Specificity and mechanism-of-action of the JAK2 tyrosine kinase inhibitors ruxolitinib and SAR302503 (TG101348)

Activating point mutations in the JAK2 kinase were identified in BCR-ABL-negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocythemia and primary myelofibrosis (MF). This encouraged the development of several small-molecule JAK2 tyrosine kinase inhibitors, of which ruxolitinib (formerly known as INCB018424) was approved by the US Food and Drug Administration for the treatment of patients with intermediate or high-risk MF, including primary MF, post-polycythemia vera MF and post-essential thrombocythemia MF. Another JAK2 inhibitor, SAR302503 (formerly known as TG101348), is in advanced clinical trials. Both drugs inhibit JAK2 kinase activity in vitro and JAK2-dependent proliferation of cell lines with IC₅₀ values in the low nanomolar concentration range. Among the four kinases of the JAK family (JAK1, JAK2, JAK3 and TYK2), SAR302503 also inhibits JAK1, Tyk2 and JAK3, albeit with ~30-, ~100- and ~300-fold weaker efficiency than JAK2, respectively. Ruxolitinib inhibits JAK1 and JAK2 equally well, and targets TYK2 > 10-fold and JAK3 ~100-fold weaker. As both drugs were only tested for inhibition of a few dozen unrelated kinases, accounting for only a small portion of the 518 human kinases, comprehensive data on their specificity are missing. In addition, no structural data of ruxolitinib or SAR302503 bound to the JAK2 kinase domain that would reveal their binding modes and molecular mechanism-of-action are available. Of note, ruxolitinib is the only FDA-approved kinase inhibitor for which no co-crystal structure with its target kinase has been published. Here, we present a near-kinome-wide survey of the specificity of ruxolitinib and SAR302503 and determine their binding modes to the JAK2 kinase domain by extensive sampling using molecular dynamics (MD) simulations.

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