WO2005047900A1 - Prion protein dye conjugates and methods of use - Google Patents

Prion protein dye conjugates and methods of use Download PDF

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Publication number
WO2005047900A1
WO2005047900A1 PCT/US2003/032764 US0332764W WO2005047900A1 WO 2005047900 A1 WO2005047900 A1 WO 2005047900A1 US 0332764 W US0332764 W US 0332764W WO 2005047900 A1 WO2005047900 A1 WO 2005047900A1
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Prior art keywords
letter
agglomeration
composition
formation
represented
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PCT/US2003/032764
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French (fr)
Inventor
Brian Zeiler
Victor Adler
Valentine Kryukov
Abraham Grossman
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Q-Rna, Inc.
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Priority to AU2003304543A priority Critical patent/AU2003304543A1/en
Priority to PCT/US2003/032764 priority patent/WO2005047900A1/en
Publication of WO2005047900A1 publication Critical patent/WO2005047900A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • Embodiments of the present invention are directed to agglomeration protein dye conjugates. Such dyes are useful for identifying cofactors participating in the formation of soluble and insoluble complexes, for applications in diagnostics, therapeutics, forensics, animal husbandry and analytical purposes.
  • nucleic acid there are two types of nucleic acid found in living cells. One is deoxyribonucleic acid (“DNA”), and the other is ribonucleic acid (“RNA”). Under normal physiological conditions, both of these nucleic acid molecules are associated with proteins and form nucleoprotein complexes. These proteins can include scaffolding proteins, enzymes, ligases, telomerases, etc. These nucleic acid binding proteins perform functions necessary for normal metabolism and cell/tissue viability. Some disease processes appear to stem from the misfolding of proteins. This misfolding is often associated with nucleic acids and binding factors. These misfolded proteins can go on to form pathological agglomerations.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • agglomerations have been shown to be associated with neuronal cell death and brain wasting diseases such as Alzheimer's and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals.
  • Spongiform encephalopathies often involved with certain neuronal cell death and brain wasting syndromes, characteristically have protein plaques or agglomerations made manifest upon dissection.
  • prion proteins are thought to be the etiologic agent.
  • Prion-based diseases result from "infectious proteins” that are cellular benign prion proteins misfolded into an infectious isoform. This infectious isoform is involved in pathological protein agglomeration. Misfolded proteins also appear to damage cells in the lung, heart, kidney, pancreas and other organs.
  • compositions and methods that can identify these binding factors involved in protein misfolding. And, there exists a need for methods and compositions which facilitate identifying agents which interfere with the formation of the misfolded proteins and/or agglomeration complex, or alter the stability of the complex.
  • SUMMARY OF THE INVENTION Embodiments of the present invention are directed to compositions, methods and kits which facilitate the detection of agglomeration complexes, compositions, methods and kits which interfere with the formation or reduce the stability of the agglomeration complex and compositions identified through the methods.
  • One embodiment of the present invention is a composition represented by the formula:
  • the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • the protein component represented by the letter A is the dominant protein associated with an agglomeration complex.
  • examples of such proteins are associated with
  • crutchfeld- Jacob disease Alzheimer's disease and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals and other spongiform encephalopathies. Examples presented herein are representative of all agglomeration diseases but are specifically directed to prion disease.
  • the fluorophor, enzyme or dye represented by the letter is amino- reactive 4- sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
  • the composition of described by A-B y is prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate.
  • the compositions of the present invention have particular application in methods of detecting the presence of a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product.
  • One method of the present invention comprises the steps of providing a composition represented by the formula: A-B y .
  • the method further comprises the step of forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. Agglomeration complexes are believed to involve, in many instances a nucleic acid component and one or more binding cof actors. The nucleic acids and binding cof actors are often difficult to identify.
  • the method further comprises the step of imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
  • the method further comprises the step of detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of the agglomeration complex.
  • the step of detecting is by monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
  • a further embodiment of the present invention features a method of identifying compositions that interfere in the formation of an agglomeration complex represented by the formula
  • the method comprises the steps of providing a composition represented by the formula:
  • the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • An admixture is formed of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition.
  • binding conditions are imposed on the admixture to form an agglomeration complex in the absence of the candidate composition.
  • the sample is monitored for the formation of the agglomeration complex.
  • the absence of the agglomeration complex indicates the candidate composition interferes with the formation thereof.
  • a further embodiment of the present invention is directed to a method of identifying compositions that reverse the formation of an agglomeration complex represented by the formula; A x - C z - D w ;
  • the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the agglomeration product can be detected by the concentration of a color or fluorophor in a specified location.
  • the method comprises the step of forming an admixture of said agglomeration product and a candidate compound.
  • the admixture is monitored for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
  • the formation of a soluble composition represented by A-B visible by color or light emission may appear in the solution by a darkening of color of the solution or by a decrease in color intensity in a band or blot on a surface.
  • compositions and derivatives thereof and such compositions having similar modes of action would also promote a decoupling of the components of the agglomeration complex.
  • compositions may have applications as therapies for the treatment of agglomeration disease.
  • Compositions with appropriate instructions for their use in the performance of the methods described herein can be bundled for use in the performance of such methods as kits.
  • kit refers to an article of manufacture comprising an assembly of compositions contained in suitable vials held together in packaging with instructions for their use.
  • One embodiment of the present invention is directed to a kit for identifying a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product.
  • the kit comprises a composition represented by the formula: A-B y .
  • the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • This composition would keep contained in a suitable containment vessel such as a vial or pouch. This composition may be held as a powder for reconstitution.
  • the kit has instructions for forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor.
  • the instruction describe binding conditions to form an agglomeration complex represented by the formula: (A-B y ) x - C z - D w ;
  • the letter C represents a binding cofactor that participates in the formation of a agglomeration complex
  • the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the instruction would describe detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex.
