Identification and Characterization of a Novel Jak2 Tyrosine Kinase Inhibitor

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Recently, a Jak2 mutation (V617F) was identified in several myeloproliferative disorders. Here, we used an in silico approach to identify novel Jak2-specific inhibitors. Specifically, we used a high-throughput program called DOCK to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. The top six scoring compounds were tested for their ability to inhibit Jak2 tyrosine kinase function in vitro. One compound, Z3, effectively inhibited Jak2-V617F and Jak2-WT autophosphorylation. Z3 inhibits total Jak2 tyrosine phosphorylation as well as phosphorylation of the critical tyrosine 1007 residue in both a dose-and time-dependent manner. Z3 reduces growth hormone-dependent Jak2 activation and is a direct inhibitor of Jak2-WT and Jak2-V617F tyrosine kinase autophosphorylation as measured by an in vitro kinase assay. In addition, Z3 inhibits proliferation of human erythroleukemia (HEL) cells, which express the Jak2-V617F mutation. Furthermore, Z3 is not cytotoxic to cells at concentrations that inhibit both Jak2-WT and Jak2-V617F autophosphorylation as measured by their ability to exclude propidium iodide. In summary, this work demonstrates proof-of-principle concept that in silico molecular modeling can be used as a means to identify specific tyrosine kinase inhibitors such as those directed against Jak2. Supported by RO1 HL67277.