3% parasitemia, while phantoms treated with rusticianin had 2.7 ± 0.8% parasitemia, a reduction of almost 50%. See Table 5. The wt azurine proteins, the mutant azurine M44KM64E, plastocyanin, cytochrome C551 human cytochrome c and cytochrome f cyanobacteria showed an parasitaemia ranging from 4.2 to 5.4%.

Table 5. Inhibition of cupredoxin and cytochrome of P. falciparum infection of red blood cell ghosts.

Treatment Parasitemia media Standard error at 40 hours Vacuum 4.6% 0.3% Azurine type 5.4% 1.0% wild Azurine M44KM64E 4.7% 0.5% Rusticianina 2.7% 0.8% Cytochrome C551 4.2% 0.4% Human cytochrome c 4.6% 0.8% Plastocyanin 4.3% 0.3% Cytochrome f 4.5% 0.9% Example 3: Structural homology between azurine and Fab fragment of monoclonal antibody G17.12 complexed with Pf MSP1-19 Previous studies have shown that cupredoxins show a structural similarity to the variable domains of members of the immunoglobulin superfamily ( Gough &Chothia, Structure 12: 917-925 (2004), Stevens et al., J. Mol. Recognition 18: 150-157 (2005)). The DALI algorithm (Holm &Park, Bioinformatics 16: 566-567 (2000)) was used to search the 3D database for the structural homologs of azurine (1JZG) of P. aeruginosa. Azurine exhibits structural similarity to the antibody Fab fragment MSP1-19 of the merozoite surface protein of MSP1 of P. falciparum. (Pizarro et al., J. Mol. Biol. 328: 1091-1103 (2003)). (Table 6). Azurine also exhibits structural similarity to ICAM-1 (Table 6), which is involved in cerebral malaria and implicated as a receptor on endothelial cells in the microvasculature of the brain and other tissues for the sequestration of erythrocytes infected with P. falciparum. (Smith et al., Proc. Nati, Acad. Sci. USA 97: 1766-1771 (2000); Franke-Fayard et al., Proc. Nati, Acad. Sci. USA 102: 11468-11473 (2005)). This example shows that cupredoxins, including azurine, demonstrate structural similarities by having two ß anti-parallel films packed face to face and linked by a disulfide bridge to the variable domains of the members of the immunoglobulin superfamily, as well as the domains extracellular adhesions of intracellular adhesion molecules (ICAM) and their ligands.

Table 6. Structural azurine similarity of P. aeruginosa with different proteins related to pathogenesis.

Azurina (ljzg) registry PDB Annotation Reference DALÍ z (1) 1VCA Molecule 1 of cell adhesion 17 3.5 Human vascular Bl, VCAM-1 1ZXQ1 The crystal structure of 19 3.3 ICAM-2 1IAM1 Structure of the two domains 20 3.0 amino terminals of, ICAM-1 10B1 Crystal structure of a Fab complex with Plasmodium 21 2.9 falciparum MSP1-19 1T0P The complex structure of the B binding domains of ICAM-3 and 22 2.5 Alphabeta2 2NCM Cell adhesion molecule 23 2.4 neural, NCAM (i) - Structural alignment to azurine was performed using DALÍ (16). The structural pairs with the registers DALÍ z < 2 are considered dissimilar.

Example 4. Cloning and expression of Laz fusion genes and azurin H.8 The laz gene of Neisseria gonorrhoeae was cloned based on its known sequence (SEQ ID NO: 22). The azurine gene of P. aeruginosa (SEQ ID NO: 1), called peace, and the sequence of the H.8 epitope of N. gonnerrhoeae laz (SEQ ID NO: 21), were used to clone in frame the epitope H.8 in the extreme 5 'and peace to produce peace H.8 or in the extreme 3' of peace to generate peace-H.8.

Table 7. Carcinogenic cells, bacterial strains, genetic constructions Cells / Characteristics Reference strains /, Relevant * plasmids P. Prototroph, FP- (minus Holloway, et al., Aeruginosa sex factor) Microbiol. Rev.