  • the instructions describe monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
  • a further embodiment of the present invention is directed to a kit for identifying compositions that interfere in the formation of an agglomeration complex represented by the formula:
  • the letter A represents a major protein component of an agglomeration product
  • the letter C represents a binding cofactor that participates in the formation of a agglomeration complex
  • the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the kit comprises a composition represented by the formula:
  • the kit further comprises instructions for forming an admixture of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. And, the instructions describe imposing binding conditions on the admixture to form an agglomeration complex represented by the formula set forth below, in the absence of said candidate composition:
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the instruction describe monitoring the sample for the formation of the agglomeration complex, the absence of which indicates the candidate composition interferes with the formation thereof.
  • the formation of a nonsoluble precipitate visible by color or light emission Preferably, the formation of a nonsoluble precipitate visible by color or light emission.
  • kits in which A-B y is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate will provide a colored band.
  • Embodiments of the present invention feature a kit for identifying compositions that reverse the formation of an agglomeration complex represented by the formula
  • the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the kit comprises a composition represented by the formula: A - B y
  • the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • This composition, at least one nucleic acid, at least one binding cofactor forms an agglomeration complex represented by the formula
  • the order above does not signify an order of binding.
  • the agglomeration product itself is provided.
  • the agglomeration product is detectable by the concentration of a color or fluorophor in a specified location.
  • the kit has instructions for forming an admixture of said agglomeration product and a candidate compound. And, the instructions describe monitoring the admixture for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
  • A-B y is prion protein amino-reactive 4-sulfo-2,3,5,6,- te rafluorophenyl (STP) ester conjugate compounds which potentially reverse the formation of the agglomeration product can be identified. These compounds which potentially reverse the formation of agglomeration disease.
  • Compounds that have been identifies include heparin and sarcin. Sarcin is an enzyme that cleaves or destroys nucleic acid. Sarcin is closely related to ricin. Heparin is an anticoagulent.
  • Figure 1 depicts a kit embodying features of the present invention.
  • Figure 2 depicts a slot blot experiment exhibiting features of the present invention.
  • Embodiments of the present invention are directed to compositions, methods and kits which facilitate the detection of agglomeration complexes, compositions, methods and kits which interfere with the formation or reduce the stability of the agglomeration complex and compositions identified through the methods.
  • One embodiment of the present invention is a composition. This description will describe such composition in detail with the understanding that the features of such composition can be applied to other proteins involved in the formation of agglomeration complexes as well.
  • composition of the present invention such composition is represented by the formula: A-B y .
  • the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • the protein component represented by the letter A is the dominant protein associated with an agglomeration complex.
  • Crutchfeld- Jacob disease, Alzheimer's disease and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals and other spongiform encephalopathies are characterized by plaques. These plaques have, as a major constituent, a protein.
  • the dominant protein of the Alzheimer's disease is a well characterized. This protein is an amyloid protein which has been sequenced.
  • the dominant protein of prion disease is also characterized by specific sequence of amino acids. Examples presented herein are representative of all agglomeration diseases but are specifically directed to prion disease.
  • the fluorophor, enzyme or dye represented by the letter is amino-reactive 4- sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
  • STP amino-reactive 4- sulfo-2,3,5,6,-tetrafluorophenyl
  • the composition of described by A-B y is prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate.
  • other labels may be utilized.
  • compositions of the present invention have particular application in methods of detecting the presence of a binding cofactor or a nucleic acid which participates in the formation of an agglomeration product.
  • One method of the present invention comprises the steps of providing a composition represented by the formula:
  • the method further comprises the step of forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. Agglomeration complexes are believed to involve, in many instances a nucleic acid component and one or more binding cof actors. The nucleic acids and binding cof actors are often difficult to identify.
  • the method further comprises the step of imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
  • the letter C represents a binding cofactor that participates in the formation of an agglomeration complex
  • the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the method further comprises the step of detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by momtoring the admixture for the presence of a fluorophor, enzyme or dye as part of the agglomeration complex.
  • the nucleic acid if unknown, can be provided as a library of nucleic acids.
  • This library can be comprised on nucleic acids having a sequences of ten or more nucleotides.
  • the nucleic acid has a sequence of twenty to forty nucleotides.
  • the library is derived from individuals who exhibit symptoms of the disease. The nucleic acid of such individuals is amplified to a high copy number. Nucleic acid sequences that are know to be common in non- disease states may be excluded.
  • Another preferred library is a library comprising nucleic acid derived from retro- viral sources.
  • the nucleic acid is an RNA.
  • the step of detecting is by momtoring the admixture for a nonsoluble precipitate visible by color or light emission.
  • the screening methods of the present invention can be performed rapidly without radiolabels.
  • a screening test of the present invention can be performed without automation in as little as twenty minutes. Multiple screens may be performed simultaneously.
  • the progression of the reactions can be monitored by color intensity over time to give important information with respect to the kinetics.
  • a further embodiment of the present invention features a method of identifying compositions that interfere in the formation of an agglomeration complex represented by the formula
  • the method comprises the steps of providing a composition represented by the formula: A-B y
  • the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • An admixture is formed of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition.
  • binding conditions are imposed on the admixture to form an agglomeration complex in the absence of the candidate composition.
  • the sample is monitored for the formation of the agglomeration complex.
  • the absence of the agglomeration complex indicates the candidate composition interferes with the formation thereof.
  • fluorphor enzyme or dye
  • the use of the fluorphor, enzyme or dye allows monitoring the admixture for the formation of a nonsoluble precipitate visible by color or light emission.
  • a further embodiment of the present invention is directed to a method of identifying compositions that reverse the formation of an agglomeration complex represented by the formula
  • the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the agglomeration product can be detected by the concentration of a color or fluorophor in a specified location.
