CN101454336A - Modified metallothionein proteins and methods for screening and treating diseases associated with oxidative stress - Google Patents

Abstract

The present invention is based on the therapeutic potential of reduced forms of thioproteins. Accordingly, the present invention relates to modified metallothionein or thionein proteins (e.g., wherein at least one sulfur atom is substituted with selenium (e.g., cysteine is substituted with selenocysteine)) and fragments thereof. The invention also relates to methods for screening candidate compounds that (i) reduce the binding of a metal (e.g., zinc) to metallothionein or thionein, and (ii) do not alter the oxidation state of metallothionein, thionein, or other proteins. The invention also relates to methods for producing modified thioproteins with reduced metal affinity, and methods for treating patients suffering from oxidative stress-related diseases.

Description

Modified metallothionein proteins and methods for screening and treating oxidative stress-related diseases

Background technique

The present invention relates to methods for treating diseases related to oxidative stress and modified metallothioneins or thioneins useful in these methods.

The metallothionein family was originally identified as proteins that bind metals, including zinc. However, although zinc is not a redox-active metal, metallothioneins and thionins can still participate in redox reactions by virtue of their characteristic grouping of cysteine residues that bind metals such as zinc . Thus, prior to the present invention, the oxidation of metallothionein was thought to be intimately linked to the release of bound metals.

Diseases associated with oxidative stress include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataractogenesis, rheumatoid arthritis, premature aging syndrome, Werner syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, Crohn's disease, macular degeneration, stroke, ischemia, and ulcerative colitis . Because many of these diseases are incurable, new approaches to treating these diseases are needed.

Summary of the invention

The present invention is based on the therapeutic potential of the reduced form of thionin.

Accordingly, the present invention relates to metallothioneins, sulfur proteins or fragments thereof in which one or more sulfur atoms have been replaced by selenium. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more cysteines The acid can be substituted with selenocysteine (eg, at any or all cysteines or methionines, as described herein). In one embodiment, the present invention relates to metals (e.g. selected from main group metals, transition metals, lanthanides and actinides, or selected from zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold , selenium, arsenic, tungsten, aluminum, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium, or lanthanum) bound metallothionein fragments (such as the alpha or beta domains of metallothionein ), wherein at least one (eg, all) of the sulfur atoms is replaced by selenium (eg, any substitution described herein). The sulfur atom in any of the polypeptides described herein may be in cysteine.

The present invention also relates to the identification and treatment of oxidative stress-related diseases (for example, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid Arthritis, Progeria, Werner Syndrome, Atherosclerosis, Diabetes, Essential Hypertension, Cystic Fibrosis, Crohn's Disease, Macular Degeneration, Stroke, Ischemia and ulcerative colitis) candidate compounds approach. The method comprises the steps of: (a) contacting a compound (e.g., a compound selected from a chemical library) with a metallothionein or a sulfur protein and a second polypeptide comprising an amino acid capable of being oxidized, and (b) measuring Metallothionein or metallothionein in the presence of metals released from the metallothionein or sulfur protein (e.g. selected from main group metals, transition metals, lanthanides and actinides, or selected from zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium , gold, selenium, arsenic, tungsten, aluminum, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium or lanthanum) and the second polypeptide (such as metallothionein or sulfur protein ) on the formation of oxidized amino acids (such as methionine sulfoxide), wherein compared to the absence of the compound, if the compound (i) increases metal release from metallothionein or sulfur protein, and (ii) does not Significantly increasing the amount of oxidized amino acids in the second polypeptide indicates that the compound is a candidate compound for treating oxidative stress-related diseases.

The present invention also relates to the identification and treatment of oxidative stress-related diseases (for example, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid Arthritis, Progeria, Werner Syndrome, Atherosclerosis, Diabetes, Essential Hypertension, Cystic Fibrosis, Crohn's Disease, Macular Degeneration, Stroke, Ischemia and ulcerative colitis) candidate compounds. The method comprises the steps of: (a) contacting a cell or cell extract with a compound (such as a compound selected from a chemical library) and (b) measuring the amount of metallothionein or sulfur protein in the cell or cell extract and the amount of the cell or the oxidation state of cell extracts (e.g. the presence of oxidized amino acids such as methionine sulfoxide), wherein if compound (i) increases sulfur proteins (e.g. The amount of metallothionein) or the amount of metallothionein is reduced, and (ii) does not significantly increase the oxidation state of the cell or cell extract, indicating that the compound is a candidate compound for the treatment of oxidative stress-related diseases .

The present invention also relates to the identification and treatment of oxidative stress-related diseases (for example, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid Arthritis, Progeria, Werner Syndrome, Atherosclerosis, Diabetes, Essential Hypertension, Cystic Fibrosis, Crohn's Disease, Macular Degeneration, Stroke, Ischemia and ulcerative colitis) for a third approach to candidate compounds. The method comprises the steps of: (a) contacting a compound, such as a compound selected from a chemical library, with a cell or cell extract comprising a polynucleotide encoding a sulfur protein, and (b) measuring Expression of sulfur proteins in the cells or cell extracts, where expression is increased in the presence of the compound compared to the absence of the compound, indicates that the compound is a candidate compound for use in the treatment of oxidative stress related diseases.

In another embodiment, the invention relates to a method of identifying a variant of a sulfur protein that has a reduced affinity for a metal. The method comprises (a) introducing point mutations, insertions or deletions into the thioprotein, or chemically altering the thioprotein, thereby producing a modified thioprotein; and (b) determining a metal (e.g., selected from a main group metal, a transition metal, Lanthanides and actinides, or elements selected from zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold, selenium, arsenic, tungsten, aluminum, manganese, iron, chromium, nickel, molybdenum, barium, strontium , bismuth, hafnium, technetium, or lanthanum) to the modified sulfur protein, wherein the reduced affinity for metals indicates that the modified sulfur protein is a variant of the sulfur protein with reduced affinity for metals. The determining step may also include measuring the reduction activity of the modified sulfur protein, wherein the reduction activity of the modified sulfur protein does not significantly decrease, indicating that the modified sulfur protein is a sulfur protein with redox activity with reduced affinity for metals. body.

The invention also relates to treatments for oxidative stress-related diseases (for example, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid Arthritis, Progeria, Werner Syndrome, Atherosclerosis, Diabetes, Essential Hypertension, Cystic Fibrosis, Crohn's Disease, Macular Degeneration, Stroke, Ischemia and ulcerative colitis) approach. In one embodiment, the method comprises administering to a patient in need thereof a sulfur protein variant identified using the methods described in the preceding embodiments. In another embodiment, the method comprises administering a chelating agent to said patient, wherein said patient suffers from the group consisting of Creutzfeldt-Jakob disease, respiratory distress syndrome, muscular dystrophy, cataracts, rheumatoid arthritis, progeria , Werner syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, Crohn's disease, macular degeneration, stroke, ischemia, or ulcerative colitis disease.

Any method of the invention may utilize any metallothionein variant, fragment or derivative (such as those described herein). In certain embodiments, the MT (metallothionein)/T (thionin) variant has a sulfur atom replaced by a selenium atom, for example comprising replacing one or more (eg, all) half Point mutations of cystine, such as cysteine as described herein.

