【作者】 王红；

【导师】 张永红；

【作者基本信息】 重庆医科大学 ， 药学（专业学位）， 2022， 硕士

【摘要】 COVID-19的大爆发,引出了突发大流行疾病临床无药可用的问题。如何快速获取潜在有效的药物?“老药新用”是科学家们关注的热点。目前,大数据、人工智能和生物信息学等都大量应用于COVID-19的“老药新用”筛选中,然而直到本研究开始,临床治疗尚无特效药。本研究拟通过构建一系列医学网络策略方法,结合传统网络药理学和高通量药物筛选方法来对现有临床药物进行筛选研究,以期为大流行病突发后提供全新的快速筛选有效“老药”方法。鉴于临床推荐中西药结合用于COVID-19治疗,且多个经典中药处方显现突出疗效,本研究对中药西药并行分析,且重点应用到了中药有效成分的筛选。第一部分,本文借助经典网络药理学方法,对临床治疗COVID-19的中药处方进行了有效成分初步分析。首先,将中药处方“甘草合剂”,“天澳1号”和“天澳2号”,分别用网络药理学方法构建“药材-成分-靶标”网络,获得了对疾病基因蛋白密切调控并且具有较高网络中心度的有效中药成分。但网络药理学存在一定局限性,其过于依赖系统调控(宿主靶标体系),并且不能基于单个蛋白快速大批量的筛选,因此,我们应用此方法进行了中药成分的初筛。第二部分,借助传统高通量药物筛选方法,对收集到的中药成分和临床用西药基于3CLpro靶标进行了分子对接,进一步虚拟筛选。首先,对三个处方的中药成分进行分子对接虚拟筛选,获得了处方中直接抑制3CLpro的中药成分。同时,将临床用2454个西药作对照筛选,将西药的分子对接打分进行了排序。由此,对比和考察了西药和中药成分的抗3CLpro的不同活性,从而评估了中药成分对单个关键病毒蛋白的有效抑制性。第三部分,本文借助网络分析技术构建了一系列医学网络策略方法,对上述研究中的西药和文献中收集的西药进行了抗COVID-19有效性打分和评估,建立起全新的系统的医学网络策略的“老药新用”快速筛选方法。首先,基于蛋白-蛋白互作网络,构建COVID-19疾病模块,应用网络邻近和网络扩散两种打分方法,分别得到两种方法的单一排名,进而综合加权得到抗COVID-19候选药物综合排名。然后,我们进行了候选药物治疗COVID-19的潜在机制探索:一方面,通过对疾病模块分析得2个COVID-19发生发展的关键靶标,将候选药物与关键靶标对接,同时也将候选药物与3CLpro和ACE2对接,评估候选药物抑制疾病关键靶标的有效性。另一方面,对疾病模块三个部分(Ⅰ)全部疾病靶标,(Ⅱ)核心疾病模块靶标,(Ⅲ)LCC进行基因富集分析,评估候选药物在COVID-19发生发展中紧密相关的通路和生物进程等中的影响,提示治疗COVID-19潜在机制。由此,本研究构建了一种医学网络策略的新型“老药新用”筛选方法。最后,将医学网络策略(指上述所构建的新型医学网络策略和已有的网络药理学方法)结合高通量筛选成功用于中药的筛选,获得了治疗COVID-19可行性高的中药成分,并探索了它们抗COVID-19的作用机制。将医学网络策略结合高通量筛选应用于临床上治疗COVID-19有效的17个中药处方中,得到quercetin,kaempferol和rosmarinic acid等15种中药成分,其中,14个分子为黄酮类及类黄酮类结构,结构决定性质,说明黄酮类结构分子在抗COVID-19药物设计方面可以先导探索。进一步将15个分子对重要靶标进行分子对接评估,发现quercetin,rosmarinic acid和kaempferol等不仅对病毒复制的关键酶3CLpro以及病毒入侵的关键酶ACE2有抑制作用,还对机体炎症免疫反应中关键蛋白TNF和IL-6有抑制作用,说明这些分子确实能在抗COVID-19中存在较好潜力。再重点对作用突出的kaempferol和rosmarinic acid进行网络重叠分析,发现二者显著调节疾病模块中特异性通路adherens junction和非特异性通路hepatitis B,从而发挥抗病毒和免疫调节作用。本文在传统的高通量药物筛选方法基础上,结合医学网络策略建立了快速有效的全新“老药新用”筛选方法,并成功应用到中药成分分析中,发现不同成分在病毒入侵和COVID-19疾病的发生和发展过程中发挥着不同作用。研究中得黄酮类或类黄酮类的中药成分是最有效的潜在中药成分,在病毒入侵、复制,机体炎症和免疫调节等过程都存在调节而实现治疗。综上所述,高通量筛选结合医学网络策略不仅能够融合多种药物筛选方法的优势,还能提供在基因,蛋白等生物分子层面上复杂机制的解释。如遇突发大流行病爆发时,本研究提供了一种快速获取潜在有效药物的新方法,该方法在“老药新用”或药物筛选中具有很大优势,尤其是在中药成分的评估中优势突显。更多还原