  • the method comprises the step of forming an admixture of said agglomeration product and a candidate compound.
  • the admixture is monitored for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
  • the formation of a soluble composition represented by A-B visible by color or light emission may appear in the solution by a darkening of color of the solution or by a decrease in color intensity in a band or blot on a surface.
  • compositions and derivatives thereof and such compositions having similar modes of action would also promote a decoupling of the components of the agglomeration complex.
  • compositions may have applications as therapies for the treatment of agglomeration disease.
  • Compositions with appropriate instructions for their use in the performance of the methods described herein can be bundled for use in the performance of such methods as kits.
  • a kit having features of the present invention is depicted in Figure 1.
  • the kit, an article of manufacture, is designated by the numeral 11.
  • the kit 11 has a composition contained in a suitable vial 13. If more than one composition is to be used, additional vials with such compositions (not shown) are placed in the kit 11. Additional vials (not shown) may also contain reagents for reconstitution or buffers.
  • the kit further contains instructions 15 describing the use of the composition with respect to one or more of the methods described herein.
  • the assembly of parts is held by suitable packaging, such as a box 17.
  • suitable packaging such as a box 17.
  • one embodiment of the present invention is directed to a kit 11 for identifying a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product.
  • the kit 11 comprises a vial 13 containing a composition represented by the formula: A-B y .
  • the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • the vial 13 may readily be substituted with any other convenient container or pouch.
  • the kit has instructions for forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor.
  • Packaging 17 holding the assembly may comprise a box, as shown or wrappings, plastic forms or envelopes (not shown).
  • the instructions describe binding conditions to form an agglomeration complex represented by the formula:
  • the letter C represents a binding cofactor that participates in the formation of a agglomeration complex
  • the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the instruction would describe detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex.
  • the instructions describe monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
  • the kit may include optical readers or film in which to record the results.
  • a further embodiment of the present invention is directed to a kit 11 for identifying compositions that interfere in the formation of an agglomeration complex represented by the formula:
  • the letter A represents a major protein component of an agglomeration product
  • the letter C represents a binding cofactor that participates in the formation of a agglomeration complex
  • the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the kit 11 comprises at least one composition or means for making such composition contained in a vial or containment vessel 13.
  • the composition is represented by the formula:
  • kit 11 further comprises instructions for forming an admixture of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. And, the instructions describe imposing binding conditions on the admixture to form an agglomeration complex represented by the formula set forth below, in the absence of said candidate composition:
  • kits in which A-B y is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate will provide a colored band.
  • the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex.
  • the order above does not signify an order of binding.
  • the letters x, z, and w represent integers from 1 to infinity.
  • the kit 11 comprises a composition represented by the formula:
  • the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
  • This composition, at least one nucleic acid, at least one binding cofactor forms an agglomeration complex represented by the formula
  • the order above does not signify an order of binding.
  • the agglomeration product itself is provided.
  • the agglomeration product is detectable by the concentration of a color or fluorophor in a specified location.
  • the kit 11 has instructions 15 for forming an admixture of said agglomeration product and a candidate compound. And, the instructions describe monitoring the admixture for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
  • A-By is a prion protein amino-reactive 4-sulfo-2,3,5,6,- teteafluorophenyl (STP) ester conjugate compounds which potentially reverse the formation of the agglomeration product can be identified. These compounds which potentially reverse the formation of agglomeration disease.
  • Compounds that have been identifies include heparin and sarcin. Sarcin is an enzyme that cleaves or destroys nucleic acid. Sarcin is closely related to ricin. Heparin is an anticoagulent.
  • Amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester reagent is conjugated to protein or peptide associated with an agglomeration complex disease.
  • the following protocol is optimized for human recombinant prion protein which was purified from E. coli and has more than 80% purity. Succimidyl esters are preferred for conjugation to proteins because they form a very stable amide bond between dye and the protein.
  • Amino-reactive reagents react with non- protonated aliphatic amine groups, including the amine terminus of proteins and the e-amino group of lysines. The e-amino group has a pKa (association constant) of around 10.5. In order to maintain this amino group in non-protonated form, the conjugation must place in buffer with slightly basic pH. It is important to avoid buffers that contain primary amines, such as Tris, as these compete for conjugation with amino-reactive compound.
  • a 0.15 M sodium bicarbonate buffer is used as a reaction buffer pH 8.5, because the terminal amine group of PrP has lower pKa than that of lysine e-amino group.
  • the 2 mg of pure hrPrP was dissolved in 150 uL of 0.15 M sodium bicarbonate buffer and 0.2 mg of Cy5 due (Amersham) in 50 ul of bicarbonate buffer pH 8.5.
  • the reaction mixture incubated for 1 hour at room temperature.
  • the reaction was stopped by adding 0.1 mL of freshly prepared 1.5 M hydroxylamine, pH 8.5.
  • the hydroxylamino-containing reaction incubated overnight at 4 °C.
  • IM, pH 7.5 is used to separate the conjugate from untreated labeling reagent.
  • the excluded fraction corresponds to the PrP fluorescent band.
  • Lanes 1, 2 - PrP and RQ without and with PK treatment Lane 3 - PrP, RQ, and serum; PK treatment. Lane 4 - 0.6 ul of 0.1 M chlorpromazine. Lanes 5, 6 - 0.1 , 1 ul (0.002, 0.02 mM) of 1 mM tetracycline. Lane 7, 8 - 0.1, 1 ul (0.02, 0.2 mM) of 10 mM tetracycline. Lanes 9, 10 - 0.1, 1 ul (0.2, 2 mM) of 100 mM tetracycline.
  • Sarcin and heparin were applied in a similar manner. And, heparin and or sarcin prevented or reversed the formation of agglomeration complexes.

