Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-27
2002-07-16
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C436S501000, C530S300000, C530S350000
Reexamination Certificate
active
06420122
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods of identifying compounds that disrupt polypeptide aggregation. The identified compound can be used to treat disorders associated with such aggregation. Huntington's disease and Alzheimer's disease are examples of these disorders.
BACKGROUND OF THE INVENTION
Huntington's disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder associated with selective neuronal cell death, occurring primarily in the cortex and striatum. The disorder is caused by a CAG codon repeat expansion in the first exon of a gene encoding a 350 kD protein, huntingtin, with unknown function (Ambrose et al.,
Somat Cell Mol. Genet.
20:27-38, 1994). CAG encodes the amino acid glutamine (“Gln” or “Q”), so CAG repeats encode polyglutamine regions within huntingtin. The polyglutamine region of huntingtin from non-HD individuals contains about 8-31 consecutive Gln residues. Huntingtin with over 37 consecutive Gln residues is associated with mild to severe HD, with the more severe cases exhibiting a polyglutamine region of up to about 68 Gln residues.
In addition to HD, at least six other inherited neurodegenerative disorders have been found to be associated with CAG expansions. Increasing the length of CAG repeats in the coding region of unrelated genes, and resulting polyglutamine regions in the encoded proteins, causes a similar pattern of neuron degeneration, indicating a similar, if not identical, mechanism of cell death. HD may be caused by abnormal protein-protein interactions mediated by elongated polyglutamines.
SUMMARY OF THE INVENTION
The invention is based, in part, on the discovery of a method for identifying compounds that disrupt the aggregation of polypeptides. These compounds are potentially useful as therapeutics for the treatment of disease conditions associated with such aggregation.
Accordingly, the invention features a method of identifying a compound which disrupts polypeptide aggregation. The method includes: providing a first polypeptide which is labelled with a detection moiety (e.g., an enzyme or a fluorescent protein) that is inactive in the presence of a denaturant, and a second polypeptide (which can be identical to the first), wherein the first and second polypeptides aggregate upon contact; contacting the first polypeptide, the second polypeptide and a test compound to form a mixture; contacting the mixture with the denaturant; and determining the activity of the detection moiety. A decrease in the activity following contact of the mixture with the denaturant indicates that the test compound has prevented at least some of the polypeptides from aggregating, thereby leaving them susceptible to inactivation by the denaturant. Such an outcome suggests that the test compound is a polypeptide aggregation disrupting compound. In the above method, the first or second polypeptide can be immobilized, or they both can be in solution. Alternatively, they can be within a cell, e.g., a cell transfected with a DNA encoding the first polypeptide and/or the second polypeptide. The first and second polypeptides can be identical or different, so long as they aggregate upon contact. The first and second polypeptides can be polypeptides that contain an extended polyglutamine region, beta-amyloid polypeptides, tau proteins, presenilins, alpha-synucleins and prion proteins. Examples of naturally occurring polypeptides that contain extended polyglutamine regions are huntingtin, atropin-1, ataxin-1, ataxin-2, ataxin-3, ataxin-7, alpha 1A-voltage dependent calcium channel, and androgen receptor. Non-naturally occurring polypeptides that contain an extended polyglutamine region are polypeptides which include at least 32 consecutive glutamine residues. In the above method, the detection moiety is preferably a fluorescent protein or an enzyme such as luciferase, and the extended polyglutamine region is preferably at least 33, 34, 35, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 80, 85, 95, 100, 104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270 or 300 glutamine residues in length.
Alternatively, the method includes: providing a fluorescently labelled first polypeptide, wherein the first polypeptide contains an extended polyglutamine region; providing a second polypeptide containing an extended polyglutamine region; contacting the first polypeptide, the second polypeptide and a test compound to form a mixture; denaturing unaggregated polypeptides in the mixture; and detecting fluorescence, wherein a decrease in fluorescence in the presence of the test compound indicates that the test compound is a polypeptide aggregation disrupting compound. The first and second polypeptides can be naturally or non-naturally occurring polypeptides that have at least 32 consecutive glutamine residues. As above, the first or the second polypeptide can be immobilized or both polypeptides can be in solution. Alternatively, they can be within a cell, e.g., a transfected cell which expresses both polypeptides.
Another method of identifying a compound which disrupts the aggregation of polypeptides containing extended polyglutamine regions includes providing a cell which is genetically modified to express a DNA encoding a heterologous polypeptide containing an extended polyglutamine region; contacting the cell with a test compound; and determining whether the test compound decreases the amount of aggregation of the polypeptide in the cell, wherein a decrease in polypeptide aggregation in the presence of the test compound indicates that the test compound is a polypeptide aggregation disrupting compound. The heterologous polypeptide can be, for example, a fusion protein comprising an antigenic tag or a label. Examples of labels include fluorescent proteins (e.g., a green fluorescent protein (GFP) or a blue fluorescent protein (BFP)) and enzymes. Where the label is a fluorescent protein or other denaturable protein, the step of determining whether the compound is an aggregation disrupting compound includes contacting the cell with a denaturant such as detergent or heat sufficient to effect denaturing of the label portion of unaggregated fusion protein, and detecting fluorescence wherein a decrease in fluorescence following contact of the cell with the denaturant, compared to fluorescence in a similar cell that is treated with the denaturant but not the test compound, indicates that the compound is a polyglutamine polypeptide aggregation disrupting compound. The expression of the DNA can be inducible, e.g., expression can be induced upon exposure of the cell to an inducing agent such as ecdysone or muristerone.
A final method of identifying a compound which disrupts the aggregation of polypeptides includes the steps of providing a cell that is genetically modified to express a DNA encoding a heterologous polypeptide, wherein molecules of the polypeptide spontaneously aggregate within the cell; contacting the cell with a test compound; and determining whether molecules of the polypeptide aggregate in the presence of the test compound, wherein a decrease in aggregation of the polypeptide molecules in the presence of the test compound indicates that the test compound is a polypeptide aggregation disrupting compound. The polypeptide can be a fusion protein comprising a label such as a fluorescent protein (e.g., a GFP or a BFP) or an enzyme. The method can further include contacting the cell with a denaturant such as a detergent or heat, and detecting fluorescence or other activity of the label, wherein a decrease in fluorescence or activity compared to a control not exposed to the test compound indicates that the compound is a polypeptide aggregation disrupting compound.
The invention features a DNA encoding a fusion protein which includes (a) at least 32 contiguous glutamine residues and (b) a label (e.g., a fluorescent protein such as GFP or BFP or an enzyme such as luciferase), wherein the sequence encoding the at least 32 glutamine residues comprises both CAG codons and CAA codons. The CAG and CAA codo
Housman David E.
Kazantsev Aleksey G.
Preisinger Elizabeth A.
Fish & Richardson P.C.
Massachusetts Institute of Technology
Stucker Jeffrey
Winkler Ulrike
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