Compositions and assays utilizing ADP or phosphate for detecting protein modulators
First Claim
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1. A high throughput assay method for rapidly identifying a compound that modulates function of a target protein wherein said target protein produces ADP or phosphate and said method comprises:
- a) adding a compound to a mixture comprising a target protein which produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate;
b) subjecting the mixture to an enzymatic reaction, wherein said enzymatic reaction uses ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized; and
c) determining NADH consumption as a measure of ADP production;
wherein a change in measure of NADH consumption between the presence and absence of said compound indicates that said compound is a modulator of said target protein, wherein up to about 10,000 assays can be performed per hour;
the assays are performed in microtiter plates having 96 wells or more; and
the assay mixture further comprises a surfactant or antifoam.
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Abstract
Described herein are methods which identify candidate agents as binding to a protein or as a modulator of the binding characteristics or biological activity of a protein. Generally, the methods involve the use of ADP or phosphate. The assays can be used in a high throughput system to obviate the cumbersome steps of using gels or radioactive materials.
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Citations
38 Claims
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1. A high throughput assay method for rapidly identifying a compound that modulates function of a target protein wherein said target protein produces ADP or phosphate and said method comprises:
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a) adding a compound to a mixture comprising a target protein which produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate;
b) subjecting the mixture to an enzymatic reaction, wherein said enzymatic reaction uses ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized; and
c) determining NADH consumption as a measure of ADP production;
wherein a change in measure of NADH consumption between the presence and absence of said compound indicates that said compound is a modulator of said target protein,wherein up to about 10,000 assays can be performed per hour;
the assays are performed in microtiter plates having 96 wells or more; and
the assay mixture further comprises a surfactant or antifoam.- View Dependent Claims (2, 3, 4, 5, 6, 18, 19, 20, 21, 22)
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7. A method of identifying a compound as a modulator of an ATPase, said method comprises:
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a) adding a compound to a mixture comprising the ATPase under conditions which normally allow for the production of ADP;
b) contacting the reaction mixture from step (a) with a kinase and a phosphorylated substrate to form ATP from ADP and a dephosphorylated substrate;
c) contacting the dephosphorylated substrate from step (b) with NADH and a dehydrogenase under oxidative-reductive conditions whereby NADH is consumed; and
d) measuring NADH consumption as a measure of ADP production;
wherein a change in measure of NADH consumption between the presence and absence of said compound indicates that said compound is a modulator of said ATPase. - View Dependent Claims (8, 9, 10, 11, 30, 31, 32, 33)
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12. A high throughput method for rapidly identifying a compound that modulates function of a target protein wherein said target protein produces ADP or phosphate and said method comprises:
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a) adding a compound to a mixture comprising a target protein which produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate, wherein said mixture further comprises a surfactant or antifoam;
b) reacting the ADP or phosphate produced in step (a) with a detectable compound; and
c) measuring ADP production indirectly by measuring said detectable compound, wherein a change in level of ADP production between the presence and absence of said compound indicates that said compound is a modulator of said target protein function, and wherein up to about 10,000 assays can be performed per hour and wherein the assays are performed in microtiter plates having 96 wells or more. - View Dependent Claims (13, 14, 15, 16, 17)
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23. A method of identifying a compound as a modulator of a target protein wherein said target protein regulates an enzyme that produces ADP or phosphate, and said method comprises:
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a) adding a compound to a mixture comprising said target protein and said enzyme under conditions which normally allow the production of ADP or phosphate;
b) utilizing the ADP or phosphate as a substrate in an enzymatic reaction; and
c) measuring NADH consumption as a measure of ADP production;
wherein a change in measure of NADH consumption between the presence and absence of said compound indicates that said compound is a modulator of said target protein. - View Dependent Claims (24, 25, 26, 27, 28, 29, 34, 35, 36, 37, 38)
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Specification
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Current AssigneeCytokinetics, Inc.
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Original AssigneeCytokinetics, Inc.
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InventorsMalik, Fady, Finer, Jeffrey T., Wood, Kenneth, Shumate, Christopher, Sakowicz, Roman
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Primary Examiner(s)Saidha, Tekchand
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Application NumberUS09/314,464Time in Patent Office1,134 DaysField of Search435/21, 435/15, 435/17, 435/18, 435/19, 435/23, 435/24, 435/196US Class Current435/21CPC Class CodesC12Q 1/42 involving phosphataseC12Q 1/48 involving transferaseC12Q 1/50 involving creatine phosphok...