In any of the compositions or methods of the invention, the MT or T utilized may contain a substitution of one or more non-cysteine residues with a different amino acid, such as a naturally occurring amino acid or a non-naturally occurring amino acid. . In addition, the MT or T utilized may contain substitutions of selenium for one or more sulfur atoms (eg, selenocysteine for cysteine residues). MT/T variants useful in the methods and compositions of the invention include one or more repeats of the primary sequence of a metallothionein alpha or beta domain (eg, separated by a spacer sequence of one or more amino acids). Other MT/T variants include alpha (e.g., 1, 2, 3, 4, 5, 8, 10, 12 or more) or beta (e.g., 1, 2, 3, 4, 5, 8, 10, 12 or more) domains, optionally with one or more spacers between said domains. The domain may also contain any non-cysteine substitutions, or selenium in place of a sulfur atom (eg, selenocysteine in place of cysteine).

"Metallothionein" refers to a protein that shares at least 50%, 60%, 70%, 80%, 90%, 95%, 98% of any one of SEQ ID NO: 1-4 or a homologue thereof , 99% or even 100% identity, and contain 7 metal atoms bound to the protein.

"Thion protein" refers to a protein that has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% identity and incorporate 6 or fewer metal atoms. "Metal-free thioprotein" refers to a thioprotein that has no bound metal atoms.

A compound that "increases the release of metals from metallothionein" refers to a compound that increases the amount of thioprotein (such as metal-free thioprotein) by at least 5%, 10%, 25%, compared to the absence of the compound %, 50%, 100%, 200%, 500%, 1000%. Alternatively, compounds that "enhance metal release from metallothionein" may increase the binding constant of MT/T to zinc (i.e. decrease the affinity) by at least 2, 5, 10, 50, 100, 1000, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ times.

A compound that "does not significantly increase the amount of the oxidized amino acid in the second polypeptide" refers to a compound that increases the amount of the oxidized amino acid by less than 1%, 2 %, 5%, 10%, 25%, 50%, 100%, or 500%. In some embodiments, the compound does not alter the amount of oxidized amino acid, or may further reduce the amount of oxidized amino acid.

A compound that "does not significantly increase the oxidation state of a cell or cell lysate" means that the compound does not increase the redox potential of said cell or cell lysate by more than 0.01, 0.05, 0.10, 0.20, 0.4, 0.5, 0.75, 1, 2, 5, 10, 25, 50, 100, 200, 500, 1000, 1500, 2000, 5000, 10,000, or 20,000 mV.

Other features and advantages of the present invention will be apparent from the following detailed description, drawings and claims.

Brief description of the drawings

Figure 1 is a set of sequences comprising human metallothionein 1, 2, 3 and 4 sequences (SEQ ID NO: 1-4).

Figures 2A-2C are schematic representations of zinc clusters in the metallothionein alpha domain (Figure 2A), the metallothionein beta domain (Figure 2B) and the GAL4 protein (Figure 2C).

Detailed description of the invention

Of the three metallothionein/thionin classes (ie, metallothionein, oxidized and reduced), we currently believe that the reduced class is important in the treatment of oxidative stress-related diseases. Accordingly, the present invention relates to modified metallothioneins and sulfur protein polypeptides, methods for increasing the level of reduced sulfur proteins, methods for producing variants of metallothioneins that favor the reduced state of metal binding and for the treatment of oxidative stress A method of reducing the binding of metal to thionin and increasing the amount of reduced thionin available in a subject for an hormone-related disease. The screening methods of the invention enable the identification of compounds that are useful in the treatment of oxidative stress-related diseases, such as those described herein.

metallothionein and sulfur protein

A sulfur protein is a protein of more than 60 amino acids containing about 20 cysteines. It contains neither aromatic nor histidine residues. Metallothionein was discovered in 1957 (Margoshes and Vallee, J. Am. Chem. Soc. 79:4813-4814, 1957). Two highly similar forms were identified: MT-1 and MT-2; more recently, a third form, MT-3, was identified as a growth inhibitor in the brains of Alzheimer's patients (Uchida et al., Neuron 7:337 -347, 1991). A fourth variant, MT-4, was also found to be expressed only in stratified squamous epithelium (Quaife et al., Biochemistry 33:7250-9, 1994). Genes encoding other (up to seventeen in total) isoforms of MT have also been identified.

Thioproteins have two domains (beta and alpha). The N-terminal β domain contains 9 cysteines and can bind 3 metal atoms (such as zinc); the C-terminal α domain contains 11 cysteines and can bind 4 metal atoms (such as zinc), thus Metallothioneins are formed (Maret et al., Proc. Natl. Acad. Sci. USA 94:2233-2237, 1997). Zinc enzymes such as GAL4 bind metals in two-zinc clusters (Fig. 2C), whereas metallothioneins bind metals in an unusual manner through tri-zinc clusters (β domains, Fig. 2B) and tetra-zinc clusters. The cluster (alpha domain, Figure 2A) binds zinc. These unusual structures are highly kinetically unstable but thermodynamically stable, which is likely related to the cellular function of MT/T in zinc regulation. In certain embodiments of the invention, the uptake or release of these clusters can be assessed as part of a domain or in a smaller portion of either domain comprising a sufficient number of amino acids (such as cysteine) to bind the metal. Metals (as described here), or the balance that controls their behavior can be evaluated. Metals used in these assays can include any of the metals described herein. Binding can be determined using isotopically labeled transition metals or Group IIb metals. Such experiments can be performed using any of MT-1, MT-2, MT-3, MT-4, or any other MT variant, derivative or fragment such as those described herein. Additional MT variants can be identified and characterized for regulation, expression or localization using any method known in the art.

cellular zinc regulation

One function of metallothioneins and sulfur proteins is to regulate cellular zinc levels (Jacob et al., Proc. Natl. Acad. Sci. USA 95:3489-3494, 1998). Zinc is an important cofactor for many enzymes. Although zinc binds MT/T strongly (binding constant of 3.2×10 ^-13 M at pH 7.4), MT/T has been shown to deliver zinc to zinc enzymes such as E. coli alkaline phosphatase and bovine carboxyl Peptidase A.

In addition to being required for enzyme activity, zinc also inhibits the activity of certain enzymes such as caspase-3, fructose 1,6-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, aldehyde dehydrogenase, tyrosine Acid phosphatase and yeast enolase (Maret et al., Proc. Natl. Acad. Sci. USA. 96:1936-1940, 1999). Enzymes inactivated by zinc showed restored activity upon addition of metal-free sulfur protein.

cellular oxidation

Previous work (Maret and Vallee, Proc. Natl. Acad. Sci. USA 95:3478-3482, 1998) showed that metallothioneins and sulfur proteins are redox active, although zinc itself is redox inert. Oxidants have been shown to reduce cysteines in MT/T, concomitantly causing the release of metals from the protein. Therefore, MT/T is likely involved in maintaining the oxidative state in cells.

