Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-30
2002-06-11
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S007800
Reexamination Certificate
active
06403325
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods and compounds for treating Alzheimer's disease.
Most current thinking about Alzheimer's disease suggests that the dementia characteristic of the disease results from aggregates formed in the brains of afflicted individuals. The aggregates are thought to be formed by a peptide of 40-43 amino acids called A&bgr;, which is derived from the amyloid precursor protein.
The A&bgr; peptide is normally found in trace amounts in blood and in cerebrospinal fluid. However, it can be found in increased levels in these fluids in patients suffering from some forms of Alzheimer's disease, and it is thought that increased concentrations of the A&bgr; peptide in these fluids contribute to its tendency to form aggregates. Because Alzheimer's disease can take many years to become clinically manifest, it is believed that over time even very small increases in circulating or cerebral spinal fluid A&bgr; levels may be damaging.
SUMMARY OF THE INVENTION
We have discovered that the addition of Wnt1 protein to A&bgr; peptide-producing cells affects the levels of A&bgr; peptide produced by these cells. Accordingly, the invention features methods of screening for compounds able to bind to the Wnt receptor and decrease the amount of A&bgr; peptide produced by these cells. The invention also features methods for treating Alzheimer's disease using compounds able to bind to the Wnt receptor and lower the amount of A&bgr; peptide produced.
In one aspect, the invention features a method of identifying a compound that inhibits expression of an A&bgr; peptide by contacting the compound with a Wnt receptor or a Wnt-binding fragment of Wnt receptor and determining the ability of the compound to bind to the Wnt receptor or Wnt-binding fragment of Wnt receptor. The binding of the compound is an indication that the compound lowers the level of A&bgr; peptide.
In one embodiment, the compound is a mammalian Wnt protein or a Wnt-receptor binding fragment of a mammalian Wnt protein, e.g., a human Wnt1 protein or a Wnt1-receptor binding fragment thereof. In another embodiment, the compound is a peptide mimetic of a Wnt protein. In yet another embodiment, the compound is a non-peptide mimetic of a Wnt protein.
In another aspect, the invention features a method of identifying a compound that inhibits expression of A&bgr; peptide by contacting a cell producing detectable levels of A&bgr; peptide with a Wnt antagonist and measuring the amount of A&bgr; peptide produced. A decrease in the amount of A&bgr; peptide produced by cell in the presence of the Wnt antagonist indicates that the Wnt antagonist is a compound that decreases A&bgr; peptide expression. The Wnt antagonist can include, e.g., a cysteine-rich domain (CRD)-containing fragment of the Wnt-receptor or the Fritz protein.
In another aspect, the invention features a method of identifying a compound that inhibits expression of A&bgr; peptide by (a) culturing a cell expressing a Wnt-receptor and A&bgr; peptide in the presence of the compound; and (b) evaluating the expression of A&bgr; peptide by the cell. A lowered level of A&bgr; peptide relative to a control cell indicates the compound inhibits expression of A&bgr; peptide.
In yet another aspect, the invention features a method of evaluating a compound for the ability to lower A&bgr; peptide levels in a mammal by contacting the compound with Wnt or a Wnt receptor-binding fragment of Wnt and Wnt-receptor or a Wnt binding fragment of Wnt receptor and determining the ability of the compound to enhance the binding of the Wnt or a Wnt receptor-binding fragment of Wnt with the Wnt receptor or a Wnt-binding fragment of Wnt receptor. An increase in the binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound lowers extracellular A&bgr; levels.
In a further aspect, the invention features a method for identifying candidate therapeutic agents for the treatment of Alzheimer's disease by (a) providing a cell which expresses a mammalian Wnt receptor, the cell comprising a reporter construct, the reporter construct comprising a sequence encoding a detectable protein, the sequence encoding the detectable protein being operably linked to a Wnt receptor responsive regulatory element; (b) contacting the cell with a test compound; (c) measuring the expression of the detectable protein in the presence of the test compound. An increase in the expression of the detectable protein in the presence of the test compound compared to the absence of the test compound indicates that the test compound is a candidate therapeutic agent for treatment of Alzheimer disease.
In one embodiment, the detectable protein is &bgr;-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo
r
, G418
r
), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding &bgr;-galactosidase), or xanthine guaninephosphoribosyltransferase (XGPRT). The reporter gene can be alkaline phosphatase.
In another aspect, the invention features a method of inhibiting A&bgr; expression in a cell by identifying a Wnt-responsive cell that expresses A&bgr; peptide and contacting the cell with a compound that binds to Wnt, or to a Wnt receptor. The binding of the compound to Wnt inhibits A&bgr; expression by the cell.
The cell can be in a mammal, e.g., in vivo, or can be a cultured cell, i.e., in vitro. The compound can inhibit secretion of A&bgr; peptide from the cell, or can inhibit intracellular accumulation of A&bgr; from the cell.
Preferably, the Wnt protein is a Wnt1 protein.
In another aspect the invention features a method of inhibiting A&bgr; expression in a cell by identifying a Wnt-responsive cell that expresses A&bgr; peptide, and contacting the cell with a compound that binds to a Wnt receptor, wherein binding of the compound to the Wnt receptor inhibits A&bgr; expression by the cell.
In a further aspect, the invention features a method of treating Alzheimer's disease by introducing into a human suspected of needing treatment for Alzheimer's disease an effective amount of a compound binding to the human Wnt receptor. Binding of the compound is associated with decreased levels of A&bgr; polypeptide levels in the patient.
In a still further aspect, the invention features a method of preventing Alzheimer's disease by introducing into a human indicating as needing prophylactic treatment for Alzheimer's disease an effective amount of a compound characterized by binding to the human Wnt receptor.
In a further aspect, the invention features a method of lowering A&bgr; peptide levels in an mammal by administering a compound characterized by binding to a Wnt receptor in an amount sufficient to lower A&bgr; peptide levels.
An “A&bgr; peptide” includes an extracellular fragment of the &bgr; amyloid precursor protein that is 40-43 amino acids in length and is detected extracellularly.
A “Wnt protein” includes a member of the Wnt family of proteins.
A “Wnt receptor” includes a macromolecule, e.g., a protein, able to confer responsiveness to a Wnt ligand when expressed on the surface of a cell.
A “peptide mimetic” includes a Wnt analog in which one or more peptide bonds have been replaced with an alternative type of covalent bond which is not susceptible to cleavage by peptidases.
A “Wnt” antagonist is a compound that inhibits the activity of a Wnt protein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specific
Kosik Kenneth S.
Morin Peter
Allen Marianne P.
Fish & Richardson P.C.
The Brigham and Women's Hospital Inc.
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