Structure-Based Drug Design for the Pseudokinase Domain of JAK2: A Novel Approach for the Treatment of Myeloproliferative Neoplasms

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The development of many myeloproliferative neoplasms (MPNs), malignant blood diseases that can develop into cancer, is caused by the oncogenic V617F mutation of the tyrosine kinase JAK2. JAK2 is a multi-domain protein that contains a kinase domain and a largely catalytically inactive pseudokinase domain that binds ATP. The pseudokinase domain regulates kinase activity by forming an autoinhibitory complex with the kinase domain. Residing on the pseudokinase domain, the V617F mutation disturbs this autoinhibitory complex and thereby constitutively hyperactivates JAK2. At present, MPNs are treated with small molecule drugs that competitively bind to the ATP binding site of the kinase domain. However, these drugs lack selective antitumor activity, show dose-limiting side effects, and do not prevent the evolution of MPNs. Thus, better therapeutic options are urgently needed. Recent mutagenesis studies revealed that ATP serves an essential structural function for the hyperactivation when it binds to the pseudokinase domain. A JAK2 V617F mutant that contains additional mutations that prevent ATP binding shows physiological activity levels. In other words, displacement of ATP from the pseudokinase domain of the disease-related JAK2 V617F mutant counteracts pathological hyperactivation. Based on this discovery, we propose a novel therapeutic approach for the treatment of MPNs by small molecules that bind with high affinity and selectivity to the JAK2 V617F pseudokinase domain and thereby competitively displace ATP. We will develop these molecules using the isolated human kinase and pseudokinase domains of JAK2 recombinantly expressed in insect cells, applying molecular modeling, synthetic organic chemistry, biological assaying, and X-ray crystallography. After potent and selective binders are obtained, we will validate our proposed therapeutic approach by a mammalian cell-based assay with full-length JAK2. The validation of the pseudokinase domain of the JAK2 V617F mutant as a drug target will reveal a promising therapeutic concept for the improved pharmacological treatment of MPNs.