Developing small molecule inhibitors of Pleckstrin-2 to treat thrombosis

项目总结 血栓形成，通常发生在不寻常的部位，如内脏静脉和动脉，是常见的并发症和 慢性骨髓增生性肿瘤(MPN)患者死亡和发病的主要原因。 开发有效的治疗MPNS血栓形成的方法还处于起步阶段。目前慢性阻塞性肺疾病的一线治疗 相MPN包括阿司匹林、羟基脲、干扰素a或阿格列德。然而，这些治疗方法 持续存在血栓形成、出血和生活质量受损的风险。目标明确 在MPNS的这个阶段，对于血栓形成的分子治疗也是缺乏的。Aplexis，Inc.是一家创业公司 专注于开发治疗MPNS慢性期血栓形成的新方法，特别是 靶向JAK2通路的下游效应器，该通路在这些疾病中通常被激活。其中之一 这些效应物是Pleckstrin-2(Plek2)，它是JAK2-STAT5途径的一个新靶点。之前的研究已经 结果表明，Plek2的缺失改善了JAK2V617F突变诱导的骨髓增殖表型，并逆转了 JAK2V617F MPN小鼠模型的血栓形成和致死性。重要的是，Plek2在骨骼中过度表达 JAK2V617F阳性慢性MPN患者的骨髓和外周血单个核细胞。给定 Plek2在MPNS血栓形成机制中的意义 用于硅基高通量筛选的Plek2小分子抑制剂的HIT化合物 和基于细胞的分析。初步数据显示，HIT化合物显著抑制Plek2的S功能 体外红细胞增殖和片状脂膜的形成。这些受攻击的化合物还能显著降低 MPN小鼠模型体内的骨髓增殖和血栓形成。初步的机理研究表明， Plek2作为支架蛋白募集PI3K效应蛋白，增强PI3K-Akt信号转导。级别2 抑制剂与Plek2结合并破坏这种募集，从而阻断PI3K-Akt信号转导并抑制细胞 过度增殖。综上所述，这些发现使我们假设Plek2抑制剂可以阻断细胞 在体外和体内的过度增殖和防止MPNS中的血栓形成。这个第一阶段SBIR项目的目标是 目的：为Plek2抑制剂在MPNS血栓形成中的治疗效果建立原则证据。在……里面 目的1、我们建议开发具有有效抑制Plek2诱导的细胞增殖作用的先导化合物 在体外使用药物化学和基于细胞的分析。在目标2中，我们将检查其有效性， 先导化合物在临床前MPN小鼠血栓形成模型中的药代动力学和毒性。Aplexis 得到了西北大学创新与新创业办公室(INVO)的大力支持 该技术的相关专利正在申请中的知识产权的独家许可。协作 与西北大学纪万昌实验室的合作将确保拟议研究的成功，这将 将Aplexis定位于生产Plek2抑制剂和前IND临床候选药物的下一步 第二阶段的调查。

PROJECT SUMMARY Thrombosis, often at unusual sites such as splanchnic vein and arteries, is common complication and one of the leading causes of mortality and morbidity in patients with chronic myeloproliferative neoplasms (MPNs). Developing effective therapies for thrombosis in MPNs is in its nascence. Current first-line therapy of chronic phase MPNs includes aspirin, hydroxyurea, interferon a, or anagrelide. However, these treatment approaches remain suboptimal with ongoing risks for thrombosis, hemorrhage, and impaired quality of life. Targeted molecular therapy at this stage of MPNs for thrombosis is also lacking. Aplexis, Inc is a startup company focusing on the development of new approaches to treat thrombosis in the chronic phase of MPNs, especially targeting the downstream effectors of the JAK2 pathway that is commonly activated in these disorders. One of these effectors is Pleckstrin-2 (Plek2) that is a novel target of the JAK2-STAT5 pathway. Previous studies have shown that loss of Plek2 ameliorated JAK2V617F mutant-induced myeloproliferative phenotypes, and reverted thrombosis and lethality of the JAK2V617F MPN mouse model. Importantly, Plek2 is overexpressed in the bone marrow and peripheral blood mononuclear cells from JAK2V617F positive chronic MPN patients. Given the significance of Plek2 in thrombosis pathogenesis in MPNs, the Ji laboratory at Northwestern University has identified hit compounds of Plek2 small molecule inhibitors using in silico-based high-throughput screenings and cell-based assays. Preliminary data show that the hit compounds significantly inhibit Plek2’s functions on erythroblast proliferation and lamellipodia formation in vitro. The hit compounds also dramatically reduce myeloproliferation and thrombosis in vivo in MPN mouse models. Preliminary mechanistic studies reveal that Plek2 functions as a scaffold protein to recruit PI3K effector proteins and enhances PI3K-Akt signaling. Plek2 inhibitors bind to Plek2 and disrupt this recruitment, which blocks PI3K-Akt signaling and inhibits cell hyperproliferation. Together, these findings lead us to hypothesize that Plek2 inhibitors block cell hyperproliferation in vitro and in vivo and prevent thrombosis in MPNs. The goal of this Phase I SBIR project is to establish proof-of-principle evidence for the therapeutic effects of Plek2 inhibitors in thrombosis in MPNs. In Aim 1, we propose to develop lead compounds with potent inhibitory effects of Plek2-induced cell proliferation in vitro using medicinal chemistry and cell-based assays. In Aim 2, we will examine the efficacy, pharmacokinetics, and toxicity of lead compounds in pre-clinical MPN mouse models with thrombosis. Aplexis has a strong support from Northwestern University’s Innovation and New Ventures Office (INVO) on an exclusive license for the associated patent-pending intellectual property from this technology. The collaboration with the Ji laboratory at Northwestern University will ensure the success of the proposed research, which will position Aplexis to the next step in the production of clinical candidate of Plek2 inhibitors and pre-IND investigation in Phase II.