PA01 43: 73-102 (1979) Cells / Characteristics Reference Strains / Relevant * plasmids E. coli Cloning and strain of Yanisch-Perron, et JM109 expression of azurine al. , Gene 33: 103-119 (1985) E. coli GST expression strain Novagen BL21 (DE3) N. Prototroph used for the American Type gonorrhoeae isolation from AD? Culture Collection F62 pUC18 Cloning vector Yanisch-Perron, et general, Apr al. , id. pUC19 Cloning vector Yanisch-Perron, et general, Apr al. , id. pUC18-laz A fragment of 1 kb of In the present AD? genome of N. gonorrhoeae F62 cloned in pUC18 Cells / Characteristics Reference strains / Relevant * plasmids pUC19 -paz A PCR fragment from Yamada, et al. , 0.55 kb of P. aeruginosa Proc. Nati Acad. PAOl cloned in HindIII Sci. USA 99: 14098- and PstI digested in 14103 (2002); pUC19, Apr Yamada, et al. , Proc. Nati Acad. Sci. USA 101: 4770-4775 (2004) pUC18-H.8- Fusion Plasmid that In the present Pa z encodes H.8 of N. gonorrhoeae and azurine of P. aeruginosa PAOl, Apr pGEX-5X-3 Amersham fusion vectors GST gene, Apr pET29a Expression vector of Novagen E. coli, Kmr pET29a-gst derived from pET29a which in the present contains the gst gene, Kmr Cells / Characteristics Reference Strains / Relevant * plasmids pGEX-5X-3- derivative of pGEX-5X-3 In the present H.8 containing H.8-coding region, Apr pET29a-gst- derivative of pET29a which in the present H.8 contains the gst-H.8 gene, Kmr * Ap, ampicillin; Km, kanamycin; GST, Glutathione S-transferase. Cloning and expression of the peace and laz genes. Cloning and hyperexpression of azurine genes have been described. (Yamada, et al., Proc. Nati, Acad. Sci. USA 99: 14098-14103 (2002); Punj, et al., Oncogene 23: 2367-2378 (2004)). The Laz coding gene (laz) from Neisseria gonorrhoeae was amplified by PCR with genomic DNA from the N. gonorrhoeae F62 strain as the DNA template. The front and reverse primers used were 5'-CCGGAATTCCGGCAGGGATGTTGTAAATATCCG-3 '(SEQ ID NO: 23) and 5' -GGGGTACCGCCGTGGCAGGCATACAGCATTTCAATCGG-3 '(SEQ ID NO: 24) wherein the introduced restriction sites additionally of the EcoRI and Kpnl sites are underlined respectively. The 1.0 kb amplified DNA fragment, digested with EcoRI and KpnI, was inserted into the corresponding sites of the pUC18 vector (Yanisch-Perron, et al., Gene 33: 103-119 (1985)), so that the gene laz was placed in the 3 'direction of the lac promoter to produce a pUC18-laz expression plasmid (Table 7). Plasmids expressing the H.8 fusion of N. gonorrhoeae Laz and P. aeruginosa azurin (Paz) were constructed by PCR with pUC19-peace and pUC18-Iaz as the templates. For fusion H.8-Paz, 3.1 kb fragment was amplified with pUC18-laz as a template and primers, 5'- (phosphorylated) GGCAGCAGGGGCTTCGGCAGCATCTGC-3 '(SEQ ID NO: 25) and 5' -CTGCAGGTCGACTCTAGAGGATCCCG -3 '(SEQ ID NO: 26) where a Sali site is underlined. 0.4 kb fragment amplified by PCR was obtained from pUC19-peace as a template and primers, 5 '- (phosphorylated) GCCGAGTGCTCGGTGGACATCCAGG-3' (SEQ ID NO: 27) and 5 '-TACTCGAGTCACTTCAGGGTCAGGGTG-3' (SEQ ID NO : 28) where an Xhol site is underlined. A SalI digested PCR fragment from pUC18-laz and an XhoI digested PCR fragment from pUC19-peace were cloned to produce a pUC18-H.8-peace expression plasmid (Table 7). E. coli JM109 was used as a host strain for the expression of azurine and its derived genes. The recombinant E. coli strains were cultured in a 2X YT medium that It contains 100 μg / mL of ampicillin, 0.1 mM IPTG and 0.5 mM CuS04 for 16 h at 37 ° C to produce the azurine proteins. When E. coli strains harboring these plasmids were grown in the presence of IPTG, the cells were lysed and the proteins were purified as described for azurin (Yamada, et al., Proc. Natl. Acad. Sci. USA 99 14098-14103 (2002); Punj, et al. , Oncogene 23: 2367-2378 (2004); Yamada, et al. , Cell. Microbiol. 7: 1418-1431 (2005)), the different azurine derivatives migrated in SDS-PAGE as the simple components, although the proteins containing H.8 (approximately 17 kDa) showed anomalous migrations, as previously reported (Cannon, Clin. Microbiol Rev. 2: S1-S4 (1989), Fisette, et al., J. Biol. Chem. 278: 46252-46260 (2003)).

