Phenotypic High-Content Assay to Evaluate Drugs
AssigneeTexas Tech University System
InventorP. Michael Conn
Life of this patent
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Abstract
The present invention includes a high throughput screen for an active agent for the treatment of comprising: plating cells at least one pathophysiologically relevant mislocated mutant form of a peroxisomal enzyme; adding a control and compound to each plate from a library of compounds; fixing the cells; contacting the cells with an agent that detects the mislocated mutant form of a peroxisomal enzyme; and imaging the cells in the wells.
Claims35
A method of determining the effectiveness of one or more drug candidates to change the intracellular localization of a target molecule, the method comprising:
- (a) incubating the one or more drug candidates with a first subset of the cells, and a control agent with a second subset of the cells;
- (b) fixing and staining the first and second subset of cells, wherein the stain detects the target molecule;
- (c) generating images of the first and second subset of cells with a camera;
- (d) measuring the difference in the intracellular localization of the target molecule in the first as compared to a second subset of cells;
- and
- (e) determining if the drug candidate modifies the localization of the intracellular localization of the target protein, wherein if the candidate drug modifies the intracellular localization of the target protein when compared to the placebo it is an effective drug candidate.
A method of determining the effectiveness of one or more candidate pharmacoperones to treat and/or prevent protein misfolding, the method comprising:
- (a) incubating the one or more candidate pharmacoperones with a first subset of the cells, and a placebo with a second subset of the cells;
- (b) fixing and staining the first and second subset of cells, wherein the stain detects anti-AGT in the cells;
- (c) generating images of the first and second subset of cells with a camera;
- (d) measuring the co-localization of AGT with the peroxisomes in the first and second subset of cells expressing a mutant form of a peroxisomal enzyme;
- (e) measuring peroxisome colocalization in the images of the first and second subset of cells;
- and
- (f) determining if the candidate pharmacoperones modifies the colocalization of the mutant form of a peroxisomal enzyme, wherein if the candidate drug modifies the colocalization of the AGT to the peroxisome it is effective when compared to the placebo.
A method of determining the effectiveness of a candidate drug to treating and/or prevent protein misfolding by one or more target-specific pharmacoperones, the method comprising:
- (a) incubating the candidate drug to a first subset of the cells, and a placebo to a second subset of the cells;
- (b) fixing and staining the first and second subset of cells, wherein the stains detects anti-alanine:glyoxylate aminotransferase (AGT) enzyme in the cells;
- (c) generating images the first and second subset of cells with a camera;
- (d) measuring the co-localization of AGT with the peroxisomes in a mammalian cell based system expressing a pathophysiologically relevant mislocated mutant form of a alanine:
- glyoxylate aminotransferase (AGT) enzyme;
- and
- (e) determining if the candidate drug modifies the colocalization of the AGT, wherein if the candidate drug modifies the colocalization of the AGT to the peroxisome it is effective when compared to the placebo.
A high throughput screen for an active agent for the treatment of comprising:
- plating cells comprising at least one mislocated mutant form of a peroxisomal enzyme;
- adding a control and compound to each plate from a library of compounds;
- fixing the cells;
- contacting the cells with an agent that detects the mislocated mutant form of a peroxisomal enzyme;
- and
- imaging the cells in the wells.
A high throughput screen for an active agent for the treatment of comprising:
- plating cells comprising at least one intracellular molecule target;
- adding a control and the active agent from a library of compounds to separate wells comprising the plated cells;
- fixing the cells;
- contacting the cells with an agent that detects the intracellular target;
- and
- imaging the cells in the wells, wherein a difference in the intracellular localization of the intracellular target in the cells treated with a control when compared to the active agent shows that the active agent is able to change the intracellular localization of the intracellular target molecule target.
An agent capable of changing the intracellular localization of a protein selected from at least one of 26-Deoxymonensin B, nigericin, salinomycin, or active derivatives thereof.
Dependencies are inferred from the claim text.