KR20080019615A - 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. ® KIPO & WIPO 2008

Description

COMPOSITIONS AND METHODS FOR TREATING MALARIA WITH CUPREDOXIN AND CYTOCHROME}

Related applications

This application claims 35 U.S.C. Under §§ 119 and 120, co-submitted U.S. submitted May 19, 2006. Patent application No. 11 / 436,591, “Compositions and Methods for Treating HIV Infection with Cupredoxin and Cytochrome c”; U.S. submitted May 19, 2006. Patent application No. 11 / 436,590; “Compositions and Methods for Treating Malaria with Cupredoxins and Cytochrome”; U.S. filed May 10, 2006. Provisional Application No. 60 / 780,868; The U.S. submission dated May 20, 2005. Provisional Application No. 60 / 682,813; U.S. filed October 7, 2004. Provisional Application No. U.S. 60 / 616,782 and filed May 13, 2005. Provisional Application No. U.S., filed November 11, 2003, claims priority on 60 / 680,500. Patent application No. Part of the continued application of 10 / 720,603, filed U.S. Patent application No. Claim priority on 11 / 244,105, supra. Patent application No. 10 / 720,603, filed U.S. Provisional Application No. U.S., filed January 15, 2002, with priority on 60 / 414,550. Patent application No. 10 / 047,710, filed part of the file, see U.S. Pat. Patent application No. 10 / 047,710, filed U.S. Provisional Application No. Claim priority on 60 / 269,133. The entire contents of these prior applications are incorporated by reference in their entirety.

Statement of Government Rights

The gist of this application was supported by Grant Number AI 16790-21, ES 04050-16, AI 45541, CA 09432, N01-CM97567 from the National Institutes of Health (NIH), Bethesda, Maryland, U.S.A. The government may assert some rights in the invention.

Technical Field of the Invention

The present invention relates to their use in the inhibition of malaria parasites, particularly parasitemia of Plasmodium falciparum , in cupredoxin and cytochrome and mammalian erythrocyte cells. The invention also relates to variants and derivatives of cupredoxin and cytochrome that maintain the ability to inhibit parasitemia by malaria parasites. Finally, the present invention provides a method for inhibiting the spread of malaria infection in insect vectors.

Approximately one quarter of the world's population is at risk of malaria, and more than millions of people die of malaria each year. Of the four types of malaria parasites that infect humans, the two major species are Plasmodium falciparum and P. vivax .

Plasmodium falciparum blood stage merozite binds to and parasitic erythrocytes using various surface proteins (Cowman et al., FEBS Lett. 476: 84-88 (2000); Baum et al. , J. Biol. Chem. 281: 5197-5208 (2006)), the major antigenic member of these surface proteins is called the Melozoite Surface Protein1 (MSP1), a 195 kDa protein. Present in all erythrocyte-invasive species of Plasmodium ) and are anchored to the cleavage surface by glycosyl-phosphatidylinositol linkage During the early stages of the erythrocyte invasion process, immediately after release from the infected erythrocytes, cleavage The body MSP1 protein undergoes proteolytic cleavage and yields the C-terminal cleavage product MSP1-42, which in turn undergoes secondary cleavage and yields 11 kDa peptide MSP1-19, which peptide It remains attached to the parasite surface during invasion of erythrocytes The formation of the cleavage product MSP1-19 is very important for successful invasion by parasites, either because of its inhibition of proteolytic formation or by monoclonal antibodies This neutralization prevents the infestation of parasites into red blood cells (Blackman et al ., J. Exptl., Med. 180: 389-393 (1994)).

The MSP1-19 peptide is one of the most important malaria vaccine candidates available. MSP1-19-specific antibodies from malaria-resistant human serum react with the antigen and carry a major erythrocyte-invasion inhibitory component (Holder & Riley, Parasitol. Today, 12: 173-174 (1996); O'Donnell et al, J. Expt Med 193:... 1403-1412 (2001)). Serum from donors in the malaria-endemic region typically exhibits strong antibody reactivity to PfMSP1-19 (Nwuba et al., Infect. Immun. 70: 5328-5331 (2002)).

Monoclonal antibody (mAb) G17.12 against recombinant PfMSP1-19 recognizes its epitope on the parasite surface, demonstrating that native MSP1 polypeptide complexes can access this region of the antigen (Pizarro et al., J Mol. Biol. 328: 1091-1103 (2003)). Interestingly, in vitro erythrocyte invasion experiments showed that this infection was not inhibited in the presence of G17.12 even at a concentration of 200 μg / ml, and G17.12 did not inhibit the in vitro secondary treatment of MSP1 ( Id ). Intrusion - the presence of antibody to block the binding of inhibiting antibodies to promote the survival of parasites was also demonstrated (... Guevara Patino et al , J. Expt Med 186: 1689-1699 (1997)), such presence of malaria (M. falciparum ) is responsible for the failure of G17.12mA in inhibition of erythrocyte invasion.

Cerebral malaria, a rare but fatal infection limited to P. falciparum invasion of brain capillary due to sequestration of parasitic erythrocytes, is the most common drug in the blood-brain barrier. It is frequently refractory because it does not reach the brain capillaries. Adhesion to cerebral capillaries of P. falciparum -infected erythrocytes is expressed on the surface of capillary endothelial cells in the parasite ligand PfEmp-1 population of proteins expressed on the surface of infected erythrocytes and in cerebrovascular vessels. Mediated by the interaction of ICAM-1 with CD36 (Smith et al., Proc. Natl. Acad. Sci. USA 97: 1766-1771 (2000); Franke-Fayard et al., Proc. Natl. Acad. Sci USA 102, 11468-11473 (2005)).

Although few drugs targeting the heme detoxification pathway, such as chloroquine, are used to treat malaria, mosquito vectors that are resistant to parasite resistance and insecticides to drugs ( The incidence of mosquito vectors is increasing. Chloroquines antagonize heme polymerization mediated by parasite-induced HRP (histidine-rich proteins), because heme monomers are extremely toxic to malaria parasites. This polymerization of heme allows for detoxification, which is reversed by chloroquine. Another drug, artemisinin, is effective against chloroquine-resistant febrile malaria ( P. falciparum ) in cerebral malaria. Artemisinin forms an adduct that carries globin-bound heme in hemoglobin, which binds to HRP to prevent heme polymerization. There is an urgent need to discover new drugs for these fatal diseases, which are particularly prevalent in Africa and Asia. Current attempts in drug development are focused on the translation of the entire parasite genomic sequence, the molecular modeling of malaria parasite proteins, and the search for new drug targets.

Summary of the invention

The present invention relates to the parasitemia of mammalian erythrocytes and other tissues infected by cupredoxin and cytochrome and malaria parasites, in particular, parasitemia of human erythrocytes caused by Plasmodium falciparum . It relates to their use alone or in combination in inhibiting the spread of blood.

One aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of cupredoxin or cytochrome and that can inhibit intracellular replication of malaria parasites in malaria-infected human erythrocytes.

Another aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of cupredoxin and is capable of binding to a protein selected from PfMSP1-19 and PfMSP1-42.

Another aspect of the invention is an isolated peptide that is a variant, derivative or structural equivalent of cupredoxin or cytochrome and which can inhibit parasitemia by malaria in malaria-infected red blood cells. Specifically, the isolated peptides can inhibit parasitemia caused by malaria in P. falciparum -infected human erythrocytes. In some embodiments, the cupredoxin is azurin, pseudoazurin, platocyanin, rusticyanin, Laz, or auracyanin. Specifically, the cupredoxin is Rusticyanine, Azurin or Laz. In some embodiments, the cupredoxin is Pseudomonas aeruginosa ), Alcaligenes faecalis ), Achromobacter xylosoxidan ) , Bordetella bronchiseptica ), Methylomonas sp . , Meningococcal bacteria ( Neisseria) meningitidis ), Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens ), Pseudomonas chlororaphis ), Xylella fastidiosa ), or enteritis Vibrio parahaemolyticus ). Specifically, cupredoxin is Thiobacillus ferrooxidans ), Pseudomonas aeruginosa ), Neisseria gonorrhoeae ) or Neisseria meningitidis .

In other embodiments of this aspect, the cytochrome is cytochrome c or cytochrome f. In particular, cytochrome c is human or Pseudomonas aeruginosa ). Cytochrome f is derived from cyanobacteria.

In another embodiment of this aspect, the isolated polypeptide is the truncation of the peptide selected from SEQ ID NOs: 1-20 and 22. In some embodiments, the isolated peptide has at least 90% amino acid sequence identity with the sequence selected from SEQ ID NOs: 1-20 and 22.

In some embodiments of this aspect, the isolated peptide is the truncation of cupredoxin or cytochrome. In some embodiments, an isolated peptide has 10 or more residues and up to 100 residues. Such isolated peptides include azurin residues 36-89. Alternatively, such isolated peptide consists of azurin residues 36-89. Alternatively, such isolated peptides include cupredoxin residues equivalent to azurin residues 36-89.

In another embodiment of this aspect, the isolated peptide is fused to the H.8 region of Laz. In another embodiment, the isolated peptide is a structural equivalent of monoclonal antibody G17.12.

Another aspect of the invention provides an isolated peptide that is at least one cupredoxin, cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome that can inhibit malaria parasiticemia in malaria-infected red blood cells. It is a pharmaceutical composition. In particular, the pharmaceutical composition is prepared for intravenous administration. The composition may contain other anti-malarial or anti-HIV agents.

In some embodiments, the cupredoxin is Pseudomonas aeruginosa ), Alcaligenes faecalis , Achromobacter xylosoxidan ) , Bordetella bronchiseptica , Methylomonas sp . , Meningococcal bacteria ( Neisseria) meningitidis ), Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens ), Pseudomonas chlororaphis ), Xylella fastidiosa ), or Vibrio parahaemolyticus . Specifically, cupredoxin is Thiobacillus ferrooxidans , Pseudomonas aeruginosa ), Neisseria gonorrhoeae ) or Neisseria meningitidis ).

In some embodiments, the cytochrome is cytochrome c or cytochrome f. Specifically, cytochrome c is derived from human or Pseudomonas aeruginosa . Cytochrome f is derived from cyanobacteria. In some embodiments, the cupredoxin or cytochrome is selected from SEQ ID NOs: 1-20 and 22.

Another aspect of the invention is a method of treating a patient suffering from infection with a malaria parasite by administering to the patient an effective amount of the composition of the invention. In certain embodiments, the peptides inhibit parasitemia caused by malaria in the malaria-infected human erythrocyte cells of the patient. In some embodiments, the malaria parasite is Plasmodium vivax or Plasmodium falciparum . In some embodiments, the patient further suffers from HIV infection. In some embodiments, the composition is administered with a second composition containing anti-malarial and / or anti-HIV agents. In some embodiments, the composition of the present invention is administered concurrently with the administration of the second composition or within 12 hours after such administration. In some embodiments, the compositions of the present invention are administered orally, inhaled, intravenously, intramuscularly or subcutaneously to a patient; In particular, the composition is administered intravenously to the patient.

Another aspect of the invention is a method of treating a patient suspected of contact with a malaria parasite, the method comprising administering to the patient an effective amount of a composition of the invention.

Another aspect of the invention is a method of preventing malaria in a mammal, the method comprising administering an effective amount of a composition of the invention to an insect vector in a population of insect vectors in which the malaria parasite is dormant.

Such aspects, advantages, and features of the present invention will become apparent from the following drawings and detailed description of specific embodiments.

Brief description of the sequence

SEQ ID NO: 1. Pseudomonas amino acid sequence of azurin from aeruginosa ) .

SEQ ID NO: 2. Pseudomonas Amino Acid Sequences of Cytochrome c ₅₅₁ from .

SEQ ID NO: 3. Neisseria meningitidis ) amino acid sequence of Laz from MC58.

SEQ ID NO: 4. Amino acid sequence of plastocyanin from Phormidium laminosum .

SEQ ID NO: 5. Thiobacillus ferrooxidans) (amino acid sequence of the non-upon Ruth T from Acidithiobacillus ferrooxidans).

SEQ ID NO: 6. Achromobacter Achromobacter amino acid sequence of pseudoazurin from cycloclastes ).

SEQ ID NO: 7. Amino acid sequence of azurin from Alcaligenes faecalis .

SEQ ID NO: 8. Achromobacter xylosoxidan ) ssp . The amino acid sequence of from the very lean Denny pecan tree's (denitrificans) I.

SEQ ID NO: 9. Bordetella Bordetella amino acid sequence of azurin from bronchiseptica ) .

SEQ ID NO: 10. Methylomonas sp . ) Amino acid sequence of from the very Lin J.

SEQ ID NO: 11.Nisseria meningitidis) amino acid sequences of Z2491 from very lean.

SEQ ID NO: 12. Pseudomonas fluorescein sense (Pseudomonas amino acid sequence of azurin from fluorescens ) .

SEQ ID NO: 13. Pseudomonas Pseudomonas amino acid sequence of azurin from .

SEQ ID NO: 14. Xylella fastidiosa) the amino acid sequence of from the very lean 9 a5c.

SEQ ID NO: 15. Cucumis amino acid sequence of stellacyanin from sativus )

SEQ ID NO: 16. Chloroflexus amino acid sequence of auracyanin A from aurantiacus ) .

SEQ ID NO: 17. Chloroflexus amino acid sequence of auracyanin B from aurantiacus ) .

SEQ ID NO: 18. Cucumis amino acid sequence of cucumber basic protein from sativus ).

SEQ ID NO: 19. Amino acid sequence of cytochrome c from Homo sapiens .

SEQ ID NO: 20. Amino acid sequence of cytochrome f from Phormidium laminosum .

SEQ ID NO: 21. Neisseria gonorrhoeae ) amino acid sequence of the H.8 region of Laz from F62.

SEQ ID NO: 22. Neisseria gonorrhoeae ) amino acid sequence of Laz from F62.

SEQ ID NO: 23 is Neisseria gonorrhoeae ) Laz-encoding gene ( laz ) is a forward primer for PCR amplification.

SEQ ID NO: 24 is Neisseria gonorrhoeae ) Laz-encoding gene ( laz ) is a reverse primer for PCR amplification.

SEQ ID NO: 25 is the forward primer for PCR amplification of a 3.1 kb fragment of pUC18- laz .

SEQ ID NO: 26 is the reverse primer for PCR amplification of a 3.1 kb fragment of puc18- laz .

SEQ ID NO: 27 is a forward primer for PCR amplification of a 0.4 kb fragment of pUC19- paz .

SEQ ID NO: 28 is the reverse primer for PCR amplification of a 0.4 kb fragment of pUC19- paz .

SEQ ID NO: 29 is the forward primer for pGST-azu 36-128.

SEQ ID NO: 30 is the reverse primer for pGST-azu 36-128.

SEQ ID NO: 31 is the forward primer for pGST-azu 36-89.

SEQ ID NO: 32 is the reverse primer for pGST-azu 36-89.

SEQ ID NO: 33 is the forward primer for pGST-azu 88-113.

SEQ ID NO: 34 is the reverse primer for pGST-azu 88-113.

SEQ ID NO: 35 is an oligonucleotide for site directed mutagenesis in the preparation of pGST-azu 88-113.

SEQ ID NO: 36 is an oligonucleotide for specific site mutagenesis in the preparation of pGST-azu 88-113.

In Figure 1, surface plastics showing the interaction of Azurin, H.8-Azurin (H.8-Az), Laz, GST-Azurin (GST-Azu) constructs and MSP1-19 and MSP1-42. The driven resonance binding titration is shown. (A) Binding curve showing the interaction of azurin with analogs thereof and MSP1-19 (MSP1-19-CM5) immobilized on a carboxymethyldextran coated gold sensor chip. Concentration-dependent binding of azurin protein to MSP1-19 was determined by injecting various concentrations (0.05-300 nM) onto the sensor surface, and the degree of binding was measured by the equilibrium resonance response measured in resonance units (RU). It was evaluated as a function of equilibrium resonance response value. H.8-Az and Laz bind somewhat stronger than Azurin, whereas no binding was observed in GST or H.8-GST. (B) In vitro binding titration of azurin and its analogs and immobilized MSP1-42 was performed in a similar manner as in MSP1-19 as shown in (A). Relative binding affinity was determined by fitting these data to the equation Req = Rmax / (1 + Kd / C), which curve fit fits the data point in the graph. Connect. MSP1-19 binding Kd values are as follows: 32.2 ± 2.4 nM (azurine), 26.2 ± 2.4 nM (Laz), 11.8 ± 0.3 nM (H.8-Az); MSP1-42 binding Kd values are as follows: 54.3 ± 7.6 nM (azurine), 45.6 ± 2.4 nM (Laz), 14.3 ± 1.7 nM (H.8-Az). (C) Binding titration results for the interaction of GST-Azu fusion proteins on the MSP1-19-CM5 sensor surface demonstrate the recognition of GST-Azu 36-128 and GST-Azu 36-89 with MSP1-19. No binding was observed in GST or GST-Azu 88-113.

2 . 2 shows the inhibition of P. falciparum parasitemia (parasite growth in RBCs) by different concentrations of azurin, H.8-azrin (H.8-Az), Laz. In these experiments, normal red blood cells are infected with schizont in the presence or absence of these proteins at different concentrations and cultured overnight, and the total number of intracellular parasites is thin blood smear and Gimessa staining. Recorded as.

Figure 3 shows surface plasmon resonance binding curves for binding of ICAMs (ICAM-1, ICAM-2, ICAM-3, NCAM, illustration) with immobilized azurin. Due to extensive nonspecific binding to the exposed Au-CM5 chips, CM5 was added to running buffer as eluent (1 mg / ml CM5 in HBS-EP buffer). Selective recognition of azurin with ICAM-3 but not ICAM-1 or ICAM-2 was observed, with a binding strength of 19.5 ± 5.4 nM. As observed in the illustrations, the Kd for the binding of NCAM to azurin was 20 ± 5.0 nM.