Construction of the plasmid for fusion GST proteins. Plasmids expressing the wt (azu) truncated azurine derivatives of glutathione S-transferase fusion were constructed by a polymerase chain reaction using test reading DNA polymerase. For pGST-azu 36-128, a fragment was introduced by amplified PCR into the BamHl and EcoRl sites of the pGEX-5X commercial GST expression vector pGEX-5X (Amersham Biosciences, Piscataway, NJ). The fragment was amplified with pUC19-azu as a template and the primers, 5'-CGGGATCC CCG GCA ACC TGC CGA AGA ACG TCA TGG GC-3 '(SEQ ID NO: 29) and 5'-CGGAATTC GCA TCA CTG CAG GGT CAG GG-3' (SEQ ID NO: 30), where the introduced BamHl and EcoRI sites are additionally underlined respectively . Truncation of the carboxyl terminus of the azu gene was performed cumulatively by introducing a stop codon using the QuickChange direct site mutagenesis kit (Stratagene, La Jolla, CA). For pGST-azu 36-89, a stop codon was introduced into Gly90. The plasmid carrying pGST-azu 36-128 was used as a DNA template. Three groups of oligonucleotides for direct site mutagenesis are shown as follows. For pGST-azu 36-89: 5 '-CCA AGC TGA TCG GCT CGT GAG AGAAGG ACT CGG TGA CC-3' (SEQ ID NO: 31), and 5 '-GGT CAC CGA GTC CTT CTC TCA CGA GCC GAT CAG CTT GG-3 (SEQ ID NO: 32). For pGST-azu 88-113, truncation of the carboxyl terminus of the azu gene was cumulatively performed by entering the stop codon using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). For pGST-azu 88-113, a stop codon was introduced in Phell4. The plasmid carrying pGST-azu 88-128 was used as the template. For pGST-azu 88-128 an amplified PCR fragment was introduced into the BamHl and EcoRl sites of the commercial GST expression vector pGEX-5X (Amersham Biosciences). The fragment was amplified with pUC19-azu as the template and the 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) wherein the BamHl and EcoRl sites herein further introduced are underlined respectively. A group of 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 XL-1-Blue strains of E. coli. Plasmid extraction was performed using a commercial kit (Qiagen, Venlo, The Netherlands) and PCR sequencing was performed to verify the insertion and transfection of the plasmid. BL21 from E. coli (DE3) was used as a host strain for the expression of gst and its fusions derivatives. XLl-Blue of the E. coli strain transformed with the plasmids pGST-azu was grown in the LB medium with ampicillin for three hours at 37 ° C, after which the IPTG induction (0.4 M) was carried out, a subsequent incubation for 2-4 h at 37 ° C to maximize expression levels. The cells were isolated by centrifugation, resuspended in 25 mL of PBS IX buffer. Subsequent cell lysis involved two sequential treatments of the cell suspension by sonication (20 minutes on ice) and heat-cold shock in an acetone-dry ice bath (using the appropriate protease inhibitors). The supernatants of the mixture of Cell lysis was isolated and passed through a 1 mL glutathione-sepharose 4B column equilibrated with PBS and packed in fresh form (Amersham Biosciences). After washing the column and subsequent elution of the GST-azu product using 10 mM glutathione in 20 mM Tris-HCl, pH 8. The purity of GST-Azu 88-113 was tested by electrophoresis using 10% of a gel of SDS-PAGE Tris-Gly stained with the Coomassie Brilliant Blue R reagent. The concentration of the protein was determined using the Bradford method.

