Jak3 Kinase Domain Crystal Structures and Implications for Jak-Specific Drug Design.

Abstract Janus (Jak) family non-receptor tyrosine kinases are critical for appropriate signaling of many growth factors and cytokines. The four vertebrate Jak kinase family members demonstrate differential receptor cytoplasmic tail binding associations and transduce discrete signals on extracellular binding of ligand to the transmembrane cytokine or growth factor receptor. On ligand binding, a rapid tyrosine phosphorylation mediated signaling cascade is initiated, culminating in translocation of cytosolic latent transcription factors, signal transducer and activator of transcription (Stat) proteins, to the nucleus and targeted activation of transcription. Dysregulation of this Jak-mediated signaling pathway is documented in a number of hematological diseases: Improper upregulation of Jak activity is seen in certain hematological malignancies [1] and inability to appropriately transduce signals from the common gamma chain, γc, through Jak3 is responsible for approximately 60% of human severe combined immunodeficiency cases [2]. In addition, Jak2 point mutation V617F is frequently documented in myeloproliferative disorders including polycythemia vera; this activating mutation may disrupt an autoinhibited conformation [3]. Therapies targeting restraint of Jak family tyrosine kinase activity may be useful for treating inappropriate activation of Jak signaling cascades or for suppressing the immune response. Advances towards structure-directed drug design of Jak-specific inhibitors were made recently with solution of the Jak3 kinase domain X-ray crystal structure [4], representing the first three-dimensional structural data for any portion of the Jak family of tyrosine kinases. Here three further crystal structures of the kinase domain of Jak3 are presented: an improved resolution co-crystal structure with staurosporine analog AFN-941 and two crystal forms of Jak3 kinase domain in complex with the kinase inhibitor small molecule compound QAD-409. Comparisons between these three solved Jak3 kinase domain crystal structures illustrate conformational flexibility between the kinase domain lobes and in the area of the catalytic cleft. Further structure analysis is also presented documenting in silico modeling of the binding of small molecule CP-690,550 [5] to the different Jak3 kinase domain crystal forms. Potential binding conformations of this inhibitor to the Jak3 kinase domain are suggested with one highly scored binding conformation predicted for all crystal forms. The crystal structures and modeling studies presented further define the extent of the Jak kinase catalytic cleft, demonstrate conformational plasticity in the active conformation Jak3 kinase domain and will aid the design of higher specificity Jak inhibitors.