Justice

As used herein, "cell" encompasses both the singular and the plural of the term unless specifically stated as a "single cell."

In this specification, "polypeptide", "peptide", "protein" are used as the same meaning, and indicate a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. The term also applies to naturally occurring amino acid polymers. “Polypeptides”, “peptides” and “proteins” are glycosylation, lipid attachment, sulfation, and gamma-carboxylation and hydroxylation of glutamic acid residues. hydroxylation, ADP-ribosylation, including but not limited to variations. Polypeptides are not always perfectly linear. For example, polypeptides are branched as a result of ubiquitination, and they are generally post-translational phenomena, including natural processing events and phenomena that do not occur naturally and are caused by human manipulation. as a result of the translation event) (either branched or unbranched). Cyclic polypeptides, branched polypeptides, branched cyclic polypeptides may be synthesized by non-translational natural processes and entirely by synthetic methods.

As used herein, “pathological condition” constitutes a damage of the normal state of a living animal or portion thereof; Disrupt or modify performance of physical functions; Various factors (such as malnutrition, industrial hazards, or climate), certain infectious pathogens (such as worms, parasites, bacteria or viruses), and genetic defects of organisms (such as genetic variants) genetic anomaly)), or a combination of these factors, covers anatomical and physiological deviations from normal.

As used herein, “disorder” encompasses anatomical and physiological deviations from normal, which constitute a steady state injury of a living animal or portion thereof and disrupt or modify the performance of body functions.

As used herein, “suffering” refers to the symptoms of a current pathological disorder and encompasses suffering from a pathological disorder, recovering from a pathological disorder, and recovering from a pathological disorder, even though there are no observable symptoms.

As used herein, “parasitemia” encompasses disorders in which parasites are present in blood and other tissues, and in particular indicates the presence of parasites with or without clinical symptoms.

As used herein, "inhibition of parasitemia" means reducing the presence of parasites in the blood of a mammal, or alleviating the rate of increase of such presence. Inhibition is a statistically significant reduction or slowdown of the rate of increase compared to control treatment.

As used herein, “treatment” encompasses preventing, decreasing, stopping or reversing the progression or severity of the disorder to be treated or the symptoms associated with the disorder. Thus, "treatment" includes appropriate medical, therapeutic and / or prophylactic administration.

As used herein, "anti-malarial activity" encompasses any activity that reduces the infectivity or reproduction of malaria parasites or inhibits the progression of the lifecycle of such parasites. "Anti-malarial activity" encompasses the inhibition of the growth of malaria infection by all means possible with current anti-malarial agents.

As used herein, "anti-malarial agent" refers to anti-malarial activity that can be used to reduce the infectivity or reproduction of malaria parasites or to inhibit the progression of the lifecycle of such parasites. It means having a drug.

As used herein, an “anti-HIV agent” is any means that includes, but is not limited to, inhibition of replication of the HIV genome, inhibition of synthesis and / or assembly of HIV envelope proteins, inhibition of HIV invasion into uninfected cells. By an agent having anti-HIV activity which reduces HIV infection in a mammal or prevents an increase in the human body.

As used herein, “substantially pure” used to modify a polypeptide or other compound refers to a polypeptide or compound in a form substantially free or free of active inhibitory agents, eg, a polypeptide isolated from a growth medium. To indicate. "Substantially pure" means at least 75% of the compound per dry weight of the separated fraction, or at least "75% substantially pure" compound. More suitably, “substantially pure” means at least 85% of the compound per building weight of the active compound, or at least “85% substantially pure” compound. Most suitably, "substantially pure" means at least 95% of the compounds per building weight of the active compound, or at least "95% substantially pure" compounds. Substantially pure cupredoxin or cytochrome c ₅₅₁ , or variants or derivatives thereof, may be combined with one or more other compounds that are substantially pure or isolated cupredoxin or cytochrome.

“Isolated”, “purified” or “biologically pure” refers to a substance that is substantially or essentially free of components normally involved in its natural state. Thus, isolated peptides according to the present invention preferably do not comprise substances typically associated with these peptides in an in situ environment. "Isolated" region refers to a region in which such region does not carry the entire sequence of the polypeptide from which it is derived. “Isolated” nucleic acids, proteins, or individual fragments thereof can include nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for nucleic acid fragments. subcloning and acquisition of proteins or protein fragments in substantially pure amounts are substantially transferred from the in vivo environment by manipulation by those skilled in the art, including but not limited to.

As used herein, “variant” refers to an amino acid sequence variant that has been substituted, deleted, or inserted into an amino acid as compared to a wild type polypeptide. The variant may be truncation of the wild type peptide. Thus, variant peptides are made by manipulation of genes encoding such polypeptides. Variants are made by changing the basic composition or properties without altering at least some of the essential activity of the polypeptide. For example, an “variant” of azurin can be a mutated azurin that maintains its ability to inhibit parasiticemia in malaria-infected human erythrocytes. In some instances, variant peptides are synthesized with non-natural amino acids such as ε- (3,5-dinitrobenzoyl) -Lys residues (Ghadiri & Fernholz, J. Am. Chem. Soc., 112: 9633). -9635 (1990)). In some embodiments, the variant has no more than 20 amino acids substituted, deleted or inserted compared to the wild type peptide. In some embodiments, the variant has 15, 14, 13, 12 or 11 amino acids substituted, deleted or inserted compared to the wild type peptide. In some embodiments, the variant has 10, 9, 8 or 7 or less amino acids substituted, deleted or inserted compared to the wild type peptide. In some embodiments, the variant has six or fewer amino acids substituted, deleted or inserted compared to the wild type peptide. In some embodiments, the variant has 5, 4 or less amino acids substituted, deleted or inserted compared to the wild type peptide. In some embodiments, the variant has three, two or less than one amino acid substituted, deleted or inserted as compared to the wild type peptide.

As used herein, “amino acid” is an amino acid moiety comprising any naturally-occurring or non-naturally occurring or synthetic amino acid residue, ie at least one carboxyl residue and one, two, three Or any moiety comprising at least one amino moiety directly linked by more than one carbon atom, typically one (α) carbon atom.

In the context of peptides, “derivatives” herein means peptides derived from target peptides. Derivation includes chemical modifications of the peptide such that the peptide still retains some of its underlying activity. For example, the "derivative" of azurin may be a chemically modified azurin that maintains its ability to inhibit parasitemia in malaria-infected red blood cells. Desired chemical modifications include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation or glycosylation of peptides. In addition, the derivative peptide may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as another peptide, a drug molecule or other therapeutic or pharmaceutical agent, or a detectable probe.

As used herein, "% amino acid sequence identity" is defined as the ratio of amino acid residues in a polypeptide that matches amino acid residues in a candidate sequence when aligning the two sequences. To determine the percent amino acid identity, the sequences are aligned and, if necessary, a gap is introduced to ensure the maximum percent sequence identity; Conservative substitutions are not considered as part of sequence identity. Amino acid sequence alignment procedures to determine percent identity are known to those skilled in the art. Publicly available computer software, such as BLAST, BLAST2, ALIGN or Megalign (DNASTAR) software, is used to align peptide sequences. In certain embodiments, Blastp (available from the National Center for Biotechnology Information, Bethesda MD) is used in the default parameters of the long complexity filter, expect 10, word size 3, existence 11, extension 1.

When aligning the amino acid sequence, the amino acid sequence identity ratio of the constant amino acid sequence A to the constant amino acid sequence B (or the constant amino acid sequence A having or including the specific amino acid sequence identity to the constant amino acid sequence B) ( %) Can be calculated as follows:

% Amino acid sequence identity = X / Y * 100

Where X is the number of amino acid residues that are recorded in the same match of A and B by sequence alignment program or algorithm alignment,

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage amino acid sequence identity of A to B will not match the percentage amino acid sequence identity of B to A. When comparing longer sequences to shorter sequences, the shorter sequence will be the “B” sequence. For example, when comparing truncated polypeptides to corresponding wild-type polypeptides, the truncated peptide will be the “B” sequence.

A “therapeutically effective amount” is an amount effective to prevent or delay the development of a particular pathological disorder in the individual being treated, or to completely relieve existing symptoms in such disorder. Determination of a therapeutically effective amount is within the ability of those skilled in the art.

General

The present invention provides compositions and methods that utilize cupredoxin and / or cytochrome to inhibit parasitemia of malaria-infected mammalian red blood cells and body tissues such as brain tissue and bone tissue.

Previously, several redox proteins belonging to a group of bronze-bearing proteins called cupredoxins, or iron-bearing proteins called cytochromes, enter mammalian cells, including cancer cells, and stop G1 in the cell cycle. (arrest) induces apoptotic cell death or leads to growth inhibition (Yamada et al., Cell Cycle 3: 752-755 (2004); Yamada et al., Cell Cycle 3: 1182-1187 (2004)). Single bacterial proteins such as cupredoxin azurin or cytochrome c ₅₅₁ produced by P. aeruginosa may exhibit one activity based on their hydrophobicity. Thus, wild type (wt) azurin induces apoptosis in murine J774 cells (Yamada et al. , Infection and Immunity, 70: 7054-7062 (2002)), while mutant M44KM64E azurin is a G1 in J774 cells. Induces cell cycle inhibition (Yamada et al., PNAS 101: 4770-4775 (2004)). In contrast, wt cytochrome c ₅₅₁ induces cell cycle inhibition at the G1 stage in J774 cells, while mutant V23DI59E cytochrome c ₅₅₁ induces apoptosis (Hiraoka et al., PNAS 101: 6427-6432 (2004)). .

According to the present invention, surprisingly, cupredoxin and cytochrome have been shown to inhibit parasitemia in human erythroid cells by the malaria parasite Plasmodium falciparum in vitro. In particular, cupredoxin azurin and Laz inhibit approximately 50% and 75% of parasitic hyperemia caused by tropical fever malaria ( P. falciparum ), respectively (see Example 6). Furthermore, Rusticyanine and cytochromes c and f inhibit parasitemia 20-30% (see Example 1). Furthermore, azurin was found to have significant structural homology to Fab fragments of G17.12 mouse monoclonal antibodies when complexed to Pf MSP1-19 fragments of MSP1 surface protein of P. falciparum (implemented) See example 2.) Without being limited to any means of inhibition mode, azurin is thought to inhibit parasiticemia of P. falciparum by interacting with the MSP1 protein on the surface of the parasite.

Surprisingly, azurin and Laz were found to bind to both PfMSP1-19 and PfMSP1-42 P. falciparum surface proteins in vitro. Furthermore, it was found that azurin amino acids 36-89 are required for binding to PfMSP1-19 and PfMSP1-42. Furthermore, Neisseria gonorrhoeae ) has been shown to increase the binding of azrin fused to the H.8 domain of Laz to PfMSP1-19 and to inhibition of parasitosis by P. falciparum (see Examples 5 and 6).

Due to the high structural homology between cupredoxins, it is contemplated that other cupredoxins will exhibit the same anti-malarial activity as Azurin, Rusticyanin, or Laz. In some embodiments, cupredoxin includes, but is not limited to, azurin, pseudoazurin, plastocyanin, auracyanin, Laz, or Rusticyanin. In certain embodiments, the cupredoxin is Laz, azurin or Rusticyanine. In another embodiment, the cupredoxin is derived from pathogenic bacteria. In a more particular embodiment, the cupredoxin is azurin. In a more specific embodiment, the azurin is Pseudomonas aeruginosa ); Alkagene genesis ( Alcaligenes) faecalis ); Achromobacter indeed in Xidan ssp. Denitrificans I; Bordetella bronchiseptica ; Methylomonas Species sp . ); Meningitis bacteria (Neisseria meningitidis ) Z2491 ; Pseudomonas fluorescein sense (Pseudomonas fluorescens ); Pseudomonas Pseudomonas chlororaphis ); Staphylococcus aureus ( Xylella) fastidiosa ) 9 a5c , or enteritis Vibrio parahaemolyticus ) . In the most specific embodiment, the azurin is Pseudomonas aeruginosa ). In another specific embodiment, the cupredoxin comprises an amino acid sequence that is SEQ ID NOs: 1, 2-18, or 21.

According to the present invention, P. aeruginosa cytochrome c ₅₅₁ , human cytochrome c, formdium laminosome ( Phormidium) laminosum ) cytochrome f has been shown to inhibit parasitemia in malaria-infected human erythrocytes. In certain embodiments, the cytochrome is cytochrome c ₅₅₁ , human cytochrome c or cytochrome f derived from P. aeruginosa . In another specific embodiment, the cytochrome comprises an amino acid sequence that is SEQ ID NO: 2, 19 or 20.

Due to this structural homology between cytochrome c, it is contemplated that other cytochromes will exhibit the same anti-malarial activity as P. aeruginosa cytochrome c ₅₅₁ and human cytochrome c. In some embodiments, the cytochrome is derived from pathogenic bacteria. In another specific embodiment, the cytochromes inhibit parasitemia in malaria-infected red blood cells, more specifically human red blood cells. In another specific embodiment, the cytochromes inhibit cell cycle progression in mammalian cancer cells, more specifically J774 cells.

Compositions of the Invention

The present invention provides peptides which are variants, derivatives or structural equivalents of cupredoxin or cytochrome. In some embodiments, the peptide is substantially pure. In other embodiments, the peptides are isolated. In some embodiments, the peptide is up to full length cupredoxin or cytochrome and retains some of the functional characteristics of the cupredoxin or cytochrome. In some embodiments, the peptide maintains the ability to inhibit parasitemia in malaria-infected red blood cells, specifically P. falciparum , in human red blood cells. In certain embodiments, the cytochrome is P. aeruginosa cytochrome c ₅₅₁ , human cytochrome c, or cyanobacterial cytochrome f, specifically SEQ ID NO: 2, 19, 20. In another specific embodiment, the peptide does not elicit an immune response in mammals, more preferably in humans.

The present invention also provides compositions containing at least one cupredoxin, cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome. The present invention also provides a pharmaceutical composition containing at least one cupredoxin or variant, derivative or structural equivalent of cupredoxin. The present invention also provides compositions containing at least one cytochrome, or variant, derivative or structural equivalent of cytochrome. In other embodiments, the composition consists essentially of these peptides. The present invention also provides pharmaceutical compositions containing at least one cupredoxin, cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome.

Due to the high structural homology between cupredoxins, other cupredoxins are associated with the inhibition of parasitic hyperemia in malaria-infected red blood cells ( Pseudomonas). aeruginosa ) is considered to exhibit the same anti-malarial activity as azurin. In some embodiments, cupredoxin includes, but is not limited to, azurin, pseudoazurin, plastocyanin, lastiyanin, Laz or auracyanin. In certain embodiments, the cupredoxin is Pseudomonas aeruginosa ); Alkagene genesis ( Alcaligenes) faecalis ); Achromobacter indeed in Xidan ssp. Denitrificans I; Bordetella bronchiseptica ; Methylomonas Species sp . ); Meningitis bacteria (Neisseria meningitidis ) Z2491 ; Neisseria gonorrhoeae ); Pseudomonas fluorescein sense (Pseudomonas fluorescens ); Pseudomonas Pseudomonas chlororaphis ); Staphylococcus aureus ( Xylella) fastidiosa ) 9 a5c , or enteritis Vibrio parahaemolyticus ) . In a more particular embodiment, the cupredoxin is Pseudomonas aeruginosa ) is derived from azurin . In another specific embodiment, the cupredoxin comprises an amino acid sequence that is SEQ ID NOs: 1, 3-18, or 22. In another specific embodiment, the cupredoxin is Neisseria meningitidis or Neisseria gonorrhoeae ) Laz protein.

The present invention provides amino acid sequence variants of cupredoxin or cytochrome with substituted, deleted or inserted amino acids as compared to wild type polypeptides. Variants of the invention may be truncated of wild type polypeptides. In some embodiments, the composition contains a peptide consisting of a region of cupredoxin or cytochrome that is a full length or less wild type polypeptide. In some embodiments, the composition contains a peptide consisting of at least 10 residues, at least 15 residues, or at least 20 residues of truncated cupredoxin or cytochrome. In some embodiments, the composition contains a peptide consisting of up to 100 residues, up to 50 residues, up to 40 residues or up to 30 residues of truncated cupredoxin or cytochrome. In some embodiments, the composition is a peptide having at least 90% amino acid sequence identity, at least 95% amino acid sequence identity or at least 99% amino acid sequence identity with cupredoxin or cytochrome, more specifically SEQ ID NOs: 1-20 or 22 It contains.

In certain embodiments, the variant of cupredoxin comprises P. aeruginosa azurin residues 36-89. In another embodiment, the variant of cupredoxin consists of P. aeruginosa azurin residues 36-89. In another specific embodiment, the variant of cupredoxin consists of the equivalent residue of cupredoxin other than azurin.

Other cupredoxin variants having an activity similar to azurin residues 36-89 may be designed. To this end, the target cupredoxin amino acid sequence is a target residue, target that is found on P. aeruginosa azurin sequence, P. aeruginosa azurin amino acid sequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR). It is aligned with the equivalent residue found in the cupredoxin sequence, thus identifying the equivalent residue of cupredoxin.

Variants also include peptides made from synthetic amino acids that do not occur naturally. For example, non-naturally occurring amino acids are incorporated into variant peptides to extend or optimize the half-life of the composition in the bloodstream. Such variants include D, L-peptide (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 with Special Amino Acids (Lee et al ., J. Pept. Res. 63 (2): 69-84 (2004)), incorporation of olefin-bearing non-natural amino acids followed by hydrocarbon stapling (Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)), ε- (3,5-dinitrobenzoyl) Peptides comprising a -Lys residue are included, but are not limited to these.

