病毒3CL蛋白酶三维结构模型及其抑制剂的虚拟筛选(英文)

目的:构建SARS病毒类3C(3CL)蛋白酶的三维结构模型,根据这一模型设计3CL蛋白酶的抑制剂。方法:用生物信息学方法从GenBank和PDB库中搜寻出具有晶体结构并与SARS病毒3CL蛋白酶有较高同源型的蛋白质,以此为模板,用同源蛋白模建方法构建SARS病毒3CL蛋白酶的三维结构模型;针对模建的三维结构模型,进行高通量虚拟筛选,从现有小分子数据库中获得具有抑制SARS病毒3CL蛋白酶活性的化合物。结果:同源性分析表明SARS病毒3CL蛋白酶与遗传性肠胃炎主蛋白酶(TGEV M~(pro)),有较高的同源性,组成底物结合口袋残基的同源性更高。因此,可以根据TGEV M~(pro)的晶体结构为模板模建SARS病毒3CL蛋白酶的三维结构。 三维结构模型表明,ARS病毒3CL蛋白酶的结构与TGEV M~(pro)的结构非常相象,两个蛋白酶活性口袋的结构和形状儿乎一样。虚拟筛选测试研究表明,MRRD数据库中的73个蛋白酶抑制剂能与两个蛋白同时作用。结论:无论是SARS病毒3CL蛋白酶还是TGEV M~(pro)的晶体结构均可以作为设计抗SARS药物的结构模型。从现有的蛋白酶抑制剂中筛选抗SARS药物可能是一条好的途径。     

SARS冠状病毒基因组序列分析与抗SARS药物设计

SARS冠状病毒是诱发SARS的主要病原体，文章综述了SARS冠状病毒基因组序列分析的最新进展，SARS冠状病毒编码的蛋白酶在病毒繁殖过程中具有重要功能，可以其作为靶点进行抗SARS冠状病毒药物的筛选。     

能作为药物目标的又一种SARS病毒蛋白

不久前曾有报道美国 Medarex公司正在开发抗 SARS病毒纤突蛋白抗体。最近确认又一种 SARS病毒蛋白——主要蛋白酶可作为药物目标 ,中国和德国的研究小组已报告它与抑制剂复合的晶体结构。　　主要蛋白酶能释放 SARS病毒复制酶基因编码的蛋白的功能性多肽。这些蛋白介导病毒复制和转录 ,从而使主要蛋白酶在病毒生活周期中起重要作用 ,并成为开发抗 SARS病毒和其他冠状病毒药物的引人关注的目标。　　科学家的新研究提供了对主要蛋白酶 (包括活性位点构象 )的初步观察。他们发现 SARS病毒主要蛋白酶具有与其他冠状病毒共同的特点 ,但…     

基于专利的全球抗冠状病毒的3CL蛋白酶抑制剂研发情况分析

3CL蛋白酶抑制剂为当前抗冠状病毒药物的研究热点，对其专利信息的分析有助于此类药物的研发和创新。本文运用专利情报分析方法依据检索得到的全球抗冠状病毒的3CL蛋白酶抑制剂相关技术的专利数据，对3CL蛋白酶抑制剂相关专利的申请趋势、专利区域分布状况、重要申请人、专利技术主题以及重点药物研发进展等因素进行了分析，梳理了相关专利技术的发展动态，有助于国内制药企业在此基础上进行创新和提早进行专利战略布局。     

感冒229E病毒3CL蛋白酶抑制剂金丝桃苷衍生物的合成及其构效关系研究

目的寻找作为感冒229E抗原型冠状病毒3CL蛋白酶抑制剂的新化学结构。方法运用分子对接方法在ACD化合物库中发现天然产物金丝桃苷是潜在的新型抑制剂,借助分子模拟的方法进行结构改造,设计并合成了5个金丝桃苷衍生物,采用表面等离子共振(SPR)法测试这些化合物与该蛋白酶的结合能力。结果与结论衍生物Ⅱ、Ⅲ、Ⅳ与蛋白酶的结合能力比原天然产物Ⅰ提高了3倍以上,它们的结合构象也明显不同于Ⅰ与SARS病毒3CL蛋白酶的结合构象。这些结合模式的差异为设计选择性更好的感冒病毒3CL蛋白酶抑制剂提供了有益的参考信息。将计算机辅助药物分子设计、有机合成和生物活性测试有机地结合起来,是发现和设计感冒229E型病毒3CL蛋白酶新型选择性抑制剂的有效途径。     

口服小分子抗新型冠状病毒药物的研究进展

新型冠状病毒肺炎是由严重急性呼吸道综合征冠状病毒2引起的急性呼吸道传染病,是近百年来人类遭遇的影响范围最广的全球性大流行疾病。抗病毒药物是治疗新型冠状病毒感染的首选。口服小分子抗新型冠状病毒药物使用方便,且适用于轻中症患者。目前小分子抗新型冠状病毒药物有血管紧张素转换酶2(ACE2)抑制剂、膜融合抑制剂、RNA聚合酶抑制剂和3CL蛋白酶抑制剂。归纳了口服小分子抗新型冠状病毒药物的研究进展,为口服小分子抗新型冠状病毒药物的研发提供思路。     

