Novel Screen for Targeted CLL Therapeutics

描述（由申请人提供）：在西方世界近84，000例白血病病例中，B细胞慢性淋巴细胞白血病（B-CLL）是最常见的，约占所有成人白血病病例的30%。其特征是单克隆成熟B细胞的不断积累。来自B-CLL患者的肿瘤细胞显示出增加的存活率，这已被证明是由于细胞凋亡抑制和参与细胞周期控制和细胞存活的基因的改变。当评估在B-CLL细胞中异常激活的信号传导途径时，证明激酶Akt是组成性激活的，并且Akt的过度刺激导致NFkB途径的激活，这导致NO的产生增加并抑制细胞凋亡。Akt和NFkB被蛋白磷酸酶-2a（PP 2a）下调。PP 2a是一种常见的肿瘤抑制因子，可被天然抑制剂抑制。最近，我们已经表明，蛋白质SET，PP 2a的天然抑制剂在B-CLL中过表达，导致B-CLL细胞中PP 2a活性降低。降低的PP 2a活性预期会导致通过Akt的异常信号传导;因此，通过抑制SET来药理学激活PP 2a可以为开发B-CLL治疗剂提供新的方法。Cognosci已经开发出在体外和体内具有强效抗炎活性的新型治疗肽。在机制研究中，我们最近证明，这些肽通过作为激活SET的SET拮抗剂抑制磷酸化和伴随的重要炎症信号的激活。在对来自患者的原代CD 19 +/CD 5 + B-CLL细胞中的几种COG化合物进行初步评价期间，我们发现这些化合物之一（COG 112）对人CLL细胞具有高度和优先的细胞毒性，其EC 50为224 nM，对正常B细胞的EC 50>20 μ M。此外，该化合物减少了CLL动物模型中的白色细胞计数，以验证SET拮抗作用作为CLL的潜在治疗方法。我们现在提出开发一种肽置换测定法，其可用于筛选化合物文库以鉴定小分子SET拮抗剂并筛选多样化的30，000个化合物文库。公共卫生相关性：在西方世界近84，000例白血病中，B细胞慢性淋巴细胞白血病（B-CLL）是最常见的，约占所有成人白血病病例的30%。其特征是单克隆成熟B细胞的不断积累。来自B-CLL患者的肿瘤细胞显示出增加的存活率，这已被证明是由于细胞凋亡抑制和参与细胞周期控制和细胞存活的基因的改变。当评估在B-CLL细胞中异常激活的信号传导途径时，证明激酶Akt是组成性激活的，并且Akt的过度刺激导致NFkB途径的激活，这导致NO的产生增加并抑制细胞凋亡。Akt和NFkB被蛋白磷酸酶-2a（PP 2a）下调。PP 2a是一种常见的肿瘤抑制因子，可被天然抑制剂抑制。最近，我们已经表明，蛋白质SET，PP 2a的天然抑制剂在B-CLL中过表达，导致B-CLL细胞中PP 2a活性降低。降低的PP 2a活性预期会导致通过Akt的异常信号传导;因此，通过抑制SET来药理学激活PP 2a可以为开发B-CLL治疗剂提供新的方法。Cognosci已经开发出在体外和体内具有强效抗炎活性的新型治疗肽。在机制研究中，我们最近证明，这些肽通过作为激活SET的SET拮抗剂抑制磷酸化和伴随的重要炎症信号的激活。在对来自患者的原代CD 19 +/CD 5 + B-CLL细胞中的几种COG化合物进行初步评价期间，我们发现这些化合物之一（COG 112）对人CLL细胞具有高度和优先的细胞毒性，其EC 50为224 nM，对正常B细胞的EC 50>20 μ M。此外，该化合物减少了CLL动物模型中的白色细胞计数，以验证SET拮抗作用作为CLL的潜在治疗方法。我们现在提出开发一种肽置换测定法，其可用于筛选化合物文库以鉴定小分子SET拮抗剂并筛选多样化的30，000个化合物文库。

DESCRIPTION (provided by applicant): Of the nearly 84,000 cases of leukemia in the Western world, B-cell chronic lymphocytic leukemia (B-CLL) is the most common and accounts for approximately 30% of all adult leukemia cases. It is characterized by the relentless accumulation of monoclonal mature B cells. Tumor cells from B-CLL patients show increased survival rates that have been shown to be due to inhibited apoptosis and alterations of genes involved in cell cycle control and cell survival. When evaluating the signaling pathways that are aberrantly activated in B-CLL cells, it was demonstrated that the kinase Akt is constitutively activated and the overstimulation of Akt results in activation of the NFkB pathway that leads to increased production of NO and inhibited apoptosis. Akt and the NFkB are downregulated by Protein Phosphatase-2a (PP2a). PP2a is a commonly known tumor suppressor that is inhibited by natural inhibitors. Recently, we have shown that the protein SET, a natural inhibitor of PP2a is overexpressed in B-CLL, leading to decreased PP2a activity in B-CLL cells. Reduced PP2a activity would be expected to lead to aberrant signaling through Akt; therefore, pharmacological activation of PP2a by inhibition of SET may provide a novel approach to development of B-CLL therapeutics. Cognosci has developed novel therapeutic peptides with potent anti-inflammatory activity in vitro and in vivo. In mechanistic studies we recently demonstrated that these peptides suppress