Cell-based high-throughput screening methods
First Claim
1. A cell-based screening method to identify compounds that reduce or inhibit the generation of one or more cellular metabolites associated with a disease or disorder, without causing non-specific cytotoxicity, comprising:
- (a) contacting cells with a test compound;
(b) determining levels of (i) a cellular precursor protein, or (ii) a conformation state of a cellular precursor protein; and
(c) determining levels of a metabolite generated from (i) or (ii) of step (b);
wherein a compound that specifically reduces or inhibits the level of the metabolite in cells shows a reduction only in the level of the metabolite relative to that in untreated cells, and a compound that is non-specifically cytotoxic shows a reduction in the levels of (i), and, optionally, associated cleavage products of (i);
or of (ii); and
of the metabolite.
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Accused Products
Abstract
The present invention describes cell-based screening methods that allow for the elimination of false-positive results due to nonspecific toxicity of test compounds, while detecting those compounds that specifically modulate cellular metabolites in metabolic pathways associated with diseases and disorders, with particular regard to Alzheimer'"'"'s disease. The methods are particularly suited to high-throughput screening techniques to identify compounds (drugs) that are effective in a cell-based system. The methods of the invention involve determining within a cell system the levels of both a metabolic precursor and a metabolite of interest and have broad application in high-throughput drug discovery and identification, particularly for precursor and metabolite molecules which are associated with disease and disease progression, such as Alzheimer'"'"'s disease.
55 Citations
61 Claims
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1. A cell-based screening method to identify compounds that reduce or inhibit the generation of one or more cellular metabolites associated with a disease or disorder, without causing non-specific cytotoxicity, comprising:
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(a) contacting cells with a test compound;
(b) determining levels of (i) a cellular precursor protein, or (ii) a conformation state of a cellular precursor protein; and
(c) determining levels of a metabolite generated from (i) or (ii) of step (b);
wherein a compound that specifically reduces or inhibits the level of the metabolite in cells shows a reduction only in the level of the metabolite relative to that in untreated cells, and a compound that is non-specifically cytotoxic shows a reduction in the levels of (i), and, optionally, associated cleavage products of (i);
or of (ii); and
of the metabolite. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 52, 53, 54, 55, 56, 57, 58, 59)
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13. A cell-based screening method to identify compounds that increase or augment the generation of one or more cellular metabolites associated with a decreased likelihood of developing, or with a decreased severity of, a disease or disorder, without causing non-specific cytotoxicity, comprising:
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(a) contacting cells with a test compound;
(b) determining levels of (i) a cellular precursor protein, or (ii) a conformation state of a cellular precursor protein; and
(c) determining levels of a metabolite generated from (i) or (ii) of step (b);
wherein a compound that specifically increases or augments the level of the metabolite in cells shows an increase in the levels of the metabolite relative to untreated cells, while not significantly changing the levels of (i) or (ii). - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A cell-based screening method for distinguishing between a compound that specifically reduces activity of cellular proteolytic pathways involved in metabolic events associated with Alzheimer'"'"'s disease, and a compound that is non-specifically toxic to cells, comprising:
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(a) treating cells with a test compound;
(b) detecting levels of one or more amyloid precursor protein (APP) metabolites;
(c) detecting levels of amyloid precursor protein (APP);
(d) comparing the effect of the compound on the levels of the one or more amyloid precursor protein (APP) metabolites and the effect of the compound on the levels of amyloid precursor protein (APP);
wherein a specific reduction in the levels of the one or more amyloid precursor protein (APP) metabolites, with no significant reduction in the levels of the amyloid precursor protein (APP), indicates that the compound specifically inhibits cellular proteolytic pathways and is not non-specifically cytotoxic. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A cell-based screening method for distinguishing between a compound that specifically reduces activity of cellular proteolytic pathways involved in metabolic events associated with Alzheimer'"'"'s disease, and a compound that is non-specifically toxic to cells, comprising:
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(a) treating cells with a test compound;
(b) detecting levels of cell-associated β
carboxy-terminal fragment (β
CTF) resulting from proteolytic processing of amyloid precursor protein APP;
(c) detecting levels of amyloid precursor protein (APP) and its associated cleavage fragments comprising α
carboxy-terminal fragment (α
CTF), β
carboxy-terminal fragment (β
CTF) and γ
carboxy-terminal fragment (γ
CTF);
(d) comparing the effect of the compound on the levels of β
CTF generated from the cleavage of APP and the effect of the compound on the levels of amyloid precursor protein (APP) and its associated cleavage, fragments;
wherein a specific reduction in the levels of β
CTF, with no significant reduction in the levels of amyloid precursor protein (APP) and its associated cleavage fragments relative to control, indicates that the compound specifically inhibits cellular proteolytic pathways and is not non-specifically cytotoxic. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42)
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43. A cell-based screening method to identify compounds that reduce or inhibit the activity of cellular metabolites involved in an Alzheimer'"'"'s disease metabolic pathway, comprising:
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(a) treating with a test compound cells plated onto a substrate coated with one or more capture antibodies directed against amyloid precursor protein (APP) and APP cell associated metabolites;
(b) disrupting the cells, wherein extracted amyloid precursor protein (APP) and APP cell associated metabolites bind to the one or more capture antibodies;
(c) detecting binding of protein to capture antibody with detection antibodies, wherein the detection antibodies comprise (i) labeled antibodies that specifically recognize one or more APP metabolites and (ii) differently labeled antibodies that specifically recognize amyloid precursor protein (APP) and, optionally, carboxy-terminal, proteolytic products thereof; and
(d) determining the levels of (i) the one or more APP metabolites, and (ii) the amyloid precursor protein (APP) and, optionally, the carboxy-terminal proteolytic products thereof;
wherein a decrease in the levels of the one or more APP metabolites, without a significant decrease in the level of the APP protein, and optionally, the carboxy-terminal proteolytic products thereof, relative to untreated cells, indicates that the compound specifically inhibits cellular proteolytic pathways and is not non-specifically cytotoxic. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51)
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60. A kit for detecting compounds which specifically reduce or increase a cellular metabolite in a pathway associated with a disease or disorder, and which are not generally toxic to cells, comprising containers comprising one or more detectable reagents to determine levels of (i) a cellular metabolic precursor protein or (ii) a conformation state of a cellular precursor protein;
- and (iii) one or more metabolites of the precursor protein of (i) or (ii); and
instructions for use. - View Dependent Claims (61)
- and (iii) one or more metabolites of the precursor protein of (i) or (ii); and
Specification