Example 5. Azurine binding to the C terminal fragments MSP1-19 and MSP1-42 of the merozoite surface protein MSP1 of P. falciparvp Given the structural similarity (Table 6) between the azurine and the fab fragment of the monoclonal antibody G17.12 in the complexation with pf MSPl-19 (Pizarro et al., id), the ability of azurine to form a complex with Pf MSP1-42 or Pf MSP1-19 was determined. Two azurine derivatives, Laz, an azurine-like protein from gonnococci and meningococci such as Neisseria meningi tidis with an additional 39 amino acid epitope called an H.8 epitope were tested.

(Gotschlich &Seiff, FEMS Microbiol, Lett 43: 253-255 (1987); Kawula et al. , Mol. Microbiol. 1: 179-185 (1987)) and H.8-azurin, where the H.8 epitope of Laz has fused in the N-terminal part of the azurine of P. aeruginosa in the framework (as described in Example 4). Protein-protein interactions in vi tro were evaluated using a Biacore X spectrometer from Biacore AB International. All experiments were conducted at 25 ° C in a running buffer HBS-EP (HEPES 0.01 M, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20 v / v) using detectors microcircuits Au-CM5 (Biacore) The protein immobilizations in the CM5 microcircuits were carried out according to the amine coupling procedure. The proteins were immobilized after pre-activation of NHS / EDC from the surface of CM5: injections of 50 μl of azurine (510 μM). Subsequent treatment of the surface of CM5 with ethanolamine (IM, pH 8.8) removed uncrosslinked proteins. The binding studies were performed by injecting the protein eluants (50 μl) on the surface of protein-CM5 at flow rates of 30 μl / minute with a time delay of 120 seconds at the end of the injections. Protein eluents included the GST-azurin fusion proteins (GST, GST-Azu 36-128, GST-Azu 36-89 and GST-Azu 88-113, as described in Example 4). Surfaces of the detector microcircuit were regenerated between injections of the protein using 100 mM NaOH (pulse injection of 10 μl). All binding studies were run in parallel against a negative flow channel with the exposed surface of the detector Au-CM5 to correct non-specific binding to microcircuits. To generate the binding constant data, the titration experiments were designed by means of injection of increased concentrations of the protein eluents (0.05-2000 nM). The SPR data were fitted to a Langmuir equilibrium binding model (1: 1) [Req = Rmax / (1 + Kd / C] as specified in the Biacore programming elements from which the data were extrapolated. binding constants (Kd) The specific interactions of the Pf proteins MSP1-19 and Pf MSP1-42 with azurine, azurine H.8 and Laz were determined by the surface plasmon resonance (SPR) analysis and the data are presented in Figure 1. SPR sensorgrams for binding of Pf and Pf MSP1-19 MSP1-42 immobilized azurin and its derivatives showed selective recognition between these proteins. While nanomolar concentrations of azurin allowed the significant binding MSP1- 19 (Fig. IA) or MSP1-42 (Fig. IB) immobilized, azurine H.8 and Laz demonstrated a higher binding affinity with the MSPl cleavage products of the merozoite surface protein, with characteristic Kd values of 32.2 nM between azurine and MSP1-19 and 54 .3 nM between azurine and MSP1-42. The Kd values between azurine H.8 and MSP1-19 and MSP1-42 were 11.8 nM and 14.3 nM, while such values between Laz and MSP1-19 and MSP1-42 ranged from 26.2 nM and 45.6 nM, respectively. To examine whether the H.8 epitope can facilitate the binding of azurine H.8 or Laz to the radicals of PfMSPl-19 or PfMSPl-42, the ability of glutathione S-transferase (GST) and a fusion derivative was tested of H.8-GST, wherein the H.8 epitope was fused at N-terminus of GST (see Example 4), to bind MSP1-19 or MSP1-42. Neither GST or H.8-GST bound to PfMSPl-19 (Fig. IA) or MSP1-42 (Fig. IB), although H.8-GST showed weak binding to MSP1-42. Glutathione S transferase (GST) and some of the fusion proteins, where parts of the azurine were fused to GST (Yamada et al., Cell, Microbiol 7: 1418-1431 (2005), and Example 4) were tested for their ability to bind MSP1-19. GST alone or GST-Azu 88-113, where the sequence of the azurine amino acid 88 to 113 by 128 amino acids of azurine, was fused to GST in the frame, showed no binding (Figure IC), while GST-Azu 36 -89 with amino acid sequence 36 to 89 and GST-Azu 36-128 with amino acid sequence 36 to 128 showed significant binding to MSP1-19 with Kd values of 20.9 nM and 25.4 nM, respectively.