【Abstract】 The outbreak of COVID-19 has raised the problem that there is no clinical drug available for sudden pandemic diseases.How to quickly obtain potentially effective drugs? And "drug repurposing" is a hot topic for scientists.At present,big data,artificial intelligence,and bioinformatics have been widely used in the screening of "drug repurposing" for COVID-19.However,until the beginning of this study,there is no specific drug for clinical treatment.Thus,this study intends to conduct research on existing clinical drugs by constructing a series of medical network strategies,and combining traditional network pharmacology and high-throughput drug screening methods,in order to provide a new rapid screening method for the outbreak of the pandemic.In view of the clinical recommendation which combined the Chinese and Western medicine for the treatment of COVID-19,and several classic Chinese medicine prescriptions displayed outstanding efficacy.This study conducted a parallel analysis of Chinese and Western medicines,and mainly focused on the screening of active herbal ingredients of Traditional Chinese medicines(TCM).Firstly,with the help of the classical network pharmacology method,a preliminary analysis of the active herbal ingredients in the TCM prescriptions was performed for the clinical treatment of COVID-19.And the "herbs-herbal ingredients-targets" networks of the Licorice Mixture,Tian’ao No.1 and No.2 were constructed respectively by the network pharmacology method,and the effective herbal ingredients were obtained,which were with close regulation for disease gene proteins and high network centrality.However,network pharmacology has some limitations.It too much relies on system regulation(host targets)and can not perform quick and high-throughput screening for one protein.Therefore,we applied this method to conduct initially screening for herbal ingredients.Secondly,with the help of traditional high-throughput drug screening methods,the collected herbal ingredients and clinical approved drugs were docked to the 3CLpro target for further virtual screening.First of all,the molecular docking virtual screening was performed on the herbal ingredients in the above three prescriptions,and the herbal ingredients that directly inhibited 3CLpro were obtained.At the same time,2454 clinical approved drugs were screened similarly as the control,and the molecular docking scores of clinical approved drugs were sorted finally.Thus,the different anti-3CLpro activities of clinical approved drugs and herbal ingredients were compared and investigated.Thus,the effective inhibitory effect of herbal ingredients on a single key viral protein was evaluated.Thirdly,with the help of network analysis technology,a series of medical network strategies were constructed.The clinical approved drugs in the above research and the drugs collected in the literature were scored to evaluate the anti-COVID-19 effectiveness,and then a new and systematic medical network strategy for rapid screening of "drug repurposing" was established.At the beginning,a COVID-19 disease module was constructed basing on the protein-protein interaction network,and two scoring methods(network proximity and network diffusion)were used to measure the relationship between drug targets and COVID-19 disease targets,and then these drugs were ranked to obtain anti-COVID-19 candidates.On the one hand,through the analysis of the disease module,two key targets for the occurrence and development of COVID-19 were obtained,and candidates were docked to these targets,at the same time,candidates were also docked with3 CLpro and ACE2,to evaluate the effectiveness of the candidates.On the other hand,gene enrichment analysis of three parts Ⅰ)all disease targets,Ⅱ)connected targets,Ⅲ)LCC in the disease modules to evaluate the impact of candidates in pathways and BPs that are closely related to the occurrence and development of COVID-19 shows a potential mechanism for the treatment of COVID-19.As a result,this study constructed a novel "drug repurposing" screening method for medical network strategies.Finally,the medical network strategy(referring to the new medical network strategy constructed above and the existing classical network pharmacology)combined with the high-throughput screening method was successfully applied to the screening of herbal ingredients.And the potential herbal ingredients with high feasibility for the treatment of COVID-19 were obtained,and their anti-COVID-19 mechanism of action was explored.The medical network strategy combined with high-throughput screening methods was applied to 17 effective Chinese medicine prescriptions which were used for the clinical treatment of COVID-19,and 15 herbal ingredients including quercetin,kaempferol and rosmarinic acid were obtained,of which 14 herbal ingredients were flavonoids.That the structure determines its property,shows that the flavonoid structure molecule can be a pioneer in the design of anti-COVID-19 drugs.The 15 herbal ingredients were further evaluated by molecular docking for key targets,and it was found that quercetin,rosmarinic acid and kaempferol not only inhibited the key enzyme3 CLpro of virus replication and the key enzyme ACE2 of virus invasion,but also inhibited the key protein TNF and IL-6 in the body’s inflammatory immune response,indicating that these herbal ingredients do have good potential in anti-COVID-19.Next,the network overlapping of kaempferol and rosmarinic acid were analyzed,and we found that kaempferol and rosmarinic acid significantly regulate the specific pathway adherens junction and the non-specific pathway Hepatitis B in the disease module,thereby exerting antiviral and immunomodulatory effects.Combining high-throughput screening methods with the medical network strategy method,a fast and effective new screening method of "drug repurposing" was established,and successfully applied to the analysis of herbal ingredients.We found different herbal ingredients play different roles in the virus invasion and the occurrence and development of COVID-19 disease.And the flavonoids in the study were identified the most effective potential herbal ingredients,and their adjustments role feature were also found in the process of virus invasion,replication,body inflammation and immune regulation to achieve treatment.In conclusion,the combination of high-throughput screening and medical network strategy can not only integrate the advantages of multiple drug screening methods,but also provide explanations of complex mechanisms at the level of biomolecules such as genes,proteins.In the sudden pandemic outbreak,this study can provide a new method to quickly obtain potentially effective drugs,which has great advantages in drug repurposing or drug screening,especially in the evaluation of herbal ingredients,of which advantage is prominent.更多还原