A substrate free in vitro protein kinase assay

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The present invention relates to methods for identifying, selecting and/or characterizing compounds capable of modulating auto-phosphorylation activity of protein-tyrosine kinases (PTKs), to compounds identified thereby and to their use in the treatment of diseases related to a protein tyrosine kinase activity, such as cancer or osteoporesis.

Protein tyrosine kinases (PTKs) are widely spread and regulate a plurality of cellular processes including proliferation and transformation, survival, differentiation and metabolism. PTKs play a key role in normal cell and tissue development. Also, PTKs are assumed to be involved in the generation and/or progression of many different types of diseases. Enhanced PTK activity, for instance, is intimately correlated with proliferative diseases, such as cancers, leukemias, psoriasis, and restenosis, as well as with immune system dysfunction and bone remodelling diseases. Accordingly, an increased need exists for suitable therapies for the treatment of diseases related to dysfunctional PTK activity. Such therapies may be based on compounds modifying or even inhibiting protein tyrosine kinases, in particular PTK trans-phosphorylation activity.

As mentioned before, PTKs, in particular Src family members, are involved in generation and/or progression of many different types of diseases and pathologic disorders. In order to develop suitable therapies for such PTK-related diseases strong efforts have been made, e.g. by identifying inhibitors of PTK-activity by means of conventional protein-tyrosine kinase assays. A typical protein-tyrosine kinase assay is based on monitoring kinase activity by determining the amount of substrate converted by the kinase. Accordingly, a PTK-substrate (e.g. peptide or a protein) is incubated along with the kinase under specific conditions, whereby phosphorylation of the substrate (due to kinase activity) is observed in the presence and absence of candidate modulators of the kinase (activity). Substrate conversion into its phosphorylated form by the kinase is measured using an appropriate detection system. Such kinase assays are typically carried out either in a homogeneous batch or on a (solid phase) surface. However, since the kinase as well as its substrate have to be supplied, it is of utmost importance to provide highly purified substrate, which may result in increased costs. Additionally, conventional assays as described above, are in general not suitable to identify test compounds, which exclusively bind to the PTK in its closed conformation(s) prior to phosphorylation of its kinase domain tyrosine residue. As a consequence, these assays are limited to the identification of compounds which are exclusively capable of binding to phosphorylated PTKs. Compounds, which bind to non-phosphorylated PTKs and also represent an interesting class of potential modulators of protein tyrosine kinases, cannot be identified by these assays. Consequently, false negative results may be obtained upon monitoring test compounds by such conventional kinase assays, since less suitable compounds may be identified than possible.

Another approach, representing a modified kinase assay, is disclosed in EP 03 028 713. EP 03 028 713 discloses a method of identifying, selecting and/or characterizing a compound modulating the activity of at least one Src family kinase member. According to EP 03 028 713, a living cell is cultivated in a first step containing at least one nucleic acid coding for a Src family kinase or a mutated Src family kinase. In a second step, the nucleic acid is expressed in the transfected cell and, in a further step, the cell is contacted with at least one test compound. A phenotype change of the cell may be determined (in the case of modulation of the activity of at least one Src family kinase by the test compound as compared to the phenotype of the cell in the preceding step. A change of phenotype indicates that the tested compound modulates the activity of at least one Src family kinase member. However, such a phenotype change as obtained by a method of EP 03 028 713 is triggered by PTK inhibitors, which merely bind to phosphorylated PTKs. Compounds binding to non-phosphorylated PTKs generally remain unidentified.

Summarizing the above, the methods known in the art merely allow to screen for compounds having binding affinity to phosphorylated PTK family members. However, at present no method is available in the art which allows to detect/identify compounds, which modulate the activity of members of the PTK family, and which bind to a non-phosphorylated PTK either typically in its open or atypically in its closed conformation. Accordingly, there is still a strong need for a method fulfilling this criterium.

It is the object of the present invention to provide more elaborate methods for identifying and characterizing compounds modulating the function of PTK family members, in particular of Src family members, and binding to a non-phosphorylated PTK either in its open or closed conformation or alternatively to a non-phosphorylated PTK in its closed conformation.

The underlying object is solved by a method for identifying, selecting and/or characterizing a compound which modulates the activity of at least one protein-tyrosine kinase (PTK) as disclosed herein. The method according to the invention comprises the steps of (a) providing a non-phosphorylated protein-tyrosine kinase; (b) providing a test compound; (c) contacting the non-phosphorylated protein-tyrosine kinase with the test compound; (d) activating auto-phosphorylation of the non-phosphorylated protein-tyrosine kinase; and (e) measuring the degree of phosphorylation of the protein-tyrosine kinase via biochemical methods.