In another embodiment, the peptide of the invention is a derivative of cupredoxin or cytochrome. Derivatives of cupredoxin and / or cytochrome are chemical modifications of the peptide such that the peptide still retains some of its essential activity. For example, the "derivative" of azurin can be a chemically modified azurin that maintains its ability to inhibit malaria parasitemia in mammalian cells. Desired chemical modifications include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation or glycosylation of peptides. In addition, the derivative peptide may be a fusion of a chemical compound, such as another peptide, pharmaceutical molecule or other therapeutic or pharmaceutical agent, or cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, to a detectable probe. It may be. Desired derivatives include, for example, 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 olefins followed by hydrocarbon stapling- Incorporation of retained non-natural amino acids (Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)) By a number of methods well known to those skilled in the art, but not limited to, the chemical modifications can be extended or optimized for half-life of the peptides and compositions of the invention in the bloodstream.

In one embodiment, the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is Neisseria meningitidis or Neisseria gonorrhoeae ) is fused to the H.8 region of Laz. One example of such a peptide is the H.8- paz fusion protein described in Example 4. In certain embodiments, H.8 is fused to the C-terminus of the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof. In other specific embodiments, the H.8 region is SEQ ID NO: 21, or a variant, derivative or structural equivalent thereof.

Peptides of the compositions of the present invention may be variants, derivatives and / or structural equivalents of one or more cupredoxins or cytochromes. For example, these peptides are truncated of PEGylated azurin, so both variants and derivatives are possible. In one embodiment, the peptide of the present invention possesses an olefin-bearing tether and is a full-hydrocarbon “staple” by ruthenium catalyzed olefin metathesis. Synthesized from the following α, α-disubstituted non-natural amino acids (Scharmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walensky et al ., Science 305: 1466-1470 (2004)). In addition, peptides that are structural equivalents of azurin are fused to other peptides, thus yielding peptides that are both structural equivalents and derivatives. These examples merely illustrate the invention and do not limit it. Variants, derivatives or structural equivalents of cupredoxin or cytochrome may or may not bind to copper.

In other embodiments, the peptide may be a structural equivalent of cupredoxin or cytochrome. Examples of studies that determine significant structural homology between cupredoxin and cytochromes and other proteins include Toth et al ., Developmental Cell 1: 82-92 (2001). Specifically, significant structural homology between cupredoxin or cytochrome and structural equivalents is determined by using the VAST algorithm (Gibrat et al., Curr Opin Struct Biol 6: 377-385 (1996); Madej et al., Proteins ) . 23: 356-3690 (1995). In certain embodiments, the VAST p value from the structural comparison of cupredoxin or cytochrome to structural equivalent is about 10 ⁻³ or less, about 10 ⁻⁵ or less, or about 10 ⁻⁷ or less. In another embodiment, significant structural homology between cupredoxin or cytochrome and structural equivalents is determined using the DALI algorithm (Holm & Sander, J. Mol. Biol. 233: 123-138 (1993)). In certain embodiments, the DALI Z score for pairwise structural comparison is at least 3.5, at least 7.0, or at least 10.0.

In another embodiment, the variant or derivative of cupredoxin has significant structural homology to Fab fragments of G17.12 mouse monoclonal antibody. An example of how to determine this structural similarity can be found in Example 3. Specifically, significant structural homology between cupredoxin and Fab fragments of G17.12 mouse monoclonal antibodies is determined using the VAST algorithm (Gibrat et al., Id ; Madej et al., Id ). In certain embodiments, the VAST p value from the structural comparison of cupredoxin to Fab fragment of G17.12 mouse monoclonal antibody is about 10 ⁻⁴ or less, about 10 ⁻⁵ or less, about 10 ⁻⁶ or less, or about 10 ⁻⁷ It is as follows. In another specific embodiment, the VAST score from the structural comparison of cupredoxin to Fab fragment of G17.12 mouse monoclonal antibody may be at least about 9, at least about 10, at least about 11, or at least about 12.

In some embodiments, any of these variants, derivatives or structural equivalents are Pseudomonas aeruginosa (P. aeruginosa) very lean, Pseudomonas aeruginosa (P. aeruginosa) functional characteristics of the cytochrome c _551, human cytochrome c, or cyanobacteria (cyanobacterial) cytochrome f Have some. In certain embodiments, the peptides of the invention are parasitic by malaria in malaria-infected erythrocytes, specifically parasites by P. falciparum in febrile malaria ( P. falciparum ) -infected human erythrocytes. Inhibits blood pressure; In the present invention, malaria-infected red blood cell parasites hyperlipidemia in, specifically, falciparum malaria (P. falciparum) - in the infected human red blood cells falciparum malaria to maintain the ability to inhibit the parasite hypertriglyceridemia by (P. falciparum) Variants, derivatives and functional equivalents of cupredoxin and cytochrome c ₅₅₁ are provided. Inhibition of parasitic hyperemia by P. falciparum in P. falciparum -infected human erythrocytes can be determined by the method described in Example 6.

Because cupredoxin and cytochromes are known to inhibit parasitemia in malaria-infected red blood cells, for example, variants, derivatives, structural equivalents of cupredoxin and cytochrome that maintain this anti-malaria activity It is possible to design. Such variants, derivatives, structural equivalents, for example, make “library” of the various variants, derivatives, structural equivalents of cupredoxin and cytochrome, and then by way of example are known in the art, for example , By investigating anti-malarial activity in each of them using the typical method described in Example 6. The resulting variants, derivatives, structural equivalents of cupredoxin and cytochrome with anti-malarial activity are described herein as cupredoxin and cytochrome. Alternatively, or in addition to cupredoxin and cytochrome, it may be used in the method of the present invention. This method of screening variants and derivatives thereof is fit in a very Pseudomonas aeruginosa (P. aeruginosa) described herein Lin, Pseudomonas aeruginosa (P. aeruginosa) cytochrome c _551, human cytochrome c, or cyanobacteria (cyanobacterial) activity of cytochrome f Can be retrofitted.

In another embodiment, the peptides of the invention inhibit intracellular replication of malaria parasites in human erythrocytes. Methods for determining intracellular replication of malaria parasites are well known in the art and one such method is described in Example 2.

In some embodiments, the peptides of the present invention bind PfMSP1-19 and / or PfMSP1-42 P. falciparum with statistically higher relative binding constants than non-binding control proteins. . Peptides can investigate this activity by using surface plasmon resonance assays as described in Example 5. Other methods known in the art to determine whether one protein binds to another protein can also be used.

In another embodiment, the peptides of the present invention bind to ICAM-3 or NCAM with statistically higher relative binding constants than non-binding control proteins. Peptides can investigate this activity by using surface plasmon resonance analysis as described in Examples 7 and 5. Other methods known in the art to determine whether one protein binds to another protein can also be used.

In some specific embodiments, the polypeptides of the invention induce apoptosis in mammalian cancer cells, more specifically J774 cells. The ability of a polypeptide to induce apoptosis was determined by a mitosensor ApoAlert confocal microscope using the MITOSENSOR ^™ APOLERT ^™ mitochondrial membrane sensor kit (Clontech Laboratories, Inc., Palo Alto, California, USA), according to Zou et al., J. Biol . Chem . 274: 11549-11556 (1999) by measuring caspase-8, caspase-9, caspase-3, and by way of example, APOLERT ^™ It can be observed by detecting apoptosis-induced nuclear DNA fragmentation using a DNA fragmentation kit (Clontech Laboratories, Inc., Palo Alto, California, USA).

In another specific embodiment, the polypeptides of the invention induce cell growth arrest in mammalian cancer cells, more specifically J774 cells. Cell growth arrest is described, for example, in Yamada et al., PNAS 101: 4770-4775 (2004), which can be determined by measuring the extent of inhibition of cell cycle progression. In another specific embodiment, the peptides of the invention inhibit cell cycle progression in mammalian cancer cells, more specifically J774 cells.

Cupredoxin

These small blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in bacterial electron transfer chains or whose function is unknown. Copper ions are bound only by protein matrices. Twisted triangular two-dimensional alignment, unique to two histidines and one cysteine around copper, results in a very unique electronic property and intense blue color of the metal sites. Many cupredoxins have been crystallographically characterized at medium to high resolution.

Cupredoxin generally has low sequence homology but high structural homology (Gough & Clothia, Structure 12: 917-925 (2004); De Rienzo et al., Protein Science 9 : 1439-1454 (2000)). For example, the amino acid sequence of azurin is 31% identical to the amino acid sequence of auracyin B, 16.3% of the amino acid sequence of Rusticyanin, 20.3% of the amino acid sequence of Plastocyanin, and 17.3% of the amino acid sequence of Pseudoazurin. (See Table 1). However, the structural similarity of these proteins is more pronounced. The VAST p value for the structural comparison of ^Azurin vs. ^auracyanin B is 10 ^-7.4 , the VAST p value for the structural comparison of ^Azurin vs. ^Rusticinine is 10 ^-5 , and the structure of the azurin vs. plastocyanin The VAST p value for the comparison is 10 ^−5.6 and the VAST p value for the structural comparison of ^azurin versus ^pseudoazurin is 10 ^−4.1 .

All cupredoxins share an eight-stranded Greek key beta-barrel or beta-sandwich fold and have a highly conserved site architecture (De Rienzo et al., Protein Science 9: 1439-1454 (2000)). Significant hydrophobic patches due to the presence of many long chain aliphatic residues such as methionine and leucine are found in azurin, amicyanin, cyanobacterial plastocyanin, Cucumber basal proteins and, to a lesser extent, are present around the copper site in pseudoazurin and eukaryotic plastocyanin ( Id. ). Hydrophobic patches are less severe, but are also observed within the stelacyanine and ruticyanine copper sites, but differ in character ( Id. ).

Sequence and structure alignment of azurin (1JZG) from P. aeruginosa against other proteins using the VAST algorithm PDB Sort Length ^One aa identity% P- value ² Score ³ RMSD ⁴ Explanation 1AOZ A 2 82 18.3 10e-7 12.2 1.9 Ascorbic acid oxidase 1QHQ_A 113 31 10e-7.4 12.1 1.9 Auracianin B 1V54 B 1 79 20.3 10e-6.0 11.2 2.1 Cytochrome c oxidase 1GY2 A 92 16.3 10e-5.0 11.1 1.8 Rusticyanine 3MSP A 74 8.1 10e-6.7 10.9 2.5 Motility Main Sperm Protein ⁵ 1IUZ 74 20.3 10e-5.6 10.3 2.3 Plastocyanin 1KGY E 90 5.6 10e-4.6 10.1 3.4 ephrinB2 1PMY 75 17.3 10e-4.1 9.8 2.3 Pseudoazurine

¹ Aligned length: The total number of equivalent pairs of C-alpha atoms superimposed between two structures, ie the total number of residues used to calculate the 3D overlap.

² P-VAL: The VAST p value is a measure of the significance of this comparison, expressed as a probability. For example, if the p value is 0.001, the odds of seeing a match of this quality by chance is 1000 to 1. The p value from VAST is adjusted for the effect of multiple comparisons using the hypothesis that there are 500 independent and unrelated types of domains in the MMDB database. The p value thus obtained corresponds to the p value for the pairwise comparison of each domain pair divided by 500.

³ score: VAST structure-similarity score. This number relates to the total number of nested secondary structural elements and the nature of such overlap. Higher VAST scores correlate with higher similarities.

⁴ RMSD: root mean square overlapping residue (in Angstrom units). This number is calculated as the square root of the mean square distance between equivalent C-alpha atoms after the best overlap of the two structures. Note that the RMSD number is proportional to the degree of structural alignment, and that size should be considered when using RMSD as a descriptor of overall structural similarity.

⁵ oocytes (oocyte) Lin F. The boy proved to be the antagonist in the mature worms (C. elegans) Major Sperm Protein (Kuwabara, 2003 "The multifaceted C.elegans major sperm Protein: an ephrin signalling antagonist in oocyte maturation" Genes and Development , 17: 155-161).

Azurin

Azurin is a copper-bearing protein consisting of 128 amino acid residues belonging to the cupredoxin population involved in electron transfer in certain bacteria. Azurin includes P. aeruginosa (SEQ ID NO: 1), Alkogen genes xylloxidans ( Alcaligenes). xylosoxidans ), alkaligenes denistypican ( Alcaligenes denitrifican ) (SEQ ID NO: 8) (Murphy et al., J. Mol. Biol. 315: 859-71 (2002)). Although amino sequence identity varies between 60-90% between azurins, these proteins exhibit strong structural homology. All azurins possess characteristic β-sandwiches containing Greek key motifs, and a single copper atom is always located in the same region of the protein. In addition, azurin essentially retains essentially neutral hydrophobic patches surrounding the copper sites (Murphy et al.).

Plastocyanin

Plastocyanin is a soluble protein of cyanobacteria, algae, and plants that has one copper molecule per molecule and is bluish in oxidized form. They are produced in chloroplasts, where they function as electron carriers. In 1978, after the determination of non-Poplar Place Toshi structure, birds (ttemok end (Scenedesmus), Parlay in (Enteromorpha), Chlamydomonas (Chlamydomonas)) and the structure of the plastic Toshi, not plants (kidney beans (French bean)) crystallographic Method, or NMR method, this poplar structure was carefully distinguished at 1.33 Å resolution. SEQ ID NO: 4 shows amino acid sequence of plastocyanin from thermophilic cyanobacteria , Phormidium laminosum .

Despite the sequence divergence (eg, 62% sequence identity between Chlamydomonas and poplar protein) between the plastocyanins of algae and vascular plants, these three-dimensional structures were preserved. (Eg, a 0.76 μs rms deviation in C alpha position between Chlamydomonas and poplar protein). Structural features include twisted tetrahedral copper binding sites, prominent negative patches, and flat hydrophobic surfaces at one end of an 8-strand antiparallel beta-barrel. . Copper sites are optimized for electron transfer function, and negative and hydrophobic patches are proposed to be involved in the recognition of physiological response partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have identified the importance of negative and hydrophobic patches in binding interactions with cytochrome f and models of two functionally important electron transfer pathways involving plastocyanin. The first putative electron transfer pathway is relatively short (approximately 4 μs) and involves solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other pathway is longer (approximately 12-15 μs) and almost conserved in the negative patch. With residue Tyr-83 (Redinbo et al., J. Bioenerg. Biomembr. 26 (1): 49-66 (1994)).

Rusticyanine

Single hold-chain polypeptide is loose tea rather than upon the TiO bacilli (Thiobacillus) bronze obtained from (currently, Acidithiobacillus). Thiobacillus ferrooxydans The oxidized X-ray crystal structure of extremely stable highly acidic cupredoxin rustiyanin (SEQ ID NO: 5) from ferrooxidans was determined by multiwavelength anomalous diffraction and finely differentiated at 1.9 Å resolution. It became. Rusticyanine consists of a core beta-sandwich fold consisting of 6- and 7-stranded β-sheets. Similar to other cupredoxins, copper ions are coordination by clusters of four conserved residues (His85, Cys138, His143, Met148) aligned within the twisted coordination site (Walter et al., J. Mol). Biol. 263: 730-51 (1996)).

Pseudoazurine

Pseudoazin is a bronze-bearing single-chain polypeptide population. The amino acid sequence of pseudoazurine obtained from Achromobacter cycloclastes is listed in SEQ ID NO: 6. X-ray structure analysis of pseudoazurine shows that pseudoazurin has a similar structure to azurin although it has low sequence homology with azurin. Two major differences exist between the overall structure of pseudoazurin and azurin. Compared to azurin, pseudoazurine has a carboxy terminal extension consisting of two alpha-helices. In the mid-peptide region, azurin has an extended loop compared to pseudoazurin, which forms a flap with a short α-helix. The only major differences at the copper atom sites are the conformation of the MET side chain and the Met-S copper bond length, which is much shorter in pseudoazurin than in azurin.

Phytocyanin

Proteins identified as phytocyanin include cucumber base protein, stelacyanin, mavicyanin, umecyanin, cucumber peeling cupredoxin, and pea pod. Estimated Bronze Protein, Arabidopsis thaliana ) include, but are not limited to, bronze proteins. With the exception of cucumber base proteins and pi-pod proteins, axial methionine ligands, which are commonly found in bronze sites, are replaced with glutamine.

Auracianin

Three small bronze proteins, named Auracyanin A, Auracyanin B-1 and Auracyanin B-2, are used for thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus ). These two B forms are glycoproteins and have almost the same properties as each other but differ from the A forms. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent monomer molecular mass was found to be 14 (A), 18 (B-2), 22 (B-1) kDa.

The amino acid sequence of auracyanin A was found to be a 139 residue polypeptide (Van Dreissche et al, Protein Science 8: 947-957 (1999)). His58, Cys123, His128, Met132 are located at a certain distance in the manner expected in the case where they are evolutionarily conserved metal ligands, such as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction was also confirmed by auracyanin Pseudomonas aeruginosa ) from azurin and poplar leaves suggesting a beta-barrel structure that is generally similar to the beta-barrel structure of plastocyanin. However, auracyanin appears to have both sequence characteristics of both small copper protein sequences. The overall similarity to the consensus sequence of azurin is almost identical to that of the consensus sequence of plastocyanin (ie, 30.5%). The N-terminal sequence regions 1-18 of auracyanin are markedly rich in glycine and hydroxy amino acids ( Id ). Chloroflexus Chloroflexus See a typical amino acid sequence SEQ ID NO: 16 (NCBI Protein Data Bank Accession No. AAM12874) for chain A of auracyanin from aurantiacus).