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Abstract

Embodiments of the present invention are directed to compositions, methods and kits for the determination of cofactors and nucleic acids which participate in the formation of agglomeration complexes and with compositions which alter or reverse such formation.

Description

PRION PROTEIN DYE CONJUGATES AND METHODS OF USE
Field of the Invention
Embodiments of the present invention are directed to agglomeration protein dye conjugates. Such dyes are useful for identifying cofactors participating in the formation of soluble and insoluble complexes, for applications in diagnostics, therapeutics, forensics, animal husbandry and analytical purposes.
Background of the Invention
Essentially there are two types of nucleic acid found in living cells. One is deoxyribonucleic acid ("DNA"), and the other is ribonucleic acid ("RNA"). Under normal physiological conditions, both of these nucleic acid molecules are associated with proteins and form nucleoprotein complexes. These proteins can include scaffolding proteins, enzymes, ligases, telomerases, etc. These nucleic acid binding proteins perform functions necessary for normal metabolism and cell/tissue viability. Some disease processes appear to stem from the misfolding of proteins. This misfolding is often associated with nucleic acids and binding factors. These misfolded proteins can go on to form pathological agglomerations. These agglomerations have been shown to be associated with neuronal cell death and brain wasting diseases such as Alzheimer's and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals. Spongiform encephalopathies, often involved with certain neuronal cell death and brain wasting syndromes, characteristically have protein plaques or agglomerations made manifest upon dissection. In spongiform encephalopathies, prion proteins are thought to be the etiologic agent. Prion-based diseases result from "infectious proteins" that are cellular benign prion proteins misfolded into an infectious isoform. This infectious isoform is involved in pathological protein agglomeration. Misfolded proteins also appear to damage cells in the lung, heart, kidney, pancreas and other organs.
It is believed that these misfolded proteins that result in agglomeration-causing disease are associated with certain RNA molecules and other binding factors. Currently, a need exists for compositions and methods that can identify these binding factors involved in protein misfolding. And, there exists a need for methods and compositions which facilitate identifying agents which interfere with the formation of the misfolded proteins and/or agglomeration complex, or alter the stability of the complex. SUMMARY OF THE INVENTION Embodiments of the present invention are directed to compositions, methods and kits which facilitate the detection of agglomeration complexes, compositions, methods and kits which interfere with the formation or reduce the stability of the agglomeration complex and compositions identified through the methods. One embodiment of the present invention is a composition represented by the formula:
A-By.
As used herein, the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
Preferably, the protein component represented by the letter A is the dominant protein associated with an agglomeration complex. Examples of such proteins are associated with
Crutchfeld- Jacob disease, Alzheimer's disease and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals and other spongiform encephalopathies. Examples presented herein are representative of all agglomeration diseases but are specifically directed to prion disease.
Preferably, the fluorophor, enzyme or dye represented by the letter is amino- reactive 4- sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester. Thus, the composition of described by A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate. The compositions of the present invention have particular application in methods of detecting the presence of a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product. One method of the present invention comprises the steps of providing a composition represented by the formula: A-By.
wherein the letter A, B, and y are as described above. The method further comprises the step of forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. Agglomeration complexes are believed to involve, in many instances a nucleic acid component and one or more binding cof actors. The nucleic acids and binding cof actors are often difficult to identify. The method further comprises the step of imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
(A-BA - Cz - Dw; As used above, the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The method further comprises the step of detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of the agglomeration complex. For example, in one preferred embodiment the step of detecting is by monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
A further embodiment of the present invention features a method of identifying compositions that interfere in the formation of an agglomeration complex represented by the formula;
A - Cz - Dw;
wherein the letter A represents a major protein component of an agglomeration product, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity. The method comprises the steps of providing a composition represented by the formula:
A-B.,
Again, as used above, the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. An admixture is formed of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. Next, binding conditions are imposed on the admixture to form an agglomeration complex in the absence of the candidate composition. The sample is monitored for the formation of the agglomeration complex. The absence of the agglomeration complex indicates the candidate composition interferes with the formation thereof.
The use of the fluorphor, enzyme or dye allows momtoring the admixture for the formation of a nonsoluble precipitate visible by color or light emission. A further embodiment of the present invention is directed to a method of identifying compositions that reverse the formation of an agglomeration complex represented by the formula; Ax - Cz - Dw;
Again, the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The method comprises the step of providing a composition represented by the formula:
A-By
wherein the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. An admixture formed of the composition, at least one nucleic acid, and at least one binding cofactor forms an agglomeration complex represented by the formula:
(A-By)x - Cz - Dw;
Again, the order above does not signify an order of binding of providing said agglomeration product of this step. The agglomeration product can be detected by the concentration of a color or fluorophor in a specified location. The method comprises the step of forming an admixture of said agglomeration product and a candidate compound. The admixture is monitored for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex. For example, the formation of a soluble composition represented by A-B visible by color or light emission may appear in the solution by a darkening of color of the solution or by a decrease in color intensity in a band or blot on a surface.
The method as applied to prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate identifies sarcin and heparin as compounds which inhibit the formation of agglomeration complexes or are capable of promoting a decoupling or decomplexation of the components. Such compositions and derivatives thereof and such compositions having similar modes of action would also promote a decoupling of the components of the agglomeration complex. Such compositions may have applications as therapies for the treatment of agglomeration disease. Compositions with appropriate instructions for their use in the performance of the methods described herein can be bundled for use in the performance of such methods as kits. As used herein, the term "kit" refers to an article of manufacture comprising an assembly of compositions contained in suitable vials held together in packaging with instructions for their use. One embodiment of the present invention is directed to a kit for identifying a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product. The kit comprises a composition represented by the formula: A-By.