Nucleotide triphosphate binding

Nucleotide triphosphates (including ATP, GTP, and ATP analogs such as adenosine 5'[γ-thio]triphosphate and AMP-PNP) bind to MT/T and cause metal release from MT/T (Jiang et al. USA 95:9146-9149, 1998). This binding is mediated through eight conserved lysine residues found in mammalian metallothioneins and sulfur proteins.

Identification of unbound sulfur proteins in cells

Although metallothioneins are usually seen in their metal-bound form, they have been detected and isolated from biological materials (Maret et al., Proc. Natl. Acad. Sci. USA. 96:1936-1940, 1999) as Endogenous chelators.

Other MT/T applications

Metallothionein or thioproteins can be isolated, purified or fractionated from any organism that naturally produces thioproteins or that has been modified to produce thioproteins. In certain embodiments, human, bovine or equine thioproteins can be isolated, purified or fractionated. The immunological properties of a metallothionein or its reactivity (eg, reactivity with any metal described herein, with any nucleotide, nucleoside, or any other compound or polypeptide) can also be assessed. In certain embodiments, the interaction of MT/T, or any variant described herein, with AMP, ADP, ATP, GSH, GSSG, or any combination thereof is studied.

还可以分析MT/T的金属载荷状态，例如使用Richarz AN.2002.Speziationsanalyse von proteingebundenen Elementen in Cytosolen alsbiologische Marker für Lebensprozesse unter besonderer Berücksichtigungder Metallothioneine im Gehirn(德国)，Technical University of Berlin ofMathematics and Natural Science：Berlin(德国) method described in the analysis.

Furthermore, detection of MT/T fractions in different cellular environments can be achieved by separation of organelles, eg, based on density centrifugation. In particular, lysosomes, peroxisomes, mitochondria, endoplasmic reticulum, Golgi apparatus, ribosomes, nucleus or any other subcellular particle may be analyzed in the method of the invention. In other embodiments, these subcellular particles from heart, liver, brain, kidney or any other organ can be analyzed. The interaction of MT/T (as any variant described herein) with AMP, ADP, ATP, GSH, GSSG, or any combination thereof can be analyzed. These methods can be performed using selenocysteine substituted MT/T, any MT/T seleno derivative, or any variant or fragment of MT/T described herein.

Selenium substituted MT/T

In one aspect, the invention relates to metallothionein or sulfur proteins in which one or more sulfur atoms have been replaced by selenium. The substituted metallothionein or sulfur protein can be bound to 0, 1, 2, 3, 4, 5, 6, 7 or more metal atoms. In certain embodiments, cysteines are substituted with selenocysteine (eg, at any or all of the cysteines described herein). Selenocysteine has a known physiological profile, is nontoxic and well tolerated. Accordingly, selenocysteine-containing proteins are useful as therapeutic agents (eg, for the treatment of oxidative stress-related diseases such as described herein).

Such proteins can be produced by any method known in the art. For example, selenocysteine can be introduced into protein sequences using peptide synthesis, such as described by Oikawa et al., Proc. In this example, a cysteine residue in the Neurospora crassa copper metallothionein protein was replaced with selenocysteine. In other embodiments, cysteine can be replaced with selenocysteine using semi-synthetic methods, as described by Hondal et al., J.Am.Chem.Soc. 123:5140-5141, 2001 or as described by Dawson and Described by Kent, Annu. Rev. Biochem. 69:923-960, 2000. Other approaches include modifying tRNAs in organisms to replace one amino acid with another, as described by Wang and Schutz, Chem. Any of these methods, or any other method known in the art, can be used to produce selenocysteine derivatives of MT/T.

Other MT/T variants

The invention also relates to MT or T variants in which one or more non-cysteine residues are substituted with a different amino acid, such as a naturally occurring or non-naturally occurring amino acid. In addition, the present invention also relates to MT or T in which one or more sulfur atoms are replaced by selenium (eg, cysteine is replaced by selenocysteine). MT/T variants comprise one or more repeats of the primary sequence of a metallothionein alpha or beta domain (eg, separated by a spacer sequence of one or more amino acids). MT/T variants may include alpha (e.g. 1, 2, 3, 4, 5, 8, 10, 12 or more) or beta (e.g. 1, 2, 3, 4, 5, 8) linked in any order , 10, 12 or more) domains, optionally with one or more spacers between said domains. This domain may also comprise substitutions of non-cysteine or cysteine substitutions with selenium-containing residues such as selenocysteine. Domains comprising alterations encompassed by the present invention are described in WO 00/50448, page 12, line 4 to page 14, line 5, which is incorporated herein by reference. Similarly, the alterations described in WO 00/50448 may also be incorporated into full length metallothionein, any fragment thereof, or any other variant described herein.

Other variants include fragments, such as any fragment of metallothionein that is capable of binding a metal atom, such as the portion of the beta or alpha domain that lacks the 1-25 amino acids at the C-terminus of the domain, lacks the 1-25 amino acids at the N-terminus of the domain, A portion of 25 amino acids or a mixture thereof. Deletion mutants capable of binding metals can be identified using molecular biology techniques known in the art, and any method can be used to determine metal binding (as described herein).

Screening Methods for Identifying Candidate Therapeutic Compounds

Based on the identification of metallothionein and thionin as zinc-binding and regulatory proteins, and their role in cellular oxidation in relation to MT/T zinc-binding, we now attempted to correlate the zinc-binding activity of MT/T with that of MT/T. Oxidation separates. Accordingly, the present invention relates to screening methods for identifying compounds that (i) reduce metal binding to MT/T and (ii) do not significantly increase oxidation of MT/T or a second polypeptide. Compounds identified by the methods of the present invention increase the availability of thioproteins available for reduction of potentially deleterious oxidized species such as metallothioneins or thioproteins containing methionine sulfoxide residues. Specific examples of diseases in which oxidized amino acids play a role in disease development include Parkinson's disease in which oxidized α-synuclein containing methionine sulfoxide has been identified (Glaser et al., Biochim. Biophys. Acta. 1703:157 -69, 2005) and Alzheimer's disease in which oxidized β-amyloid containing methionine sulfoxide residues has been identified (Schoneich, Biochim. Biophys. Acta. 1703:111-9, 2005). Other examples of oxidative stress related diseases are also described herein. Therefore, the compounds identified by the screening method of the present invention can be used in the treatment of diseases related to oxidative stress.

Screening assays can be performed by standard methods to identify compounds that reduce metal binding to MT/T without significantly increasing MT/T or oxidation of the second polypeptide. Such screening methods may include high-throughput techniques. Additionally, these screening techniques can be performed in cultured cells or organisms such as worms, flies or yeast.

