新型抗动脉粥样硬化TLR3高选择性抑制剂的结构优化及作用机制研究

Transmembrane protein TLR3 is a new target of anti-atherosclerosis (AS) drugs, but the bottleneck of this kind of drugs is that the inhibitors are limited and also with low selectivity. Therefore, the discovery and design of highly selective inhibitors have become the key problems which need to be solved urgently in AS and related immune diseases. By targeting TLR3/dsRNA interactions and using cell based screening of 20,000 compounds, we recently found a class of indole-isoquinoline polycyclic natural products with advantage of novel structure skeleton, selective inhibition of TLR3 in vitro and anti-AS in vivo. As a continue of our existing work, a series of novel indole-isoquinoline compounds were designed and synthesized by using various drug design methods for the key residues of TLR3/dsRNA. In view of their selectivity, activity in HEK-TLR cell lines, and anti-AS efficacy in ApoE-/- gene knockout mice, as well as preliminary safety properties, 2-3 lead structures will be selected. The interaction between the lead structures and target also will be evaluated to disclose the key residues and reveal the bioactivity by means of mutation and bioassay. This project will elucidate the molecular mechanism of TLR3 selective inhibitor recognition, and provide more adequate scientific basis for the design of high selective TLR3 inhibitors, also offer a useful basis for clinical drug development.

免疫跨膜蛋白TLR3是当前备受关注的抗动脉粥样硬化（AS）药物新靶标，该类药物的主要瓶颈是抑制剂非常有限且亚型选择性低。因而，发现、设计高选择性抑制剂已成为AS及相关免疫疾病研究亟待解决的关键问题。我们近期通过基于细胞（过表达TLR3）的筛选办法，从2万个化合物中发现了一类吲哚并异喹啉天然产物，具备骨架结构新颖、体外选择性抑制TLR3、体内抗AS的特点。本项目拟在已有基础上，定向TLR3/dsRNA关键残基，使用多种药物设计方法，设计合成出系列新型吲哚异喹啉类化合物；综合其在HEK-TLR过表达细胞中对TLR各亚型的选择性、TLR3抑制效应、ApoE-/-基因敲除小鼠体内抗AS药效和初步安全性等性质，优化出2-3个先导结构；开展先导结构与靶标的相互作用研究，通过点突变揭示影响抑制活性的关键残基，并阐明TLR3识别选择性抑制剂的分子机制，为TLR3高选择性抑制剂的设计提供充分的科学依据。

Toll样受体3（TLR3）作为一种重要的模式识别受体（PRR），参与先天性免疫和获得性免疫反应调节多种急性或慢性炎症疾病。动脉粥样硬化被证实是一类炎症性疾病，炎症反应参与到动脉粥样硬化疾病的发生和发展过程中。然而，TLR3对动脉粥样硬化的作用尚未明确。本项目中，通过对交通意外事故的动脉粥样硬化患者的动脉血管样本分析，我们发现在动脉粥样斑块部位TLR3上调明显。此外，动物体内激活TLR3显著促进粘附因子和趋化因子等促动脉粥样硬化因子形成。相反，沉默TLR3可抑制巨噬细胞摄取氧化低密度脂蛋白(Ox-LDL)，显著减少泡沫细胞形成，而泡沫细胞是形成动脉粥样硬化的关键。鉴于以上TLR3功能异常对动脉粥样硬化发展的影响，我们提出TLR3可以作为临床动脉粥样硬化治疗的新靶点。通过对近2万个化合物进行筛选，我们发现了一个新型玫瑰树生物碱具有抑制TLR3的效果；经合成优化后得到最佳生物碱衍生物SMU-CX24，体外能够特异性抑制TLR3 (IC50 = 18.87 ± 2.21 nM)，与TLR3结合紧密，呈现出低毒性和良好的生物利用度。体内可以缓解高脂饮食喂养的Apoe-/-小鼠的动脉粥样硬化，伴随病变部位TLR3和炎症浸润显著减少。通过本研究，我们证实TLR3参与动脉粥样硬化的形成，且首次发现利用TLR3特异性小分子抑制剂通过药物负调节TLR3表达可以缓解动脉粥样硬化疾病进展，为动脉粥样硬化治疗提供新的策略和候选药物分子。