As used above, the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. This composition would keep contained in a suitable containment vessel such as a vial or pouch. This composition may be held as a powder for reconstitution. The kit has instructions for forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. The instruction describe binding conditions to form an agglomeration complex represented by the formula: (A-By)x - Cz - Dw;
As used above, the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The instruction would describe detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex. Preferably, the instructions describe monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
A further embodiment of the present invention is directed to a kit for identifying compositions that interfere in the formation of an agglomeration complex represented by the formula: As used above, the letter A represents a major protein component of an agglomeration product, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. Again, the order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The kit comprises a composition represented by the formula:
A-By
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A. The letter y represents an integer from 1 to infinity. This composition is held in a suitable containment vessel. The kit further comprises instructions for forming an admixture of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. And, the instructions describe imposing binding conditions on the admixture to form an agglomeration complex represented by the formula set forth below, in the absence of said candidate composition:
(A-By)x - Cz - Dw.
As used above, the order above does not signify an order of binding. The letters x, z, and w represent integers from 1 to infinity. The instruction describe monitoring the sample for the formation of the agglomeration complex, the absence of which indicates the candidate composition interferes with the formation thereof. Preferably, the formation of a nonsoluble precipitate visible by color or light emission.
For example, a kit in which A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate will provide a colored band.
Embodiments of the present invention feature a kit for identifying compositions that reverse the formation of an agglomeration complex represented by the formula;
A - Cz - Dw.
As used above, the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The kit comprises a composition represented by the formula: A-By
Again, the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. This composition, at least one nucleic acid, at least one binding cofactor forms an agglomeration complex represented by the formula
(A-By)x - Cz - Dw;
The order above does not signify an order of binding. In the alternative the agglomeration product itself is provided. The agglomeration product is detectable by the concentration of a color or fluorophor in a specified location. The kit has instructions for forming an admixture of said agglomeration product and a candidate compound. And, the instructions describe monitoring the admixture for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
For example where the A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- te rafluorophenyl (STP) ester conjugate compounds which potentially reverse the formation of the agglomeration product can be identified. These compounds which potentially reverse the formation of agglomeration disease. Compounds that have been identifies include heparin and sarcin. Sarcin is an enzyme that cleaves or destroys nucleic acid. Sarcin is closely related to ricin. Heparin is an anticoagulent.
Brief description of the Drawings Figure 1 depicts a kit embodying features of the present invention; and,
Figure 2 depicts a slot blot experiment exhibiting features of the present invention.
Detailed Description of the Invention Embodiments of the present invention are directed to compositions, methods and kits which facilitate the detection of agglomeration complexes, compositions, methods and kits which interfere with the formation or reduce the stability of the agglomeration complex and compositions identified through the methods. One embodiment of the present invention is a composition. This description will describe such composition in detail with the understanding that the features of such composition can be applied to other proteins involved in the formation of agglomeration complexes as well.
Turning now to one composition of the present invention, such composition is represented by the formula: A-By.
As used herein, the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
Preferably, the protein component represented by the letter A is the dominant protein associated with an agglomeration complex. Crutchfeld- Jacob disease, Alzheimer's disease and Parkinson's disease in humans, scrapie, mad cow disease and chronic wasting diseases in animals and other spongiform encephalopathies are characterized by plaques. These plaques have, as a major constituent, a protein. The dominant protein of the Alzheimer's disease is a well characterized. This protein is an amyloid protein which has been sequenced. The dominant protein of prion disease is also characterized by specific sequence of amino acids. Examples presented herein are representative of all agglomeration diseases but are specifically directed to prion disease.
Preferably, the fluorophor, enzyme or dye represented by the letter is amino-reactive 4- sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester. Thus, the composition of described by A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate. However, other labels may be utilized.
The compositions of the present invention have particular application in methods of detecting the presence of a binding cofactor or a nucleic acid which participates in the formation of an agglomeration product. One method of the present invention comprises the steps of providing a composition represented by the formula:
A-By.
wherein the letter A, B, and y are as described above. The method further comprises the step of forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. Agglomeration complexes are believed to involve, in many instances a nucleic acid component and one or more binding cof actors. The nucleic acids and binding cof actors are often difficult to identify. The method further comprises the step of imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
(A-By)x - Cz - Dw;
As used above, the letter C represents a binding cofactor that participates in the formation of an agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The method further comprises the step of detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by momtoring the admixture for the presence of a fluorophor, enzyme or dye as part of the agglomeration complex.
The nucleic acid, if unknown, can be provided as a library of nucleic acids. This library can be comprised on nucleic acids having a sequences of ten or more nucleotides. Preferably, the nucleic acid has a sequence of twenty to forty nucleotides. Preferably the library is derived from individuals who exhibit symptoms of the disease. The nucleic acid of such individuals is amplified to a high copy number. Nucleic acid sequences that are know to be common in non- disease states may be excluded. Another preferred library is a library comprising nucleic acid derived from retro- viral sources. Preferably, the nucleic acid is an RNA. In one preferred embodiment the step of detecting is by momtoring the admixture for a nonsoluble precipitate visible by color or light emission. The screening methods of the present invention can be performed rapidly without radiolabels. For example, a screening test of the present invention can be performed without automation in as little as twenty minutes. Multiple screens may be performed simultaneously. The progression of the reactions can be monitored by color intensity over time to give important information with respect to the kinetics.
A further embodiment of the present invention features a method of identifying compositions that interfere in the formation of an agglomeration complex represented by the formula;
K - CZ - ΌV
wherein the letter A represents a major protein component of an agglomeration product, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity. The method comprises the steps of providing a composition represented by the formula: A-By
Again, as used above, the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. An admixture is formed of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. Next, binding conditions are imposed on the admixture to form an agglomeration complex in the absence of the candidate composition. The sample is monitored for the formation of the agglomeration complex. The absence of the agglomeration complex indicates the candidate composition interferes with the formation thereof.