The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of auracyanin B from Chloroflexus aurantiacus was investigated (Bond et al., J. Mol. Biol. 306: 47-67 (2001)). With the exception of the additional N-terminal strands, the molecule is very similar to the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands is located on strand 4 of the polypeptide and the other three are located along the large loop between strands 7 and 8. Cu site geometry is discussed with reference to amino acid spacing between the three latter ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably surrounding the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other electron-transfer proteins that are membrane-bound. Bound to the periplasmic side The amino acid sequence of form B is described by McManus et al., J Biol Chem. 267: 6531-6540 (1992). See the typical amino acid sequence SEQ ID NO: 17 (NCBI Protein Data Bank Accession No. 1QHQA) for chain B of auracianin from Chloroflexus aurantiacus .

Stellacyanine

Stellacyanin is a ubiquitous population of plant cupredoxins, a subclass of phytocyanin. Typical sequences of stellacyanine herein are listed in SEQ ID NO: 15. Stellacyanine, Umecyanin (Koch et al., J. Am. Chem . Soc . 127: 158-166 (2005)) and Cucumber Stellacyanine (Hart et al., Protein ) from Horseradish Roots Science 5: 2175-2183 (1996)) is also known. The protein has a total fold similar to other phytocyanins. The ephrinB2 protein ectodomain tertiary structure has significant similarity to stelacyanin (Toth et al., Developmental Cell 1: 83-92 (2001)). A typical amino acid sequence of Stellacyanin is found in SEQ ID NO: 15 (National Center for Biotechnology Information Protein DataBank Accession No. 1JER).

Cucumber Basic Protein

Typical amino acid sequences from cucumber based proteins are listed herein in SEQ ID NO: 18. The crystal structure of the type 1 bronze protein, cucumber basal protein (CBP), was carefully differentiated at 1.8 resolution. The molecule is similar to other bronze proteins in that it has a Greek key beta-barrel structure, but the barrel is open on one side and is "beta-sandwich" or "beta-taco". (Guss et al., J. Mol. Biol. 262: 686-705 (1996)). This ephrinB2 protein ectodomain tertiary structure has high similarity to cucumber basal proteins (rms deviation 1.5 for 50 α carbon) (Toth et al., Developmental Cell 1: 83-92 (2001)).

Cu atoms have a bond length Cu-N (His39) = 1.93, Cu-S (Cys79) = 2.16, Cu-N (His84) = 1.95, Cu-S (Met89) = 2.61, and the normal bronze NNSS 'isotopic (co- ordination). Disulfide linkages, (Cys52) -SS- (Cys85), seem to play an important role in stabilizing this molecular structure. Polypeptide folding is typical for a sub-family of bronze proteins (phytocyanins) and for non-metalloprotein, ragweed allergen Ra3, which has high sequence homology to CBP. Proteins currently identified as phytocyanin are presumably bronze proteins from CBP, Stellacyanin, Maviscyanin, Umecyanin, Cucumber Husk Cupredoxin, Piperde, Arabidopsis thaliana ) is a bronze protein. With the exception of CBP and P-Pad proteins, the axial methionine ligands commonly observed at bronze sites are replaced with glutamine. Typical sequences for cucumber basal proteins are found in SEQ ID NO: 18 (NCBI Protein Data Bank Accession No. 2CBP).

Cytochrome

Cytochrome c ₅₅₁

Cytochrome C ₅₅₁ (Pa-C551) from P. aeruginosa is a physiological electron donor of nitrite reductase, which is responsible for the 82 amino acid residues (SEQ ID NO: 2) involved in the denitrification. Monomeric redox protein. The functional characteristics of Pa-C551 have been extensively investigated. Reactions with non-physiological small inorganic redox reactants and reactions with other macromolecules such as blue copper protein, eukaryotic cytochrome c, physiological relative nitrite reductase, have been shown to affect protein-protein electron transfer. The test was provided.

The three-dimensional structure of Pa-C551, a member of the bacterial class I cytochrome, is a single row with His-Met ligation and a typical polypeptide fold that keeps the edges of the pyrrole rings II and III of heme exposed. -Single low-spin heme (Cutruzzola et al., J. Inorgan . Chem . 88: 353-61 (2002)). The absence of a 20-residue omega loop present in mammalian class I cytochrome results in additional exposure of the heme edge at the level of propionate 13. The distribution of charged residues on the surface of Pa-C551 is extremely anisotropic: one is rich in acidic residues, while the other is a hydrophobic patch surrounding the heme crevice. A positive side chain located mainly at the border of the (hydrophobic patch), mainly a ring of lysine. Such patches include residues Gly11, Val13, Ala14, Met22, Va123, Pro58, Ile59, Pro60, Pro62, Pro63, Ala65. Anisotropic charge distribution leads to the formation of large dipolar moments that are important for electron transfer complexes.

The charge distribution described above for Pa-C551 has also been reported in other electron transfer proteins and their electron acceptors. In addition, modifications by specific site mutagenesis of residues within hydrophobic or charged patches demonstrate the importance of surface complementarity for binding and electron transfer in different proteins. As an example, evidence of the relevance of the hydrophobic patch to the electron transfer properties of azurin from P. aeruginosa has been obtained from studies conducted on mutants of residues Met44 and Met64 that have been changed to positive and negatively charged amino acids. ( Id ).

Cytochrome The c-type domain has a fold consisting of a series of alpha helix and reverse turn that serve to enclose covalently bound heme in a hydrophobic pocket. Such domains can be observed in monodomain cytochrome c proteins such as cytochrome c6, cytochrome c ₅₅₂ , cytochrome c ₄₅₉ , mitochondrial cytochrome c. Such cytochrome c-type domains include many other proteins, for example cytochrome cd1-nitrite reductase as the N-terminal heme c domain, quinoprotein alcohol dehydrogenase as the C-terminal domain, domains 1 and 2 Quinohemoprotein amine dehydrogenase A chain, cytochrome bc ₁ Cytochrome bc ₁ as domain Appear within the complex. Structure Analysis with VAST Algorithm ( Pseudomonas as Query In cytochrome c ₅₅₁ from aeruginosa asa, significant structural neighbors (P values of 10 ^−10.3 to 10 ^−4.5 ) were identified only for cytochromes.

How to use

The present invention provides a method of treating a patient at risk of or suffering from malaria, or inhibiting the spread of malaria parasites. These methods include administering to a patient or to an insect vector a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, that inhibits parasitemia of malaria-infected mammalian cells. Such inhibition of parasitic hyperemia can be determined by several methods well known in the art. One method is described in Example 6, which determines the inhibition of parasitic hyperemia in malaria-infected human erythrocytes. In another embodiment, the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof inhibits intracellular replication of malaria parasites in human erythrocytes and is administered to a patient or insect vector. Methods for determining intracellular replication of malaria parasites are well known in the art, and one such method is described in Example 2. The modality of the present invention is not limited to any specific mechanism, and inhibition of parasitemia may include inhibition of replication of parasites in infected blood cells, inhibition of parasites infecting uninfected blood cells, inhibition of parasite growth cycles, Inhibition from invasion into mammalian cells results from a number of factors, including but not limited to these.

The present invention provides a method for treating a patient suffering from infection with a malaria parasite by administering an effective amount of at least one protein that is cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof. A patient that can be treated in this way is any mammal, in particular a human patient, that can be infected by malaria parasites. Malaria parasites that are known to infect mammals include febrile malaria ( Plasmodium falciparum ), P. vivax , P. berghei (rodent-specific), and P. yoelli ( P. yoelli ). (Murine-specific), P. cynomolgi , P. knowlesi (monkey-specific), but are not limited to these.

In addition, cupredoxin and cytochrome c ₅₅₁ have been found to be effective against HIV-1 infection, as disclosed in co-submitted applications (“Compositions And Methods For Treating HIV Infection With Cupredoxin And Cytochrome c,” US Provisional Patent Application Serial) No). Furthermore, co-infection with HIV and malaria is very common in many parts of the world, particularly in sub-Saharan Africa. In some embodiments, the patient suffering from infection with a malaria parasite is also infected with HIV. In some embodiments, the methods of treatment of the present invention also include administering an anti-HIV agent. In some embodiments, the anti-HIV agent is co-administered.

The present invention also provides a method for treating a patient suspected of contact with a malaria parasite, wherein the method comprises at least one of cupredoxin and / or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome. Administering an effective amount of the peptide to the patient. Patients may be suspected of contact with malaria parasites, for example, if they have lived or traveled in areas where malaria infections are frequent in other individuals of this patient's species. Treatment by this method can begin immediately before or after the patient has contacted the Marilla parasite. Since contact with malaria parasites is almost always caused by contact with insect vectors such as mosquitoes, areas where these insects and malaria parasites are prevalent are considered to be areas where patients are likely to come in contact with malaria parasites. Such regions around the world include, but are not limited to, Africa, Asia, and Latin America. Furthermore, the patient may be suspected of contact with the malaria parasite if it comes into contact with blood infected with the malaria parasite, intentionally exposed to the malaria parasite, or if an accidentally infused blood or drug contaminated with the malaria parasite.

Cupredoxin or cytochrome, or variants, derivatives or structural equivalents of cupredoxin or cytochrome may be administered to patients in various regimes by a variety of routes well known to those skilled in the art. In certain embodiments, the cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome is administered orally, topically, inhaled, injected, more specifically, intravenously, intramuscularly or subcutaneously.

In one embodiment, these methods comprise a composition comprising one unit dose of a composition containing cupredoxin or cytochrome, or a variant, derivative or structural equivalent thereof and an anti-malarial and / or anti-HIV agent Co-administering a unit dose of 1 to the patient sequentially. These compositions can be administered at about the same time, or within a fixed time after administration of another agent, for example, within about 1 minute to 60 minutes after administration of another agent, or about 1 minute to 60 minutes, or about 1 hour after administration of another agent. It can also be administered within 12 hours.

In the present invention, at least one of a cupredoxin or cytochrome, or a variant, derivative or structural equivalent of cupredoxin or cytochrome in an effective amount to reduce the infectivity of parasites in a coexisting mammalian population. A method of inhibiting the spread of malaria parasites within an insect vector population in which malaria parasites are parasitic by administering to insect vectors within the population is provided. In certain embodiments, the insect vector is a mosquito, more specifically, a mosquito from Anopheles gambiae species. In this method, administration of cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin or cytochrome can be achieved by placing these peptides in a composition consumed by such insect vector, Any method of contacting such peptides with malaria parasites in the intestine is contemplated. Various methods of administering chemicals to insect populations that perform such consumption are known in the art.

In another embodiment, a transmissible genetic element that migrates from one mosquito to another mosquito is operably linked to a cupredoxin coding sequence operably linked to a structural promoter, such a cupredoxin or cytochrome, Or a variant, derivative or structural equivalent of cupredoxin or cytochrome is produced in the Anopheles gambiae infected with P. falciparum and will interfere with its replication / survival in the mosquito.

Other methods of administering these peptides to insect vectors include the fusing of a cupredoxin or cytochrome, or a variant, derivative or structural equivalent gene of cupredoxin or cytochrome to a gene from other proteins commonly consumed by these insects. Included, but not limited to. The amount of peptide administered with an insect vector should be an effective amount that reduces the degree of infection of malaria parasites in the mammal when such insect vector comes into contact with the mammal. In certain embodiments, the amount administered should be effective to reduce the infectivity of malaria parasites when such insect vectors come into contact with humans.

Mosquito larvae are suitable for use in the present invention, and the promoters used are preferably strong promoters. Two alternative categories of promoters can be used: inducible and constitutive promoters. Inducible promoters include, for example, heat shock promoters. Suitably, the heat shock promoter is a Drosophila melanogaster capable of attracting the expression of genes in heterologous organisms, including insect heat shock promoters such as the Mediterranean fruit worm. hsp 70 promoter. The present invention also encompasses the use of this Mediterranean fruit worm hsp70 promoter (Papadimitriou et al., Insect Mol Biol 7: 279-90 (1998)). An alternative system is based on induction with the antibiotic tetratracycline (Heinrich and Scott, PNAS 97: 8229-8232, (2000)).

Heat shock promoters are induced by raising the temperature of the conditions under which Mediterranean fruit worms are cultured. For example, at 23-25 ° C., the hsp70 promoter exhibits low or no activity. This allows insect larvae to develop without the stress induced by the production of heterologous proteins. But at higher temperatures, for example 37-42 ° C., the hsp70 promoter is induced and expresses heterologous proteins at high levels.

Inducible promoters can be constructed based on known inducible gene control elements. For example, inducible promoters are constructed by binding elements that respond to drugs or hormones administered in the diet. In a preferred embodiment, when the insect is transformed with a second coding sequence that expresses a human estrogen receptor, a human estrogen responsive element (ERE) is used to regulate the expression of the protein of interest.

Structural promoters can also be used to express such proteins and / or other proteins required by insect larvae. For example, the structural promoter is a cytoplasmic actin promoter. D. melanogaster cytoplasmic actin promoter was cloned (Act5C), which shows high activity in mosquitoes (Huynh and Zieler, J. Mol. Biol. 288: 13-20 (1999)).

The cytoplasmic actin gene and these promoters may be isolated from other insects, including Mediterranean fruit worms. Other examples include cytoplasmic tubulin promoters, such as the Mediterranean fruit worm cytoplasmic tubulin promoter.

Promoters that control secreted polypeptides can optionally be used with appropriate signal sequences to direct secretion of the protein to hemolymph. For example, a larval serum protein promoter is used (Benes et al ., Mol. Gen. Genet 249 (5): 545-556 (1995)).

Mass rearing techniques for mosquitoes are highly developed. For protein production, larval cultures started at different time points can be synchronized with an appropriate temperature shift regime. This is possible because the growth rate depends on the ambient temperature; Larvae growth rate is reduced by approximately 50% at 18 ° C.

Cupredoxin  or Cytochrome , Or objection Variant , Derivative or structural Equivalent  Pharmaceutical Compositions Containing

Pharmaceutical compositions containing a cupredoxin or cytochrome, or variant, derivative or structural equivalent of cupredoxin thereof may be prepared in any conventional manner, such as conventional mixing, degradation, granulation, dragee-making, emulsions. It may be prepared by encapsulation, encapsulation, capture, or freeze drying. Substantially pure cupredoxin and / or cytochrome, or variants, derivatives or structural equivalents thereof, can be readily mixed with pharmaceutically acceptable carriers known in the art. These carriers enable the preparation in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Suitable excipients may include, for example, fillers and cellulose preparations. Other excipients may include, for example, fragrances, colorants, detackifiers, thickness and other acceptable additives, adjuvants or binders. In some embodiments, the pharmaceutical composition is substantially free of preservatives. In another embodiment, the pharmaceutical composition contains at least one preservative. Overall methods for pharmaceutical forms are found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore MD (1999)).

Compositions containing cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, used in the present invention may be used for injection (eg, intradermal, subcutaneous, intramuscular, intraperitoneal, etc.), inhalation, topical administration, suppositories, transdermal patches. Or in various ways, including orally. For general information about drug delivery systems, see Ansel et al., Id. See. In some embodiments, compositions containing cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, may be prepared and directly used as injectables, in particular for subcutaneous injection and intravenous injection. Such injectable preparations can be advantageously used to treat patients who are at high risk for, or suffering from, such infections, in particular at risk of malaria-infection. Compositions containing cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, may also be taken orally after mixing with a protective or similar coating agent such as polypropylene glycol. have.

When administered by injection, cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, are prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline. The solution contains formulary agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is formulated in powder form for constitution with a suitable vehicle, eg pyrogen-free sterile water, prior to use. In some embodiments, the pharmaceutical composition does not contain an adjuvant or any other substance added to enhance the immune response stimulated by such peptide. In some embodiments, the pharmaceutical composition contains a substance that inhibits an immune response to such peptide.

When administered as an intravenous fluid, the intravenous fluid used to administer cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, may consist of crystalloids or colloids. . Crystalline herein is an aqueous solution of an inorganic salt or other water soluble molecule. Colloids herein include large insoluble molecules such as gelatin. Intravenous fluid is sterile.

Crystalloid fluids used for intravenous administration include, as described in Table 2, normal saline (sodium chloride solution at 0.9% concentration), Ringer's lactate or Ringer's solution, dissolved in water. % Dextrose solution (aka D5W) is included, but is not limited to these.

Composition of common crystalline solution solution Other designations [Na ⁺ ] [Cl ^- ] [Glucose] D5W 5% dextrose 0 0 252 2/3 & 1/3 3.3% dextrose / 0.3% brine 51 51 168 Semi-normal brine 0.45% NaCl 77 77 0 Normal saline 0.9% NaCl 154 154 0 Ringer's Lactate * Ringer's solution 130 109 0

^* Ringer's lactate is 28 mmol / ℓ ^{lactate, 4 mmol / ℓ K +,} 3 mmol / ℓ Ca 2 + is also contained.

When administered by inhalation, cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, may be prepared using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas. It is delivered in the form of an aerosol spray from a pressurized pack or sprayer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. By way of example, capsules and cartridges of gelatin used in an inhaler or blower are prepared to contain a powder mixture of protein and a suitable powder base, such as lactose or starch.

When administered topically, cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, are prepared as solutions, gels, ointments, creams, suspensions and the like, as is well known in the art. In some embodiments, administration is accomplished by transdermal patches. When administered as a suppository (eg rectal or vaginal), cupredoxin and / or cytochrome c, or variants, derivatives or structural equivalents thereof, are prepared in the form of compositions containing conventional suppository bases.

When administered orally, cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, can be used as pharmaceutically acceptable, such cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof in the art. It can be easily prepared by mixing with a carrier. Solid carriers such as mannitol, lactose, magnesium stearate and the like; These carriers may be prepared into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for oral ingestion by a subject to whom the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is treated. do. Excipients suitable for oral solid preparations such as powders, capsules, tablets include fillers (eg, sugars), cellulose preparations, granulating agents, binders and the like.

