针对B细胞相关恶性肿瘤的激酶抑制剂的药物学研究

发布时间：2018-07-26 20:38

【摘要】：1.针对B细胞恶性肿瘤的高活性高选择性PI3Kδ激酶抑制剂以及PI3Kδ/VPS34双重抑制剂的发现研究PI3Kδ主要在白细胞中表达,并且在B细胞相关恶性肿瘤细胞如慢性淋巴细胞性白血病,急性髓细胞性白血病中过量表达,有研究结果表明,其与B-细胞相关癌症有密切的关系,因此PI3Kδ激酶在近年来受到药物研究领域的广泛关注。本论文从两个方面对其从药物发现到药物作用机制进行了研究。第一章：针对B细胞恶性肿瘤中PI3Kδ激酶选择性抑制剂PI3KD-IN-015的发现研究。利用计算机辅助设计和药物化学方法,我们发现了一个ATP竞争性的PI3Kδ抑制剂PI3KD-IN-015,生化实验和细胞内实验结果均表明PI3KD-IN-015对PI3Kδ具有较好的选择性,同时对激酶组中绝大多数激酶没有抑制活性。通过抑制PI3Kδ介导的信号通路,PI3KD-IN-015能够在一定程度上抑制各种B细胞相关癌症细胞系的增殖,并且能够诱导B细胞恶性肿瘤细胞系凋亡和自噬,另外,与自噬抑制剂Bafilomycin的组合用药,能够增强PI3KD-IN-015的抗细胞增殖作用。PI3KD-IN-015能够抑制慢性淋巴细胞性白血病人和急性髓性白血病人原代细胞的增殖。以上结果均表明,PI3KD-IN-015可能会是一个潜在的针对B细胞相关恶性肿瘤的候选药物。第二章：靶向B细胞恶性肿瘤中PI3Kδ/VPS34激酶双重抑制剂PI3KD/V-IN-01的发现研究。尽管第一个选择性的PI3Kδ抑制剂CAL1 01已经取得了临床成功,但是临床试验和基础研究结果均表明抑制PI3Kδ本身对B细胞恶性肿瘤细胞系并没有较强的杀伤效果,在对PI3KD-IN-015的表征上也观测到了类似的现象。一个可能的原因就是抑制PI3Kδ能够诱导细胞自噬,从而保护细胞逃避死亡。由于Ⅲ类P13K亚型PIK3C3/Vps34在自噬的起始和发展中起到重要调控作用,因此我们判断一个PI3Kδ和Vps34双重抑制剂有可能会增强我们所观察到的PI3K8靶向抑制剂的抗增殖活性。通过基于结构的药物设计方法,我们发现了一个高活性的ATP竞争性的PI3Kδ/Vps34双重抑制剂PI3KD/V-IN-01,该化合物对PI3K8和Vps34的IC50分别是6 nM和19 nM。对其他的P13K亚型,PI3KD/V-IN-01表现出10-1500倍的选择性,而且并不抑制激酶组中其它激酶。在细胞环境中,PI3KD/V-IN-01对PI3Kδ和其它Ⅰ类P13K亚型中表现出了30-300倍的选择性。跟选择性的PI3K8抑制剂CAL-101和选择性的Vps34抑制剂VPS34-IN-1单独用药结果相比,PI3KD/V-IN-01表现出更好的抗慢性淋巴细胞性白血病细胞系,急性髓性白血病细胞系Burkitt淋巴瘤细胞系的增殖活性。此外我们还观察到FLT3-ITD急性髓细胞性白血病细胞系要比表达野生型FLT3的急性髓性白血病细胞系对PI3KD/V-IN-01敏感。在急性髓性白血病细胞系MV4-11接种的异种移植瘤小鼠模型中,PI3KD/V-IN-01表现出剂量依赖性的的抗肿瘤增殖效果。这些结果提示我们,双重抑制PI3Kδ和Vps34可能会是一种行之有效的改善P13K8抑制剂抗肿瘤效果的方法。2.针对慢性骨髓白血病的高活性高选择性BCR-ABL激酶抑制剂CHMFL-ABL-053的发现研究BCR-ABL融合酪氨酸激酶肿瘤蛋白是诱发费城染色体阳性慢性骨髓白血病(Ph+CML)的关键因素,作为新药研发靶点,人们已经对BCR-ABL进行了大量的探索。但是目前绝大多数针对BCR-ABL激酶的抑制剂均为多靶点抑制剂,特别是他们都能够强烈的抑制与ABL激酶结构相近的cKIT的活性。为了给进一步探究ABL激酶的生理和病理作用提供一个更有选择性的抑制剂,我们从一个多靶点ABL抑制剂GNF-7的二氢嘧啶并嘧啶核心骨架开始,使用药物化学的方法对其进行修饰,发现了一个高活性高选择性的BCR-ABL抑制剂CHMFL-ABL-053,它对ABL1激酶的ICso是70 nM。在DiscoveRx's KinomeScanTM选择性试验中,CHMFL-ABL-053在激酶组中表现出很高的选择性,S得分(1)=0.02。此外CHMFL-ABL-053对临床上常用的BCR-ABL激酶抑制剂Imatinib, Nilotinib, Botutinib和Dasatinib的共同副靶点c-KIT激酶没有明显抑制活性。通过有效抑制BCR-ABL的自身磷酸化(ECso约100 nM)及其下游STAT5, Crkl和ERK的磷酸化,CHMFL-ABL-053能够抑制慢性骨髓白血病细胞系K562 (GI50:14 nM), KU812 (GI50:25 nM), MEG-01 (GI50:16 nM)的增殖,并将细胞周期进程阻滞在G0/G1期,同时也能够诱导细胞凋亡。在大鼠上的药代动力学研究结果显示CHMFL-ABL-053的半衰期大于4h,具有24%的生物利用度。在接种K562细胞的异种移植瘤小鼠模型中,50mg/kg/天剂量给药,几乎完全抑制小鼠体内肿瘤生长。作为一个潜在的有效的慢性骨髓白血病候选药物,CHMFL-ABL-053目前正在进行大量的临床前期成药性评估。
