Discovery of human coronaviruses pan-papain-like protease inhibitors using computational approaches

The papain-like protease(PLpro)is vital for the replication of coronaviruses(Co Vs),as well as for escaping innate-immune responses of the host.Hence,it has emerged as an attractive antiviral drug-target.In this study,computational approaches were employed,mainly the structure-based virtual screening coupled with all-atom molecular dynamics(MD)simulations to computationally identify specific inhibitors of severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)PLpro,which can be further developed as potential pan-PLprobased broad-spectrum antiviral drugs.The sequence,structure,and functional conserveness of most deadly human Co Vs PLprowere explored,and it was revealed that functionally important catalytic triad residues are well conserved among SARS-Co V,SARS-Co V-2,and middle east respiratory syndrome coronavirus(MERS-Co V).The subsequent screening of a focused protease inhibitors database composed of^7,000 compounds resulted in the identification of three candidate compounds,ADM13083841,LMG15521745,and SYN15517940.These three compounds established conserved interactions which were further explored through MD simulations,free energy calculations,and residual energy contribution estimated by MM-PB(GB)SA method.All these compounds showed stable conformation and interacted well with the active residues of SARS-Co V-2 PLpro,and showed consistent interaction profile with SARS-Co V PLproand MERS-Co V PLproas well.Conclusively,the reported SARS-Co V-2 PLprospecific compounds could serve as seeds for developing potent pan-PLprobased broad-spectrum antiviral drugs against deadly human coronaviruses.Moreover,the presented information related to binding site residual energy contribution could lead to further optimization of these compounds.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people,leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment.Here,we screened about 1.8 million small molecules against the main protease(M^(pro))and papain like protease(PL^(pro)),two major proteases in severe acute respiratory syndrome-coronavirus 2 genome,and identified 1851M^(pro)inhibitors and 205 PL^(pro)inhibitors with low nmol/l activity of the best hits.Among these inhibitors,eight small molecules showed dual inhibition effects on both M^(pro)and PL^(pro),exhibiting potential as better candidates for COVID-19 treatment.The best inhibitors of each protease were tested in antiviral assay,with over 40%of M^(pro)inhibitors and over 20%of PL^(pro)inhibitors showing high potency in viral inhibition with low cytotoxicity.The X-ray crystal structure of SARS-CoV-2 M^(pro)in complex with its potent inhibitor 4a was determined at 1.8Åresolution.Together with docking assays,our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

血清CST1、JAM2在非小细胞肺癌中的表达及其诊断、预后价值

目的探讨非小细胞肺癌(NSCLC)患者血清半胱氨酸蛋白酶抑制剂1(CST1)、连接黏附分子2(JAM2)的表达及其对NSCLC诊断及预后评估的价值。方法选取2019年2月—2021年2月中国人民解放军联勤保障部队第九二六医院肿瘤科收治的NSCLC患者112例作为NSCLC组,以肺良性疾病患者60例为非NSCLC组,医院同期健康体检者60例作为健康对照组。采用ELISA法检测血清CST1、JAM2水平;Kaplan-Meier曲线比较不同CST1、JAM2表达水平NSCLC患者的预后差异;Cox回归分析NSCLC患者生存预后的独立危险因素;受试者工作特征曲线(ROC)分析血清CST1、JAM2对NSCLC的诊断价值。结果与非NSCLC组和健康对照组比较,NSCLC组患者血清CST1水平较高,而血清JAM2水平较低,差异均有统计学意义(F/P=1154.772/<0.001,354.830/<0.001);与TNM分期Ⅰ~Ⅱ期、无淋巴结转移患者比较,TNM分期Ⅲ期、淋巴结转移患者血清CST1较高、血清JAM2较低,差异均有统计学意义(t/P=8.842/<0.001,10.070/<0.001,18.243/<0.001,23.365/<0.001);CST1高表达亚组及低表达亚组3年总生存率分别为50.00%(30/60)、71.15%(37/52),2亚组比较差异有统计学意义(Log Rankχ^(2)=5.897,P=0.015);JAM2高表达亚组及低表达亚组3年生存率分别为72.73%(40/55)、47.37%(27/57),2亚组比较差异有统计学意义(Log Rankχ^(2)=7.299,P=0.007)。肿瘤分期Ⅲ期、淋巴结转移、血清CST1高是NSCLC患者不良预后的危险因素,JAM2高是保护因素[HR(95%CI)=1.610(1.007~2.505),2.263(1.151~6.100),2.522(1.406~4.563),0.557(0.368~0.844)];血清CST1、JAM2及二者联合预测NSCLC预后的AUC分别为0.816、0.862、0.924,二者联合大于血清CST1、JAM2各自单项的AUC(Z=5.123、4.012,P均<0.001)。结论NSCLC患者血清CST1升高,JAM2降低,与不良临床病理参数有关,两者联合对NSCLC的预后具有较高的诊断价值,是评估NSCLC患者预后的血清标志物。

