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US 2010/0143937 A1Published application
ASSAY METHODS FOR IDENTIFYING AGENTS THAT MODIFY THE ACTIVITY OF NAPE-PLD OR Abh4
AssigneeSanofi-Aventis
InventorsJane Vivienne Peppard, Shujaath Mehdi, Zhuyin Li, Mei S. Duguid
Life of this patent
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Abstract
The present invention provides methods for the detection of agents that modify the activity of the N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) enzyme or the Alpha/beta-hydrolase-4 (Abh4) enzyme. The disclosed methods are configured in an assay format amenable to high throughput screening applications.
Claims23
A method of identifying an agent that modifies the activity of NAPE-PLD or Abh4, the method comprising:
- a) adding NAPE-PLD or Abh4 to at least one receptacle in an array of receptacles;
- b) adding an agent to at least one receptacle in the array of receptacles;
- c) adding a substrate to at least one receptacle in the array of receptacles wherein the substrate comprises a donor moiety and a quencher moiety wherein intramolecular proximity of the donor moiety and the quencher moiety results in a quenched detectable signal;
- d) incubating the agent, the NAPE-PLD or Abh4 and the substrate in the array of receptacles for a predetermined period of time;
- e) measuring the amount of detectable signal from the donor moiety following removal of the quencher moiety from the substrate by NAPE-PLD or Abh4 in the array of receptacles;
- and
- f) comparing the amount of detectable signal in a first receptacle comprising the agent, the substrate and the NAPE-PLD or Abh4 to the amount of detectable signal in a second receptacle comprising the substrate and the NAPE-PLD or Abh4
- thereby identifying said agent as a modifying agent when the difference exceeds a predetermined threshold.
5 more elements
A method of identifying an agent that modifies the activity of NAPE-PLD or Abh4, the method comprising:
- a) adding NAPE-PLD or Abh4 to at least one receptacle in an array of receptacles;
- b) adding an agent to at least one receptacle in the array of receptacles;
- c) adding a substrate to at least one receptacle in the array of receptacles wherein the substrate comprises a donor moiety and a quencher moiety wherein intramolecular proximity of the donor moiety and the quencher moiety results in a quenched detectable signal;
- d) measuring the amount of detectable signal from the donor moiety following removal of the quencher moiety from the substrate by NAPE-PLD or Abh4 in the array of receptacles at a first time point;
- e) incubating the agent, the NAPE-PLD or Abh4 and the substrate in the array of receptacles for a predetermined period of time;
- f) measuring the amount of detectable signal from the donor moiety following removal of the quencher moiety from the substrate by NAPE-PLD or Abh4 in the array of receptacles at a second time point;
- and
- g) comparing the amount of detectable signal at the first time point to the amount of detectable signal at the second time point,
- thereby identifying said agent as a modifying agent when the difference exceeds a predetermined threshold.
6 more elements
A method of identifying an agent that modifies the activity of NAPE-PLD or Abh4, the method comprising:
- a) adding NAPE-PLD or Abh4 to at least one receptacle in an array of receptacles;
- b) adding an agent to at least one receptacle in the array of receptacles;
- c) adding a substrate to at least one receptacle in the array of receptacles wherein the substrate comprises a donor moiety and an acceptor moiety;
- d) incubating the agent, the NAPE-PLD or Abh4 and the substrate for a predetermined period of time;
- e) measuring the amount of detectable signal wherein the detectable signal measured is selected from the group consisting of the donor moiety, the acceptor moiety, the ratio of donor moiety to acceptor moiety and the donor-acceptor moiety complex;
- and
- f) comparing the amount of detectable signal in a first receptacle comprising the agent, the substrate and the NAPE-PLD or Abh4 to the amount of detectable signal in a second receptacle comprising the substrate and the NAPE-PLD or Abh4
- thereby identifying said agent as a modifying agent when the difference exceeds a predetermined threshold.
5 more elements
A method of identifying an agent that modifies the activity of NAPE-PLD or Abh4, the method comprising:
- a) adding NAPE-PLD or Abh4 to at least one receptacle in an array of receptacles;
- b) adding an agent to at least one receptacle in the array of receptacles;
- c) adding a substrate to at least one receptacle in the array of receptacles wherein the substrate comprises a donor moiety and an acceptor moiety;
- d) measuring the amount of detectable signal at a first time point wherein the detectable signal measured is selected from the group consisting of the donor moiety, the acceptor moiety, the ratio of donor moiety to acceptor moiety and the donor-acceptor moiety complex;
- e) incubating the agent, the NAPE-PLD or Abh4 and the substrate for a predetermined period of time;
- f) measuring the amount of detectable signal at a second time point wherein the detectable signal measured is selected from the group consisting of the donor moiety, the acceptor moiety, the ratio of donor moiety to acceptor moiety and the donor-acceptor moiety complex;
- and
- g) comparing the amount of detectable signal at the first time point to the amount of detectable signal at the second time point
- thereby identifying said agent as a modifying agent when the difference exceeds a predetermined threshold.
6 more elements
The method of 12 wherein the buffer comprises about 0.05% Triton X-100.
An assay system for identifying an agent that modifies the activity of NAPE-PLD or Abh4, the assay system comprising:
- a) an array of receptacles;
- b) NAPE-PLD or Abh4;
- c) a substrate comprising a donor moiety and a quencher moiety wherein the donor moiety is capable of detection;
- and
- d) at least one component wherein the component is selected from the group consisting of the agent(s), an inducer(s), an inhibitor, a modulator(s), a modulator(s) of the inducer(s), a modulator(s) of the inhibitor(s) and control(s).
2 more elements
An assay system for identifying an agent that modifies the activity of NAPE-PLD or Abh4, the assay system comprising:
- a) an array of receptacles;
- b) NAPE-PLD or Abh4;
- c) a substrate comprising a donor moiety and an acceptor moiety wherein the donor moiety or the acceptor moiety or both are capable of detection;
- and
- d) at least one component wherein the component is selected from the group consisting of the agent(s), an inducer(s), an inhibitor, a modulator(s), a modulator(s) of the inducer(s), a modulator(s) of the inhibitor(s) and control(s).
2 more elements
Dependencies are inferred from the claim text.