Whole cell assay systems for cell surface proteases
First Claim
1. A method of assaying for the activity of an ADAM (a disintegrin and metalloprotease) in a whole cell system, comprising:
- (a) selecting a soluble substrate that is specifically cleavable by the ADAM;
(b) combining the soluble substrate with the whole cell system under conditions that allow processing of the substrate to a product by the ADAM; and
(c) determining the amount of the product as an indication of the ADAM activity.
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Abstract
The activity of a cell surface protease, particularly an ADAM, is determined in a rapid and sensitive assay employing a whole cell system. The assays are effective to identify effector molecules that affect the activity of a cell surface protease directly or indirectly, and to screen for therapeutic agents that modulate those effector molecules. The assays can also be used to screen for therapeutic agents that modulate the activity of a cell surface protease associated with a disease or medical condition. Kits comprising at least one component of the present assays are also provided.
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Citations
47 Claims
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1. A method of assaying for the activity of an ADAM (a disintegrin and metalloprotease) in a whole cell system, comprising:
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(a) selecting a soluble substrate that is specifically cleavable by the ADAM;
(b) combining the soluble substrate with the whole cell system under conditions that allow processing of the substrate to a product by the ADAM; and
(c) determining the amount of the product as an indication of the ADAM activity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for determining the effect of a candidate effector on the activity of an ADAM in a whole cell system, comprising:
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(a) selecting a soluble substrate that is specifically cleavable by the ADAM;
(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the candidate effector;
(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;
(d) determining the amount of the product formed in each mixture; and
(e) comparing the amount of product formed in the separate mixtures to determine the effect of the candidate effector on the ADAM activity. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for determining the effect of a candidate ligand on a receptor in a whole cell system comprising an ADAM, wherein the receptor is known to modulate the activity of said ADAM, comprising:
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(a) selecting a soluble substrate that is specifically cleavable by the ADAM;
(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the candidate ligand;
(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;
(d) determining the amount of the product formed in each mixture; and
(e) comparing the amount of product formed in the separate mixtures, to determine the effect of the candidate ligand on the receptor. - View Dependent Claims (27, 28, 29, 30, 31, 32)
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33. A method of determining the effect of a G-protein coupled receptor (GPCR) on the activity of an ADAM in a whole cell system, comprising:
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(a) selecting a ligand known to modulate activity of the GPCR and a soluble substrate that is specifically cleavable by the ADAM;
(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the ligand;
(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;
(d) determining the amount of the product formed in each mixture; and
(e) comparing the amount of product formed in the separate mixtures to determine the effect of the GPCR on the ADAM activity. - View Dependent Claims (34, 35, 36, 37)
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38. A method for determining the effect of a compound on cleavage of the β
- -amyloid precursor protein (APP) at the α
-secretase site in a whole cell system, comprising;
(a) selecting an enzyme that is capable of cleaving the β
-amyloid precursor protein at the α
-secretase site in the whole cell system;
(b) selecting a soluble substrate having a cleavable α
-secretase site;
(c) preparing two mixtures of the soluble substrate and the whole cell system, wherein only one of the mixtures contains the compound;
(d) incubating the mixtures under conditions that allow cleavage of the substrate at the α
-secretase site;
(e) determining the amount of cleavage of the substrate in each mixture; and
(f) comparing the amount of cleavage in the separate mixtures, to determine the effect of the compound on cleavage of APP. - View Dependent Claims (39, 40, 41, 42)
- -amyloid precursor protein (APP) at the α
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43. A kit comprising
a soluble substrate for an ADAM and a candidate effector for ADAM activity.
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46. A method of assaying for the activity of a plurality of cell surface proteases in a whole cell system, comprising:
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(a) providing, for each protease being assayed, a soluble substrate specifically cleavable by the protease, (b) combining the substrates with the whole cell system under conditions that allow processing of each substrate to a product by the respective protease; and
(c) electrophoretically separating and determining the amount of each product, as an indication of the activity of the corresponding protease;
wherein each substrate is an electrophoretic probe comprising an electrophoretic tag, said tag having distinct optical or separation properties with respect to the electrophoretic tag of every other probe used in the assay; and
each said probe is cleavable by the corresponding protease to release the electrophoretic tag. - View Dependent Claims (47)
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Specification