运用点击化学优势片段组合及原位筛选技术发现HIV双靶点抑制剂

Emergence of drug-resistant HIV-1 mutants and side effects impede the long-term usage of drugs, requiring a significant need for new strategies or protocols of drug discovery. Design of multitarget-directed ligands (MTDLs) has gained increasing acceptance; Meanwhile, fragment-based assembly using Huisgen Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC, a paradigm of click chemistry) combined with in situ screening has become a widely used strategy for high-throughput drug discovery and chemical space exploration of lead compounds. In order to improve drug resistance profile, druggability and lower cytotoxicity of reported hit molecules (hydroxynaphthyridinones and hydroxyisoquinoline-1,3-diones) with RNase H-IN dual inhibition, in this project, based on the two-metal binding pharmacophore model of RNase H-IN dual inhibitors, we start an analoging-based optimization program aimed at the exploration of the chemical diversity in metal binding group (MBG) and hydrophobic group (HG) by constructing 1,2,3-triazole linked molecular library using the CuAAC click reaction. By the hierarchical multiple-filter (binding affinity, drug-like profiles) virtual database searching strategy to predict the favorable molecules from a large collection of 1,4-disubstituted 1,2,3-triazoles, the top-ranking compounds will be selected and be dissected into a set of alkyne-containing MBGs and azide-containing HGs fragments. Initially, a library of 120-membered triazoles between 10 alkynes and 12 azides is rapidly constructed using CuAAC reaction and tested against RNase H and IN without purification (in situ screening). From the preliminary inhibition data, the most potent library members were individually resynthesized in large-scale for complete characterization and further assay of their anti-HIV activities in vitro and in vivo and their preliminary evaluation of drug-like properties. The binding modes of selected bioactive compounds and QSAR will be investigated by molecular simulations. Our approach thus lays the foundation for discovery of novel RNase H-IN dual inhibitors with improved drug resistance profile and favorable druggability.

多靶点药物设计是研发高效抗耐药HIV抑制剂的新策略；基于CuAAC点击化学的片段组装及原位筛选是具有广阔应用前景的药物先导物发现新技术。本课题为提高萘啶酮及N-羟胺类HIV RNase H-IN双靶点抑制苗头化合物的抗病毒活性及成药性、调节对靶点的特异性，根据RNase H与 IN三维结构、抑制剂药效团及抗耐药策略，运用多靶点药物设计策略及基于CuAAC点击化学高效合成筛选技术，对苗头化合物的金属螯合区及疏水作用区进行结构多样性的改造，构建以三唑为Linker的聚焦型RNase H-IN双靶点虚拟化合物库；通过多层级虚拟筛选确定目标化合物结构，优选取代炔或叠氮为优势片段实施化学合成并构建组合库，经原位抑酶活性筛选发现活性分子；继而进行扩大量合成、抗HIV活性及成药性评价发现抗HIV药物侯选物，并借助计算机技术探讨构效关系和结合模式。经几轮设计，为开发具有自主知识产权的抗HIV新药奠定基础。

多靶点药物设计是研发高效抗耐药HIV抑制剂的新策略。本研究以RNase H及整合酶催化位点为双靶点抑制剂设计的靶标，基于对HIV-1 IN与RNase H的结构类似性及其抑制剂结构类似性的认识，依据药效团模型特征，根据生物电子等排原理、骨架跃迁、优势结构再定位以及基于药效团模型特征及天然产物结构简化策略，对金属螯合区进行结构变换，同时对可修饰区域进行结构多样性的改造，设计合成聚焦型化合物库，进而进行抑酶活性筛选、细胞水平的抗病毒活性筛选及初步的成药性评价，探讨和揭示活性化合物产生活性的结构因素和作用机理。经多轮循环设计，发现了5-羟基吡啶[2,3-b]吡嗪-6（5H）-酮、羟基喹唑啉、吡啶骈嘧啶酮、多酚等多类HIV-1 RT-RNase H/IN双靶点或选择性抑制剂先导化合物，以及三唑类HIV衣壳蛋白抑制剂及逆转录酶抑制剂，并形成该领域的关键技术，为开发具有自主知识产权的抗艾滋病新药奠定基础。. 在本项目的资助下，在J Med Chem、Eur J Med Chem、Drug Discov Today. 本领域等权威期刊发表SCI文章11篇，其中6分以上文章5篇，项目申请人均为第一或通讯作者，依托单位山东大学均为第一单位。前期研究成果获授权专利2项，新申请4项，含一项PCT专利。指导硕士研究生毕业4人，博士毕业生1人，另有5位在读研究生从事该课题研究。. 项目负责人展鹏副教授入选2016年山东大学青年学者未来计划，获第十九届中国药学会-施维雅青年药物化学奖（2016.07）、山东大学齐鲁医学院银丰优秀青年教师科研奖（2017.12），受邀在全国性会议报告成果10次，会议论文12篇。获中国药物化学学术会议优秀青年报告奖（2019.8）、中国药学会第十三届青年药学科研成果交流会优秀论文二等奖（2016.9）、第十四届中国药学会青年药学论坛优秀论文一等奖（2018.7）。以本项目成果及建立的关键技术为基础，申报山东省重点研发计划（山东省重大科技创新工程项目），已获批立项（2019.01-2021.12），立项经费350万。