The use of the fluorphor, enzyme or dye allows monitoring the admixture for the formation of a nonsoluble precipitate visible by color or light emission.
A further embodiment of the present invention is directed to a method of identifying compositions that reverse the formation of an agglomeration complex represented by the formula;
V C, - DW;
Again, the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The method comprises the step of providing a composition represented by the formula:
A-By
wherein the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. An admixture formed of the composition, at least one nucleic acid, and at least one binding cofactor forms an agglomeration complex represented by the formula:
(A-Bv)x - Cz - Dw
Again, the order above does not signify an order of binding of providing said agglomeration product of this step. The agglomeration product can be detected by the concentration of a color or fluorophor in a specified location. The method comprises the step of forming an admixture of said agglomeration product and a candidate compound. The admixture is monitored for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex. For example, the formation of a soluble composition represented by A-B visible by color or light emission may appear in the solution by a darkening of color of the solution or by a decrease in color intensity in a band or blot on a surface. The method as applied to prion protein amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester conjugate identifies sarcin and heparin as compounds which inhibit the formation of agglomeration complexes or are capable of promoting a decoupling or decomplexation of the components. Such compositions and derivatives thereof and such compositions having similar modes of action would also promote a decoupling of the components of the agglomeration complex. Such compositions may have applications as therapies for the treatment of agglomeration disease. Compositions with appropriate instructions for their use in the performance of the methods described herein can be bundled for use in the performance of such methods as kits. A kit having features of the present invention is depicted in Figure 1. The kit, an article of manufacture, is designated by the numeral 11. The kit 11 has a composition contained in a suitable vial 13. If more than one composition is to be used, additional vials with such compositions (not shown) are placed in the kit 11. Additional vials (not shown) may also contain reagents for reconstitution or buffers. The kit further contains instructions 15 describing the use of the composition with respect to one or more of the methods described herein. The assembly of parts is held by suitable packaging, such as a box 17. For example, one embodiment of the present invention is directed to a kit 11 for identifying a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product. The kit 11 comprises a vial 13 containing a composition represented by the formula: A-By.
As used above, the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. Individuals skilled in the art will readily recognize that the vial 13 may readily be substituted with any other convenient container or pouch. The composition may be held as a powder for reconstitution or be ready for use. The kit has instructions for forming an admixture of the composition and at least one nucleic acid and at least one binding cofactor. Packaging 17 holding the assembly may comprise a box, as shown or wrappings, plastic forms or envelopes (not shown). The instructions describe binding conditions to form an agglomeration complex represented by the formula:
(A-By)x - Cz - Dw; As used above, the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The instruction would describe detecting the presence or absence at least one of the group selected from the binding cofactor and the nucleic acid by monitoring the admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex.
Preferably, the instructions describe monitoring the admixture for a nonsoluble precipitate visible by color or light emission. The kit may include optical readers or film in which to record the results.
A further embodiment of the present invention is directed to a kit 11 for identifying compositions that interfere in the formation of an agglomeration complex represented by the formula:
Aχ - Cz - Dw;
As used above, the letter A represents a major protein component of an agglomeration product, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. Again, the order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The kit 11 comprises at least one composition or means for making such composition contained in a vial or containment vessel 13. The composition is represented by the formula:
A-By
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A. The letter y represents an integer from 1 to infinity. The kit 11 further comprises instructions for forming an admixture of the composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition. And, the instructions describe imposing binding conditions on the admixture to form an agglomeration complex represented by the formula set forth below, in the absence of said candidate composition:
(A-By)x - Cz - Dw. As used above, the order above does not signify an order of binding. The letters x, z, and w represent integers from 1 to infinity. The instructions 15 describe monitoring the sample for the formation of the agglomeration complex, the absence of which indicates the candidate composition interferes with the formation thereof.
Preferably, the formation of a nonsoluble precipitate visible by color or light emission. For example, a kit in which A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate will provide a colored band. Embodiments of the present invention feature a kit 11 for identifying compositions that reverse the formation of an agglomeration complex represented by the formula;
A. - C, - Dw.
As used above, the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex. The order above does not signify an order of binding. And, the letters x, z, and w represent integers from 1 to infinity. The kit 11 comprises a composition represented by the formula:
A-By
Again, the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity. This composition, at least one nucleic acid, at least one binding cofactor forms an agglomeration complex represented by the formula
(A-By)x - Cz - Dw;
The order above does not signify an order of binding. In the alternative the agglomeration product itself is provided. The agglomeration product is detectable by the concentration of a color or fluorophor in a specified location. The kit 11 has instructions 15 for forming an admixture of said agglomeration product and a candidate compound. And, the instructions describe monitoring the admixture for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
For example where the A-By is a prion protein amino-reactive 4-sulfo-2,3,5,6,- teteafluorophenyl (STP) ester conjugate compounds which potentially reverse the formation of the agglomeration product can be identified. These compounds which potentially reverse the formation of agglomeration disease. Compounds that have been identifies include heparin and sarcin. Sarcin is an enzyme that cleaves or destroys nucleic acid. Sarcin is closely related to ricin. Heparin is an anticoagulent.
Other advantages of the present invention will apparent from a reading of the examples below.
Example 1
Amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester reagent is conjugated to protein or peptide associated with an agglomeration complex disease. The following protocol is optimized for human recombinant prion protein which was purified from E. coli and has more than 80% purity. Succimidyl esters are preferred for conjugation to proteins because they form a very stable amide bond between dye and the protein. Amino-reactive reagents react with non- protonated aliphatic amine groups, including the amine terminus of proteins and the e-amino group of lysines. The e-amino group has a pKa (association constant) of around 10.5. In order to maintain this amino group in non-protonated form, the conjugation must place in buffer with slightly basic pH. It is important to avoid buffers that contain primary amines, such as Tris, as these compete for conjugation with amino-reactive compound.