Example 6. Inhibition of parasitaemia of Plasmodium falcxparum by azurine, Azurin H.8 and Laz. The degree of parasitaemia was determined using the parasites of the schizont stage and normal red blood cells (RBC). Normal red blood cells (RBC's) were washed twice in serum-free medium and resuspended at 10% hematocrit in complete RPMI. 200 μl of 10% hematocrit RBCs were added to each of the 24 wells in addition to 300 μl of complete RPMI with or without azurine, H.8 azurine or Laz at different concentrations. Parasites of the schizont stage of P. falciparum were prepared by centrifuging a culture in the late stage through a Percoll pad at 3200 rpm for 10 minutes. For infection, 4xl06 parasites per well in a volume of 500 μl were added at time zero. The plate was incubated overnight (approximately 16 h) and then recorded by thin blood smears and Giemsa staining at that time. Azurine, H.8 azurine or Laz demonstrated a significant inhibition of parasitemia in a dose-dependent manner (Figure 2), albeit at relatively high concentrations (approximately 50 μM). These high concentrations presumably reflect the multiple ways in which malaria parasites invade the erythrocytes (Cowman et al., FEBS Lett 476: 84-88 (2000), Baum et al., J. Biol. Chem. 281: 5197 -5208 (2006)) and a high concentration of azurine or Laz is necessary to interfere with the entry process. As indicated by their improved binding affinities to MSP1- 19, the azurine protein H.8 and Laz de Neisseria showed a higher level of inhibition of parasitemia by P. falciparum, compared with azurine (Figure 2). When azurine was labeled with the fluorescent red fluorescent dye 568 from Alexa and used during the invasion test, very little red fluorescence was detected within RBC, suggesting that azurine appears to enter the RBC as part of MSP1-19 united, or more likely, that the RBCs that showed the presence of schizonts were the only ones when the azurine did not bind with MSP1-19. These data are in full agreement with our previous observation (Yamada et al., Cell, Microbiol., 7: 1418-1431 (2005)) that azurine does not enter normal cells, such as macrophages, mast cells, etc. and the effect of azurine, azurin H.8 or Laz is at the entry level instead of the intracellular replication of the parasite. Taken together, the data in Figure 2 demonstrate the potential antimalarial action of azurin, azurin H.8 and Laz through interference in the invasion of RBC by parasites.

Example 7. Azurine binding to ICAMs There is an interesting structural similarity between azurine and ICAMs (Table 6) which is known to be involved as receptors for erythrocytes infected with P. falciparum (Wassmer et al., PloS Med. 2: 885-890 (2005); Dormeyer et al. , Antimicrob. Agents Chemotherap. 50: 724-730 (2006)) promoted the analysis of the test of protein-protein interactions as measured by SPR between azurine and ICAMs, such as ICAM-1, ICAM-2, ICAM-3 and NCAM. With the azurine immobilized in the microcircuit of CM5, ICAM-3 (Fig. 3, Kd = 19.5 + 5.4 nM) and NCAM (Fig. 3, insect), but interestingly not for ICAM-1 and ICAM-2, showed strong binding. While not limited to the manner in which the invention operates, part of the effect of azurine on the inhibition of parasitemia by P. falciparum could also be mediated through its interaction with ICAM-3 or NCAM.

Example 8. Treatment of patients who are probably exposed or exposed to malaria. Clinical use for the prevention of malaria, a pharmacist comprising one or more of cupredoxin and / or a cytochrome is administered to a patient. Fifteen healthy male volunteers, aged 22-50, who do not have a history of pre-existing antibodies to parasites of P. falciparum stage blood, as determined by the immunofluorescent test, but reside in an area where malaria is endemic, will be injected with a purified cupredoxin pharmaceutical preparation and purified cytochrome. Two men will serve as untreated controls.

