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

许多骨髓增生性肿瘤（MPN）的发展，恶性血液疾病，可发展成癌症，是由致癌V617F突变的酪氨酸激酶JAK2。JAK2是一种多结构域蛋白，其包含激酶结构域和结合ATP的大部分无催化活性的假激酶结构域。假激酶结构域通过与激酶结构域形成自抑制复合物来调节激酶活性。V617F突变位于假激酶结构域，干扰了这种自抑制复合物，从而组成性地过度激活JAK2。目前，用竞争性结合激酶结构域的ATP结合位点的小分子药物治疗MPN。然而，这些药物缺乏选择性抗肿瘤活性，显示出剂量限制性副作用，并且不能阻止MPN的演变。因此，迫切需要更好的治疗选择。最近的诱变研究表明，ATP作为一个必不可少的结构功能的超激活时，它结合到假激酶结构域。JAK2 V617F突变体含有阻止ATP结合的额外突变，显示出生理活性水平。换句话说，疾病相关JAK2 V617 F突变体的假激酶结构域的ATP置换抵消了病理性超活化。基于这一发现，我们提出了一种新的治疗方法，用于通过小分子治疗MPN，所述小分子以高亲和力和选择性与JAK2 V617F假激酶结构域结合，从而竞争性地取代ATP。我们将开发这些分子使用分离的人激酶和假激酶结构域的JAK2重组表达在昆虫细胞中，应用分子建模，合成有机化学，生物测定和X射线晶体学。在获得有效的和选择性的结合剂后，我们将通过使用全长JAK2的基于哺乳动物细胞的测定来验证我们提出的治疗方法。JAK2 V617F突变体的假激酶结构域作为药物靶标的验证将揭示用于改进MPN的药理学治疗的有希望的治疗概念。

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.