In vitro fluorescence polarization assay

#The invention relates to materials and methods for the identification of inhibitors of intermolecular interactions, especially those involved in cellular signal transduction.

#Cellular signal transduction, i.e., the series of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states. Numerous proteins that function as signal transducing molecules have been identified, including receptors, docking or recruiting proteins and enzymes such as receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with either proteins or other signaling molecules (e.g. lipids) to form a signaling complex that can alter cell activity.

#Signaling proteins often contain domain(s) of conserved sequence, which serve as non-catalytic modules that direct protein-protein interactions during signal transduction. Such domains include among others, SH2, phosphotyrosine interaction (“PI”), WW and SH3 domains. SH2 and PI domains recognize, i.e., bind to, proteins containing characteristic peptide sequences which include one or more phosphorylated tyrosine residues. WW and SH3 domains recognize proteins containing characteristic peptide sequences which need not contain phosphotyrosine residues. Significant information related to such domains, proteins containing them, the production of proteins containing such domains (including protein fragments and fusion proteins), the characteristic peptide sequences which they recognize and the biological and/or clinical role played by the interactions of such proteins has been described in the scientific literature.

#Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

#FIG. 1 depicts the structure of a fluorescent probe FMT1, described in Example 2.

#FIG. 2A is the saturation curve of FMT1 binding to Src SH2, which plots bound Src/total tracer vs. total Src concentration. Non-linear least squares fit for a saturation experiment between probe FMT1 and the Src-SH2 domain. Calculated Kd is 0.24 with an error of 0.008 M, (Chi)2 of 0.0027, R of 0.9987.

#Various binding assays and methods have been designed for determining the presence and/or concentration in biological fluids of a known substance of interest, e.g. a therapeutic drug, a metabolite thereof, an illicit drug, etc. The present invention addresses a different problem: the identification of a substance, of known or unknown structure, which inhibits the intermolecular association of two assay components, typically a protein and either a protein or non-protein ligand therefor. Said differently, this invention provides a method and materials for characterizing the ability of a test substance (of known or unknown structure) to inhibit the previously mentioned intermolecular association. Our invention does so by providing a fluorescence polarization (FP)-based assay for identifying and measuring the capacity of a test substance to disrupt or inhibit the association between a pair of molecules, e.g. proteins. The novel assay methods and materials disclosed herein have the advantages of being robust, non-radioactive, and amenable to varying degrees of automation.

#To facilitate understanding of this invention, the following descriptions of the components of the assay are provided:

#A. The Intemiolecular Interaction