具有抗心肌纤维化作用的ATX抑制剂的发现、优化与作用机制探索研究

Myocardial fibrosis (MF) turned out to be the critical factor which may contribute to the development of various major illnesses such as myocardial infarction and diastolic heart failure. To date, there are absent of effective therapeutic drugs targeting fibrosis. Autotaxin (ATX) is a secreted lysophophosholipase D that converts lysophosphatidycholine (LPC) to the bioactive lysophosphatidic acid (LPA), and thus promotes the process of myocardial fibrosis. ATX has become a potential target for myocardial fibrosis treatment. In our previous study, we successfully uncovered the correlation between the ATX expression and myocardial fibrosis through the myocardial infarction (MI) model. Moreover, a lead compound XSC-71 was discovery and optimization with an ATX IC50 of 29.1 nM. With the aim to overcome the deficiency of the metabolic instability and a relatively higher Log D value (3.91) of XSC-71, this project will design and synthesize six kinds of more stabilized molecules based on design strategies of "fragonomics" and "mix-n-match". All the target compounds will be evaluated for their activity by employing various biological assays. As a result, this project will identify 2 to 4 promising lead compounds at nanomolar level as ATX inhibitors. Furthermore, the mechanisms of action of lead compounds will be illuminated by means of diverse biological techniques, such as western blot, RT-PCR, immunofluorescence and morphological detections. The project would resolve two questions. One is how to design the effective druggable molecules against ATX and evaluate their activity on myocardial fibrosis; the other is how to identify the mechanisms of the title compounds on ATX and the downstream related proteins so as to ascertain the feasibility of ATX as effective target for myocardial fibrosis treatment. As a result, this project may provide scientific basis to identify of novel ATX selective inhibitors.

心肌纤维化（MF）是导致心肌梗死、心衰等重大疾病的关键因素，目前尚无针对性的有效治疗药物。自分泌运动因子（ATX）可以通过诱导产生LPA而促进纤维化的进程，成为纤维化治疗的潜在有效靶点。申请人前期在心梗模型中已证实了ATX的表达与心肌纤维化的相关性，并获得了先导物XSC-71（ATX：IC50=29.1nM）。针对改善XSC-71代谢不稳定及LogD值偏高的缺点，本课题将运用“片段组学”结合“Mix-n-match”策略，设计并合成6类目标分子，通过多水平的活性筛选，得到2-4个nM级别的ATX选择性抑制剂；应用Western blot、PCR、免疫荧光及形态学检测等多种技术，初步阐明目标分子的作用机制。解决：①如何设计更具成药性的ATX抑制剂并评价其活性；②如何阐明其抑制ATX及下游蛋白的分子机制，确定ATX作为MF治疗靶点的可行性这两个关键问题，为新型ATX抑制剂的发现提供科学依据。

本课题按照申报书研究计划，重点开展了新型ATX抑制剂的分子设计、合成与生物活性筛选及成药性评价。借鉴先导物XSC-71及GLPG1690与ATX的作用模式，基于HTS所得先导片段Indole-1，综合采用优势骨架拼合、片段生长等设计策略，设计了四氢吡啶并[4,3-d]嘧啶、咪唑并[1,2-a]吡啶及吲哚类共三个系列的ATX抑制剂，合成全新化合物约450个，构建结构多样的小分子化合物库。以FS-3及LPC为底物，测试目标化合物的ATX酶抑制活性，优选出活性突出的候选分子LHR-118（C-36，0.41 nM）及LHR-144（C-28，1.01 nM）等。建立了小鼠心肌梗死模型、博来霉素介导的小鼠肺纤维化模型和CCl4诱导的肝纤维化模型，开展病理学（Masson 染色、HE 染色）研究，完成候选分子的急性毒性、心脏毒性评价及药代动力学研究（t1/2、Cmax、AUC），深入评价了成药性水平。基于体内动物模型，采用RT-PCR 及免疫荧光技术，考察目标分子对ATX下游通路相关蛋白（TGF-β、TNF-α、IL-6及α-SMA）基因表达的影响，初步阐明了作用机制。本课题深入挖掘了变构型ATX抑制剂的结构类型，为新型ATX抑制剂的合理化设计提供了更充分的科学依据；同时通过多样化体内纤维化模型验证了优选分子的抗纤维化分子机制，获得6个具有自主知识产权且极具开发潜力的候选分子，其中对LHR-144的深入开发获得2020年辽宁省重点研发计划项目支持，为深入推进临床前研究奠定了基础，对后续抗纤维化药物的开发具有鲜明的指导意义。.本课题取得了丰硕研究成果。项目负责人作为第一申请人申请中国发明专利4项、PCT专利1项，其中2项获得授权（ZL201810149223.2、ZL202010043108.4）。作为通讯作者发表课题相关SCI论文13篇，包含药物化学领域Top期刊Journal of Medicinal Chemistry等中科院一区论文6篇；于2021年中国药物化学学术会议上做大会分会场报告1次，发表会议论文3篇。培养博士生1名、硕士生5名，获评辽宁省优秀博士学位论文、省优秀毕业生、国家奖学金等荣誉，人才培养效果显著。课题负责人近3年获评兴辽英才计划科技创新领军人才、辽宁省首届优秀研究生导师、省优秀博士/硕士学位论文指导教师、省学位与研究生教育先进个人等诸多省级荣誉称号。