Peptidomimetic ligands for the tandem SH2 domain of Syk kinase

The Spleen tyrosine kinase (Syk) protein functions as a switch in a number of receptor signaling cascades. One of these cascades is the high affinity IgE receptor (Fc?RI) signaling pathway. Fc?RI consists of an ?-, ?- and two ?-chains. The ?- and ?-chains have intracellular an Immunoreceptor Tyrosine based Activation Motif (ITAM). The ITAM sequence consists of Tyr-Xxx-Xxx-(Leu/Ile)-(Xxx6-8)-Tyr-Xxx-Xxx-(Leu/Ile), in which Xxx can be any amino acid and the Tyr-Xxx-Xxx-(Leu/Ile) sequences are, if phosphorylated, the binding epitopes of the SH2 domains of Syk. Upon IgE stimulation of the receptor, the ITAM motive is doubly phosphorylated. After this phosphorylation, Syk is recruited to the membrane where it becomes active. The first event in this activation process is the binding of the tandem SH2 domain (tSH2) of Syk to the phosphorylated ITAM motive on the ?-chain. Syk activation eventually leads to degranulation of the cell and thus release of mediators. Overstimulation of this pathway leads to allergic responses and therefore Syk is an interesting target for anti-allergic therapy. Recently, we have found that binding of Syk tSH2 to an ITAM peptide causes a significant change in the dynamics of tSH2. This change could imply that a change in the inter SH2 distance is necessary for Syk activation. To gain more insight into the functioning of Syk tSH2 we decided to replace the amino acid residues between the two binding epitopes of ITAM. Replacement with rigid building blocks preserved the high binding affinity. This rigid ITAM mimic was used in a pull-down approach to identify the proteins binding to it. Furthermore, the ITAM mimic was conjugated to different cell penetrating peptides, to evaluate the intracellular effects. The ITAM mimic proved to be a stimulator of mast cells degranulation. A rigid ITAM mimic with a larger linker between the SH2 binding epitopes was found to be an inhibitor of Syk kinase activity, as found by microarray experiments. In addition, a series of photoswitchable azobenzene-containing ITAM mimics was prepared. The interaction of the cis – and trans isomers with Syk tSH2 was assayed with SPR binding studies. For the smallest photoswitchable peptide a large difference in binding affinity between the cis – and trans isomer was found. Furthermore, one ITAM-derived tetrapeptidic SH2 binding epitope was conjugated to dendrimers via click chemistry to create a series of functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent dendrimer. The tetra- and octavalent dendrimer had high nanomolar affinity for Syk tSH2, indicating a multivalency effect.