[Abstract]:1. the discovery of high activity, high selective PI3K delta kinase inhibitors and PI3K Delta /VPS34 double inhibitors for B cell malignancies and the discovery of PI3K delta mainly expressed in leukocytes and overexpression in B cell related malignant tumor cells such as chronic lymphoblastic leukemia and acute myelocytic leukemic disease. B- cell related cancer has a close relationship, so PI3K delta kinase has been widely concerned in the field of drug research in recent years. This paper has studied its mechanism from drug discovery to drug action from two aspects. Chapter 1: the discovery of PI3K delta kinase selective inhibitor PI3KD-IN-015 for B cell malignant tumor. In computer aided design and drug chemical methods, we found a competitive ATP PI3K delta inhibitor PI3KD-IN-015. Both biochemical experiments and intracellular experimental results showed that PI3KD-IN-015 had better selectivity to PI3K Delta and did not inhibit most of the kinases in the kinase group. By inhibiting the PI3K delta mediated signal pathway, PI3KD- IN-015 can inhibit the proliferation of various B cell related cancer cell lines to a certain extent, and can induce apoptosis and autophagy in the malignant tumor cell lines of B cells. In addition, the combination of the autophagy inhibitor Bafilomycin can enhance the anti proliferation effect of PI3KD-IN-015 to inhibit the chronic lymphocytic leukemia. The proliferation of primary cells in human and acute myeloid leukocytes. These results suggest that PI3KD-IN-015 may be a potential candidate for B cell related malignancies. Second chapter: the present study of the PI3K Delta /VPS34 kinase double inhibitor PI3KD/V-IN-01 in the target B cell malignant tumor, although the first selective PI3K delta suppression. The preparation CAL1 01 has achieved clinical success, but the results of clinical trials and basic studies show that inhibition of PI3K delta itself has no strong killing effect on the cell lines of B cells, and a similar phenomenon has been observed in the characterization of PI3KD-IN-015. One possible reason is the inhibition of PI3K delta to induce cell autophagy. Protective cells escape death. Since class III P13K subtype PIK3C3/Vps34 plays an important role in the initiation and development of autophagy, we judge that a double inhibitor of PI3K Delta and Vps34 may enhance the antiproliferative activity of the PI3K8 targeting inhibitor we observed. A highly active ATP competitive PI3K Delta /Vps34 double inhibitor PI3KD/V-IN-01, which is 6 nM and 19 nM. for the other P13K subtypes of PI3K8 and Vps34, shows 10-1500 times the selectivity for PI3KD/V-IN-01, and does not inhibit other kinases in the kinase group. The type I P13K subtype showed a 30-300 fold selectivity. Compared with the selective PI3K8 inhibitor CAL-101 and the selective Vps34 inhibitor VPS34-IN-1, the PI3KD/V-IN-01 showed a better anti chronic lymphocytic leukemia cell line and the proliferation activity of the acute myeloid leukemia