phosphorylation and the accompanying activation of important inflammatory signaling by acting as antagonists of SET that activate SET. During a preliminary evaluation of several COG compounds in primary CD19+/CD5+ B-CLL cells from patients, we discovered that one of these compounds, (COG112) is highly and preferentially cytotoxic for human CLL cells with an EC50 of 224 nM and an EC50 for normal B-cells of >20 uM. Furthermore, this compound reduced white cell counts in an animal model of CLL to validate SET antagonism as a potential therapeutic approach for CLL. We now propose to develop a peptide displacement assay that can be used to screen compound libraries to identify small molecule SET antagonists and screen a diverse 30,000 compound library. PUBLIC HEALTH RELEVANCE: Of the nearly 84,000 cases of leukemia in the Western world, B-cell chronic lymphocytic leukemia (B-CLL) is the most common and accounts for approximately 30% of all adult leukemia cases. It is characterized by the relentless accumulation of monoclonal mature B cells. Tumor cells from B-CLL patients show increased survival rates that have been shown to be due to inhibited apoptosis and alterations of genes involved in cell cycle control and cell survival. When evaluating the signaling pathways that are aberrantly activated in B-CLL cells, it was demonstrated that the kinase Akt is constitutively activated and the overstimulation of Akt results in activation of the NFkB pathway that leads to increased production of NO and inhibited apoptosis. Akt and the NFkB are downregulated by Protein Phosphatase-2a (PP2a). PP2a is a commonly known tumor suppressor that is inhibited by natural inhibitors. Recently, we have shown that the protein SET, a natural inhibitor of PP2a is overexpressed in B-CLL, leading to decreased PP2a activity in B-CLL cells. Reduced PP2a activity would be expected to lead to aberrant signaling through Akt; therefore, pharmacological activation of PP2a by inhibition of SET may provide a novel approach to development of B-CLL therapeutics. Cognosci has developed novel therapeutic peptides with potent anti-inflammatory activity in vitro and in vivo. In mechanistic studies we recently demonstrated that these peptides suppress phosphorylation and the accompanying activation of important inflammatory signaling by acting as antagonists of SET that activate SET. During a preliminary evaluation of several COG compounds in primary CD19+/CD5+ B-CLL cells from patients, we discovered that one of these compounds, (COG112) is highly and preferentially cytotoxic for human CLL cells with an EC50 of 224 nM and an EC50 for normal B-cells of >20 uM. Furthermore, this compound reduced white cell counts in an animal model of CLL to validate SET antagonism as a potential therapeutic approach for CLL. We now propose to develop a peptide displacement assay that can be used to screen compound libraries to identify small molecule SET antagonists and screen a diverse 30,000 compound library.