The sterile pharmaceutical preparation is in the form of single dose ampoules of 0.5 mL of the sterile Pseudomonas aeruginosa azurine in a pharmaceutical preparation designed for intravenous administration, as will be well known to those skilled in the art. The pharmaceutical preparation is stored at 4 ° C and protected from light before administration. In a clinical trial, azurine is prepared at five different concentrations: 10 μg, 30 μg, 100 μg, 300 μg and 800 μg of azurine per dose of 0.5 mL. The pharmaceutical preparation is administered intravenously to thirteen volunteers for every 10 doses. The volunteers receive a primary treatment on day 0 and subsequent doses in identical doses each day for thirteen weeks. Volunteers are observed for immediate toxic effects for twenty-five minutes after injection. Twenty-four and forty-eight hours later, they were examined for evidence of fever, local sensitivity, erythema, burning, induration and lymphadenopathy and were asked about the complications of headache, fever, chills, malaise, local pain, nausea and articulations pain. Before each dose, blood and urine samples were taken for the complete laboratory examination. The profiles of the serum chemistry and the whole blood count were checked again two days after each dose. The presence of the malaria parasite is determined by microscopic examination (ME) light test kits from stained blood smears or ICT Malaria P. f. / P. v. (Binax, Inc., Portland, ME). The results demonstrate the effectiveness of the therapy.

Example 9. Control of malaria infection of insects A transmissible genetic element passing from one mosquito to another will be operably linked to the cupredoxin coding sequence operably linked to a constitutive promoter. Therefore, P. aeruginosa azurin will be produced within Anopheles gambiae infected with P. falciparum and will interfere with its replication / survival in the mosquito. This mosquito will then be introduced into an endemic area, so that the genetic element that hosts the azurine will spread to other A. gambiae mosquitoes infected with P. falciparum, which inhibit the growth or survival of P. falciparum.

Example 10. Treatment of patients infected with malaria The clinical use of a malaria therapy, comprising one or more of cupredoxin and / or cytochrome, for the treatment of malaria infection in humans.

Fifteen healthy male volunteers aged 22-50, who exhibit a history of pre-existing antibodies to stage-in-blood parasites of P. falciparum, as determined by the immunofluorescent test, are injected with a purified P. aeruginosa azurine pharmaceutical preparation. . Two men served as the treated controls. The sterile pharmaceutical preparation is in the form of single dose ampoules of 0.5 mL sterile P. aeruginosa azurine in a pharmaceutical preparation designed for intravenous administration, as will be well known to those skilled in the art. The pharmaceutical preparation is stored at 4 ° C and protected from light before administration. In a clinical trial, the azurine of P. aeruginosa is prepared as five different concentrations: 10 μg, 30 μg, 100 μg, 300 μg and 800 μg of azurine / cytochrome c55? (1: 1 on the molecular basis) per 0.5 ml of dose. The pharmaceutical preparation is administered intravenously to thirteen volunteers for every 10 doses. The volunteers receive a primary treatment on day 0 and subsequent doses in identical doses each day for thirteen weeks. Volunteers are observed for immediate toxic effects for twenty-five minutes after injection. Twenty-four and forty-eight hours later, they were examined for evidence of fever, local sensitivity, erythema, burning, induration and lymphadenopathy and were asked about the complications of headache, fever, chills, malaise, local pain, nausea and joint pain. Before each dose, blood and urine samples are taken for full laboratory examination. The profiles of the serum chemistry and the whole blood count were checked again two days after each dose. The presence of the malaria parasite is determined by microscopic examination (ME) light test kits from stained blood smears or ICT Malaria P. f. / P. v. (Binax, Inc., Portland, ME). The results demonstrate the effectiveness of the therapy.