A 0.15 M sodium bicarbonate buffer is used as a reaction buffer pH 8.5, because the terminal amine group of PrP has lower pKa than that of lysine e-amino group. The 2 mg of pure hrPrP was dissolved in 150 uL of 0.15 M sodium bicarbonate buffer and 0.2 mg of Cy5 due (Amersham) in 50 ul of bicarbonate buffer pH 8.5. The reaction mixture incubated for 1 hour at room temperature. The reaction was stopped by adding 0.1 mL of freshly prepared 1.5 M hydroxylamine, pH 8.5. The hydroxylamino-containing reaction incubated overnight at 4 °C. A Sephadex G-25 gel filtration column (10x300 mm) equilibrated with PBS in MORS buffer (0. IM, pH 7.5) is used to separate the conjugate from untreated labeling reagent. The excluded fraction corresponds to the PrP fluorescent band. This fraction was purified additionally by extensive dialysis against 0.1 M MOPS buffer pH 7.5. The protein was aliquated and store at 70°C (1 ug PrP protein = 70 Fu, 590 nm emf). Protection assays were carried out by incubating 380 picomoles ( 11.4 ug) of blue PrP
1.5 picomoles (97.6 ng) of RQ, and different types of blood fractions. The reactants were mixed at 4°C and the reactions took place in a total volume of 10 ul for 60 minutes at RT. 2 ul of protease K (250 ng/ul) were added to the samples, which were incubated for 30 minutes at 37°C. Then 1 ul of 0.1 M PMSF, 2 ul of 20% SDS, and 10 ul of a 2x Sample buffer were added and the samples incubated for 10 minutes at 95°C and 2 minutes in ice. 20 ul were loaded in each well of 12 % PAAG (NOVEX); the electrophoresis took 60 minutes at 150 V. The quantitative change will registered by direct gel scanning Fluoroimagerscanner, Bio-Rad). Protection assays were carried out by incubating 380 picomoles (11.4 ug) of blue prp, 1.5 picomoles (97.6 ng) of RQ, 86.2 ug of human serum, and different concentrations of tetracycline for 30 minutes at 4°C and 30 minutes at RT. The reactions took place in a total volume of 10 ul (water and 2 ul of binding buffer mixture). The reactants were added in the following order: H20, mixture of PrP and RQ in the correct proportion, tetracycline, serum, bbmix. 2 ul of protease K (250 ng/ul) were added to the samples, which were incubated for 30 minutes at 37°C. 1 ul of PMSF, 2 ul of 20% SDS, and 10 ul of a 2x buffer were added and the samples incubated for 10 minutes at 95°C and 2 minutes in ice. 20 ul were loaded in each well; the electrophoresis took 60 minutes at 150 V.
Turning to figure 2, Lanes 1, 2 - PrP and RQ without and with PK treatment. Lane 3 - PrP, RQ, and serum; PK treatment. Lane 4 - 0.6 ul of 0.1 M chlorpromazine. Lanes 5, 6 - 0.1 , 1 ul (0.002, 0.02 mM) of 1 mM tetracycline. Lane 7, 8 - 0.1, 1 ul (0.02, 0.2 mM) of 10 mM tetracycline. Lanes 9, 10 - 0.1, 1 ul (0.2, 2 mM) of 100 mM tetracycline.
Sarcin and heparin were applied in a similar manner. And, heparin and or sarcin prevented or reversed the formation of agglomeration complexes.
Thus, preferred embodiments of the present invention have been disclosed with reference to the figures, description and examples with the understanding that the teaching of the present disclosure can be modified by those skilled in the art without departing from the sperit of the invention. Therefore, the present invention should not be limited to the immediate disclosure but should encompass such subject matter that is directed to the following claims.

Claims

Claims
1. A composition represented by the formula:
A-By
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity.
2. The composition of claim 1 wherein said protein component represented by the letter A is a prion protein.
3. The composition of claim 1 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
4. As a composition of matter, prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
5. A method of detecting the presence of a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product, comprising the steps of
a) providing a composition represented by the formula:
A-By
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity;
b) forming an admixture of said composition and at least one nucleic acid and at least one binding cofactor;
c) imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
(A-By)x - Cz - Dw; wherein the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
c) detecting the presence or absence at least one of the group selected from said binding cofactor and said nucleic acid by monitoring said admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex.
6. The method of claim 5 wherein said step of detecting is by monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
8. The method of claim 5 wherein said protein component represented by the letter A is a prion protein.
9. The method of claim 5 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
10. The method of claim 8 wherein A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
11. A method of performing identifying compositions that interfere in the formation of an agglomeration complex represented by the formula;
(A-By)x - Cz - Dw;
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
comprising the steps of
a) providing a composition represented by the formula:
A-By wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y 2o represents an integer from 1 to infinity;
b) forming an admixture of said composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition;
c) imposing binding conditions on said admixture to form said agglomeration complex in the absence of said candidate composition;
d) monitoring the sample for the formation of the agglomeration complex, the absence of which indicates the candidate composition interferes with the formation thereof.
12. The method of claim 11 wherein said monitoring the admixture detects the formation of a nonsoluble precipitate visible by color or light emission.
13. The method of claim 11 wherein said protein component represented by the letter A is a prion protein.
14. The method of claim 11 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
15. The method of claim 14 wherein A-By is prion protein amino-reactive 4-sulfo-2,3 ,5 ,6,- tetrafluorophenyl (STP) ester conjugate.
