High Throughput Screen for JAK2V617F Mutant Selective Inhibitors

描述（由申请人提供）：相同的功能获得性JAK2V617F等位基因存在于大多数骨髓增殖性疾病（MPD）真性红细胞增多症（PV）、原发性血小板增多症（ET）和原发性骨髓纤维化（PMF）患者中，体外和体内数据证明了该激活突变在这些疾病发病机制中的核心作用。虽然JAK2激酶活性的小分子抑制剂正在开发中，但这些化合物同时抑制野生型和突变型JAK2激酶活性。JAK2信号在一系列细胞过程中的中心作用表明，这些化合物可能具有造血和非造血毒性。本提案的目的是实现JAK2V617F激酶突变选择性抑制剂的高通量筛选，并使用二级分析优化该筛选的探针。本提案的目的是：a .开发、转移和筛选旨在识别JAK2V617F突变激酶抑制剂的HTS试验。B.使用在EPO存在下生长的Ba/F3-EPOR-JAK2细胞进行计数筛选，以鉴定对JAk2V617F突变激酶具有选择性的化合物。在二级分析中测试化合物，并在必要时进行优化，以生成适合Aim C. C.的化学探针。使用体外和体内试验对JAK2V617F突变选择抑制剂进行表征，以证明突变选择性激酶抑制的有效性概念，随后对JAK2V617F抑制剂进行优化和临床前开发，证明其在细胞培养和动物模型系统中具有活性。突变选择性抑制剂的鉴定具有生物学和临床重要性，因为这将为分子靶向治疗的设计提供一种新方法，并将为开发具有改善mpd和人类恶性肿瘤毒性特征的候选化合物提供一种方法。

DESCRIPTION (provided by applicant): The identical gain-of-function JAK2V617F allele is present in the majority of patients with the myeloproliferative disorders (MPD) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and in vitro and in vivo data demonstrate the central role of this activating mutation in the pathogenesis of these disorders. Although small molecule inhibitors of JAK2 kinase activity are being developed, these compounds inhibit both wild-type and mutant JAK2 kinase activity. The central role of JAK2 signaling in a spectrum of cellular processes suggests these compounds may have hematopoietic and non-hematopoietic toxicities. The aims in this proposal are designed to implement a high throughput screen for mutant selective inhibitors of the JAK2V617F kinase, and to optimize the probes from this screen using secondary assays. The aims of this proposal are: A. Develop, transfer, and screen a HTS assay designed to identify inhibitors of the JAK2V617F mutant kinase. B. Perform a counter screen using Ba/F3-EPOR-JAK2 cells grown in the presence of EPO in order to identify compounds that are selective for the JAk2V617F mutant kinase. Test the compounds in secondary assays and optimize if necessary to generate a chemical probe suitable for Aim C. C. Characterization of JAK2V617F mutant select inhibitors using in vitro and in vivo assays, in order to demonstrate proof of concept for efficacy for mutant selective kinase inhibition, followed by optimization and preclinical development of JAK2V617F inhibitors that demonstrate activity in cell culture and animal model systems. The identification of mutant selective inhibitors is of biologic and clinical importance, as this would provide a novel approach to the design of molecularly targeted therapies and would offer a way to develop candidate compounds with an improved toxicity profile for MPDs and for human malignancies in general.