Metallothionein

The screening methods of the present invention may involve the use of any MT/T protein, eg, a protein homologous to human MT protein (eg, MT protein from mouse, rat or rabbit). Any form of MT/T (such as MT-3 and those described herein) can be used in the methods of the invention. In some embodiments, the metallothionein or sulfur protein used in the screening methods of the invention may comprise selenium in place of one or more sulfur atoms in the wild-type protein (e.g., selenocysteine in place of one or more cysteine acid). In other embodiments, the screening method uses any of the metallothioneins or variants of a sulfur protein described herein. Any concentration of MT/T that allows detection of metal release can be used in this screening method. Any metal capable of binding to sulfur proteins (including metals selected from the main group metals, transition metals, lanthanides, and actinides, and metals selected from the group consisting of zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold, selenium , arsenic, tungsten, aluminum, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium and lanthanum) can be used to form metallothionein. In some embodiments, a metal-binding (eg, zinc-binding) fragment of MT/T (eg, any of the fragments described herein comprising a β-domain or an α-domain) is used in the screening method. Any of the MT/T variants, derivatives, fragments described herein may also be used in the methods of the invention.

Detection of decreased metal binding to metallothionein

The screening methods of the present invention include the step of determining metal release from MT/T or any fragment or variant thereof described herein. Any method known in the art for measuring metal release can be used.

In one embodiment, the method described by Maret and Vallee (Proc. Natl. Acad. Sci. USA 95:3478-3482, 1998) is used. Briefly, zinc complex dyes such as 4-(2-pyridylazo)resorcinol (PAR) or 2-carboxy-2'-hydroxy-5'-sulfophenylazo (zinc reagent )) to measure the release of zinc from MT/T, since these dyes change their spectroscopic properties upon binding to zinc. In a specific example, a buffer containing 100 μM PAR or zinc reagent is incubated with 1.3 μM zinc-MT. A test compound is added to this solution and the change in absorbance (at 500 nm for PAR and 620 nm for zinc reagent) can be measured using a spectrophotometer and compared to the absorbance in the absence of the test compound, where an increase in absorbance indicates the change in zinc from MT/T release. If necessary, the absorbance measurement can be corrected for the absorbance produced by the test compound. Other molecules that can be used to detect free cellular zinc include Zinpyr-1 analogs described, for example, in Goldsmith and Lippard, Inorg. Chem. 45:555-561, 2006 and Woodroofe et al., Inorg. Chem. .

In another embodiment, copper-MT is used in the screening methods of the invention. At this point, the copper-MT was contacted with the test compound and 4-(1,4,7,10-tetrathia-13-aza-cyclopentadecane-13-yl)-benzene (CTAP-1) to monitor copper release. CTAP-1 exhibits enhanced emission at 480 nm after excitation at 365 nm in the presence of copper compared to the absence of copper (Yang et al., Proc. Natl. Acad. Sci. USA. 102:11179-11184, 2005), and Also suitable for screening methods using cells or cell extracts.

It is also possible to analyze changes in the relative amounts of metallothionein and thionin and the number of metal atoms bound to the thionin, e.g. using Richarz AN. The method described in University of Berlin of Mathematics and Natural Science: Berlin (Germany) was used for analysis. A decrease in the metal-loading state of metallothionein or thionin or an increase in the amount of metal-free thionin in a biological system following exposure to a test compound can indicate that the compound reduces the ability of MT or T to bind metal and can be determined using the methods described. detection. In addition, detection of MT/T fractions in different cellular environments can be achieved by separation of organelles, eg, based on density centrifugation. In particular, lysosomes, peroxisomes, mitochondria, endoplasmic reticulum, Golgi apparatus, ribosomes, nucleus or any other subcellular particle may be analyzed in the method of the invention. In other embodiments, these subcellular particles from heart, liver, brain, kidney or any other organ can be analyzed. The interaction of MT/T (as any variant described herein) with AMP, ADP, ATP, GSH, GSSG, or any combination thereof can be analyzed. Such a method can be performed using MT/T substituted with selenocysteine or any other selenium derivative of MT/T.

Detection of amino acid oxidation and cellular oxidative status

The screening methods of the invention also include measuring amino acid oxidation in a second polypeptide (e.g., MT, T, or any fragment or variant described herein) or determining whether the test compound significantly increases the oxidized state of the cell (e.g., formation of oxidized amino acids or active oxygen species).

For the detection of oxidized amino acids, any method known in the art can be used in the screening method of the present invention. Exemplary detection methods are disclosed in Shacter, Drug Metab. Rev. 32:307-26,2000. The specific method used for amino acid detection will depend on the specific type of oxidized amino acid being detected.

Oxidation of methionine can result in the formation of methionine sulfoxide, and in one embodiment, such residues are detected. Detection of methionine sulfoxide is particularly useful since nearly all proteins, including sulfur proteins and metallothioneins, have an N-terminal methionine residue. Methionine sulfoxide can be detected by the method described by Sochaski et al. (Anal. Chem. 73:4662-7, 2001). At this point, the sample was hydrolyzed with methanesulfonic acid. The hydrolyzed sample was then separated on a cation exchange column to derivatize the amino acid to its trimethylsilyl ester. The sample is then detected for the presence of methionine sulfoxide by selected ion monitoring gas chromatography/mass spectrometry, as known in the art. Compounds that reduce metal (eg zinc) binding to MT/T but do not increase methionine sulfoxide formation (eg at the N-terminal methionine of MT/T) are considered useful in the present invention.

Changes in redox potential in methods of the invention performed with cells or cell extracts may also be detected using any method known in the art. For example, commercially available kits (eg, Image-iT ^™ LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen)) can also be used to determine the presence of reactive oxygen species. Other methods for measuring redox potential in situ are described in Hanson et al., J. Biol. Chem. 279:13044-13053, 2004. In this case, green fluorescent protein (GFP) was modified to contain cysteine. The formation of disulfide bonds in the modified GFP results in changes in the protein's fluorescence in response to changes in the redox potential, which can be used to monitor changes in the redox potential in the cellular environment.

Compounds that reduce the binding of metals (eg, zinc) to metallothionein or sulfur proteins without significantly reducing the ability of the metallothionein or sulfur proteins to participate in redox chemistry are considered useful in the present invention.

Screening for increased thioprotein expression

There are a variety of methods that can be used to perform screening assays to identify compounds that increase expression of sulfur proteins such as MT-3. According to one approach, a candidate compound is added at various concentrations to the culture medium of cells expressing a polynucleotide encoding a metallothionein. Gene expression is then measured, for example, by standard Northern blot analysis (Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 1997) using any suitable fragment prepared from the polynucleotide as a hybridization probe. The expression level of the gene in the presence of the candidate compound is compared to the level measured in a control medium without the candidate molecule. Compounds that promote increased expression of sulfur proteins, such as MT-3, are contemplated as being useful in the present invention, for example, such molecules are useful as therapeutic agents for diseases associated with oxidative stress, such as those described herein.

The effect of candidate compounds can also be measured against the level of polypeptide production, if necessary, using the same general methods and standard immunological techniques (such as western blotting or immunoprecipitation using antibodies specific for metallothionein or sulfur proteins). For example, immunoassays can be used to detect or monitor the expression of metallothionein or sulfur proteins. Polyclonal or monoclonal antibodies that bind such polypeptides can be used in any standard immunoassay format (eg, ELISA, western blot, or RIA assay) to measure metallothionein levels. Compounds that promote increased expression of metallothionein or sulfur proteins are believed to be particularly useful. Likewise, such molecules are useful, for example, as therapeutic agents for diseases related to oxidative stress.

