【作者】 郑伟；

【导师】 陈俐娟；

【作者基本信息】 四川大学 ， 人类重大疾病生物治疗， 2022， 硕士

【摘要】 恶性肿瘤严重威胁人类生命健康,我国恶性肿瘤的发生率和死亡率在过去20年中明显上升。而随着临床肿瘤学新技术的不断发展,研究人员对分子靶点的认识日益加深,使得小分子靶向药物在抗肿瘤领域越来越受到重视。本课题组一直致力于开发新型小分子靶向抑制剂,以获得药效更好、毒性更小的活性分子,为肿瘤临床治疗提供更多选择。本论文基于肿瘤治疗,对课题组前期自主研发的基于c-Met的多激酶抑制剂16o和PI3Kδ/γ双靶点抑制剂PD106开展了研究,以期为肿瘤治疗提供更多的候选药物。第一部分基于c-Met的多激酶抑制剂16o治疗肝细胞癌的药效学评价及作用机制研究c-Met是原癌基因Met编码的蛋白,是肝细胞生长因子(HGF)的受体,具有酪氨酸激酶活性。c-Met的异常激活在几乎所有类型的实体肿瘤中都有发生,能够促进肿瘤细胞的增殖、加速新生血管的生成和影响肿瘤细胞的侵袭性生长。同时,c-Met和HGF的过度表达也与肝癌的转移或预后不良相关。因此,c-Met是治疗肝癌的重要靶标之一。16o是课题组前期自主合成的新型c-Met抑制剂,在体内外对肝细胞癌具有良好的抗肿瘤活性,本课题以肝细胞癌(Hepatocellular carcinoma,HCC)为研究对象,对16o进行了抗肿瘤活性评价和作用机制研究。首先,我们在体外激酶活性研究中发现化合物16o对c-Met具有较好的活性,抑制活性为8 n M。接下来,在体外抗肿瘤活性研究中,我们发现16o具有较好的抗肝细胞癌细胞增殖的作用,同时,对正常细胞具有较低的毒性。而后,在体外抗肿瘤机制研究中,我们发现化合物16o可以显著下调c-Met蛋白磷酸化水平及其异常激活的下游信号通路,能够将HCC细胞周期阻滞在G2/M期,并通过影响HCC细胞中Bcl-2家族蛋白的表达、介导线粒体功能受损,诱导HCC细胞凋亡。而且,16o还可以阻滞新生血管的生成,影响HCC细胞的侵袭和迁移。最后,在体内抗肿瘤活性研究中,我们发现16o可以明显抑制Sk-hep-1肺转移模型中肿瘤细胞的转移。综上,我们认为16o可以作为治疗HCC的潜在药物。第二部分PI3Kδ/γ双靶点抑制剂PD106治疗弥漫大B细胞淋巴瘤的药效学评价及作用机制研究弥漫性大B细胞淋巴瘤(Diffuse Large B-cell Lymphoma,DLBCL)是最常见的侵袭性非霍奇金淋巴瘤亚型,占所有成人非霍奇金淋巴瘤患者的30%～40%。目前针对DLBCL患者标准的一线治疗方法为利妥昔单抗联合环磷酰胺、阿霉素、长春新碱和泼尼松的多药化疗(R-CHOP)方案。但仍有约40%的患者在接受一线化疗后,无反应、疗效差或复发。近年来,靶向CD19的CAR-T细胞治疗已经批准用于治疗复发难治的DLBCL,能有效延长患者的生存期。但由于细胞产品价格昂贵,物流困难,以及CAR-T的独特的毒性作用,包括细胞因子风暴和神经毒性,限制了CAR-T的使用。因此,仍需寻找新的治疗方案,尤其是个体化的靶向治疗方法。小分子靶向抑制剂由于其具有更明确的靶向性,更低的毒副作用和更好的治疗效果,成为了目前研究的重点之一。DLBCL细胞中普遍存在PI3Kδ/γ的高表达,PI3Kδ/γ也是DLBCL细胞中PI3K-AKT信号通路激活的主要因素,所以PI3Kδ/γ可以作为DLBCL的治疗靶点。但是单独使用PI3Kδ/γ抑制剂治疗DLBCL的效果并不十分明显,因此可以将同样是BCR下游的BTK作为增敏靶点,与PI3Kδ/γ抑制剂进行联合治疗。针对PI3Kδ/γ靶点,课题组前期合成了一系列的小分子抑制剂,我们展开了优选分子的广谱筛选和体内活性研究。综合考虑酶活性和细胞活性,最终选择了PD106作为优选化合物,并围绕弥漫性大B细胞淋巴瘤进行了单药和联合用药的药理药效研究。体外抗肿瘤活性实验中,流式周期、凋亡实验及Western Blot实验均证明PD106具有良好的抑制增殖和诱导肿瘤细胞凋亡的作用。但是单独使用PD106在小鼠体内SU-DHL-6弥漫大B细胞淋巴瘤异种移植模型上的抑瘤效果并不理想。基于此结果,考虑将PD106与BTK抑制剂Ibrutinib联用。在体外,PD106和Ibrutinib联用CI指数小于1,说明两者具有协同抑制肿瘤细胞增殖的作用。而且,联合应用PD106和Ibrutinib可以协同阻滞周期并促进细胞凋亡,通过Western Blot实验证明这种协同效应是通过抑制AKT/ERK蛋白的磷酸化产生的。在体内SU-DHL-6小鼠异种移植瘤模型,发现联用PD106和Ibrutinib的抑瘤效果,明显大于单独用药,且联用的耐受性良好。综上,本课题的研究为PI3Kδ/γ抑制剂的单用及联用策略提供了理论依据。更多还原

【Abstract】 Malignant tumors have always been one of the major diseases that threaten human health.The incidence and mortality of malignant tumors in our country have increased significantly in the past 20 years.Statistics released by the National Cancer Center in February 2018 showed that the estimated number of cases is 3.804 million,with an average of more than 10,000 people diagnosed with cancer every day,and 7people are diagnosed with cancer every minute.At the same time,with the continuous development of new technologies in clinical oncology,researchers’ understanding of molecular targets is also deepening,which makes small molecule targeted drugs more and more important.Our research group has been committed to the development of new small molecule targeted inhibitors to obtain active molecules with better efficacy and less toxicity,providing