Scintillation proximity assay for type II fatty acid biosynthetic enzymes
AssigneeVirginia Commonwealth University
InventorsKevin A. Reynolds, Xin He
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Abstract
The present invention provides scintillation proximity assays (SPAs) for measuring the activity of enzymes involved in type II fatty acid biosynthesis. Enzymes which can be assayed in this manner include KASI, KASII, KAS III and MAT. The present invention also provides methods of assaying for ketoacyl synthase activity, malonyl transferase activity, and acyl transferase activity in enzymes using the SPA format. Because the SPA format allows the direct detection of a radiolabeled product, these assay methods are appropriate for use in high throughput screening techniques. The present invention also provides methods for assessing a compound's ability to modulate the activity of a type II fatty acid biosynthetic enzyme.
Claims68
A method to assay an enzymatic activity of a type II fatty acid biosynthetic enzyme, comprising the steps of:
- forming a reaction mixture under suitable reaction conditions by combining
- a) said type II fatty acid biosynthetic enzyme;
- b) a tagged substrate which has been tagged with a ligand, wherein said substrate is ACP or MACP;
- and
- c) a radiolabeled acyl or malonyl thioester
- exposing said reaction mixture to a Scintillation Proximity Assay (SPA) support system, wherein said SPA support system comprises trapped scintillant and a receptor for said ligand;
- and
- measuring a level of scintillation that correlates with a level of activity of said enzyme.
A method for assessing a compound's ability to modulate the enzymatic activity of a type II fatty acid biosynthetic enzyme, comprising the steps of:
- generating a test reaction mixture and a control reaction mixture, wherein said test reaction mixture comprises
- a) said type II fatty acid biosynthetic enzyme;
- b) a tagged substrate which has been tagged with a ligand, wherein said substrate is ACP or MACP;
- c) a radiolabeled acyl or malonyl thioester
- d) said compound,
- and wherein said control reaction mixture comprises
- a) said type II fatty acid biosynthetic enzyme;
- b) a tagged substrate which has been tagged with a ligand, wherein said substrate is ACP or MACP;
- and
- c) a radiolabeled acyl or malonyl thioester
- exposing said test reaction mixture and said control reaction mixture to a SPA support system, wherein said SPA support system comprises trapped scintillant and a receptor for said ligand, wherein said step of exposing is carried out under conditions which allow said ligand and said receptor to bind;
- measuring a first and a second level of scintillation which respectively corresponds to said test reaction mixture and said control reaction mixture
- determining a difference between said first and second levels of scintillation, wherein said difference is correlated to said ability of said compound to modulate said enzymatic activity of said type II fatty acid biosynthetic enzyme.
A kit for assaying enzymatic activity of a type II fatty acid biosynthetic enzyme, comprising
- a) a tagged substrate which has been tagged with a ligand, wherein said substrate is ACP or MACP;
- b) a radiolabeled acyl or malonyl thioester;
- and
- c) a SPA support system.
A method for assaying ketoacyl synthase activity in an enzyme, comprising,
- forming a reaction mixture under suitable reaction conditions by combining
- a) said enzyme;
- b) malonyl acyl carrier protein, wherein said malonyl acyl carrier protein has been tagged with a ligand
- c) a radiolabeled acyl thioester
- exposing said reaction mixture to a Scintillation Proximity Assay (SPA) support system, wherein said SPA support system comprises trapped scintillant and a receptor for said ligand, and wherein said step of exposing is carried out under conditions which allow said ligand and said receptor to bind;
- and
- measuring a level of scintillation that correlates with a level of ketoacyl synthase activity of said enzyme.
A method for assaying malonyl transferase activity in an enzyme, comprising,
- forming a reaction mixture under suitable reaction conditions by combining
- a) said enzyme;
- b) acyl carrier protein, wherein said acyl carrier protein has been tagged with a ligand;
- and
- c) a radiolabeled malonyl CoA;
- exposing said reaction mixture to a Scintillation Proximity Assay (SPA) support system, wherein said SPA support system comprises trapped scintillant and a receptor for said ligand, and wherein said step of exposing is carried out under conditions which allow said ligand and said receptor to bind;
- and
- measuring a level of scintillation that correlates with a level of malonyl transferase activity of said enzyme.
A method for assaying acyl transferase activity in an enzyme, comprising,
- forming a reaction mixture under suitable reaction conditions by combining
- a) said enzyme;
- b) acyl carrier protein, wherein said acyl carrier protein has been tagged with a ligand;
- and
- c) a radiolabeled acyl thioester;
- exposing said reaction mixture to a Scintillation Proximity Assay (SPA) support system, wherein said SPA support system comprises trapped scintillant and a receptor for said ligand, and wherein said step of exposing is carried out under conditions which allow said ligand and said receptor to bind;
- and
- measuring a level of scintillation that correlates with a level of acyl transferase activity of said enzyme.
A method of evaluating enzymatic activity in a type II fatty acid biosynthetic pathway, said method comprising:
- (a) contacting a test sample comprising a target enzyme with a test compound under suitable conditions that allow the components to interact;
- (b) adding to the components a radiolabeled acyl or malonyl thioester substrate and either an acyl carrier protein or malonyl acyl carrier protein;
- and
- (c) comparing the enzymatic activity of the target enzyme in the test sample to the enzymatic activity of the target enzyme in a sample not contacted with said test compound, wherein the difference in enzymatic activity in the test sample is indicative of the effect of said test compound on said target enzyme.
A method for identifying a test compound that modulates KAS isozyme activity, said method comprising:
- contacting a test sample comprising KAS isozyme;
- a biotinylated malonylacyl-acyl carrier protein, and a tritiated acyl-CoA with a test compound under conditions suitable to allow the components to interact;
- and
- comparing the KAS isozyme activity in the test sample to KAS isozyme activity in a sample not contacted with said test compound, wherein the difference in KAS isozyme activity in the test sample is indicative of the effect of said test compound.
An assay for measuring the activity of compounds that affect the activity of β-Ketoacyl-ACP Synthase III (KASIII) comprising, mixing the following components in solution:
- (a) KASIII;
- (b) the compound, whose effect on KASIII is to be measured;
- (c) conjugate comprising a high affinity moiety and an acyl carrier protein;
- (d) radioactive acyl thioester substrate;
- and (e) Scintillation Proximity Assay (SPA) support system, where the SPA support system is coated with proteins having high affinity for the affinity moiety of the conjugate;
- and measuring the radioactivity incorporated into the SPA support system.
An assay test kit for measuring the activity of compounds that affect the activity of a KAS isozyme comprising:
- (a) KAS isozyme composition;
- (b) conjugate comprising a high affinity moiety and an acyl carrier protein;
- (c) radioactive acyl CoA substrate;
- and (d) SPA support system.
A rapid assay for measuring the activity of compounds that inhibit KAS from a type II fatty acid synthase comprising, mixing the following components in solution:
- a test compound;
- KAS;
- radiolabeled acyl thioester substrate;
- a tagged malonyl acyl carrier protein;
- a product capture element;
- and measuring for a signal produced when the radiolabeled acyl thioester substrate is sufficiently close to the product capture element.
Dependencies are inferred from the claim text.