Test compounds and extracts

In general, compounds capable of treating oxidative stress-related diseases are identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the art of drug discovery will appreciate that the particular source of the test extract or compound is not critical to the screening methods of the invention. Thus, essentially any chemical extract or compound can be screened using the methods of the invention. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic, or animal based extracts, fermentation broths, and synthetic and modified existing compounds. There are also a large number of methods available for the random generation or directed synthesis (e.g. semi- or total synthesis) of any compound, including but not limited to those based on sugars, lipids, peptides and polynucleotides such as siRNA or microRNA. Other compounds useful in the screening methods of the invention include any of the compounds described herein (e.g., chelators, modified sulfur proteins, and selenium compounds (e.g., selenocysteine, phenylselenoyl chloride, and phenylselenite). Any of these compounds are chemically modified using standard methods in the art.

Synthetic compound libraries are commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available. In addition, naturally and synthetically produced libraries, where desired, are generated according to methods known in the art (eg, by standard extraction and fractionation methods). Furthermore, any library or compound is conveniently modified, if desired, using standard chemical, physical or biochemical methods.

In addition, those skilled in the art of drug development will readily understand that methods for exclusion of duplications (e.g., taxonomic exclusion, biological exclusion, and chemical exclusion, or any combination thereof) should be used as much as possible or to eliminate known Methods for duplicating materials active in treating oxidative stress-related diseases.

When a crude extract is found to have the desired activity (such as reduced metal binding to metallothionein or thionin or increased expression of thionin), further fractionation of the positive lead extract is necessary to isolate the The chemical composition of the effect. Therefore, the purpose of the extraction, fractionation and purification steps is to characterize and identify chemical entities in the crude extract that have activity useful in the treatment of oxidative stress related diseases. Methods for fractionation and purification of these heterogeneous extracts are known in the art. Compounds shown to be useful in the treatment of oxidative stress-related diseases are chemically modified, if desired, according to methods known in the art.

Modified sulfur protein with reduced metal binding

The present invention also relates to methods of producing modified sulfur proteins (T) with reduced binding to metals, such as zinc. In certain embodiments, the modified sulfur protein also retains the ability to participate in redox reactions compared to wild-type sulfur protein. These modified sulfur protein molecules can be used to treat oxidative stress-related diseases. Modifications can be made by any method known in the art. Methods of introducing sequence changes such as point mutations, insertions, deletions or any combination thereof are well known to those skilled in the art.

modified sulfur protein

Thioproteins can be modified at any residue (e.g. by chemical derivatization or point mutation) and by insertion or deletion of one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20) amino acids for modification. Any modification of the sulfur protein described herein can be used in the methods herein. For example, such modifications can be achieved using standard molecular biology techniques. In certain embodiments, lysine residues are altered to reduce binding to metals (eg, zinc). Such alterations may include the addition or substitution of mercapto, sulfenic, sulfinic, sulfonic, sulfonate, sulfoxide or sulfone moieties. These lysine residues include 8 lysines at positions 20, 22, 25, 30, 31, 43, 51 and 56 in MT1 or MT2 (SEQ ID NO: 1 and 2), MT3 (SEQ ID NO: 3 ) in 21, 26, 31, 32, 44, 47, 52 and 63 lysine residues, or in MT4 (SEQ ID NO: 4) in 21, 28, 32, 44, 52 or 57 Lysine residues because these residues are involved in ATP binding in an as yet unknown order (see Jiang et al., supra). As noted above, binding to ATP reduces the affinity of MT for metals. Thus, one can modify a lysine residue in a thioprotein (as described above), a residue adjacent to a lysine residue, or a residue close to a lysine residue in the three-dimensional structure of a thioprotein, and test the relationship with Enhancement of binding of AMP, ADP, ATP, GSH or GSSG or any combination thereof. Modifications that increase ATP binding or binding to other nucleotide triphosphates or analogs thereof can reduce the affinity of sulfur proteins for metals, such as zinc, and thus may be particularly useful in the methods of the invention.

Other exemplary modifications may include substituting selenium for any sulfur atoms. For example, any of the 20 cysteine residues in MT/T can be substituted for methionine or selenocysteine. Specifically, the 5th, 7th, 13th, 15th, 19th, 21st, 24th, 26th, 29th, 33rd, 34th, 36th, 37th, 41st, 44th, 48th, 50th, 57th, 59th or 60th in MT1 or MT2 can be modified Residue at position 6, 8, 14, 16, 20, 22, 25, 27, 30, 34, 35, 37, 38, 42, 45, 49, 51, 64, 66, or 67 in MT3 or residues 6, 8, 14, 16, 20, 22, 25, 27, 30, 34, 35, 37, 38, 42, 45, 49, 51, 58, 60, or 61 in MT4. These modifications reduce the affinity of the sulfur protein for metals, but in certain embodiments allow the modified sulfur protein to participate in redox reactions.

In certain embodiments, all cysteine residues are substituted with selenocysteine. Such modified MT/T proteins can be obtained using solid phase peptide synthesis or using any technique known in the art or described below. Likewise, these methods can also utilize any MT/T variant (such as the variants described herein).

Determination of metal binding

Determination of affinity and binding to metals can be performed as described herein or as known in the art. These assays can be used to determine which sulfur protein variants show reduced metal (e.g. zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold, selenium, arsenic, tungsten, aluminum, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium or lanthanum).

Determination of redox activity

In certain embodiments, the redox activity of the modified sulfur protein is further determined. The exact method used is not critical to the invention and any method known in the art can be used to make such measurements. For example, the redox state of the N-terminal methionine can be determined by the methods described above, including measuring the redox potential using modified GFP or a commercially available kit (as described herein). In other embodiments, electrodes, such as those available from Broadley James Corporation, Irvine, Calif., can be used to measure the redox potential of proteins in solution.

Peptide production

Modified thioprotein polypeptides can be produced by transforming a suitable host cell with a polynucleotide molecule encoding all or part of the thioprotein in a suitable expression vector, or a fragment thereof. Those skilled in the art of molecular biology will appreciate that a number of expression systems can be used to provide the thionin polypeptides. The exact host cell used is not critical to the invention. Modified sulfur proteins can be produced in prokaryotic hosts such as E. coli or eukaryotic hosts such as Saccharomyces cerevisiae, insect cells such as Sf21 cells, or mammalian cells such as NIH 3T3, HeLa or preferably COS cells. These cells are available from a number of sources (eg American Type Culture Collection, Rockland, Md., see also eg Ausubel et al., supra). The method of transformation or transfection and the choice of expression vector will depend on the host system chosen. Transformation and transfection methods are described in Ausubel et al. (supra); expression vectors can be selected from, for example, those provided in Cloning Vectors: A Laboratory Manual (Pouwels, P.H. et al., 1985, 1987 supplement).

Thionins and thionin fragments (particularly those containing such as selenocysteine) can also be produced by chemical synthesis (e.g. by Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, III. method described).

Modified sulfur proteins with specific properties such as enhanced ATP binding or reduced affinity for metals such as zinc may also include chemical modifications such as derivatization of side chain groups, as is known in the art.