cell line Burkitt lymphoma cell line. In addition, we also observed that the FLT3-ITD acute myeloid leukemia cell line is more sensitive to PI3KD/V-IN-01 than the acute myeloid leukemia cell lines expressing wild type FLT3. In the xenograft mice model of acute myeloid leukemia cell line MV4-11, PI3KD/V-IN-01 showed a dose dependent anti tumor effect. The results suggest that double inhibition of PI3K Delta and Vps34 may be an effective method to improve the antitumor effect of P13K8 inhibitors..2. for the high activity and high selectivity of BCR-ABL kinase inhibitor CHMFL-ABL-053 for chronic myelocytic leukemia, BCR-ABL fusion tyrosine kinin tumor protein is the cause of chromosome positive in Philadelphia The key factors of sexual myelocytic leukemia (Ph+CML), as a target for new drug development, have been a great deal of exploration for BCR-ABL. However, most of the inhibitors of BCR-ABL kinase are multiple target inhibitors, especially they can strongly inhibit the activity of cKIT similar to the ABL kinase structure. In order to further explore A The physiological and pathological effects of BL kinase provide a more selective inhibitor. We begin to modify the two pyrimidine and pyrimidine core skeleton of a multi target ABL inhibitor, GNF-7, and modify it by drug chemistry. A highly active and highly selective BCR-ABL inhibitor, CHMFL-ABL-053, is found, and the ICso of ABL1 kinase is 70. NM. in the DiscoveRx's KinomeScanTM selective test, CHMFL-ABL-053 has a high selectivity in the kinase group. S score (1) =0.02. and CHMFL-ABL-053 have no obvious inhibitory activity on the common side target of Imatinib, Nilotinib, Botutinib, and Dasatinib, which are commonly used as the BCR-ABL kinase inhibitors. CR-ABL's autophosphorylation (ECso about 100 nM) and its downstream STAT5, Crkl and ERK phosphorylation, CHMFL-ABL-053 can inhibit the proliferation of chronic myelogenous leukemia cell line K562 (GI50:14 nM), KU812 (GI50:25), and also induce cell apoptosis. At the same time, it can also induce apoptosis. The results of the kinetic study showed that the half-life of CHMFL-ABL-053 was greater than that of 4H and had 24% bioavailability. In the mouse model of xenograft tumor inoculated with K562 cells, the dose of 50mg/kg/ day dose almost completely inhibited the growth of tumor in mice. As a potential and effective candidate for chronic myelogenous leukemia, CHMFL-ABL-053 is currently being used. A large number of pre clinical drug resistance assessments were carried out.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5

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