Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-22
2001-03-27
Park, Hankyel (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C436S501000, C549S223000
Reexamination Certificate
active
06207397
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to materials and methods for the identification of inhibitors of intermolecular interactions, especially those involved in cellular signal transduction.
BACKGROUND OF THE INVENTION
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.
In cases in which the interaction of a particular protein molecule with a binding partner is associated with the cause or symptoms of a disease or pathological condition, compounds capable of interfering with that interaction may be useful in preventing or treating the disease or condition in mammals, including human patients.
Critical tools for the discovery of such inhibitors of protein:protein or other intermolecular interactions are binding assays. The well-known two-hybrid interaction/binding assay described by Song and Fields,
Nature
, 340:245-247 (1989) has been used to study the interactions of protein-protein interacting partners [See, Fields et al, U.S. Pat. No. 5,283,173 (Feb. 1, 1994)]. Such approaches have also been used to identify presumed SH2 dependent interactions using yeast [Xing, Z. et al.,
Mol. Biol. Cell
, 5:413-421 (1994); and Osborne, M. A. et al.,
Biotechnol
., 13:1474-1478 (1995)] or to detect the inhibition of two-hybrid formation in yeast [Chaudhuri, B. et al.,
FEBS Lett
., 357:221-226 (1995)]. See, also, International patent application No. PCT/US95/03208, incorporated herein by reference for background information on SH3 domains and their ligands including information on the design and preparation of proteins containing various SH3 domains, preparation of peptide ligands for an SH3 domain of interest, and biological/clinical roles of SH3 mediated interactions. See, PCT/US97/02635, incorporated herein by reference, for information on receptor domains (e.g., SH2 and PI domains) for phosphotyrosine-containing ligands, including the design and preparation of proteins containing various SH2 domains, preparation of peptide ligands for an SH2 domain of interest, and biological/clinical roles of SH2-mediated interactions.
Competitive binding assays have been described for detecting test substances which interfere with the association of proteins containing an SH2 domain with their phosphotyrosine containing ligands. See, e.g., Pawson, U.S. Pat. No. 5,352,660. More recently reported binding assays have utilized surface plasmon resonance (Biacore) [see, e.g., Panayotou et al,
Mol. Cell. Biol
., 13:3567-3576 (1993)] or radioactive ligand based assays. The former has a relatively low throughput, while the latter requires cumbersome filtration manipulations and generates radioactive waste, an increasingly difficult disposal issue.
The availability of materials and methods designed for the rapid and effective identification of inhibitors of protein:protein interactions would be a boon for drug discovery efforts aimed at a wide variety of target protein mediators. It would permit higher-throughput and more efficient identification and development of new pharmaceutical compositions containing inhibitors of intermolecular interactions linked to undesirable or pathological conditions.
SUMMARY OF THE INVENTION
The present invention addresses this need by providing novel materials and methods for in vitro competitive binding assays for identifying substances which inhibit or interfere with the binding together of pairs of molecules capable of mutual association, i.e., binding, to form binding complexes. Such associations include, among others, protein:protein interactions as described above, protein:lipid interactions (see, for example, Rameh et al,
Cell
83(5), 821-830 (1995)), or protein:small molecule interactions. One class of small molecules capable of binding to signal transducing molecules, specifically SH2 domains, is described in detail in WO 97/12903.
Of particular interest are assays for identifying compounds capable of inhibiting the binding of intracellular proteins or protein domains, especially those involved in cellular signal transduction with binding partners therefor The binding partner may be a naturally occurring ligand for the protein or protein domain of interest, may be derived from such a natural ligand, or may be a surrogate therefor. Such proteins include, for instance, proteins which contain one or more SH2 domains, PI domains, SH3 domains, or WW domains, each with a respective ligand.
In one aspect, the invention provides an in vitro assay method for identifying a test substance which inhibits the mutual association of two molecules. In a preferred case the two molecules are polypeptides, and in fact, protein:protein embodiments such as are disclosed in detail below are useful for illustrating the general class of intermolecular associations. The method includes the steps of preparing a mixture containing the first protein, the second protein bearing a covalently linked fluorophore, and at least one test substance. The mixture is irradiated with polarized light of a suitable wavelength permitting excitation of the fluorophore as indicated by emission of polarized light. The degree of polarization of the emission is measured and the effect of the presence or concentration of the test substance is determined. Inhibitory activity of the test substance is shown by a decrease in the observed emission polarization values of the mixture of the first and second proteins in the presence of the test substance as compared with the same protein mixture in the absence of the test substance.
Inhibition of protein:protein association can result from binding a test substance to the first protein or to the labeled ligand protein or peptide. Thus, the assay method can be viewed as a method for identifying a test substance which competitively binds to either member of the binding pair. As above, the degree of polarization of the emission is measured, and the effect of the presence or concentration of the test substance in decreasing the observed emission polarization is observed and compared with a mixture in the absence of the test substance. Competitive binding of the test substance correlates with decreased depolarization values.
In still another aspect, the invention provides an inhibitor of the association of a first protein with a second protein, first identified by the methods above.
In yet another aspect, the invention provides components or reagents, e.g., a protein bearing a covalently linked fluorophore, useful in the methods of the invention. One or more of the components or reagents can further be packaged in a
Lynch Berkley A.
MacNeil Ian
Zoller Mark
ARIAD Pharmaceuticals, Inc.
Berstein David L.
Park Hankyel
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