High Throughput Screen for JAK2V617F Mutant Selective Inhibitors

描述（由申请方提供）：相同的功能获得性JAK 2 V617 F等位基因存在于大多数骨髓增生性疾病（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JAK 2激酶活性的小分子抑制剂正在开发中，但这些化合物抑制野生型和突变型JAK 2激酶活性。JAK 2信号在一系列细胞过程中的核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JAK 2 V617 F激酶突变选择性抑制剂的高通量筛选，并使用二次测定优化来自该筛选的探针。本建议的目的是：A。开发、转移和筛选旨在鉴定JAK 2 V617 F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-JAK 2细胞进行计数筛选，以鉴定对JAK 2 V617 F突变激酶具有选择性的化合物。在二次试验中检测化合物，并在必要时进行优化，以生成适用于目标C的化学探针。C.使用体外和体内试验表征JAK 2 V617 F突变体选择性抑制剂，以证明突变体选择性激酶抑制的功效的概念证明，然后优化和临床前开发JAK 2 V617 F抑制剂，其在细胞培养和动物模型系统中证明活性。突变体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疗法的设计提供新的方法，并将提供一种方法来开发具有改善的MPD和一般人类恶性肿瘤毒性特征的候选化合物。

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.