16. A method of performing identifying compositions that reverse the formation of an agglomeration complex represented by the formula;
Aχ - Cz - Dw;
wherein the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
comprisingthe steps of
a) providing a composition represented by the formula: A-By
wherein the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity, and forming an admixture of said composition, at least one nucleic acid, at least one binding cofactor to form an agglomeration complex represented by the formula
(A-By)x - Cz - Dw;
wherein the order above does not signify an order of binding or providing said agglomeration product of this step, said agglomeration product detectable by the concentration of a color or fluorophor in a specified location;
b) forming an admixture of said agglomeration product and a candidate compound;
c) monitoring the admixture for the solubilization of A-B, C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
17. The method of claim 16 wherein said momtoring the admixture detects the formation of a soluble composition represented by A-B visible by color or light emission.
18. The method of claim 16 wherein said protein component represented by the letter A is a prion protein.
19. The method of claim 16 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
20. The method of claim 15 wherein A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
21. A kit for identifying a binding cofactor or an nucleic acid which participates in the formation of an agglomeration product, comprising:
a) a composition represented by the formula:
A-By wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity;
b) and instructions for
i.) forming an admixture of said composition and at least one nucleic acid and at least one binding cofactor;
ii) imposing binding conditions on said admixture to form an agglomeration complex represented by the formula;
(A-By)x - Cz - Dw;
wherein the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of 20 binding and the letters x, z, and w represent integers from 1 to infinity;
iii) detecting the presence or absence at least one of the group selected from said binding cofactor and said nucleic acid by monitoring said admixture for the presence of a fluorophor, enzyme or dye as part of said agglomeration complex.
22. The kit of claim 21 wherein said step of detecting is by monitoring the admixture for a nonsoluble precipitate visible by color or light emission.
23. The kit of claim 21 wherein said protein component represented by the letter A is a prion protein.
24. The kit of claim 21 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
25. The kit of claim 24 wherein A-By is prion protein arnino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
26. A kit for identifying compositions that interfere in the formation of an agglomeration complex represented by the formula;
Aχ - Cz - Dw; wherein the letter A represents a major protein component of an agglomeration product, and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
comprising:
a) a composition represented by the formula:
A-By
wherein the letter A represents a major protein component of an agglomeration product and the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity;
b) instructions for i) forming an admixture of said composition, at least one nucleic acid, at least one binding cofactor and at least one candidate composition;
ii) imposing binding conditions on said admixture to form an agglomeration complex represented by the formula set forth below, in the absence of said candidate composition:
(A-By)x - Cz - Dw;
wherein the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
iii) monitoring the sample for the formation of the agglomeration complex, the absence of which indicates the candidate composition interferes with the formation thereof.
27. The kit of claim 26 wherein said monitoring the admixture detects the formation of a nonsoluble precipitate visible by color or light emission.
28. The kit of claim 26 wherein said protein component represented by the letter A is a prion protein.
29. The kit of claim 26 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
30. The kit of claim 26 wherein A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
31. A kit for identifying compositions that reverse the formation of an agglomeration complex represented by the formula;
A. - C. - D.,;
wherein the letter A represents a major protein component of an agglomeration product and the letter C represents a binding cofactor that participates in the formation of a agglomeration complex, and the letter D represents a nucleic acid that participates in the formation of an agglomeration complex, the order above does not signify an order of binding and the letters x, z, and w represent integers from 1 to infinity;
comprising:
a) a composition represented by the formula:
A-By
wherein the letter B represents a fluorophor, enzyme, or dye covalently bonded to A and y represents an integer from 1 to infinity, and forming an admixture of said composition, at least one nucleic acid, at least one binding cofactor to form an agglomeration complex represented by the formula
(A-By)x - Cz - Dw;
wherein the order above does not signify an order of binding or providing said agglomeration product of this step, said agglomeration product detectable by the concentration of a color or fluorophor in a specified location;
b) instructions for:
i) forming an admixture of said agglomeration product and a candidate compound; ϋ) monitoring the admixture for the solubilization of A-B , C or D apart from the agglomeration complex, by migration of the composition represented by A-B from the agglomeration complex.
32. The kit of claim 31 wherein said momtoring the admixture detects the formation of a soluble composition represented by A-B visible by color or light emission.
33. The kit of claim 31 wherein said protein component represented by the letter A is a prion protein.
34. The kit of claim 31 wherein said flurophor, enzyme or dye represented by the letter is amino-reactive 4-sulfo-2,3,5,6,-tetrafluorophenyl (STP) ester.
35. The kit of claim 31 wherein A-By is prion protein amino-reactive 4-sulfo-2,3,5,6,- tetrafluorophenyl (STP) ester conjugate.
36. A composition identified by the method of claims 11 or 16.
37. The composition of claim 36 wherein said composition is an enzyme that cleaves or destroys nucleic acid.
38. The composition of claim 36 wherein said composition is an anticoagulent.
39. The composition of claim 36 comprising sarcin, ricin and heparin.
PCT/US2003/032764 2003-10-15 2003-10-15 Prion protein dye conjugates and methods of use WO2005047900A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040039201A1 (en) * 2001-11-14 2004-02-26 Lugade Ananda G. Functionalized compositions for improved immobilization

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040039201A1 (en) * 2001-11-14 2004-02-26 Lugade Ananda G. Functionalized compositions for improved immobilization

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN J.Q. ET AL.: "In vivo evaluation of 99mTc/188R-labeled linear alpha-melanocyte stimulating hormone analogs for specific melanoma targeting", vol. 26, 1999, pages 687 - 693, XP001062750, DOI: doi:10.1016/S0969-8051(99)00032-3 *
NANDI P.K. ET AL.: "DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid", J. MOL. BIOL., vol. 322, September 2002 (2002-09-01), pages 153 - 161, XP004449834, DOI: doi:10.1016/S0022-2836(02)00750-7 *

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