Other conventional carriers well known in the art also include multivalent carriers such as bacterial capsular polysaccharides, dextran or genetically engineered vectors. In addition, sustained release formulations containing cupredoxin or cytochrome, or variants, derivatives, or structural equivalents thereof, release such cupredoxin or cytochrome, or variants, derivatives, or structural equivalents thereof for extended periods of time. In the absence of an agent, cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, are removed from the subject's tissues and / or degraded by, for example, proteases and simple hydrolysis prior to inducing or enhancing the therapeutic effect. do.

The half-life in the bloodstream of the compositions of the present invention is circularized peptide (Monk et al., BioDrugs 19 (4): 261-78, (2005); DeFreest et al., J. Pept. Res. 63 ( 5): 409-19 (2004)), D, L-peptide (diastereomer) (Futaki et al., J. Biol. Chem. Feb 23; 276 (8): 5836-40 (2001); Papo et al., Cancer Res. 64 (16): 5779-86 (2004); Miller et al, Biochem. Pharmacol. 36 (1): 169-76, (1987)), peptides with special 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)), hydrocarbon stapling (Schafmeister et al. , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)) It may be extended or optimized in a number of ways well known to those skilled in the art. In particular, modification of peptide stability by d-isomerization (substitution) and D-substitution or L-amino acid substitution and hydrocarbon stapling is noted.

In various embodiments, the pharmaceutical composition comprises a carrier and excipients (buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids (eg glycine), antioxidants, bacteriostatic agents, chelating agents, suspending agents, thickening agents and / or preservatives). ), Water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliaries required to approximate physiological conditions (e.g. buffers, tonicity adjusting agents, wetting agents) ), Etc.). Although any carrier known to those skilled in the art can be used to administer the compositions of the present invention, the type of carrier depends on the mode of administration. The compound may be encapsulated in liposomes using well known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present 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; 5,942,252.

These pharmaceutical compositions may be sterilized by conventional well known sterilization techniques, or may be sterile filtered. The resulting aqueous solution is packaged intact or lyophilized, which freeze-dried preparation is mixed with sterile solution prior to administration.

Cupredoxin  or Cytochrome , Or objection Variant , Derivative or structural Equivalent  administration

Cupredoxin or cytochromes, or variants, derivatives or structural equivalents thereof are, for example, by any suitable route, eg, oral, buccal, inhaled, sublingual, rectal, vaginal, transurethral, nasal, Topical, transdermal, that is to say transdermal or parenteral (including intravenous, intramuscular, subcutaneous, coronary). Pharmaceutical formulations thereof may be administered in an effective amount to achieve the intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is generally approximately 0.01-20 mg / day per kg body weight.

Compounds comprising cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, alone or in combination with other active agents, are useful for the treatment and / or prevention of malaria infections. Appropriate doses will vary depending upon, for example, the compound of the cupredoxin or cytochrome c _{551 used} , or variant, derivative or structural equivalent thereof, host, mode of administration, and the nature and severity of the disease being treated. In general, however, satisfactory results in humans are indicated to be achieved at a daily dosage of approximately 0.01 to 20 mg / kg body weight. The daily dose indicated in humans is cupredoxin or cytochrome c _{551 in the} range of approximately 0.7 mg to 1400 mg, which is conveniently administered, for example, in daily, weekly, monthly, and / or continuous dosing. , Or variants, derivatives or structural equivalents thereof. Daily doses may be administered discontinuously from 1 to 12 times per day. Alternatively, the dose may be administered in increments of one every two days, every three days, every four days, every five days, every six days, every week, and similarly up to 31 or more days. Alternatively, the dosing can be a continuous dosing using a patch, iv administration, or the like.

The method of introducing a cupredoxin or cytochrome, or variant, derivative, or structural equivalent thereof into a patient, in some embodiments, includes a cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, and an agent used to treat malaria. Co-administration. These methods are well known in the art. In certain embodiments, the cupredoxin and / or cytochrome c are part of a cocktail comprising other malaria therapeutics, or co-administered with these therapeutics. Treatments for malaria include proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, and pyri Pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halophantrine, quinine, quinidine, amodiaquine, amopyro Amopyroquine, sulphonamide, artemisinin, artemisinin, arteflene, arteterene, artemether, artesunate, primaquine, pyronaridine , Proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halophantrine, quinine (quinine), proguanil, chloroquine, maple Mefloquine, 1,16-hexadecamethylenebis (N-methylpyrrolidinium) dibromide, and combinations thereof, including but not limited to these.

Ku single frame or cytochrome c _551, or a variant, derivative or structural equivalents to the method introduced in the patient In some embodiments, the pre-ku single or cytochrome c _551, or a variant, derivative or structural equivalents and anti -HIV A drug, such as a protease-inhibitor-containing cocktail, is introduced at the same time. These methods are well known in the art. In certain embodiments, the cupredoxin or cytochrome c ₅₅₁ , or variant, derivative or structural equivalent thereof, is part of a cocktail comprising an anti-HIV therapeutic agent, or co-administered with these therapeutic agents. Anti-HIV agents include reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), and d4T (stavudine). stavudine [Zerit]), 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): delavirdine (Rescriptor) and nevirapine ( nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), allopinavir and ritonavir combination (Kaletra), saquinavir (Inquinase) ), Indinavir sulphate (Crixivan), amprenavir (Agenerase), nelfinavir (Viracept), but are not limited to these. In some embodiments, a combination of several agents, called highly active antiretroviral therapy (HAART), is used to treat these patients.

The exact formulation, route of administration, and dose are determined by the attending physician taking into account the patient's condition. Dosage and resting periods can be adjusted individually to provide an active cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, at a plasma level sufficient to maintain a therapeutic effect. In general, the desired cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, is administered in a mixture with the intended route of administration and a pharmaceutical carrier selected according to standard pharmaceutical practice.

In one aspect, the cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof is delivered as DNA and such polypeptides are produced in situ . In one embodiment, DNA is described by way of example in Ulmer et al., Science, Vol. 259, pp-1745-49 (1993) and discussed in Cohen, Science, vol. 259, pp-1691-92 (1993). As it is, it is "naked." Uptake of naked DNA can be increased by coating the DNA on a carrier, eg, biodegradable beads, which are effectively transported into the cell. In this method, DNA is present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial expression systems, viral expression systems. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art (WO 90/11092, WO 93/24640, WO 93/17706, US Pat. No. 5,736,524).

Vectors used to transfer genetic material from organisms to organisms can be broadly divided into two classes: Cloning vectors are replicating plasmids or phages that are essential for proliferation in suitable host cells and that contain regions in which foreign DNA can be inserted. to be; Foreign DNA replicates and multiplies as if it is a component of the vector. Expression vectors (eg, plasmids, yeast or animal viral genomes) are used to introduce foreign genetic material into host cells or tissues for transcription and translation of foreign DNA such as cupredoxin and / or cytochrome DNA. In 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 particularly useful, such as inducible promoters that regulate gene transcription in response to certain factors. By operably linking the cupredoxin or cytochrome, or variant, derivative or structural equivalent polynucleotide thereof to the inducible promoter, the expression of the cupredoxin or cytochrome and variants and derivatives thereof can be controlled in response to certain factors. Examples of classical inducible promoters are promoters that respond to α-interferon, heat shock, heavy metal ions and steroids such as glucocorticoids (Kaufman, Methods Enzymol. 185: 487-511 (1990)) and tetracycline. Other preferred inducible promoters are those which are not endogenous to the cells into which these constructs are inserted but which react in these cells when induction agents are supplied from outside. In general, useful expression vectors are mainly plasmids. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, adeno-associated viruses) are also contemplated.

Vector selection depends on the organism or cell employed and the desired purpose of the vector. In general, vectors include signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters, transcription termination sequences. For example, the cupredoxin or cytochrome gene can be cloned into a vector delivered to malaria parasite-latent mosquitoes to prevent parasites from replicating into mosquitoes. This transmissibility of the vector will enable diffusion of cupredoxin / cytochrome to neighboring mosquitoes infected with malaria parasites.

The exact formulation, route of administration, and dose are determined by the attending physician taking into account the patient's condition. Doses and resting periods can be adjusted individually to provide an active cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, at a plasma level sufficient to treat the patient and / or maintain the therapeutic effect. In general, the desired cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof, may be administered in a mixture with the intended route of administration and a pharmaceutical carrier selected according to standard pharmaceutical practice. Pharmaceutical compositions for use in the present invention include excipients and auxiliaries that facilitate processing of cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof; For use in therapy in a conventional manner using one or more physiologically acceptable carriers containing an active substance that inhibits / promotes the secretion of cupredoxin or cytochrome, or variants, derivatives or structural equivalents thereof, or mixtures thereof It can be formulated into a formulation that can be.

Cupredoxin  or Cytochrome , Or objection Variants and  Derivative or structural Equivalent  Containing Kit

In one aspect, the invention provides a biologically active composition containing (1) cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof in a package or container; (2) pharmaceutically acceptable adjuvants or excipients; (3) carriers for administration, for example syringes; (4) Provide a kit containing one or more of the instructions for administration. Also contemplated are embodiments in which two or more of the components (1)-(4) are found in the same vessel.

In another aspect, the invention provides a biologically active composition containing (1) cupredoxin or cytochrome, or variant, derivative or structural equivalent thereof in a package or container; (2) proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumephantrine, atovaquone, pyri Pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halophantrine, quinine, quinidine, amodiaquine, amopyro Amopyroquine, sulphonamide, artemisinin, artemisinin, arteflene, arteterene, artemether, artesunate, primaquine, pyronaridine , Proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halophantrine, quinine (quinine), proguanil, chloroquine, mefloquine, 1,16- Antimalarial agents, including but not limited to hexadecamethylenebis (N-methylpyrrolidinium) dibromide; (3) pharmaceutically acceptable adjuvants or excipients; (4) a vehicle for administration, eg a syringe; (5) Provide a kit containing one or more of the instructions for administration. Also contemplated are embodiments in which two or more of the components (1)-(5) are found in the same package or container.

In some embodiments, such kits further comprise an anti-HIV therapeutic agent in a package or container. Anti-HIV therapeutics include reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), and d4T (stavudine stavudine [Zerit]), 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): delavirdine (Rescriptor) and nevirapine ( nevirapine (Viramune), protease inhibitors: ritonavir (Norvir), allopinavir and ritonavir combination (Kaletra), saquinavir (Inquinase) ), Indinavir sulphate (Crixivan), amprenavir (Agenerase), nelfinavir (Viracept), but are not limited to these. In some embodiments, a combination of several agents is included in the kit, referred to as highly active antiretroviral therapy (HAART).

When a kit is supplied, different components of the composition can be packaged in separate containers and mixed just prior to use. Such individual packaging of these components allows for long term storage without loss of function of the active component.

The reactants included in the kit are supplied in a constant container so that the lifetime of the different components is preserved and not absorbed or deformed by the container material. For example, a sealed glass ampoule may contain a lyophilized polypeptide or polynucleotide of cupredoxin or cytochrome c, or a variant, derivative or structural equivalent thereof, or a buffer packaged under a neutral non-active gas such as nitrogen. Include. The ampoule consists of any suitable material, such as glass, organic polymers (eg, polycarbonates, polystyrenes, etc.), ceramics, metals, or any other material typically used to hold similar reactants. Another example of a suitable container is a simple bottle made from an ampoule-like material and a lid holding a foiled interior, such as aluminum or alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container holds a stoppered bottle that can be penetrated by a sterile access port, for example a hypodermic needle. The other container has two compartments separated by a membrane that are easily removable and allow components to be mixed after removal. Removable membranes are glass, plastic, rubber, and the like.

Instructions may also be provided with the kit. The instructions may be printed on paper or other substrate and / or provided on an electronically readable medium, such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape, flash memory device, or the like. Can be. Detailed instructions may not be physically attached to the kit; Instead, the user may be directed to an Internet web site specified by the manufacturer or distributor of such a kit, or provided with instructions for use as an e-mail.

Cupredoxin  And / or Cytochrome  transform

Cupredoxin or cytochrome can be chemically modified or genetically altered to produce variants and derivatives as described above. Such variants and derivatives can be synthesized by standard techniques.

In addition to naturally-occurring allelic variants of cupredoxin and cytochromes, it significantly alters the ability of cupredoxins or cytochromes to cause modifications in the amino acid sequence of encoded cupredoxins or cytochromes but inhibit parasitemia in malaria-infected red blood cells. Unchanged changes can be introduced into the cupredoxin or cytochrome coding sequence by mutation. “Non-essential” amino acid residues are those residues that can be modified from the wild-type sequence of cupredoxin without altering the biological activity, while “essential” amino acid residues are necessary for this biological activity. For example, amino acid residues that are conserved between cupredoxins are particularly unmodified and are therefore expected to be “essential”.

Amino acids for which conservative substitutions that do not change the activity of the polypeptide can be achieved are well known in the art. Useful conservative substitutions are shown in Table 3, “Preferred Substitutions”. Conservative substitutions in which one class of amino acids are substituted with other amino acids of the same type are within the scope of the present invention unless such substitutions physically alter the biological activity of the compound.

Preferred Substitution first Residue Typical substitution Preferred Substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norleucine Leu

Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as β-sheet or α-helix structure, (2) charge, (3) hydrophobicity, or (4) the size of the target site side chain Can alter virulence factor function. The residues are divided into several groups based on common side chain properties, as shown in Table 4. Non-conservative substitutions involve the replacement of members of one class with members of another class. Substitutions can be introduced into conservative substitution sites, or more specifically, into non-conserved sites.

Amino acid class Bracket amino acid Hydrophobic Norleucine, Met, Ala, Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr acid Asp, Glu Basic Asn, Gln, His, Lys, Arg Disturbed chain structure Gly, Pro Aromatic Trp, Tyr, Phe

Variant polypeptides can be made using methods known in the art, such as oligonucleotide-mediated (specific site) mutagenesis, alanine scanning, PCR mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237: 1-7 (1986); Zoller and Smith, Methods Enzymol., 154: 329-50 (1987)), cassette mutagenesis, restriction screening mutagenesis ( restriction selection mutagenesis) (Wells et al., Gene 34: 315-23 (1985)), or other known techniques, are performed on cloned DNA to give cupredoxin or cytochrome c ₅₅₁ Variant DNA can be calculated.

A known mutation in the ku single frame and cytochrome c ₅₅₁ may also be used to produce a mutant pre-ku single and cytochrome c ₅₅₁ is used in the method of the present invention. For example, the C112D and M44KM64E mutants of azurin are known to exhibit different cytotoxic and growth arrest activity than native azurin, which may be useful in the methods of treatment of the present invention. One embodiment of these methods of the present invention utilizes cupredoxin and / or cytochromes and variants and derivatives thereof that retain the ability to inhibit the growth of malaria infections in mammalian cells. In another embodiment, the methods of the invention utilize cupredoxin variants, such as the M44KM64E variant, which have the ability to induce cellular growth arrest.

A more complete understanding of the invention may be achieved by reference to the specific examples below. These examples are described for illustrative purposes only and do not limit the scope of the invention. Changes in form and substitution of equivalents are considered to be shortcuts. Although specific terms have been used herein, these terms are for illustrative purposes and do not limit the invention. Modifications of the invention are possible without departing from the spirit and scope of the invention, and therefore the invention is limited only by the appended claims.

Example  One: Cupredoxin Cytochrome  by Tropical heat  malaria( P. falciparum ) Parasitic In vitro  Inhibition

Cupredoxin Bacteria wt Azurin, M44KM64E Azurin, Rusticinine, Cyanobacterial Plastocyanin and Cytochrome Pseudomonas aeruginosa ) cytochrome c ₅₅₁ , human cytochrome c, Phormidium laminosum ) cytochrome f was examined at normal red blood cell (RBC) assay at 30 ㎍ / ml at 30 hr post inoculation. In these experiments, normal RBCs were washed twice in serum free medium and resuspended in 10% hematocrit in complete RPMI. In addition to 30 μl complete RPMI containing recombinant cupredoxin or cytochrome protein at 666 for a final concentration of 200, 200 μl of 10% Hct RBC was added to each of the 24 wells (final 2% Hct at 1 mL). Cleavage-stage parasites were prepared by centrifugation of the late-stage cultures through a Percoll cushion for 10 minutes at 3200 rpm. For infection, 4 × 10 ⁶ parasites / wells were added at t = 0 hr in 500 μl volume. Plates were incubated for 30 hours and recorded at this time with thin blood smear and Giemsa staining.

Control was 9.5% parasiticemia (standard error 1.3%), wt azurin 6.9% parasiticemia (se 1.4%), M44KM64E azurin 9.1% parasiticemia (se 1.0%), Rusticyanin 7.2% parasiticemia (se 0.7%), cytochrome c ₅₅₁ 7.5% parasitemia (se 1.5%), human cytochrome c 8.4% parasitemia (se 0.4%), platocyanin 8.1% parasitemia (se 1.3%), cytochrome f 6.6% (se 1.0%), wt Cupredoxins such as azurin and rustyyanin and cytochromes such as cytochrome f or cytochrome c ₅₅₁ suggest 20 to 30% inhibition of parasitemia.

Examine the effects of these cupredoxins at various stages of the parasite life cycle (0-24 hours, ring formation; 24-36 hours, trophozoite; 36-48 hours, schizont) The control showed 0.1% mean ring formation, 9.4% nutrient formation, wt azurin no ring formation, 6.9% nutrient formation, and cytochrome f showed 0.2% ring formation and significantly lower (6.3%) nutrient formation. . Interestingly, Rusticyanine showed very high (2.0%) ring formation and significantly reduced (5.2%) nutrient formation. The rest did not show a significant effect. The parasites appear to be diseased and dying in the Rusticinine-treated samples as compared to the rest of the samples, demonstrating significant inhibition and toxic effects of Rusticyanine on parasite development.