Treatment of oxidative stress-related diseases

The present invention relates to methods of treating a subject suffering from a disease related to oxidative stress. Compounds for use in the treatment methods of the invention can be, for example, compounds identified using the screening methods described herein, modified sulfur proteins such as those described herein, or chelating agents.

Oxidative Stress Related Diseases

Oxidative stress-related diseases include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, progeria, Werner syndrome syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, Crohn's disease, macular degeneration, stroke, ischemia, and ulcerative colitis.

modified sulfur protein

A modified sulfur protein (eg, a sulfur protein identified by the methods of the invention or a sulfur protein described herein) can be administered to a subject for the treatment of oxidative stress-related diseases. Providing a subject with a modified sulfur protein can treat these oxidative stress-related diseases by reducing the effects of oxidative stress.

Gene therapy

In addition to administering a modified thioprotein, the expression of a polynucleotide encoding a thioprotein (such as a modified thioprotein as described herein) can be induced to treat oxidative stress-related diseases by introducing a gene vector into a subject. Any standard gene therapy vector and method can be used for such administration.

Metal binding/chelating agents

Chelating agents that remove metals (such as zinc) from MT are also useful in the treatment of oxidative stress-related diseases such as Creutzfeldt-Jakob disease, respiratory distress syndrome, muscular dystrophy, cataracts, rheumatoid arthritis, progeria, Weir Nathan syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, Crohn's disease, macular degeneration, stroke, ischemia, and ulcerative colitis. Such chelators remove metals from MTs, allowing apoprotein T to participate in redox reactions and relieve oxidative stress.

Any chelating agent may be used in the treatment method of the present invention, including EDTA, EGTA, 1,10-phenanthroline, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN ), diethyldithiocarbamate (DEDTC), 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonate, sodium diethyldithiocarbamate and 2, 2'-bipyridine. Other chelating agents (e.g., agents that bind zinc or copper) including agents of the Zinpyr family are described, e.g., in Goldsmith and Lippard, Inorg.Chem.45:555-561, 2006; Woodroofe et al., Inorg.Chem.44:3112-3120, 2005; Woodroofe and Lippard, J.Am.Chem.Soc.125:11458-11459, 2003; Burdette et al., J.Am.Chem.Soc.125:1778-1787, 2003; Boerzel et al., Inorg.Chem.42: 1604-1615, 2003; Nolan and Lippard, Inorg.Chem.43:8310-8317; 2004; Nolan et al., Inorg.Chem.43:2624-2635, 2004 and Kuzelka et al., Inorg.Chem.43:1751-1761, 2004. Bis(thiosemicarbazone) reagents that form complexes with zinc (eg, diacetylbis(4-pyrrolidinyl-3-thiosemicarbazone)) are also useful in the methods of the invention. These reagents are described in more detail, eg, in Cowley et al. (Chem. Commun. (Camb). 2005(7): 845-847, 2005).

Formulation of pharmaceutical compositions

Administration of any compound described herein or identified using the screening methods of the invention may be by any suitable method that produces concentrations of the compound that treat oxidative stress-related diseases. The compound may be included in any suitable carrier material in any suitable amount, usually present in an amount of 1-95% by weight of the total composition. The composition may be suitable for oral, parenteral (e.g., intravenous, intramuscular, intracranial, intrathecal), rectal, epidermal, intranasal, vaginal, inhalation, dermal (patch), intraocular or intracranial administration. Dosage forms of the drug route are provided. The pharmaceutical composition can be formulated according to conventional pharmaceutical practice (see for example Remington: The Science and Practice of Pharmacy, Twentieth Edition, 2000, edited by A.R.Gennaro, Lippincott Williams & Wilkins, Philadelphia and Encyclopedia of Pharmaceutical Technology, J.Swarbrick and Edited by J.C. Boylan, 1988-1999, Marcel Dekker, New York).

The pharmaceutical compositions may be formulated to release the active compound immediately after administration, or at any predetermined time or period of time after administration. Compositions of the latter type are commonly referred to as "controlled release formulations" and include (i) formulations that produce a substantially constant concentration of an agent of the invention in vivo over an extended period of time; (ii) after a predetermined lag period Formulations that produce substantially constant concentrations of agents of the invention in vivo over extended periods of time; (iii) by maintaining relatively constant, efficacious levels of agent in vivo while disabling adverse effects associated with fluctuations in plasma levels of the agent (saw-tooth kinetic pattern) Formulations that minimize side effects while maintaining the action of the agent for a predetermined period of time; (iv) formulations that limit the area of action of the agent (eg, spatially place the controlled release composition at or near the diseased tissue or organ); (v) Formulations for ease of dosing (eg weekly or bi-weekly administration of the composition); and (vi) formulations to target the action of the agent by using carriers or chemical derivatives to deliver the compound to specific target cell types. Administration of compounds in a controlled-release formulation is particularly preferred for compounds that have a narrow window of absorption in the gastrointestinal tract or that have a relatively short biological half-life.

Various strategies can be followed to obtain controlled release at a rate higher than the metabolic rate of the compound of interest. In one example, controlled release is achieved by appropriate selection of formulation parameters and ingredients including, for example, various types of controlled release compositions and coatings. Accordingly, the compounds are formulated with suitable excipients in pharmaceutical compositions which release the compounds in a controlled manner after administration. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches and liposomes.

parenteral compositions

Compositions containing compounds described herein or identified using the methods of the invention may be administered by injection, infusion or implantation in dosage forms, formulations containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants, or via suitable delivery devices or implants. Injection (subcutaneous, intravenous, intramuscular, intraperitoneal, intracranial, intrathecal, etc.) for parenteral administration. The formulation and preparation of such compositions are well known to those skilled in the pharmaceutical arts.

Parenteral compositions for use in the methods of the invention may be in a form suitable for sterile injection. To prepare such compositions, the appropriate active agent is dissolved or suspended in a parenterally acceptable liquid carrier. Acceptable vehicles and solvents that may be used are water, water adjusted to the appropriate pH by adding appropriate amounts of hydrochloric acid, sodium hydroxide, or a suitable buffer, 1,3-butanediol, Ringer's solution, dextrose solution, and isotonic chloride sodium solution. The aqueous formulations may also contain one or more preservatives (eg methyl, ethyl or n-propylparaben). In the case where one of the compounds is very low or slightly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may contain 10-60% (w/w) propylene glycol or the like.

Nervous system administration

In many instances, it will be desirable to limit the administration of the compound to the tissues affected by the particular disease afflicting the subject. In the case of diseases affecting the nervous system, such as Alzheimer's disease or Parkinson's disease, delivery to the affected area of the nervous system can be achieved, for example, by the following methods.

Treatment of neurodegenerative diseases can be hampered by the inability of active therapeutic compounds to cross the blood-brain barrier (BBB). Strategies for delivering compounds of the invention (e.g., modified thioproteins) in these diseases and disorders include strategies to bypass the BBB (e.g., intracranial and intrathecal administration via craniotomy) as well as strategies across the BBB ( Examples include the use of compounds that increase BBB permeability in conjunction with systemic administration of therapeutic compounds, and the modification of compounds to increase their permeability or transport across the blood-brain barrier).

