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），可发展为癌症的恶性血液疾病，是由酪氨酸激酶JAK2的致癌V617F突变引起的。JAK2是一种多结构域蛋白，包含一个激酶结构域和一个结合ATP的催化活性很大的假激酶结构域。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。位于假激酶结构域的V617F突变扰乱了这种自身抑制复合体，从而构成性地过度激活JAK2。目前，mpn是用竞争性结合激酶结构域ATP结合位点的小分子药物治疗的。然而，这些药物缺乏选择性抗肿瘤活性，表现出剂量限制的副作用，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，当ATP结合到假激酶结构域时，它在过度激活中起着重要的结构作用。JAK2 V617F突变体包含阻止ATP结合的额外突变，显示出生理活性水平。换句话说，疾病相关的JAK2 V617F突变体的伪激酶结构域的ATP位移抵消了病理性的过度激活。基于这一发现，我们提出了一种新的治疗方法，通过高亲和力和选择性结合JAK2 V617F假激酶结构域的小分子来治疗mpn，从而竞争性地取代ATP。我们将利用在昆虫细胞中重组表达的JAK2的分离的人激酶和假激酶结构域，应用分子建模、合成有机化学、生物分析和x射线晶体学来开发这些分子。在获得有效和选择性的结合物后，我们将通过基于哺乳动物细胞的全长JAK2检测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点的验证将为改善MPNs的药物治疗揭示一个有希望的治疗概念。

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.