SARS-3CL蛋白酶基因在杆状病毒载体中的构建及其转染

目的:构建SARS冠状病毒3CL蛋白酶基因的杆状病毒重组供体质粒,包装重组3CL蛋白酶的杆状病毒,感染昆虫细胞进行表达.方法:首先扩增含3cl-Teagy和pFagtBac HTh的转化菌,用酶切连接法构建重组转座质粒pFB HTb-3cl.将该质粒转化E.coli DH10Bac感受态菌,在菌体内进行重组,并经三重抗性和蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HTb-3cl,对重组质粒Bacmid-HTB-3cl进行纯化并转染St9昆虫细胞包装杆状病毒,利用病毒感染St9昆虫细胞并进行蛋白表达.利用SDS-PAGE检测蛋白表达情况.结果:成功构建了重组表达载体并得到了重组杆状病毒,SDS-PAGE检测到有3CL蛋白酶表达.结论:3CL蛋白酶在昆虫细胞中的表达为蛋白活性的检测及抑制剂的筛选奠定了基础.     

SARS治疗的相关信息

氨基肽酶N抑制剂和SARS　目前 ,紧迫问题是缺乏有效对付冠状病毒的抗病毒药物 ,如果药物能有效调节如病毒入侵靶细胞等病毒复制的关键步骤 ,将有更好的治疗效果。一些有关人类冠状病毒受体的研究可帮助找到阻止病毒扩散的方法。研究发现 ,人类细胞膜结合的金属蛋白酶、氨基肽酶N(CD13)是冠状病毒 2 2 9E的受体 ,也是肠道冠状病毒的受体 ,这种细胞表面糖蛋白在人体包括肺脏的多种组织中有表达 ,而且在免疫细胞和炎症部位活化的血管内皮中高表达 ,从而为病毒的入侵提供了潜在位点。目前 ,已研制的选择性受体抑制剂乌苯美司 (ubenimex)是一…     

3CL蛋白酶抑制剂在抗新型冠状病毒领域的专利申请状况分析

3CL蛋白酶是抗新型冠状病毒(SARS-CoV-2)药物研发的理想靶点,本文对3CL蛋白酶抑制剂在抗SARS-CoV-2领域的专利申请状况进行分析,重点分析了涉及SARS-CoV-2 3CL蛋白酶抑制剂的专利申请时间分布、重点申请人情况以及重点产品专利申请情况等,反映该类药物全球专利布局格局,揭示我国在该类药物研发上的特点和壁垒,为我国药物研发攻关以及宏观决策提供支撑。结合专利分析结果建议我国申请人基于中医药资源拓展研发思路,同时注重海外专利布局,加快产学研深度融合。     

SARS病毒蛋白中caveolin推测的结合位点

目的：获得SARS冠状病毒蛋白与caveolin-1蛋白相互作用的信息,识别病毒蛋白中可能的caveolin-1结合位点,为SARS冠状病毒蛋白的功能研究以及设计抗SARS病毒的药物和疫苗提供线索．方法：基于3个caveolin结合基序,采用氨基酸基序搜索方法预测SARS冠状病毒蛋白中可能与caveolin-1相互作用的区域,并用分子模拟和分子对接的方法进一步证实它们之间的相互作用。结果：通过生物信息学分析,在SARS冠状病毒蛋白中发现了36个caveolin结合基序。经过分子模拟和分子对接,获得了SARS冠状病毒蛋白与caveolin-1相互作用的8个结合位点。这些caveolin-1结合位点分别位于replicase 1AB、S蛋白、ofr3蛋白和M蛋白．结论：caveolin-1可能是SARS冠状病毒蛋白结合的一个靶点,它们之间的相互作用可能与SARS冠状病毒的感染、复制、装配和释放有关。     

Discovery of a novel family of SARS-CoV protease inhibitors by virtual screening and 3D-QSAR studies

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3C-like protease (3CL(pro) or M(pro)) is an attractive target for the development of anti-SARS drugs because of its crucial role in the viral life cycle. In this study, a compound database was screened by the structure-based virtual screening approach to identify initial hits as inhibitors of SARS-CoV 3CL(pro). Out of the 59,363 compounds docked, 93 were selected for the inhibition assay, and 21 showed inhibition against SARS-CoV 3CL(pro) (IC(50) 相似文献   

