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US 6,808,873 B2Granted
Screening assays using intramitochondrial calcium
AssigneeMitoKor
InventorsAnne N. Murphy, Amy K. Stout
Life of this patent
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Abstract
The invention provides methods for screening for agents that modulate mitochondrial function and in particular mitochondrial regulation of intracellular calcium. The methods may be used to detect agents that bind to a mitochondrial calcium uniporter and may also detect inhibitors or uncouplers of mitochondrial respiration. Agents identified using the screens provided herein have application in the prevention and treatment of a variety of diseases associated with abnormal mitochondrial function.
Claims71
A method of identifying an agent that alters mitochondrial function, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- and
- (c) comparing the signal generated by the calcium indicator molecule at one or more of said time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of said time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
4 more elements
A method of identifying an agent that uncouples oxidative phosphorylation from ATP production, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- (c) repeating steps (a) and (b) at least once;
- and
- (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that uncouples oxidative phosphorylation from ATP production.
5 more elements
A method of identifying an agent that that is a respiratory inhibitor, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- (c) repeating steps (a) and (b) at least once;
- and
- (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that is a respiratory inhibitor.
5 more elements
A method of identifying an agent that alters a mitochondrial calcium uniporter, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- (c) repeating steps (a) and (b) at least once;
- and
- (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point following a contacting step in the presence of the agent, compared to the level of calcium in the cytosol following a contacting step in the absence of the agent, indicates that the agent alters a mitochondrial calcium uniporter.
5 more elements
A method of identifying an agent that alters mitochondrial function, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a cell containing a mitochondrion, cytosol and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with
- (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- and
- (c) comparing the signal generated by the calcium indicator molecule at one or more of said time points in the absence of the candidate agent, to the signal generated by the calcium indicator molecule at one or more of said time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
3 more elements
A method of identifying an agent that alters mitochondrial function, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising a permeabilized cell depleted of cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cell;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- and
- (c) comparing the signal generated by the calcium indicator molecule at one or more of said time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of said time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
4 more elements
A method of identifying an agent that alters mitochondrial function, comprising:
- (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array,
- (i) a biological sample comprising one or more isolated mitochondria and a calcium indicator molecule in a medium, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the biological sample;
- (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- and
- (c) comparing the signal generated by the calcium indicator molecule at one or more of said time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of said time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
4 more elements
A method of identifying an agent that alters mitochondrial function, comprising:
- (a) contacting
- (i) a biological sample comprising one or more isolated mitochondria and a calcium indicator molecule in a medium, under conditions that permit maintenance of mitochondrial membrane potential, with
- (ii) a source of calcium cations,
- wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the biological sample;
- (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points, under conditions that permit identification of (i) mitochondrial calcium uniporter activity, wherein a decrease over time in the signal that is proportional to the level of calcium in the cytosol indicates mitochondrial calcium uniporter activity, and (ii) mitochondrial uncoupler or respiratory inhibitor activity, wherein an increase over time in the signal that is proportional to the level of calcium in the cytosol without repeating step (a) indicates mitochondrial uncoupler or respiratory activity;
- and
- (c) comparing the signal generated by the calcium indicator molecule at one or more of said time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of said time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
4 more elements
Dependencies are inferred from the claim text.