Claims (1)

1. CLAIMS 1. An isolated peptide, characterized in that it is a variant, derivative, truncated or structural equivalent of a cupredoxin or cytochrome, and can inhibit parasitaemia by malaria in red blood cells infected by malaria. 2. The isolated peptide according to claim 1, characterized in that it can inhibit parasitaemia by malaria in the red blood cells of human infected with P. falciparum. 3. An isolated peptide, characterized in that it is a variant, truncated or structural equivalent of a cupredoxin or cytochrome, and that it can inhibit the intracellular replication of a malaria parasite in the red blood cells of human infected with malaria. 4. An isolated peptide, characterized in that it is a variant, derivative, truncation or structural equivalent of a cupredoxin and that it can bind a protein selected from the group consisting of PfMSPl-19 and PfMSPl-42. 5. The isolated peptide according to claim 1, characterized in that the cupredoxin is selected from the group consisting of azurine, pseudoazurin, plastocyanin, rusticianin, Laz and auracyanin. 6. The isolated peptide according to claim 1, characterized in that the cupredoxin is an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. I. The isolated peptide according to claim 1, characterized in that the cytochrome is selected from the group consisting of cytochrome c and cytochrome f. 8. The isolated peptide according to claim 7, characterized in that the cytochrome c is an organism selected from the group consisting of human and Pseudomonas s a eruginosa. 9. The isolated peptide according to claim 7, characterized in that the cytochrome f is from a cyanobacterium. 10. The isolated peptide according to claim 1, characterized in that a truncation of a peptide selected from the group consisting of SEQ ID NOs. NOS: 1-20 and 22. II. The isolated peptide according to claim 1, characterized in that a sequence is selected from the group consisting of SEQ ID NOS: 1-20 and 22 having at least 90% identity of the sequence of amino acid 12. The isolated peptide according to claim 1, characterized in that the peptide is more than about 10 residues and no more than about 100 residues. The isolated peptide according to claim 1, characterized in that it comprises the azurine residues 36-89 of SEQ ID NO: 1. 14. The isolated peptide according to claim 1, characterized in that it consists of the amino acid residues. 36-89 of SEQ ID NO: 1. The isolated peptide according to claim 1, characterized in that it is fused to a H.8 region of Laz. 16. The isolated peptide according to claim 1, characterized in that it is a structural equivalent of the monoclonal antibody G17.12. 17. A composition, characterized in that it comprises a cupredoxin, cytochrome or peptide isolated according to claim 1 in a pharmaceutical composition. 18. The composition according to claim 17, characterized in that the pharmaceutical composition is formulated for intravenous administration. 19. The composition according to claim 17, characterized in that it also comprises another anti- malaria. 20. The composition according to claim 17, characterized in that it also comprises an anti-HIV drug. The composition according to claim 17, characterized in that the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus. 22. The composition according to claim 17, characterized in that the cytochrome is selected from the group consisting of cytochrome c and cytochrome f. 23. The composition according to claim 22, characterized in that the cytochrome c is from an organism selected from the group consisting of human and Pseudomonas aeruginosa. 24. The composition according to claim 22, characterized in that the cytochrome f is from a cyanobacterium. 25. The composition according to claim 17, characterized in that the cupredoxin or cytochrome c is selected from the group consisting of SEQ ID NOS: 1-20 and 22. 26. A method for treating a patient suffering from an infection by a malaria parasite, characterized in that it comprises administering to the patient an effective amount of the composition according to claim 17. 27. A method for treating a patient who is suspects that it has contact with a malaria parasite, characterized in that it comprises administering to the patient an effective amount of the composition according to claim 17. 28. A method for preventing malaria in mammals, characterized in that it comprises administering to an insect vector in a population of insect vectors harboring a malaria parasite, an amount of the composition according to claim 17. 29. The method according to claim 28, characterized in that the peptide inhibits parasitemia by malaria in red blood cells. blood of humans infected with malaria. 30. The method according to claim 26, characterized in that the malaria parasite is selected from the group consisting of Plasmodium vivax and Plasmodium falciparum. 31. The method according to claim 26, characterized in that the patient is suffering additionally of HIV infection. 32. The method according to claim 26, characterized in that the composition is administered with a second composition comprising an effective ingredient of the group consisting of an anti-malaria drug and an anti-HIV drug. 33. The method according to claim 32, characterized in that the composition according to claim 21 is administered within 0 minutes to 12 hours after the administration of a second composition. 34. The method according to claim 26, characterized in that the composition is administered to the patient by a method selected from the group consisting of orally, by inhalation, intravenously, intramuscularly and subcutaneously. 35. The method according to claim 34, characterized in that the composition is administered to the patient intravenously. 36. The method according to claim 28, characterized in that the composition is administered to the insect vector orally.