Therapeutic compounds can be delivered to the brain using methods known in the art - craniotomy. In this method, an incision is made in the subject's skull and a compound is delivered through a catheter. This approach can be used to target compounds to specific regions in the brain (eg, the substantia nigra for Parkinson's disease treatment or the cortex for Alzheimer's disease treatment).

Intrathecal administration provides another method for drug delivery that bypasses the blood-brain barrier. Briefly, drugs are administered to the spinal cord through a lumbar puncture or through the use of a device such as a pump. Lumbar punctures are preferred for single or infrequent dosing, while frequent and/or chronic dosing may use any commercially available pump (e.g., Medtronic (Minneapolis, Minn.) pump and catheter) to achieve.

For delivery across the BBB, compositions of the invention may be administered with compounds that induce a transient increase in blood-brain barrier permeability. Such compounds include mannitol, ceropati (Cereport, RMP-7) and KB-R7943 (a Na ⁺ /Ca ⁺⁺ exchange blocker).

In other embodiments, compounds (e.g., compounds identified using the screening methods of the invention) can be modified (e.g., lipidate, acetylated) using standard chemical modifications in the art to enhance systemic delivery ( Transport across the blood-brain barrier after parenteral administration). In one embodiment, a compound of the invention is conjugated to a peptide carrier that transports across the BBB. For example, a compound can be conjugated to a monoclonal antibody directed against the human insulin receptor as described by Partridge (Jpn. J. Pharmacol. 87:97-103, 2001), thereby allowing the compound to be transported across BBB. For example, compounds such as those identified using the screening methods of the invention can be conjugated to such peptide carriers using biotin-streptavidin technology. In the case of treatment with gene therapy vectors, promoters that restrict expression to specific neuronal subpopulations may be used instead or in addition to limiting the range of vector delivery. For example, expression of gene therapy vectors can be restricted to dopaminergic neurons in the treatment of PD by using the tyrosine hydroxylase promoter.

dose

The dosage of any compound described herein or identified using the methods described herein will depend on several factors including: the method of administration, the condition being treated, the severity of the condition or disease, whether the condition or disease is being treated or prevented, and The age, weight and health status of the subject to be treated.

With respect to the methods of treatment of the present invention, it is not intended that the administration of the compound to a subject be limited to a particular mode of administration, dosage or frequency of administration, and all modes of administration (including intracranial, intrathecal, intramuscular, intravenous intraperitoneal, intracapsular, intra-articular, subcutaneous or any other route sufficient to provide a dose sufficient to treat oxidative stress-related diseases) are within the contemplation of the present invention. The compounds can be administered to a subject in single or multiple doses. For example, a compound described herein or identified using the screening methods of the invention may be administered once a week, eg, for 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more weeks. It will be understood that for any particular subject, the specific dosing regimen will be adjusted according to the needs of that individual and the professional judgment of the administerer or instructor who is administering the compound. For example, the dose of the compound may be increased if lower doses do not provide adequate treatment. Conversely, if the disease is attenuated or eliminated, the dose of the compound may be reduced.

A therapeutically effective amount of a compound described herein (such as a modified sulfur protein with reduced zinc binding) or identified using the screening method of the invention may be, for example, 0.0035 μg to 20 μg/kg body weight, although the appropriate amount and dosage regimen will ultimately be determined by the attending physician /day or 0.010μg to 140μg/kg body weight/week. A desirable therapeutically effective amount is 0.025 μg to 10 μg/kg body weight administered daily, every other day or twice weekly, for example at least 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 , 3.5, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 μg/kg body weight. Additionally, a therapeutically effective amount may be 0.05 μg to 20 μg/kg body weight administered weekly, every other week or monthly, for example at least 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0 or 18.0 μg/kg body weight. In addition, a therapeutically effective amount of the compound may be, for example, 100 μg/m ² to 100,000 μg/m ² administered every other day, once a week, or every other week. In desirable embodiments, the therapeutically effective amount is 1000 μg/m ² to 20,000 μg/m ² (e.g., at least 1000, 1500, 4000, or 14,000 μg /m ² ) compounds.

The following examples are intended to illustrate but not limit the invention.

Example

Synthesis of modified metallothionein or sulfur proteins

In one example, selenocysteine-substituted metallothioneins are synthesized substantially as described in WO00/50448. Briefly, the method comprises the following steps: (a) synthesizing MT or T using a solid support and at least two alpha amino acids having alpha amino groups selected from the group having side chains containing aliphatic groups (e.g. hydrogen or alkyl), amino acids with side chains containing aromatic groups, amino acids with side chains containing sulfur groups (such as mercapto or thioether), amino acids with side chains containing hydroxyl groups, amino acids with side chains containing amine groups An amino acid having a side chain containing a guanidine group, an amino acid having a side chain containing a carboxyl group, and an amino acid having a side chain containing an amide group. The alpha amino acids are protected with Fmoc, t-Boc or CBZ. The carboxyl group is protected with tert-butyl ester or benzyl ester. The hydroxyl group is protected with tert-butyl ether or dimethyl phosphate. The amine group is protected with t-Boc or CBZ. The thiol group is protected with acetimidomethyl. After step (a), step (b) of the method comprises cleaving the peptide synthesized in step (a) from said solid support and removing the non-acetimidomethyl protecting group. Step (c) comprises purifying the peptide obtained in step (b) and step (d) comprises precipitating the peptide obtained in step (c). Step (e) involves removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt. The primary amino acid sequence of MT or T may differ from the wild-type sequence. For example, the amino acid sequence may comprise substitutions of one or more non-cysteine residues with any naturally occurring or non-naturally occurring amino acid. In certain embodiments, one or more cysteines are replaced with selenocysteine. Other modifications include the addition of one or more repeats of the alpha or beta domain primary sequence in MT. These repeats can be fused together sequentially or in any arrangement of the alpha and beta domains. The domains may be separated by a spacer sequence comprising one or more amino acids. In repeat domains, one or more cysteine residues may be substituted with selenocysteine.

Step (a) of the method can be accomplished using an automated solid phase synthesizer. The α-amino group can be protected with Fmoc protecting group, the carboxyl group can be protected with tert-butyl ester protecting group, the hydroxyl group can be protected with tert-butyl ether protecting group, and the amine group can be protected with t-Boc protecting group.

Cutting step (b) can use about 75 parts (by weight) phenol, about 28 parts (by weight) ethanedithiol, about 53 parts (by weight) sulfide anisole, about 50 parts (by weight) in water and about 142 parts (by weight) of trifluoroacetic acid; the purification step (c) is accomplished by gel filtration chromatography using a gel prepared from beads containing dextran, which The polysaccharide has been cross-linked with epichlorohydrin under alkaline conditions, wherein the dried beads have a diameter of about 20 microns to about 150 microns, and wherein the gel is prepared and washed with an aqueous solution containing 0.1% trifluoroacetic acid take off. Removal step (e) can be accomplished using a solution containing silver(I) nitrate in acetic acid.

The metallothionein or sulfur protein produced may be metal-containing or metal-free. When metals are present, the metals can be selected from main group metals, transition metals, lanthanides and actinides. The metal can also be zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technetium, lanthanum, or combinations thereof .