Example  2: Rusticyanine  by Tropical heat  malaria( P. falciparum ) Intracellular  Replica In vitro  Inhibition

To determine whether bacterial redox proteins can inhibit intracellular replication of malaria parasites, erythrocytes were transferred to intracellular recombinant proteins at a concentration of 200 μg / ml using a hypotonic ghost preparation. It dripped. The cells were then washed, resuspended and infected with the meristem-stage parasite ( P. falciparum ), as described in Example 1. These erythrocyte ghost cells were incubated for 19 and 40 hours and made gimsa smears.

Compared to infection of normal erythrocyte cells in Example 1, total parasiticemia was reduced in ghost cell cultures loaded only with Rusticinine. At 19 hours, invasion and ring formation were 5.0 ± 0.4% in empty ghost cells and 4.5 ± 1.0% in Rusticinine-loaded phantom cells, with no significant difference observed. However, at 40 hours, Rusticinine-loaded phantom cells had lower infection levels. At 19 hours no major effects by these bacterial proteins were observed. However, at the 40 hour time point, untreated control ghost cells showed 4.6 ± 0.3% of parasitemia, while Rusticyanin-treated ghost cells showed a nearly 50% reduction of 2.7 ± 0.8% of parasitemia ( See Table 5). Wt azurin, M44KM64E mutant azurin, plastocyanin, cytochrome c ₅₅₁ , human cytochrome c, cynobacterial cytochrome f proteins showed varying parasitemia in the range of 4.2 to 5.4%.

Cupredoxin and cytochrome inhibition of P. falciparum infection of erythrocyte ghost cells. process Average at 40 hr Parasitic hyperemia Standard error Lack 4.6% 0.3% Wild Type Azurin 5.4% 1.0% M44KM64E Azurin 4.7% 0.5% Rusticyanine 2.7% 0.8% Cytochrome c ₅₅₁ 4.2% 0.4% Human cytochrome c 4.6% 0.8% Plastocyanin 4.3% 0.3% Cytochrome f 4.5% 0.9%

Example  3. Azurin  And Pf MSP1 Combined with -19 G17 .12 Monoclonal  Antibody Fab  Structural similarity between fragments

In previous studies (Gough & Chothia, Structure 12: 917-925 (2004); Stevens et al. , J. Mol. Recognit. 18: 150-157 (2005)), cupredoxin is a variable domain of immunoglobulin versus members Have been shown to exhibit structural similarity. A 3D database was searched for the structural homolog of azurin (1JZG) from P. aeruginosa using the DALI algorithm (Holm & Park, Bioinformatics 16: 566-567 (2000)). Azurin shows structural similarity to Fab fragments of G17.12 monoclonal antibodies complexed with PfMSP1-19 fragments of MSP1 cleavage surface proteins of P. falciparum (Pizarro et al., J. Mol. Biol. 328: 1091-1103 (2003)) (Table 6). Azurin is also involved as cerebral malaria and as a receptor on endothelial cells in the brain's microvasculture and other tissues for sequestering P. falciparum -infected red blood cells. Functional ICAM-1 (Table 6) also shows structural similarities (Smith et al ., Proc. Natl. Acad. Sci. USA 97: 1766-1771 (2000); Franke-Fayard et al. , Proc. Natl. Acad. Sci. USA 102: 11468-11473 (2005)).

In this embodiment, cupredoxins, including azurin, are bound to face each other, and disulfide bridges in the extracellular domains of variable domains and intercellular adhesion molecules (ICAMs) and their ligands of immunoglobulin family members. It demonstrates structural similarity in that it has two antiparallel β sheets connected by).

Structural Similarity of P. aeruginosa Azurin and Various Pathogenesis-Related Proteins PDB Remark Reference Azurin (1 jzg ) DALI  z Score ^(One) 1VCA B1 Human vascular cell adhesion molecule-1, VCAM-1 17 3.5 1ZXQ1 Crystal Structure of ICAM2 19 3.3 1IAM1 Structure of the Two Amino-terminal Domains of ICAM-1 20 3.0 1OB1 A1 Crystal Structure of Fab Complex with Plasmodium falciparum MSP1-19 21 2.9 1TOP B Complex structure of the binding domain of ICAM-3 and alphabeta2 22 2.5 2NCM Neutral cell adhesion molecule, NCAM 23 2.4

¹ structure alignment of very lean was performed using a DALI (16). Structure pairs with a DALI z score <2 are considered different.

Example  4. Laz And H.8- Azurin  Fusion gene Cloning and  Expression.

Neisseria gonorrhoeae ) from laz The gene was cloned based on its known sequence (SEQ ID NO: 22). The sequence of H.8 epitope of laz (SEQ ID NO: 21) from P. aeruginosa azurin gene (SEQ ID NO: 1) ( paz ) and gonococcus ( N. gonnerrhoeae ) yields H.8- paz to the clones frame (in frame) H.8 epitope gene in the 3'-end of paz to calculate, or paz -H.8 in the 5'-end of paz was used to.

Cancer Cells, Bacterial Strains and Gene Constructs Cell / strain / Plasmid Related Characteristics * references P. aeruginosa PAO1 Protrophic, FP- (gender of gender factor) Holloway, et al ., Microbiol. Rev. 43: 73-102 (1979) E. coli JM109 Cloning and Azurin Expression Strains Yanisch-Perron, et al. , Gene 33: 103-119 (1985) E. coli BL21 (DE3) GST expressing strain Novagen N. gonorrhoeae F62 Protonutrients Used for DNA Separation American Type Culture Collection pUC18 Common Cloning Vector, Ap ^r Yanisch-Perron, et al., Id. pUC19 Common Cloning Vector, Ap ^r Yanisch-Perron, et al., Id. pUC18- laz 1 kb PCR fragment from genomic DNA of N. gonorrhoeae F62 cloned into pUC18 This specification pUC19- paz 0.55 kb PCR fragment from P. aeruginosa PAO1 cloned into HindIII and PstI cleaved pUC19, Ap ^r Yamada, et al ., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Yamada, et al. , Proc. Natl. Acad. Sci. USA 101: 4770-4775 (2004) pUC18-H.8- paz Fusion plasmids encoding azurin from H.8 and P. aeruginosa PAO1 from N. gonorrhoeae , Ap ^r This specification pGEX-5X-3 GST gene fusion vector, Ap ^r Amersham pET29a E. coli expression vector, Km ^r Novagen pET29a-gst pET29a derivative carrying the gst gene, Km ^r This specification pGEX-5X-3-H.8 PGEX-5X-3 derivatives bearing the H.8-encoding region, Ap ^r This specification pET29a- gst -H.8 pET29a derivative carrying the gst-H.8 gene, Km ^r This specification

Ap, ampicillin; Km, kanamycin; GST, glutathione S-transferase.

Cloning and Expression of pza and laz Genes . Cloning and hyperexpression of the azurin gene is described in Yamada, et al ., Proc. Natl. Acad. Sci. USA 99: 14098-14103 (2002); Punj, et al. , Oncogene 23: 2367- 2378 (2004). Neisseria Laz- encoding genes gonorrhoeae) (laz) was amplified by PCR of genomic DNA of Neisseria gonorrhoeae (N. gonorrhoeae) strain F62 into the template DNA. The forward and reverse primers used were 5'CCG GAATTC CGGCAGGGATGTTGTAAATATCCG-3 '(SEQ ID NO: 23) and 5'GG GGTACC GCCGTGGCAGGCATACAGCATTTCAATCGG-3' (SEQ ID NO: 24), where EcoRI and KpnI Additional introduced restriction sites of the sites are each underlined. 1.0 kb amplified DNA fragments digested with EcoRI and KpnI were inserted into the corresponding sites of the pUC18 vector (Yanisch-Perron, et al. , Gene 33: 103-119 (1985)) to laz The expression plasmid pUC18- laz was calculated by placing the gene downstream of the lac promoter (Table 7).

Plasmids expressing a fusion of H.8 of N. gonorrhoeae Laz and Azurin (Paz) of P. aeruginosa were constructed by PCR using pUC19- paz and pUC18- laz as templates. In the case of the H.8-Paz fusion, the 3.1 kb fragment is pUC18- laz as a template. And a primer, 5 '(phosphorylated) GGCAGCAGGGGCTTCGGCAGCATCTGC-3' (SEQ ID NO: 25) and 5'CTGCAG GTCGAC TCTAGAGGATCCCG-3 '(SEQ ID NO: 26), wherein SalI Sites are indicated as underscores. PCR amplified 0.4 kb fragments were obtained from pUC19- paz and primers, 5 '(phosphorylated) GCCGAGTGCTCGGTGGACATCCAGG-3' (SEQ ID NO: 27) and 5'TA CTCGAG TCACTTCAGGGTCAGGGTG-3 '(SEQ ID NO: 28) as templates. Where XhoI Sites are indicated as underscores. pUC18- laz from SalI Truncated PCR Fragments and XhoI from pUC19- paz The cleaved PCR fragment was cloned to yield the expression plasmid pUC18-H.8- paz (Table 7).

E. coli JM109 was used as a host strain for expression of azurin and its derivative genes. Recombinant E. coli strains were cultured for 16 hours at 37 ° C. in 2 × YT medium containing 100 μg / ml ampicillin, 0.1 mM IPTG, 0.5 mM CuSO ₄ to produce azurin protein.

E. coli strains carrying these plasmids were grown in the presence of IPTG, cells were lysed and the proteins purified as described in 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), in previous literature ( Cannon, Clin. Microbiol. Rev. 2: S1-S4 (1989); Fisette, et al. , J. Biol. Chem. 278: 46252-46260 (2003)). Except for 8 retained proteins (approximately 17 kDa), various azurin derivatives migrated as single components on SDS-PAGE

Plasmid Construction for Fusion GST Proteins . Plasmids expressing fused glutathione S-transferase (GST) -truncated wt-azurin (azu) derivatives were constructed by polymerase chain reaction using proofreading DNA polymerase. In the case of pGST-azu 36-128, the amplified PCR fragments were introduced into the BamHl and EcoRl sites of the commercial GST expression vector pGEX-5X (Amersham Biosciences, Piscataway, NJ). These fragments are shown as pUC19-azu and primers, 5'-C GGGATCC CCG GCA ACC TGC CGA AGA ACG TCA TGG GC-3 '(SEQ ID NO: 29) and 5'-CG GAATTC GCA TCA CTT CAG GGT CAG GG- Amplified to 3 ′ (SEQ ID NO: 30), where additionally introduced BamHl and EcoRI sites are underlined, respectively. azu The carboxy-terminal truncation of the gene was cumulative by introducing a stop codon using the QuickChange specific site mutagenesis kit (Stratagene, LaJolla, Calif.).

In the case of pGST-azu 36-89, a stop codon was introduced into Gly90. Plasmids bearing pGST-azu 36-128 were used as template DNA. Three sets of oligonucleotides for specific site mutagenesis are shown below. For pGST-azu 36-89: 5'-CCA AGC TGA TCG GCT CGT GAG AGA AGG 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).

in pGST-azu 88-113, azu The carboxy-terminal truncation of the gene was cumulative by introducing a stop codon using the QuickChange specific site mutagenesis kit (Stratagene, LaJolla, Calif.). In the case of pGST-azu 88-113, a stop codon was introduced into Phe114. Plasmids bearing pGST-azu 88-128 were used as templates. in the case of pGST-azu 88-128, the expression of the amplified PCR fragment commercial GST vector pGEX-5X (AmershamBiosciences) BamH1 and EcoR1 for Introduced into the site. These fragments are composed of pUC19-azu and primers, 5'-C GGGGATCC CCG GCT CGG GCG AGA AGG AC-3 '(SEQ ID NO: 33) and 5'-CGG GAATTC TCC ACT TCA GGG TCA GGG TG-3' Amplified by SEQ ID NO: 34, where the additionally introduced BamH1 and EcoR1 sites are each underlined.

One set of oligonucleotides for specific site mutagenesis that allows the preparation of pGST-azu 88-113 is as follows: 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). E. coli XL-1 Blue strain was transformed with pGST-azu 88-113. Plasmid extraction was performed using a commercial kit (Qiagen, Venlo, The Netherlands), and PCR sequencing was performed to assess plasmid insertion and transfection.

E. coli BL21 (DE3) was used as a host strain for the expression of gst and its fusion derivatives. E. coli strain XL1-Blue transformed with pGST-azu plasmid was grown for 3 hours at 37 ° C. in LB medium containing ampicillin, and then IPTG induction (0.4 mM) was induced to maximize expression levels. And further incubated at 37 ° C. for 2-4 hours. Cells were separated by centrifugation and resuspended in 25 ml IX PBS buffer. Subsequent cell lysis entails two sequential treatments of the cell suspension via sonication (20 min on ice) and heat-cold shock in an acetone-dry ice electrolyzer (using the appropriate protease inhibitor). It was. The supernatant of the cell lysis mixture was separated and passed through a freshly charged and PBS equilibrated 1 ml glutathione-Sepharose 4B (Amersham Biosciences) column. After column washing of GST-azu product with 10 mM glutathione dissolved in 20 mM Tris-HCl pH 8 and subsequent elution, electrophoresis using 10% SDS-PAGE Tris-Gly gel stained with Coomassie Brilliant Blue R reagent. The purity of GST-Azu 88-113 was investigated. Protein concentration was determined using the Bradford method.

Example  5. Azurin  Pseudomonas aeruginosa ( P. falciparum ) Fission body  Cleavage protein MSP1 C-terminal fragment MSP1 -19 and MSP1 Binds to -42

Considering the structural similarities (Table 6) between the Fab fragment (Pizarro et al., Id ) of the monoclonal antibody G17.12 complexed with azurin and PfMSP1-19, it is possible to form a complex with pfMSP1-42 or PfMSP1-19. Lin's ability was determined.

Azurin, Laz, with an additional 39 amino acid epitopes and are either gonnococci or meningococci (eg, Neisseria) azurin-like protein (H.8 epitope) derived from meningitidis (Gotschlich & Seiff, FEMS Microbiol. Lett. 43: 253-255 (1987); Kawula et al. , Mol. Microbiol. 1: 179-185 ( 1987)), two derivatives were examined in H.8-azrin (H.8 epitopes of Laz are fused in-frame within the N-terminal portion of P. aeruginosa azurin; see Example 4).

In vitro protein-protein interactions were evaluated using a Biacore X spectrometer from Biacore AB International. All experiments were carried out using Au-CM5 sensor chip (Biacore) in HBS-EP working buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) at 25 ° C. Was performed. Protein immobilization on the CM5 chip was performed according to the amine coupling procedure. Protein was fixed after NHS / EDC preactivation of CM5 surface: 50 μl injection of azurin (510). Subsequent treatment of the CM5 surface with ethanolamine (1M, pH 8.8) removed non-crosslinked protein. Binding studies were performed by injecting protein eluent (50 μl) at a flow rate of 30 μl / min on the protein-CM5 surface, which was accompanied by a 120 second time delay at the end of the injection. Protein eluents included GST-Azurin fusion proteins (GST, GST-Azu 36-128, GST-Azu 36-89, GST-Azu 88-113; see Example 4). Sensor chip surfaces were regenerated between protein injections using 100 mM NaOH (10 μl injection pulse). All binding studies were conducted on negative flow channels with exposed Au-CM5 sensor surfaces to compensate for nonspecific binding to the chip. To generate binding constant data, titration experiments were designed and data collected through injection of increasing concentrations of protein eluent (0.05-2000 nM). These SPR data can be fitted to the Langmuir equilibrium binding model [Req = Rmax / (1 + Kd / C)] as specified in the Biacore software, from which the binding constant (Kd) was inferred.

Specific interactions of azurin, H.8-azrin, and Laz with Pf MSP1-19 and Pf MSP1-42 proteins were determined by surface plasmon resonance (SPR) analysis, and the data are shown in FIG. 1. do. SPR sensorgrams for the binding of immobilized Pf MSP1-19 with Pf MSP1-42 azurin and its derivatives indicated selective recognition between these proteins. Azurin at nanomolar concentrations allows significant binding with immobilized MSP1-19 (FIG. 1A) or MSP1-42 (FIG. 1B), but both H.8-Azurin and Laz have azurin and MSP1- Characteristic Kd values of 32.2 nM between 19 and 54.3 nM between azurin and MSP1-42 showed higher affinity in binding to the cleavage surface protein MSP1 cleavage product. The Kd values between H.8-Azurin and MSP1-19 and MSP1-42 are 11.8 nM and 14.3 nM, respectively, while these values between Laz and MSP1-19 and MSP1-42 are 26.2 nM and 45.6 nM, respectively. It was.

Glutathione S-, which binds to MSP1-19 or MSP1-42, to investigate whether the H.8 epitope promotes the binding of H.8-azurine or Laz to the PfMSP1-19 or PfMSP1-42 moiety. The ability of the fusion derivative H.8-GST (see Example 4) in which transferase (GST) and H.8 epitope were fused within the N-terminus of GST was investigated. GST and H.8-GST did not bind PfMSP1-19 (FIG. 1A) or MSP1-42 (FIG. 1B), although H.8-GST showed weak binding with MSP1-42.

Some fusion proteins (Yamada et al. , Cell. Microbiol. 7: 1418-1431 (2005); see Example 4; Glutathione S Transferase (GST) and a portion of azurin fused to GST) are described in MSP1-19. The ability to bind was investigated. GST alone, or GST-Azu 88-113, in which the azurin amino acid sequences 88-113 are in-frame fused to GST out of 128 amino acids of azurin, show no binding (FIG. 1C), while they retain amino acid sequences 36-89. GST-Azu 36-89 and GST-Azu 36-128 with amino acid sequences 36-128 showed significant binding with MSP1-19, with Kd values of 20.9 nM and 24.5 nM, respectively.

