Anaplastic lymphoma kinase assay, reagents and compositions thereof

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Discloses a method for detecting ALK tyrosine kinase activity and identification of compounds for regulating the activity of the ALK peptide substrates, reagents, and combinations thereof.

The present invention provides an inter-measurement anaplastic lymphoma kinase (anaplastic lymphoma kinase) (ALK) kinase activity assay. More particularly, the present invention is used for detecting ALK tyrosine phosphorylation activity methods, reagents and compositions. The assay is particularly useful for in vitro screening and identification of potential ALK inhibitors.

Anaplastic lymphoma kinase between (ALK) is a receptor tyrosine kinase, is thought to play an important role in the development and function of the nervous system areas. ALK is normally expressed in the central nervous system, and in the neonatal period a weak expression. However, due to chromosomal translocations, ALK may also be in the form of oncogenic fusion proteins abnormal expression or activation in some cancers. In all non-Hodgkin (Hodgkin) lymphomas, about 5-10% due to the ALK fusion protein induced. The annual incidence of ALK-positive lymphoma in the world for about 100,000 new cases in the European Union (EU) countries every year for 2000-3000. Because ALK plays a fundamental role in the carcinogenicity of expression in normal and basically confined to the central nervous system, it becomes an excellent candidate for therapeutic intervention. Therefore, ALK inhibitors specific treatment will be effective ALK positive lymphomas, and almost with no clinical side effects. For the identification of potential ALK inhibitors, most need to develop a direct assessment of the inhibitory effect on the activity of compounds suitable assay.

According to the invention, there is provided an in vitro kinase specific for ALK and ALK activity for compound screening adjusting assays. The assay is based on detection using readily phosphorylated ALK peptide substrates and subsequent phosphorylation of the resultant product. In one preferred embodiment of the present invention, the assay is ELISA, wherein the phosphorylated product is detected by immunochemical reactions.

In order to generate a selective ALK substrate, synthesized and tested reproduction ALK activation loop: peptide (amino acids 1274-1294 ALK_HUMAN, Q9UM73, swiss-PROT) sequence. Peptides ARDIYRASFFRKGGCAMLPVK (SEQ ID NO.1) and ARDIYRASYYRKGGCAMLPVK (SEQ ID NO.2) is particularly effective as ALK substrates, exhibit higher than the degree of phosphorylation of poly Glu / Tyr, and poly-Glu / Tyr is a random poly material, which is known for most tyrosine kinases are excellent substrates. Therefore a first object of the present invention is a peptide selected from SEQ ID NO.1 and SEQ ID NO.2 of the amino acid sequence.

Still object of the present invention is to detect ALK tyrosine kinase activity assay, which generally includes the steps: i) The peptide ALK protein or a functional derivative thereof selected from SEQ ID NO.1 and 2 is suitable for peptide phosphorylation Under the conditions of incubation; ii) detecting the formation of phosphorylated peptide.

As used herein, "ALK functional derivative" means any modified form of ALK protein, for example, which maintains the catalytic activity of unmodified ALK truncated or conjugated form or a fragment thereof. Full ALK catalytic domain across functional derivative should preferably contain ALK sequence (Q9UM73) in 1116-1392 residues. Preferably used to extend from residues Leu1073 part of ALK protein Ala1459. When produced by recombinant gene technology using the baculovirus-based expression system to produce, ALK fragment exhibit correct folding (confirmed by circular dichroism spectra) and an effective catalytic activity.

Further, use of the article containing a constitutively active form of ALK replaced purified protein or a functional derivative thereof. Such products preferably a cell lysate, ALK cells in order to assess cell lysis solution ALK kinase activity assay, cell lysates may be used to a) assess the expression of ALK or ALK fusion proteins expressed by the whole cell treated with different compounds The ALK activity and b) assess the potential ALK inhibitors on intracellular effects.