All patents, patent applications (including U.S. Patent Application Nos. 60/787,400, filed March 30, 2006, and 60/839,582, filed August 23, 2006) and publications mentioned herein are incorporated by reference Herein, to the same extent as if each individual patent, patent application, or publication was specifically indicated to be incorporated herein by reference.

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Claims (33)

1. comprise the polypeptide with metallothionein(MT) or the essentially identical aminoacid sequence of sulfoprotein, wherein at least one sulphur atom is replaced by selenium.

2. the polypeptide of claim 1, wherein said sulphur atom is arranged in the cysteine residues of described polypeptide.

3. the polypeptide of claim 2,10 cysteine residues in the wherein said polypeptide are replaced by seleno-cysteine.

4. the polypeptide of claim 2, the whole cysteine residues in the wherein said polypeptide are replaced by seleno-cysteine.

5. comprise the segmental polypeptide of metallothionein(MT) or sulfoprotein, wherein at least one sulphur atom is replaced by selenium, and described fragment can with melts combine.

6. the polypeptide of claim 5, wherein said sulphur is arranged in the cysteine residues of described polypeptide.

7. the polypeptide of claim 5, the whole cysteine residues in the wherein said polypeptide are replaced by seleno-cysteine.

8. the polypeptide of claim 5, wherein said fragment comprises the αJie Gouyu or the beta structure territory of metallothionein(MT) or sulfoprotein.

9. the polypeptide of claim 5, wherein said metal is a zinc.

10. be used to identify the method for the candidate compound for the treatment of the oxidative stress relative disease, said method comprising the steps of:

(a) make compound contacting metal sulfoprotein and comprise can be oxidized amino acid whose second polypeptide; And

(b) measure the amino acid whose formation of oxidized form in the amount of metal in the presence of described compound, from described metallothionein(MT), discharge and described second polypeptide, do not compare when wherein not existing with described compound, compound (i) raising discharges metal and does not (ii) significantly improve the amino acid whose amount of oxidized form described in described second polypeptide from metallothionein(MT), represent that then described compound is the candidate compound that is used for the treatment of the oxidative stress relative disease.

11. the method for claim 10, wherein said compound is selected from the chemical library.

12. the method for claim 10, wherein said oxidized form amino acid is methionine sulfoxide.

13. the method for claim 10, wherein said metal are zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold, selenium, arsenic, tungsten, aluminium, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium or lanthanum.

14. the method for claim 13, wherein said metal are zinc.

15. the method for claim 10, wherein said second polypeptide is metallothionein(MT) or sulfoprotein.

16. the method for claim 15, wherein said oxidized form amino acid is methionine sulfoxide.

17. the method for claim 10, wherein said disease are selected from alzheimer's disease, Parkinson's disease, creutzfeldt-jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, senium praecox, werner's syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, regional ileitis (Crohn's disease), macular degeneration, apoplexy, local asphyxia and ulcerative colitis.

18. be used to identify the method for the candidate compound for the treatment of the oxidative stress relative disease, said method comprising the steps of:

(a) make cell or cell extract contact compound; And

(b) measure metallothionein(MT) or the amount of sulfoprotein and the state of oxidation of this cell or cell extract in this cell or the cell extract, wherein compare with cell that does not contact this compound or cell extract, compound (i) improves the amount of sulfoprotein or reduces the amount of metallothionein(MT), and (ii) do not significantly improve the state of oxidation of this cell or cell extract, represent that then this compound is the candidate compound that is used for the treatment of the oxidative stress relative disease.

19. the method for claim 18, wherein said compound is selected from the chemical library.

20. the method for claim 18, the wherein said measurement state of oxidation comprise the oxidized form occurrence of amino acid situation that detects.

21. the method for claim 20, wherein said oxidized form amino acid is methionine sulfoxide.

22. the method for claim 18, wherein said disease are selected from alzheimer's disease, Parkinson's disease, creutzfeldt-jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, senium praecox, werner's syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, regional ileitis (Crohn's disease), macular degeneration, apoplexy, local asphyxia and ulcerative colitis.

23. be used to identify the method for the candidate compound for the treatment of the oxidative stress relative disease, said method comprising the steps of:

(a) make compound exposing cell or cell extract, comprise the polynucleotide of the sulfoprotein of encoding in described cell or the cell extract; And

(b) measure the expression of sulfoprotein in this cell or the cell extract, do not compare when wherein not existing with this compound, the raising of expressing in the presence of this compound represents that this compound is the candidate compound that is used for the treatment of the oxidative stress relative disease.

24. the method for claim 23, wherein said compound is selected from the chemical library.

25. the method for claim 23, wherein said disease are selected from alzheimer's disease, Parkinson's disease, creutzfeldt-jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, senium praecox, werner's syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, regional ileitis (Crohn's disease), macular degeneration, apoplexy, local asphyxia and ulcerative colitis.

26. be used to identify the method for the sulfoprotein variant that the avidity to metal reduces, said method comprising the steps of:

(a) in sulfoprotein, introduce point mutation, insertion or disappearance, perhaps chemically change sulfoprotein, thereby produce modified sulfoprotein; And

(b) avidity of the described metal of mensuration and this modified sulfoprotein wherein represents that to the avidity reduction of described metal this modified sulfoprotein is the sulfoprotein variant to the avidity reduction of metal.

27. the method for claim 26, wherein said determination step (b) also comprises the reducing activity of measuring this modified sulfoprotein, and the reducing activity of wherein said modified sulfoprotein does not significantly reduce represents that this modified sulfoprotein is the redox active sulfoprotein variant to the avidity reduction of metal.

28. the method for claim 26, wherein said point mutation comprise by the point mutation of cysteine mutation to seleno-cysteine.

29. the method for claim 26, wherein said metal are zinc, copper, cadmium, lead, silver, gadolinium, cobalt, calcium, gold, selenium, arsenic, tungsten, aluminium, manganese, iron, chromium, nickel, molybdenum, barium, strontium, bismuth, hafnium, technetium or lanthanum.

30. the method for claim 29, wherein said metal are zinc.

31. be used for the treatment of the method for oxidative stress relative disease, described method comprises the sulfoprotein variant of the patient that these needs are arranged being used the method evaluation of using claim 26.

32. the method for claim 31, wherein said disease are selected from alzheimer's disease, Parkinson's disease, creutzfeldt-jakob disease, amyotrophic lateral sclerosis, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, senium praecox, werner's syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, regional ileitis (Crohn's disease), macular degeneration, apoplexy, local asphyxia and ulcerative colitis.

33. be used for the treatment of the patient's who suffers from the oxidative stress relative disease method, described method comprises uses sequestrant to described patient, and wherein said disease is selected from creutzfeldt-jakob disease, respiratory distress syndrome, muscular dystrophy, cataract, rheumatoid arthritis, senium praecox, werner's syndrome, atherosclerosis, diabetes, essential hypertension, cystic fibrosis, regional ileitis (Crohn's disease), macular degeneration, apoplexy, local asphyxia and ulcerative colitis.