more options for clinical tumor treatment.Based on tumor treatment,this paper studies the c-Met-related multi-kinase inhibitor 16 o and dual PI3Kδ/γ inhibitor PD106 independently developed by the research group in the early stage,in order to provide more candidate drugs for tumor treatment.Part Ⅰ.Antitumor activity and mechanism of a c-Met-related multi-kinase inhibitor in the treatment of hepatocellular carcinoma.The receptor tyrosine kinase c-Met is a receptor for hepatocyte growth factor(HGF).c-Met and its natural ligand,hepatocyte growth factor(HGF),play a role in normal human development,organ formation and homeostasis and other physiological processes.Abnormal activation of c-Met occurs in almost all types of solid tumors,often causing tumor cell proliferation,angiogenesis,and aggressive tumor cell growth.Meanwhile,overexpression of c-Met and HGF is also associated with tumor metastasis or poor prognosis.Therefore,c-Met is one of the important targets for the treatment of liver cancer.16 o is a new c-Met inhibitor independently designed and synthesized by our research group before.In this project,hepatocellular carcinoma(HCC)was taken as the research object,and the anti-tumor activity and mechanism of 16 o were evaluated.Compound 16 o was found to have good activity against c-Met in the kinase activity study in vitro,and the inhibitory activity was 8 n M.Firstly,we found that16 o has a good anti-tumor cell proliferation effect in the anti-tumor activity study in vitro,while has a low toxicity to normal cells.Then,in the study of anti-tumor mechanism in vitro,we found that compound 16 o can significantly down-regulate the phosphorylation level of c-Met protein and its abnormally activated downstream signaling pathways,arrest the HCC cell cycle in G2/M phase,and by influencing the expression of Bcl-2 family proteins,mediating impaired mitochondrial function,which induces apoptosis in HCC cells.At the same time,16 o can block the formation of angiogenesis and affect the invasion and migration of HCC cells in vitro.Finally,in the antitumor activity study in vivo,we found that 16 o could significantly inhibit the metastasis of tumor cells in the Sk-hep-1 lung metastasis model.In conclusion,we believe that 16 o can be a potential drug for the treatment of HCC.Part Ⅱ.Antitumor activity and mechanism of selective PI3Kδ/γ inhibitor PD106 in the treatment of diffuse large B-cell lymphoma.Diffuse Large B-cell Lymphoma(DLBCL)is the most common histopathological type in non-Hodgkin Lymphoma,accounting for 30% to 40% of all new nonHodgkin Lymphoma patients,and is the largest group of aggressive Lymphoma.Currently,the standard first-line treatment for DLBCL patients is cyclophosphamide,doxorubicin,vincristine,and prednisone multidrug chemotherapy combined with CD20 monoclonal antibody(R-CHOP).However,about 40% of DLBCL patients