Example  6. Azurin , H.8- Azurin , Laz On by Tropical heat  malaria( Plasmodium falciparum Inhibition of parasites

The degree of parasitemia was determined using mitotic stage parasites and normal red blood cells (RBCs). Normal red blood cells (RBCs) were washed twice in serum-free medium and resuspended with 10% hematocrit in complete RPMI. In addition to 300 μl complete RPMI with or without azurin, H.8-azrin or Laz at various concentrations, 200 μl of 10% hematocrit RBC was added to each of the 24 wells. Cleavage-stage P. falciparum parasites were prepared by centrifugation of the late-stage culture via a Percoll cushion for 10 minutes at 3200 rpm. For infection, 4 × 10 ⁶ parasites / wells were added at t = 0 hr in 500 μl volume. Plates were incubated overnight (approximately 16 hours) and recorded at this time with thin blood smear and Giemsa staining.

Azurin, H.8-Azurin, and Laz all significantly inhibited parasitemia in a dose-dependent manner at relatively high concentrations (approx. 50) (FIG. 2). Perhaps this high concentration reflects the different ways in which malaria parasites invade red blood cells (Cowman et al. , FEBS Lett. 476: 84-88 (2000); Baum et al., J. Biol. Chem. 281: 5197 -5208 (2006)), high concentrations of azurin or Laz are required to interfere with this invasion process. As indicated by enhanced binding affinity for MSP1-19, both H.8-azurin and Neisserial Laz proteins have higher levels of P. falciparum parasites compared to azurin . Inhibition was shown (FIG. 2).

When azurin is labeled with the red fluorescent dye Alexa fluor 568 and used during this invasion test, little red fluorescence is detected in the RBC, which enters RBC as part of the azurin-bound MSP1-19. RBCs that do not, or show the presence of these cleavage, suggest that Azurin has failed to bind MSP1-19. These data indicate that azurin does not enter normal cells such as macrophages, mast cells, etc., and that the effects of azurin, H.8-azurin, or Laz are exerted at the level of invasion rather than intracellular replication of parasites. This is in perfect agreement with the observations (Yamada et al. , Cell. Microbiol. 7: 1418-1431 (2005)). Taken together, the date in Figure 2 demonstrates the potential antimalarial action of Azurin, H.8-Azurin, Laz via interference in RBC invasion by parasites.

Example  7. Azurin ICAM To combine.

Interesting structural similarities between ICAMs (Table 6), which are known to be involved as receptors for azurin and P. falciparum -infected red blood cells (Wassmer et al. , PloS Med. 2: 885-890 (2005); Dormeyer et al. , Antimicrob.Agents Chemotherap. 50: 724-730 (2006)) are proteins that are measured by SPR between azurin and ICAM, such as ICAM-1, ICAM-2, ICAM-3, NCAM. Triggered assay analysis of protein interactions. For azurin immobilized on the CM5 chip, ICAM-3 (FIG. 3, Kd = 19.5 + 5.4 nM) and NCAM (FIG. 3, illustration) showed strong binding, while ICAM-1 and ICAM-2 did not. Without limiting the manner in which the invention works, some of the effects of azurin on the inhibition of P. falciparum parasitic hyperemia may be mediated through interaction with ICAM-3 or NCAM.

Example  8. Treatment of patients exposed to or likely to be exposed to malaria

A pharmaceutical composition clinically used for the prevention of malaria and containing one or more cupredoxins and / or cytochromes is administered to a patient.

Healthy 22-year-old healthy male volunteers aged 15 to 50 years who live in areas where malaria is prevalent, although there is no history of existing antibodies against blood-stage P. falciparum parasites as measured by immunofluorescent assay Is injected with a pharmaceutical preparation of purified cupredoxin and purified cytochrome. Two of these serve as untreated controls.

Sterile pharmaceutical formulations, sterile Pseudomonas aeruginosa contained in a pharmaceutical formulation designed for the intravenous administration as is well known to those skilled in the art (Pseudomonas aerugninosa ) in the form of a 0.5 ml single-dose ampoule of azurin . Such pharmaceutical preparations are stored at 4 ° C. and protected from sunlight prior to administration. In one clinical trial, azurin was administered in five different concentrations: 10 μg, 30 μg, 100 μg, 300 μg, 800 μg azurin per 0.5 ml dose.

These pharmaceutical formulations are given intravenously to 13 volunteers, respectively, for 10 doses. Volunteers receive primary care on day 0, followed by the same dose every other day for three weeks. Volunteers are tested for immediate toxic effects 20 minutes after injection. After 24 and 48 hours, they are examined for evidence of fever, local tenderness, erythema, warmth, induration, and lymphadenopathy, headache, fever, chills, discomfort, Have a questionnaire about local pain, nausea and complaints of joint pain. Prior to each dose, blood and urine samples are taken for full laboratory examination. The complete blood count and serum chemistry profile are reviewed two days after each dose. The presence of malaria parasites is determined by light microscopic examination (ME) of stained blood smears or by the ICT Malaria Pf / Pv test kit (Binax, Inc., Portland, ME). These results demonstrate the efficacy of this therapy.

Example  9: Control of Malaria Infection of Insects

A transmissible genetic element that migrates from one mosquito to another will be operably linked to the cupredoxin coding sequence operably linked to the structural promoter. Through this, P. aeruginosa azurin will be produced in Anopheles gambiae infected with P. falciparum and will interfere with its replication / survival in the mosquito. This mosquito then enters the contagious area, so that the azurin-bearing gene element spreads to other P. falciparum -infected A. gambiae to grow or survive P. falciparum . Will inhibit.

Example  10. Treatment of patients infected with malaria

Pharmaceutical compositions used clinically for the treatment of malaria and containing one or more cupredoxins and / or cytochromes are administered in humans for the treatment of malaria infections.

Purified P. aeruginosa from 15 22-50-year-old healthy male volunteers showing the power of existing antibodies against blood-stage P. falciparum parasites as measured by immunofluorescent assay. A pharmaceutical formulation of azurin is injected. Two of these serve as treated controls.

Sterile pharmaceutical preparations are in the form of 0.5 ml single-dose ampoules of sterile P. aerugninosa azurin contained in pharmaceutical preparations designed for intravenous administration, as is well known to those skilled in the art. Such pharmaceutical preparations are stored at 4 ° C. and protected from sunlight prior to administration. In one clinical trial, P. aerugninosa azurin was administered at five different concentrations: 10 μg, 30 μg, 100 μg, 300 μg, 800 μg azurin / cytochrome c ₅₅₁ (at molecular basis) per 0.5 ml dose. 1: 1).

These pharmaceutical formulations are given intravenously to 13 volunteers, respectively, for 10 doses. Volunteers receive primary care on day 0, followed by the same dose every other day for three weeks. Volunteers are tested for immediate toxic effects 20 minutes after injection. After 24 and 48 hours, they are examined for evidence of fever, local tenderness, erythema, warmth, induration, and lymphadenopathy, headache, fever, chills, discomfort, Have a questionnaire about local pain, nausea and complaints of joint pain. Prior to each dose, blood and urine samples are taken for full laboratory examination. The complete blood count and serum chemistry profile are reviewed two days after each dose. The presence of malaria parasites is determined by light microscopic examination (ME) of stained blood smears or by the ICT Malaria Pf / Pv test kit (Binax, Inc., Portland, ME). These results demonstrate the efficacy of this therapy.

