Inhibition of intracellular signal transduction by 14-3-3-binding peptides
AssigneeWashington University
InventorsAndrey S. Shaw, Anthony J. Muslin
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Abstract
Compositions and methods for inhibition of the intracellular activation of a signal transducing protein comprising phosphoserine containing peptides derived from the Raf-1 protein are disclosed. The peptides bind to 14-3-3 protein and thereby prevent its association with the signal transducing peptide to block signal transduction. Also provided is an assay method for identifying a pharmacologic agent that can bind 14-3-3 protein and block signal transduction.
Claims27
A method for identifying a pharmacologic agent that inhibits the activation of a signaling protein of the type that binds to a 14-3-3 protein, the method comprising:
- forming a mixture comprising a construct containing a 14-3-3 protein or derivative thereof which binds to full-length Raf-1, wherein the protein or derivative is immobilized to a substrate, a labeled peptide of about 6 to about 30 amino acids containing a sequence derived from a Raf-1 sequence surrounding serine-259 or serine-621, where the serine residues in the peptide that correspond to serine-259 and serine-621 are phosphorylated, and said pharmacologic agent;
- subjecting the mixture to conditions under which presence of the pharmacologic agent decreases the binding of the labeled peptide to the construct;
- and
- detecting the binding of the labeled peptide to the construct wherein a decrease in binding of the labeled peptide indicates a binding of the pharmacologic agent to the construct.
A method for identifying a pharmacologic agent that inhibits the activation of a signaling protein of the type that binds to a 14-3-3 protein, the method comprising:
- forming a mixture comprising a construct containing a peptide of about 6 to about 30 amino acids containing a sequence derived from a Raf-1 sequence surrounding serine-259 or serine-621 immobilized to a substrate, where the serine residues in the peptide that correspond to serine-259 and serine-621 are phosphorylated, a labeled 14-3-3 protein or derivative thereof which binds to full-length Raf-1, and said pharmacologic agent;
- subjecting the mixture to conditions under which presence of the pharmacologic agent decreases the binding of the construct to the labeled 14-3-3 protein or derivative thereof;
- and
- detecting the binding of the construct to the labeled 14-3-3 protein or derivative thereof wherein a decrease in binding of the labeled 14-3-3 protein or derivative thereof indicates a binding of the pharmacologic agent to the 14-3-3 protein or derivative thereof.
A method for identifying a pharmacologic agent that inhibits the activation of a signaling protein, the method comprising:
- forming a mixture comprising a 14-3-3 protein or derivative thereof which binds to full-length Raf-1, a peptide containing a sequence derived from a Raf-1 sequence surrounding a phosphorylated serine-259 or a phosphorylated serine-621, and said pharmacologic agent, where either the 14-3-3 protein or derivative thereof or the peptide is immobilized to a substrate to form a construct, and where the non-immobilized entity is labeled;
- subjecting the mixture to conditions under which presence of the pharmacologic agent decreases the binding of the labeled entity to the construct;
- and
- detecting the binding of the labeled entity to the construct wherein a decrease in binding of the labeled entity indicates a binding of the pharmacologic agent to the 14-3-3 protein or derivative thereof.
Dependencies are inferred from the claim text.