新型LSD1/HDACs双靶点抑制剂的设计、合成及抗肿瘤活性评价

LSD1 and HDACs are overexpressed in many human cancers, resulting in aberrant silencing of tumor suppressor genes, downregulation of their expression or inhibition of their activity can prevent cancer growth, migration and invasion. LSD1 and HDACs have been validated as potential target for the discovery of antitumor agents. Moreover, there are intimate functional cross-talks between the LSD1 and HDACs. LSD1 cooperates with HDACs to participate in a multistep process allowing the transition from an active to a repressed state. Simultaneous inhibition of LSD1 and HDACs exhibits cooperation and synergy in regulating gene expression and growth inhibition, and represent a promising and novel approach for epigenetic therapy of cancers. In our previous work, novel compounds with LSD1/HDACs dual inhibition activities were creatively designed and synthesized, preliminary bioactivity evaluation results showed that some compounds exhibited good anticancer activity in vitro. On the basis of this work, this project will focus on the systematic synthesis and antitumor activity evaluation of these compounds. At the same time, a series of novel resveratrol-type dual LSD1/HDACs inhibitor will be designed and synthesized according to the molecular docking results. The bioactivity of these new compounds will be evaluated by experiments of LSD1/HDACs inhibition and antitumor effects in vitro and vivo, and the lead compounds with dual targets will be found out. The antineoplastic molecular mechanism will be preliminarily explored, including expression of some key proteins and genes related to LSD1/HDACs signaling pathways. The complement of the present proposal will help us to develop LSD1/HDACs dual inhibitors as novel anticancer drugs.

LSD1和HDACs在多种恶性肿瘤中过度表达并异常激活，导致抑癌基因异常沉默，抑制其活性或下调表达量可抑制肿瘤的生长、侵袭和转移，是目前抗肿瘤药物研发的热点靶标。LSD1和HDACs之间存在密切的串话关联，二者相互作用调控基因表达状态从活化转为抑制，同时抑制LSD1和HDACs具有协同抗肿瘤效应，是发展肿瘤表遗传学治疗的新策略。在本课题前期工作中，合成得到一类具有LSD1/HDACs双重抑制活性的新型化合物，部分化合物显示出较好的体外抗肿瘤活性。在此基础上，本课题计划对该类化合物进行系统的合成和活性研究；同时根据前期分子对接结果，设计合成一类兼具LSD1/HDACs抑制剂特征的白藜芦醇衍生物，通过LSD1、HDACs抑制活性和体内外抗肿瘤活性评价，筛选出高活性LSD1/HDACs双靶点抑制剂，并研究其作用于双靶点后关键蛋白的表达变化，初步探索其抗肿瘤作用机制，为研发新型抗肿瘤药物奠定基础。

LSD1和HDACs在多种恶性肿瘤中过度表达并异常激活，导致抑癌基因沉默，抑制其活性可抑制肿瘤的生长、侵袭和转移。LSD1和HDACs之间存在密切的串话关联，同时抑制LSD1和HDACs具有协同抗肿瘤效应，设计合成LSD1/HDACs双靶点抑制剂是发展肿瘤表观遗传学治疗的新策略。本项目的工作主要包括以下几个方面：. 1、利用分子杂合原理，设计合成了一系列具有羟肟酸结构的苯环丙胺衍生物。化合物I-7对HDAC1/2、LSD1的IC50分别是15nM、23nM和1.20μM。I-7对所测试的四种癌细胞的抑制活性均优于SAHA，其中对MGC-803的抑制活性是SAHA的10余倍。I-7可以显著的上调MGC-803细胞内H3K4、H3K9的甲基化水平和H3的乙酰化水平，降低线粒体膜电位，诱导细胞凋亡。分子对接显示I-7可以在LSD1及HDAC2的活性位点稳定结合。I-7良好的体外抗肿瘤活性使其可作为先导化合物进一步开发高活性的LSD1/HDACs双靶点抑制剂。. 2、基于白藜芦醇的LSD1抑制活性，设计合成了一系列白藜芦醇类LSD1抑制剂。化合物II-4e和II-4m可逆性抑制LSD1，IC50分别是121nM和123nM。II-4e和II-4m可以显著上调MGC-803细胞内H3K4Me2、CD86的表达，而对LSD1的表达没有影响。在此基础上，设计合成了一系列二苯乙烯类LSD1抑制剂。化合物III-9c为可逆性的FAD竞争性的LSD1抑制剂，IC50为283nM。III-9c浓度依赖性的上调THP-1细胞内CD86的表达，对MOLM-13和THP-1细胞具有较好的抑制活性，5.0μmol/L的III-9c可以显著的抑制THP-1细胞的克隆形成。分子对接显示III-9c稳定结合在FAD所在空腔，阻碍LSD1与FAD结合。. 3、在二苯乙烯类LSD1抑制剂基础上，设计合成了一系列二苯乙烯类LSD1/HDACs双靶点抑制剂。化合物IV-4i可逆性抑制LSD1，对LSD1、 HDAC1/2的IC50分别为1.28μM，0.203μM和0.428μM。IV-4i能有效抑制A-549细胞内LSD1、HDACs 活性，上调H3K4Me2、乙酰化H3、CD86以及Bax的表达，下调Bcl-2的表达，显著诱导细胞凋亡，对所测试的三种癌细胞的抑制活性均优于SAHA.