Characterization and inhibition of SARS-coronavirus main protease

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV). During the 2003 epidemic, the disease rapidly spread from its origin in southern China to other countries and affected almost 8000 patients, which resulted in about 800 fatalities. A chymotrypsin-like cysteine protease named 3C-like protease (3CLpro) is essential for the life cycle of the SARS-CoV. This main protease is responsible for maturation of functional proteins and represents a key anti-viral target. HPLC and fluorescence-based assays have been used to characterize the protease and to determine the potency of the inhibitors. The fluorogenic method monitoring the increase of fluorescence from the cleavage of a peptide substrate containing an Edans-Dabcyl fluorescence quenching pair at two ends has enabled the use of high throughput screening to speed up the drug discovery process. Several groups of inhibitors have been identified through high throughput screening and rational drug design approaches. Thus, alpha,beta-unsaturated peptidomimetics, anilides, metal-conjugated compounds, boronic acids, quinolinecarboxylate derivatives, thiophenecarboxylates, phthalhydrazide-substituted ketoglutamine analogues, isatin and natural products have been identified as potent inhibitors of the SARS-CoV main protease. The different classes of inhibitors reported in these studies are summarized in this review. Some of these inhibitors could be developed into potential drug candidates, which may provide a solution to combat possible reoccurrence of the SARS and other life-threatening viruses with 3CL proteases.     

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease

Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chemical database containing 58,855 compounds followed by the testing of potential compounds for SARS-CoV M(pro) inhibition leads to two hit compounds. The core structures of these two hits, defined by the docking study, are used for further analogue search. Twenty-one analogues derived from these two hits exhibited IC50 values below 50 microM, with the most potent one showing 0.3 microM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV M(pro) were solved by X-ray crystallography. They bind to the protein in a distinct manner compared to all published SARS-CoV M(pro) complex structures. They inhibit SARS-CoV M(pro) activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.     

Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

AIM: To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CL(pro)) and its four site-directed mutants. METHODS: Based on the fluorescence resonance energy transfer (FRET) principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CL(pro) proteolytic activity. RESULTS: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 micromol.L(-1), kcat=1.08 min(-1), and kcat/Km=2.7 mmol(-1).L.min(-1). SARS-CoV 3CL(pro) showed substantial pH and temperature-triggered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CL(pro) revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. CONCLUSION: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CL(pro). This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CL(pro) putative inhibitors.     

Thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus

The papain-like protease of severe acute respiratory syndrome coronavirus (PLpro) (EC 3.4.22.46) is essential for the viral life cycle and therefore represents an important antiviral target. We have identified 6MP and 6TG as reversible and slow-binding inhibitors of SARS-CoV PLpro, which is the first report about small molecule reversible inhibitors of PLpro. The inhibition mechanism was investigated by kinetic measurements and computer docking. Both compounds are competitive, selective, and reversible inhibitors of the PLpro with K(is) values approximately 10 to 20 microM. A structure-function relationship study has identified the thiocarbonyl moiety of 6MP or 6TG as the active pharmacophore essential for these inhibitions, which has not been reported before. The inhibition is selective because these compounds do not exert significant inhibitory effects against other cysteine proteases, including SARS-CoV 3CLpro and several cathepsins. Thus, our results present the first potential chemical leads against SARS-CoV PLpro, which might be used as lead compounds for further optimization to enhance their potency against SARS-CoV. Both 6MP and 6TG are still used extensively in clinics, especially for children with acute lymphoblastic or myeloblastic leukemia. In light of the possible inhibition against subset of cysteine proteases, our study has emphasized the importance to study in depth these drug actions in vivo.     

Development of antiviral therapy for severe acute respiratory syndrome

A new disease, the severe acute respiratory distress syndrome (SARS), caused by the SARS coronavirus (SARS-CoV), emerged at the beginning of 2003 and rapidly spread throughout the world. Although the disease had disappeared in June 2003 its re-emergence cannot be excluded. The development of vaccines against SARS-CoV may take years. Therefore, the availability of effective antiviral drugs against SARS-CoV may be crucial for the control of future SARS outbreaks. In this review, experimental and clinical data about potential anti-SARS drugs is summarised and discussed. Animal model studies will be needed to help to determine which interventions warrant controlled clinical testing.     

Isatin compounds as noncovalent SARS coronavirus 3C-like protease inhibitors

A series of isatin derivatives were synthesized and tested against SARS CoV 3C-like protease. Substitutions at the N-1 and C-5 positions were examined to elucidate the differences in substrate binding sites of the rhinovirus 3C protease and SARS CoV 3C-like protease. Compound 5f shows significant inhibition with an IC(50) of 0.37 microM. Further study showed that, unlike the irreversible covalent binding of isatin derivatives to human rhinovirus 3C protease, the compounds tested in this study are all noncovalent reversible inhibitors.     

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

SARS coronavirus main protease (SARS-CoV M^pro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M^pro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M^pro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M^pro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M^pro, with K_i values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M^pro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M^pro, with K_i = 2.24 ± 0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV M^pro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.     

SARS疫苗研究进展

严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是由SARS相关冠状病毒(SARS-associated coronavirus,SARS-CoV)引起的一类严重的急性呼吸系统传染病.目前尚未研制出治疗SARS的有效药物,防范SARS-CoV感染最有效的方法是使用疫苗....     

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.