Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-04-03
2004-03-02
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S023000, C435S024000, C435S007100, C435S029000, C435S183000, C435S195000, C435S212000, C514S002600, C530S300000, C530S350000
Reexamination Certificate
active
06699681
ABSTRACT:
TECHNICAL FIELD
This invention relates to Alzheimer's disease, and more particularly, to catabolism of A&bgr;.
BACKGROUND
Alzheimer's disease (AD) is the most common cause of dementia in the elderly and is characterized pathologically by the accumulation of &bgr;-amyloid polypeptides (A&bgr;) in the brain in the form of senile plaques. A&bgr; is produced from the amyloid precursor protein (APP) through the combined proteolytic actions of &bgr; and &ggr; secretase and is then secreted into the extracellular milieu.
SUMMARY
The invention identifies endothelin-converting enzymes (ECE) as A&bgr; degrading enzymes. The invention features methods of identifying compounds that increase expression or activity of an ECE. Methods are also provided to increase the catabolism of A&bgr; in a cell, for example by decreasing or abolishing the activity of an ECE inhibitor or, alternatively, by reducing degradation of ECE. In addition, methods of treating an individual with AD are provided. As ECE inhibitors are currently being evaluated for anti-hypertension activity, the invention also features methods of identifying anti-hypertension compounds that do not cause an increase in the level of A&bgr;. Mutants of ECE polypeptides are also provided by the invention, as are mutant ECE nucleic acids. Such mutant ECE nucleic acids and polypeptides can be used to diagnose an individual with AD or those having a predisposition for AD.
In one aspect, the invention provides methods of identifying a compound that increases expression of an ECE nucleic acid, including contacting cells comprising an ECE nucleic acid with a compound and detecting the amount of ECE RNA or ECE polypeptide. An increase in the amount of ECE RNA or polypeptide in the presence of the compound compared to the amount produced in the absence of the compound indicates that the compound increases expression of an ECE nucleic acid. ECE RNA and ECE polypeptides are typically detected using Northern blots and Western blots, respectively. Representative examples of cells include H4 neuroglioma cells, CHO cells and HUVEC cells. Alternatively, the effect of a compound on the expression of an ECE nucleic acid also can be determined using a cell-free transcription/translation system.
The invention further provides methods of identifying compounds that increase activity of an ECE polypeptide, including contacting A&bgr; with an ECE polypeptide in the presence of the compound and detecting the amount of unhydrolyzed A&bgr;. Generally, a decrease in the amount of unhydrolyzed A&bgr; produced in the presence of the compound compared to the amount produced in the absence of the compound is an indication that the compound increases activity of an ECE polypeptide. In one embodiment, the ECE nucleic acid or polypeptide and the A&bgr; are together in a cell. Generally, the unhydrolyzed A&bgr; is detected using one of many immunoassays available in the art.
In another aspect, the invention provides methods of identifying a compound that increases catabolism of A&bgr;, including performing a big-ET conversion assay in the presence or absence of the compound. Typically, an increase in the amount of mature ET in the presence of the compound compared to the amount in the absence of the compound indicates a compound that increases catabolism of A&bgr;.
In yet another aspect, the invention provides methods of identifying a compound that has anti-hypertension activity but does not cause an increase in the level of A&bgr;. The methods include contacting A&bgr; with an ECE nucleic acid or polypeptide in the presence of the compound, detecting the amount of unhydrolyzed A&bgr;, and determining the anti-hypertension activity of the compound. Generally, a lack of an increase in the amount of unhydrolyzed A&bgr; produced in the presence of the compound compared to the amount produced in the absence of the compound is an indication that the compound does not cause an increase in ECE activity. If necessary, the anti-hypertension activity of a compound can be determined using an animal model, such as a spontaneously hypersensitive rat (SHR).
In still another aspect of the invention, there are provided methods of determining that an anti-hypertension compound or candidate compound does not cause an increase in the level of A&bgr;, including contacting A&bgr; with an ECE nucleic acid or polypeptide in the presence of the anti-hypertension compound or candidate compound and detecting the amount of unhydrolyzed A&bgr;. A lack of an increase in the amount of unhydrolyzed A&bgr; produced in the presence of the compound compared to the amount produced in the absence of the compound is an indication that the compound does not cause an increase in ECE activity. The anti-hypertension compound or candidate compound can be, for example, an ECE inhibitor.
In an aspect of the invention, there are provided methods of increasing intracellular or extracellular A&bgr; catabolism, including administering a compound to a cell. A compound can decrease or abolish the activity of an inhibitor of an ECE polypeptide or can decrease the degradation of an ECE polypeptide.
In another aspect of the invention, there are provided methods of decreasing the amount of A&bgr; in a cell by administering a vector. In one embodiment, a vector includes an ECE nucleic acid and elements necessary for expression operably linked to the ECE nucleic acid. The ECE nucleic acid can be introduced to cells in vitro or in vivo.
In another aspect of the invention, there are provided methods of treating an individual having AD, including administering a vector to the individual. The vector typically includes an ECE nucleic acid and elements necessary for expression operably linked to the ECE nucleic acid. In one embodiment, the vector is targeted to the brain of the individual.
In yet another aspect of the invention, there are provided methods of treating an individual having AD, including administering an ECE polypeptide to the individual. An ECE polypeptide can be administered directly into the brain of the individual.
The invention further provides ECE-selective inhibitors. It is preferred that cells or the blood-brain barrier be impermeable to such ECE-selective inhibitors.
It is another aspect of the invention to provide isolated mutant ECE polypeptides. In one embodiment, mutant ECE polypeptides exhibit altered A&bgr; hydrolysis properties compared to a corresponding wild-type ECE polypeptide. Such altered A&bgr; hydrolysis properties can result in decreased catabolism of A&bgr; or an accumulation of A&bgr;, and/or result in or contribute to AD. Altered A&bgr; hydrolysis properties also can be associated with an increased risk for AD in an individual. Also provided by the invention are isolated mutant ECE nucleic acids. Mutant ECE nucleic acids can include promoter and/or regulatory sequences and additionally or alternatively can include coding sequences.
In yet another aspect, the invention provides methods of diagnosing an individual having AD or having an altered predisposition for AD. The methods include detecting the presence or absence of one or more mutant ECE nucleic acid molecules in a biological sample from the individual. The presence of the mutant ECE nucleic acid is indicative of an individual having AD or having an altered predisposition (e.g., an increased predisposition) for AD. Representative biological samples include blood, serum, cerebrospinal fluid, brain and skin.
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 pertains. 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In ca
Eckman Christopher B.
Eckman Elizabeth A.
Chernyshev Olga N.
Fish & Richardson P.C. P.A.
Mayo Foundation for Medical Education and Research
Ulm John
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