                         SEQUENCE LISTING <110> The Board of Trustees of the University of Illinois        Chakrabarty, Ananda        Das Gupta, Tapas        Chaudhari, Anita        Fialho, Arsenio        Hong, Chang Soo        Yamada, Tohru   <120> COMPOSITIONS AND METHODS FOR TREATING MALARIA WITH CUPREDOXIN AND         CYTOCHROME <130> 51282-00063 <150> 60 / 780,868 <151> 2006-03-10 <150> 60 / 682,813 <151> 2005-05-20 <150> 11 / 244,105 <151> 2005-10-06 <150> 60 / 616,782 <151> 2004-10-07 <150> 60 / 680,500 <151> 2005-05-13 <150> 10 / 720,603 <151> 2003-11-11 <150> 60 / 414,550 <151> 2003-08-15 <150> 10 / 047,710 <151> 2002-01-15 <150> 60 / 269,133 <151> 2001-02-15 <160> 36 <170> PatentIn version 3.3 <210> 1 <211> 128 <212> PRT <213> Pseudomonas aeruginosa <400> 1 Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln Met Gln Phe Asn 1 5 10 15 Thr Asn Ala Ile Thr Val Asp Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met     50 55 60 Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys             100 105 110 Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys         115 120 125 <210> 2 <211> 82 <212> PRT <213> Pseudomonas aeruginosa <400> 2 Glu Asp Pro Glu Val Leu Phe Lys Asn Lys Gly Cys Val Ala Cys His 1 5 10 15 Ala Ile Asp Thr Lys Met Val Gly Pro Ala Tyr Lys Asp Val Ala Ala             20 25 30 Lys Phe Ala Gly Gln Ala Gly Ala Glu Ala Glu Leu Ala Gln Arg Ile         35 40 45 Lys Asn Gly Ser Gln Gly Val Trp Gly Pro Ile Pro Met Pro Pro Asn     50 55 60 Ala Val Ser Asp Asp Glu Ala Gln Thr Leu Ala Lys Trp Val Leu Ser 65 70 75 80 Gln lys          <210> 3 <211> 166 <212> PRT <213> Neisseria meningitidis <400> 3 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Ala 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp                 85 90 95 Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ala Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Glu     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 4 <211> 105 <212> PRT <213> Phormidium laminosum <400> 4 Glu Thr Phe Thr Val Lys Met Gly Ala Asp Ser Gly Leu Leu Gln Phe 1 5 10 15 Glu Pro Ala Asn Val Thr Val His Pro Gly Asp Thr Val Lys Trp Val             20 25 30 Asn Asn Lys Leu Pro Pro His Asn Ile Leu Phe Asp Asp Lys Gln Val         35 40 45 Pro Gly Ala Ser Lys Glu Leu Ala Asp Lys Leu Ser His Ser Gln Leu     50 55 60 Met Phe Ser Pro Gly Glu Ser Tyr Glu Ile Thr Phe Ser Ser Asp Phe 65 70 75 80 Pro Ala Gly Thr Tyr Thr Tyr Tyr Cys Ala Pro His Arg Gly Ala Gly                 85 90 95 Met Val Gly Lys Ile Thr Val Glu Gly             100 105 <210> 5 <211> 155 <212> PRT <213> Thiobacillus ferrooxidans <400> 5 Gly Thr Leu Asp Thr Thr Trp Lys Glu Ala Thr Leu Pro Gln Val Lys 1 5 10 15 Ala Met Leu Glu Lys Asp Thr Gly Lys Val Ser Gly Asp Thr Val Thr             20 25 30 Tyr Ser Gly Lys Thr Val His Val Val Ala Ala Ala Val Leu Pro Gly         35 40 45 Phe Pro Phe Pro Ser Phe Glu Val His Asp Lys Lys Asn Pro Thr Leu     50 55 60 Glu Ile Pro Ala Gly Ala Thr Val Asp Val Thr Phe Ile Asn Thr Asn 65 70 75 80 Lys Gly Phe Gly His Ser Phe Asp Ile Thr Lys Lys Gly Pro Pro Tyr                 85 90 95 Ala Val Met Pro Val Ile Asp Pro Ile Val Ala Gly Thr Gly Phe Ser             100 105 110 Pro Val Pro Lys Asp Gly Lys Phe Gly Tyr Thr Asp Phe Thr Trp His         115 120 125 Pro Thr Ala Gly Thr Tyr Tyr Tyr Val Cys Gln Ile Pro Gly His Ala     130 135 140 Ala Thr Gly Met Phe Gly Lys Ile Val Val Lys 145 150 155 <210> 6 <211> 124 <212> PRT <213> Achromabacter cycloclastes <400> 6 Ala Asp Phe Glu Val His Met Leu Asn Lys Gly Lys Asp Gly Ala Met 1 5 10 15 Val Phe Glu Pro Ala Ser Leu Lys Val Ala Pro Gly Asp Thr Val Thr             20 25 30 Phe Ile Pro Thr Asp Lys Gly His Asn Val Glu Thr Ile Lys Gly Met         35 40 45 Ile Pro Asp Gly Ala Glu Ala Phe Lys Ser Lys Ile Asn Glu Asn Tyr     50 55 60 Lys Val Thr Phe Thr Ala Pro Gly Val Tyr Gly Val Lys Cys Thr Pro 65 70 75 80 His Tyr Gly Met Gly Met Val Gly Val Val Gln Val Gly Asp Ala Pro                 85 90 95 Ala Asn Leu Glu Ala Val Lys Gly Ala Lys Asn Pro Lys Lys Ala Gln             100 105 110 Glu Arg Leu Asp Ala Ala Leu Ala Ala Leu Gly Asn         115 120 <210> 7 <211> 128 <212> PRT <213> Alcaligenes faecalis <400> 7 Ala Cys Asp Val Ser Ile Glu Gly Asn Asp Ser Met Gln Phe Asn Thr 1 5 10 15 Lys Ser Ile Val Val Asp Lys Thr Cys Lys Glu Phe Thr Ile Asn Leu             20 25 30 Lys His Thr Gly Lys Leu Pro Lys Ala Ala Met Gly His Asn Val Val         35 40 45 Val Ser Lys Lys Ser Asp Glu Ser Ala Val Ala Thr Asp Gly Met Lys     50 55 60 Ala Gly Leu Asn Asn Asp Tyr Val Lys Ala Gly Asp Glu Arg Val Ile 65 70 75 80 Ala His Thr Ser Val Ile Gly Gly Gly Glu Thr Asp Ser Val Thr Phe                 85 90 95 Asp Val Ser Lys Leu Lys Glu Gly Glu Asp Tyr Ala Phe Phe Cys Ser             100 105 110 Phe Pro Gly His Trp Ser Ile Met Lys Gly Thr Ile Glu Leu Gly Ser         115 120 125 <210> 8 <211> 129 <212> PRT <213> Achromobacter xylosoxidans ssp. denitrificans I <400> 8 Ala Gln Cys Glu Ala Thr Ile Glu Ser Asn Asp Ala Met Gln Tyr Asn 1 5 10 15 Leu Lys Glu Met Val Val Asp Lys Ser Cys Lys Gln Phe Thr Val His             20 25 30 Leu Lys His Val Gly Lys Met Ala Lys Val Ala Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Glu Ala Asp Lys Gln Gly Val Ala Thr Asp Gly Met     50 55 60 Asn Ala Gly Leu Ala Gln Asp Tyr Val Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Pro Gly Glu Ala Tyr Ala Tyr Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Met Met Lys Gly Thr Leu Lys Leu Ser         115 120 125 Asn      <210> 9 <211> 129 <212> PRT <213> Bordetella bronchiseptica <400> 9 Ala Glu Cys Ser Val Asp Ile Ala Gly Thr Asp Gln Met Gln Phe Asp 1 5 10 15 Lys Lys Ala Ile Glu Val Ser Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Lys His Thr Gly Lys Leu Pro Arg Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile     50 55 60 Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Leu Ala His Thr Lys Val Leu Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ala Lys Leu Ala Gly Asp Asp Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Gly Ala Leu Met Lys Gly Thr Leu Lys Leu Val         115 120 125 Asp      <210> 10 <211> 129 <212> PRT <213> Methylomonas sp. J. <400> 10 Ala Ser Cys Glu Thr Thr Val Thr Ser Gly Asp Thr Met Thr Tyr Ser 1 5 10 15 Thr Arg Ser Ile Ser Val Pro Ala Ser Cys Ala Glu Phe Thr Val Asn             20 25 30 Phe Glu His Lys Gly His Met Pro Lys Thr Gly Met Gly His Asn Trp         35 40 45 Val Leu Ala Lys Ser Ala Asp Val Gly Asp Val Ala Lys Glu Gly Ala     50 55 60 His Ala Gly Ala Asp Asn Asn Phe Val Thr Pro Gly Asp Lys Arg Val 65 70 75 80 Ile Ala Phe Thr Pro Ile Ile Gly Gly Gly Glu Lys Thr Ser Val Lys                 85 90 95 Phe Lys Val Ser Ala Leu Ser Lys Asp Glu Ala Tyr Thr Tyr Phe Cys             100 105 110 Ser Tyr Pro Gly His Phe Ser Met Met Arg Gly Thr Leu Lys Leu Glu         115 120 125 Glu      <210> 11 <211> 166 <212> PRT <213> Neisseria meningitidis <400> 11 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Ala 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Thr Ser Met Gly His Asn Ile Val Ile Gly Lys Thr Glu Asp Met Asp                 85 90 95 Gly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Le Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Glu     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 12 <211> 128 <212> PRT <213> Pseudomomas fluorescens <400> 12 Ala Glu Cys Lys Thr Thr Ile Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Ala Ile Glu Ile Asp Lys Ala Cys Lys Thr Phe Thr Val Glu             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Leu         35 40 45 Val Ile Ser Lys Gln Ala Asp Met Gln Pro Ile Ala Thr Asp Gly Leu     50 55 60 Ser Ala Gly Ile Asp Lys Asn Tyr Leu Lys Glu Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Ala Gly Glu Lys Asp Ser Leu Thr                 85 90 95 Ile Asp Val Ser Lys Leu Asn Ala Ala Glu Lys Tyr Gly Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Ile Ser Met Met Lys Gly Thr Val Thr Leu Lys         115 120 125 <210> 13 <211> 128 <212> PRT <213> Pseudomonas chlororaphis <400> 13 Ala Glu Cys Lys Val Asp Val Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Glu Ile Thr Ile Asp Lys Ser Cys Lys Thr Phe Thr Val Asn             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Lys Ser Ala Asp Met Ala Gly Ile Ala Thr Asp Gly Met     50 55 60 Ala Ala Gly Ile Asp Lys Asp Tyr Leu Lys Pro Gly Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Ile Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Ala Gly Glu Ser Tyr Glu Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Asn Ser Met Met Lys Gly Ala Val Val Leu Lys         115 120 125 <210> 14 <211> 129 <212> PRT <213> Xylella fastidiosa 9a5c <400> 14 Lys Thr Cys Ala Val Thr Ile Ser Ala Asn Asp Gln Met Lys Phe Asp 1 5 10 15 Gln Asn Thr Ile Lys Ile Ala Ala Glu Cys Thr His Val Asn Leu Thr             20 25 30 Leu Thr His Thr Gly Lys Lys Ser Ala Arg Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Thr Asp Met Gln Ala Val Ala Leu Ala Gly Leu     50 55 60 His Ala Thr Leu Ala Asp Asn Tyr Val Pro Lys Ala Asp Pro Arg Val 65 70 75 80 Ile Ala His Thr Ala Ile Ile Gly Gly Gly Glu Arg Thr Ser Ile Thr                 85 90 95 Phe Pro Thr Asn Thr Leu Ser Lys Asn Val Ser Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Leu Met Lys Gly Thr Leu Asn Phe Gly         115 120 125 Gly      <210> 15 <211> 138 <212> PRT Cucumis sativus <400> 15 Met Gln Ser Thr Val His Ile Val Gly Asp Asn Thr Gly Trp Ser Val 1 5 10 15 Pro Ser Ser Pro Asn Phe Tyr Ser Gln Trp Ala Ala Gly Lys Thr Phe             20 25 30 Arg Val Gly Asp Ser Leu Gln Phe Asn Phe Pro Ala Asn Ala His Asn         35 40 45 Val His Glu Met Glu Thr Lys Gln Ser Phe Asp Ala Cys Asn Phe Val     50 55 60 Asn Ser Asp Asn Asp Val Glu Arg Thr Ser Pro Val Ile Glu Arg Leu 65 70 75 80 Asp Glu Leu Gly Met His Tyr Phe Val Cys Thr Val Gly Thr His Cys                 85 90 95 Ser Asn Gly Gln Lys Leu Ser Ile Asn Val Val Ala Ala Asn Ala Thr             100 105 110 Val Ser Met Pro Pro Pro Ser Ser Pro Pro Ser Ser Val Met Pro         115 120 125 Pro Pro Val Met Pro Pro Pro Ser Pro Ser     130 135 <210> 16 <211> 162 <212> PRT <213> Chloroflexus aurantiacus <400> 16 Met Lys Ile Thr Leu Arg Met Met Val Leu Ala Val Leu Thr Ala Met 1 5 10 15 Ala Met Val Leu Ala Ala Cys Gly Gly Gly Gly Ser Ser Gly Gly Ser             20 25 30 Thr Gly Gly Gly Ser Gly Ser Gly Pro Val Thr Ile Glu Ile Gly Ser         35 40 45 Lys Gly Glu Glu Leu Ala Phe Asp Lys Thr Glu Leu Thr Val Ser Ala     50 55 60 Gly Gln Thr Val Thr Ile Arg Phe Lys Asn Asn Ser Ala Val Gln Gln 65 70 75 80 His Asn Trp Ile Leu Val Lys Gly Gly Glu Ala Glu Ala Ala Asn Ile                 85 90 95 Ala Asn Ala Gly Leu Ser Ala Gly Pro Ala Ala Asn Tyr Leu Pro Ala             100 105 110 Asp Lys Ser Asn Ile Ile Ala Glu Ser Pro Leu Ala Asn Gly Asn Glu         115 120 125 Thr Val Glu Val Thr Phe Thr Ala Pro Ala Ala Gly Thr Tyr Leu Tyr     130 135 140 Ile Cys Thr Val Pro Gly His Tyr Pro Leu Met Gln Gly Lys Leu Val 145 150 155 160 Val asn          <210> 17 <211> 140 <212> PRT <213> Chloroflexus aurantiacus <400> 17 Ala Ala Asn Ala Pro Gly Gly Ser Asn Val Val Asn Glu Thr Pro Ala 1 5 10 15 Gln Thr Val Glu Val Arg Ala Ala Pro Asp Ala Leu Ala Phe Ala Gln             20 25 30 Thr Ser Leu Ser Leu Pro Ala Asn Thr Val Val Arg Leu Asp Phe Val         35 40 45 Asn Gln Asn Asn Leu Gly Val Gln His Asn Trp Val Leu Val Asn Gly     50 55 60 Gly Asp Asp Val Ala Ala Ala Val Asn Thr Ala Ala Gln Asn Asn Ala 65 70 75 80 Asp Ala Leu Phe Val Pro Pro Pro Asp Thr Pro Asn Ala Leu Ala Trp                 85 90 95 Thr Ala Met Leu Asn Ala Gly Glu Ser Gly Ser Val Thr Phe Arg Thr             100 105 110 Pro Ala Pro Gly Thr Tyr Leu Tyr Ile Cys Thr Phe Pro Gly His Tyr         115 120 125 Leu Ala Gly Met Lys Gly Thr Leu Thr Val Thr Pro     130 135 140 <210> 18 <211> 96 <212> PRT Cucumis sativus <400> 18 Ala Val Tyr Val Val Gly Gly Ser Gly Gly Trp Thr Phe Asn Thr Glu 1 5 10 15 Ser Trp Pro Lys Gly Lys Arg Phe Arg Ala Gly Asp Ile Leu Leu Phe             20 25 30 Asn Tyr Asn Pro Ser Met His Asn Val Val Val Val Asn Gln Gly Gly         35 40 45 Phe Ser Thr Cys Asn Thr Pro Ala Gly Ala Lys Val Tyr Thr Ser Gly     50 55 60 Arg Asp Gln Ile Lys Leu Pro Lys Gly Gln Ser Tyr Phe Ile Cys Asn 65 70 75 80 Phe Pro Gly His Cys Gln Ser Gly Met Lys Ile Ala Val Asn Ala Leu                 85 90 95 <210> 19 <211> 104 <212> PRT <213> Homo sapiens <400> 19 Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln 1 5 10 15 Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu             20 25 30 His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr         35 40 45 Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu     50 55 60 Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met 65 70 75 80 Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala                 85 90 95 Tyr Leu Lys Lys Ala Thr Asn Glu             100 <210> 20 <211> 338 <212> PRT <213> Phormidium laminosum <400> 20 Met Asn Phe Lys Val Cys Ser Phe Pro Ser Arg Arg Gln Ser Ile Ala 1 5 10 15 Ala Phe Val Arg Val Leu Met Val Ile Leu Leu Thr Leu Gly Ala Leu             20 25 30 Val Ser Ser Asp Val Leu Leu Pro Gln Pro Ala Ala Ala Tyr Pro Phe         35 40 45 Trp Ala Gln Gln Asn Tyr Ala Asn Pro Arg Glu Ala Thr Gly Arg Ile     50 55 60 Val Cys Ala Asn Cys His Leu Ala Ala Lys Pro Ala Glu Ile Glu Val 65 70 75 80 Pro Gln Ala Val Leu Pro Asp Ser Val Phe Lys Ala Val Val Lys Ile                 85 90 95 Pro Tyr Asp His Ser Val Gln Gln Val Gln Ala Asp Gly Ser Lys Gly             100 105 110 Pro Leu Asn Val Gly Ala Val Leu Met Leu Pro Glu Gly Phe Thr Ile         115 120 125 Ala Pro Glu Asp Arg Ile Pro Glu Glu Met Lys Glu Glu Val Gly Pro     130 135 140 Ser Tyr Leu Phe Gln Pro Tyr Ala Asp Asp Lys Gln Asn Ile Val Leu 145 150 155 160 Val Gly Pro Leu Pro Gly Asp Gln Tyr Glu Glu Ile Val Phe Pro Val                 165 170 175 Leu Ser Pro Asn Pro Ala Thr Asn Lys Ser Val Ala Phe Gly Lys Tyr             180 185 190 Ser Ile His Leu Gly Ala Asn Arg Gly Arg Gly Gln Ile Tyr Pro Thr         195 200 205 Gly Glu Lys Ser Asn Asn Ala Val Tyr Asn Ala Ser Ala Ala Gly Val     210 215 220 Ile Thr Ala Ile Ala Lys Ala Asp Asp Gly Ser Ala Glu Val Lys Ile 225 230 235 240 Arg Thr Glu Asp Gly Thr Thr Ile Val Asp Lys Ile Pro Ala Gly Pro                 245 250 255 Glu Leu Ile Val Ser Glu Gly Glu Glu Val Ala Ala Gly Ala Ala Leu             260 265 270 Thr Asn Asn Pro Asn Val Gly Gly Phe Gly Gln Lys Asp Thr Glu Ile         275 280 285 Val Leu Gln Ser Pro Asn Arg Val Lys Gly Arg Ile Ala Phe Leu Ala     290 295 300 Ala Ile Thr Leu Thr Gln Ile Leu Leu Val Leu Lys Lys Lys Gln Val 305 310 315 320 Glu Arg Val Gln Ala Gly Arg Asp Asp Leu Leu Lys Ala Ala Phe Ile                 325 330 335 Ala gly          <210> 21 <211> 39 <212> PRT <213> Neisseria gonnorrhoeae <400> 21 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala         35 <210> 22 <211> 166 <212> PRT <213> Neisseria gonorrhoeae F62 <400> 22 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp                 85 90 95 Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Asp     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 23 <211> 33 <212> DNA <213> artificial sequence <220> <223> forward primer for Laz <400> 23 ccggaattcc ggcagggatg ttgtaaatat ccg 33 <210> 24 <211> 38 <212> DNA <213> artificial sequence <220> PCR primers <400> 24 ggggtaccgc cgtggcaggc atacagcatt tcaatcgg 38 <210> 25 <211> 27 <212> DNA <213> artificial sequence <220> PCR primers <400> 25 ggcagcaggg gcttcggcag catctgc 27 <210> 26 <211> 26 <212> DNA <213> artificial sequence <220> PCR primers <400> 26 ctgcaggtcg actctagagg atcccg 26 <210> 27 <211> 25 <212> DNA <213> artificial sequence <220> PCR primers <400> 27 gccgagtgct cggtggacat ccagg 25 <210> 28 <211> 27 <212> DNA <213> artificial sequence <220> PCR primers <400> 28 tactcgagtc acttcagggt cagggtg 27 <210> 29 <211> 37 <212> DNA <213> Artificial sequence <220> <223> forward primer for pGST-azu 36-128 <400> 29 cgggatcccc ggcaacctgc cgaagaacgt catgggc 37 <210> 30 <211> 28 <212> DNA <213> Artificial sequence <220> <223> reverse primer for pGST-azu 36-128 <400> 30 cggaattcgc atcacttcag ggtcaggg 28 <210> 31 <211> 38 <212> DNA <213> Artificial sequence <220> <223> forward oligonucleotide for pGST-azu 36-89 <400> 31 ccaagctgat cggctcgtga gagaaggact cggtgacc 38 <210> 32 <211> 38 <212> DNA <213> Artificial sequence <220> <223> reverse oligonucleotide for pGST-azu 36-89 <400> 32 ggtcaccgag tccttctctc acgagccgat cagcttgg 38 <210> 33 <211> 29 <212> DNA <213> artificial sequence <220> PCR primers <400> 33 cggggatccc cggctcgggc gagaaggac 29 <210> 34 <211> 29 <212> DNA <213> artificial sequence <220> PCR primers <400> 34 cgggaattct ccacttcagg gtcagggtg 29 <210> 35 <211> 29 <212> DNA <213> artificial sequence <220> PCR primers <400> 35 gttcttctgc acctagccgg gccactccg 29 <210> 36 <211> 29 <212> DNA <213> artificial sequence <220> PCR primers <400> 36 cggagtggcc cggctaggtg cagaagaac 29  

Claims (41)

An isolated peptide that is a variant, derivative or structural equivalent of cupredoxin or cytochrome and capable of inhibiting parasitemia caused by malaria in malaria-infected red blood cells. The isolated peptide of claim 1, which is capable of inhibiting parasitemia caused by malaria in Plasmodium falciparum -infected human red blood cells. An isolated peptide that is a variant, derivative or structural equivalent of cupredoxin or cytochrome and capable of inhibiting intracellular replication of malaria parasites in malaria-infected human red blood cells. An isolated peptide that is a variant, derivative or structural equivalent of cupredoxin and is capable of binding to a protein selected from PfMSP1-19 or PfMSP1-42. The method according to claim 1, wherein the cupredoxin is selected from azurin, pseudoazurin, platocyanin, rusticyanin, Laz, or auracyanin Isolated peptides. The isolated peptide of claim 5, wherein the cupredoxin is selected from Lustyyanine, Azurin, or Laz. The method of claim 1, wherein the cupredoxin is Pseudomonas aeruginosa ), Alcaligenes faecalis ), Achromobacter xylosoxidan ) , Bordetella bronchiseptica ), Methylomonas sp . ), Neisseria meningitidis , Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens ), Pseudomonas chlororaphis ), Xylella fastidiosa ), or Vibrio parahaemolyticus . An isolated peptide characterized in that it is derived from an organism selected from. The method according to claim 6, Cupredoxin is Thiobacillus ( Tobacillus ferrooxydans) ferrooxidans ), Pseudomonas aeruginosa ), Neisseria An isolated peptide, characterized in that it is derived from an organism selected from gonorrhoeae ) or meningitis ( Nisseria meningitidis ). The isolated peptide of claim 1, wherein the cytochrome is cytochrome c or cytochrome f. The method of claim 9, wherein the cytochrome c is human or Pseudomonas isolated peptide derived from the organism selected from aeruginosa ). The isolated peptide of claim 9, wherein the cytochrome f is derived from cyanobacteria. The isolated peptide of claim 1, which is truncated of the peptide selected from SEQ ID NOs: 1-20 and 22. The isolated peptide of claim 1, having at least 90% amino acid sequence identity with the sequence selected from SEQ ID NOs: 1-20 and 22. The isolated peptide of claim 1, which is a truncation of cupredoxin or cytochrome. The isolated peptide of claim 1, wherein the peptide has at least 10 residues and up to 100 residues. The isolated peptide of claim 1, comprising azurin residues 36-89. The isolated peptide of claim 1, consisting of azurin residues 36-89. The isolated peptide of claim 1, comprising a cupredoxin residue equivalent to azurin residues 36-89. The isolated peptide of claim 1, wherein the peptide is fused to the H.8 region of Laz. The isolated peptide of claim 1, which is a structural equivalent of monoclonal antibody G17.12. Pharmaceutical composition containing at least one cupredoxin, cytochrome, or the isolated peptide of claim 1 The pharmaceutical composition of claim 21, which is prepared for intravenous administration. The pharmaceutical composition of claim 21, further comprising another anti-malarial agent. The pharmaceutical composition of claim 21, further comprising an anti-HIV agent. The method of claim 21, wherein the cupredoxin is Pseudomonas aeruginosa ), Alcaligenes faecalis ), Achromobacter xylosoxidan ) , Bordetella bronchiseptica ), Methylomonas sp . ), Neisseria meningitidis , Neisseria gonorrhoeae), Pseudomonas fluorescein sense (Pseudomonas fluorescens ), Pseudomonas chlororaphis ), Xylella fastidiosa ), or enteritis Vibrio ( Vibrio parahaemolyticus ). The method of claim 25, wherein the cupredoxin is Thiobacillus ferrooxidans ), Pseudomonas aeruginosa ), Neisseria pharmaceutical composition, characterized in that it is derived from a microorganism selected from gonorrhoeae ) or meningitis bacteria ( Nisseria meningitidis ). The pharmaceutical composition of claim 21, wherein the cytochrome is cytochrome c or cytochrome f. The method of claim 27, wherein the cytochrome c is human or Pseudomonas aeruginosa ) pharmaceutical composition, characterized in that it is derived from an organism selected from. The pharmaceutical composition of claim 27, wherein cytochrome f is derived from cyanobacteria. The pharmaceutical composition of claim 21, wherein the cupredoxin or cytochrome is selected from SEQ ID NOs: 1-20 and 22. A method of treating a patient suffering from infection with a malaria parasite, comprising administering to the patient an effective amount of the pharmaceutical composition of claim 21. A method of treating a patient suspected of contact with a malaria parasite, the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 21. A method of preventing malaria in a mammal, the method comprising administering to the insect vector an effective amount of the pharmaceutical composition of claim 21 in a population of insect vectors to which the malaria parasite is dormant. The method of claim 33, wherein the peptide inhibits parasitemia caused by malaria in the malaria-infected human erythrocyte cells of the patient. The method of claim 31, wherein the malaria parasite is selected from Plasmodium vivax or Plasmodium falciparum . The method of claim 31, wherein the patient further suffers from HIV infection. The method of claim 31, wherein the pharmaceutical composition is administered with a second composition containing an active ingredient selected from an anti-malarial agent or an anti-HIV agent or both. The method of claim 37, wherein the pharmaceutical composition of claim 21 is administered concurrently with the administration of the second composition or within 12 hours after such administration. The method of claim 31, wherein the pharmaceutical composition is administered orally, inhaled, intravenously, intramuscularly or subcutaneously to the patient. The method of claim 39, wherein the pharmaceutical composition is administered intravenously to the patient. The method of claim 33, wherein the pharmaceutical composition is administered orally to an insect vector.

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