Pan-kinase activation and evaluation of signaling pathways
AssigneesBeckman Coulter, Inc., University Health Network
InventorsDavid Hedley, Sue Chow, T. Vincent Shankey
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Abstract
Methods and reagents are provided for determining the activation state of a signal transduction pathway signaling protein. There exists a need in the art for methods that can monitor the efficacy of a signal transduction inhibitor in a patient. Other needs exist for detecting and monitoring certain disease or disorders that are associated with aberrant activation of a signal transduction pathway signaling protein. The present assay provides a highly sensitive assay that is also useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates.
Claims24
A method of identifying and assessing the efficacy of an inhibitor of ribosomal S6 pathway activation, said method comprising the steps of:
- a) contacting a population of cells comprising a protein activated by the ribosomal S6 pathway with a first inhibitor, wherein said first inhibitor is a putative inhibitor of extracellular signal-related kinase (ERK) or phosphoinositide 3-kinase (PI3K);
- b) contacting said population of cells with a second inhibitor known to inhibit lipopolysaccharide activation of the ribosomal S6 pathway wherein said second inhibitor is an ERK inhibitor when said first inhibitor is a PI3K inhibitor and said second inhibitor is a PI3K inhibitor when said first inhibitor is an ERK inhibitor, wherein complete inhibition of LPS induced activation of ribosomal S6 pathway requires both of the ERK inhibitor and the PI3K inhibitor;
- c) contacting said population of cells with a lipopolysaccharide to activate said protein;
- d) contacting said activated population of cells with a preservative;
- e) unmasking intracellular epitopes of said preserved population of cells;
- f) contacting said preserved and unmasked population of cells with a fluorescently labeled capture molecule being specific for the activated form of said protein;
- g) detecting the fluorescence of the first putative inhibitor exposed cells indicative of binding of said labeled capture molecule to the activated form of said protein;
- and
- h) comparing the detected fluorescence of the first putative inhibitor exposed cells to detected fluorescence of a control cell sample comprising the protein that was not exposed to the first putative inhibitor but subjected to steps b) to f),
- wherein said first putative inhibitor is assessed to be an effective inhibitor of ribosomal S6 pathway activation if the detected fluorescence of the first putative inhibitor exposed cells is decreased relative to the detected fluorescence of the control sample not exposed to the first putative inhibitor.
Dependencies are inferred from the claim text.