still have no response,poor efficacy,or relapse after chemotherapy.In particular,salvage treatment after relapse is worse.In recent years,CAR-T cell therapy targeting CD19 has been approved for the treatment of relapsed and refractory DLBCL,which can effectively prolong the survival of patients.However,the use of CAR-T is limited due to the high price of cell products,difficult logistics,and the unique toxic effects of CAR-T,including lethal immunological and neurotoxicity.Therefore,it is indeed necessary to find new therapeutic schemes,especially individualized targeted therapies,while small-molecule targeted inhibitors have become one of the focuses of current research due to their clearer targeting,lower toxicity and better therapeutic effects.The high expression of PI3Kδ/γ is ubiquitous in hematological tumor cells,and PI3Kδ/γ is also the main driver of the PI3K-AKT signaling pathway in hematological tumor cells.Abnormal activation of this signaling pathway can easily lead to multidrug resistance of tumors,thereby causing tumor cells resistance to chemoradiotherapy.For the PI3Kδ/γ target,the research group synthesized a series of structural PI3Kδ/γ inhibitors before.Therefore,we carried out broad-spectrum screening and activity studies of the preferred molecules in vivo.Considering the enzymatic activity and cell activity,we finally chose PD106 as the choice to study pharmacology and pharmacodynamics around diffuse large B-cell lymphoma.PD106 is a novel and highly selective PI3Kδ/γ inhibitor synthesized by our research group,whose activity is better than the latest FDA-approved similar compound IPI-145.The anti-tumor activity experiments,flow cycle,apoptosis experiments and Western Blot experiments all proved that PD106 has a good effect on inhibiting proliferation and inducing tumor cell apoptosis in vitro.However,the inhibitory effect of PD106 alone in the SU-DHL-6 diffuse large B-cell lymphoma xenograft model in mice is not satisfactory.Therefore,based on this result,the combination of PD106 and Ibrutinib,an inhibitor that is also a downstream BTK target of BCR,is considered.In the studies in vitro,the CI index of PD106 combination with Ibrutinib at low doses is less than 1,indicating that the two drugs have synergy inhibitory effects on tumor cell proliferation.The combination of PD106 and Ibrutinib can synergistically block the cycle and promote cell apoptosis,and Western Blot experiments show that this synergistic effect is produced by inhibiting the phosphorylation of AKT/ERK protein.Finally,in the SU-DHL-6mouse xenograft tumor model,it was found that the combination of PD106 and Ibrutinib had a significantly greater tumor-inhibitory effect than the single drug,while the combination was well tolerated.In conclusion,this study provides a theoretical basis for single use and combined use of PI3Kδ/γ inhibitors.更多还原