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3- TBK1 complex

SARS coronavirus （SARS-CoV） develops an antagonis- tic mechanism by which to evade the antiviral activities of interferon （IFN）. Previous studies suggested that SARS-CoV papain-like protease （PLpro） inhibits activa- tion of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism（s） used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro effi- ciently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain （PLpro-TM） from SARS-CoV inhibits STING/ TBKl/IKKE-mediated activation of type I IFNs and dis- rupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBKI. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKK~, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activa- tion of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is dis- rupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3- TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction withSTING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

The papain-like protease(PL^(pro)) of Middle-East respiratory syndrome coronavirus(MERS-CoV) has proteolytic,deubiquitinating,and de ISGylating activities.The latter two are involved in the suppression of the antiviral innate immune response of the host cell.To contribute to an understanding of this process,we present here the X-ray crystal structure of a complex between MERS-CoV PL^(pro) and human ubiquitin(Ub) that is devoid of any covalent linkage between the two proteins.Five regions of the PL^(pro) bind to two areas of the Ub.The C-terminal five residues of Ub,RLRGG,are similar to the P5–P1 residues of the polyprotein substrates of the PL^(pro) and are responsible for the major part of the interaction between the two macromolecules.Through sitedirected mutagenesis,we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PL^(pro).The enzyme appears not to be optimized for catalytic efficiency; thus,replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities.Ubiquitin binding by MERS-CoV PL^(pro) involves remarkable differences compared to the corresponding complex with SARS-CoV PL^(pro).The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PL^(pro).

甜荞木瓜类半胱氨酸蛋白酶基因FeRD21的克隆与表达分析（英文）

该研究以旱区小杂粮作物甜荞(Fagopyrum esculentum)为材料,采用同源克隆、RACE技术和实时荧光定量RT-PCR方法,对其半胱氨酸蛋白酶基因(FeRD21)进行了分离和表达分析。结果表明:(1)FeRD21基因cDNA全长1 750bp,包含1个1 407bp的完整开放阅读框,编码468个氨基酸。(2)蛋白序列比对发现,甜荞FeRD21全酶包括信号肽、N末端自主抑制前体区域、蛋白酶、脯氨酸富含结构域和C末端颗粒体蛋白结构域,同时,其蛋白酶结构域包含1个木瓜类蛋白酶家族保守的催化三连体活性位点:Cys168-His304-Asn324。(3)分子系统发生分析证实,其与拟南芥的RD21一致性最高,属类RD21半胱氨酸蛋白酶类。(4)基因表达分析表明,FeRD21能被干旱、高盐、ABA和衰老胁迫诱导。

五味子多糖对人脑胶质瘤U87细胞活力的影响及其机制

目的观察五味子多糖(CPSC)对人脑胶质瘤U87细胞活力的影响,并探讨其可能作用机制。方法取适量对数生长期人脑胶质瘤U87细胞,分为A、B、C组和对照组,A、B、C组分别加入终浓度为200、400、800μg/m L的CPSP培养,对照组加入等体积细胞培养液培养,分别于培养24、48、72 h时观察各组细胞活力;培养48 h时取各组培养液上清,检测半胱氨酸天冬氨酸蛋白酶3(Caspase-3)。结果培养24、48、72 h时,A组细胞存活率分别为1.17±0.04、1.09±0.05、1.39±0.05,B组分别为1.01±0.03、1.01±0.03、1.21±0.02,C组分别为0.90±0.02、0.76±0.02、0.98±0.03,对照组分别为1.28±0.03、1.16±0.03、1.45±0.01;培养72 h时,B组细胞存活率与对照组相比,P<0.05;培养24、48、72 h时,C组细胞存活率与对照组相比,P均<0.05。培养48 h时A、B、C组及对照组培养液上清Caspase-3蛋白的相对表达量分别为0.79±0.02、0.96±0.01、1.13±0.01、0.78±0.01,B、C组与对照组相比,P均<0.05。结论高浓度CPSC可抑制U87细胞的活力,其机制可能是与CPSC促进细胞Caspase-3表达有关。

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

A new coronavirus(SARS-CoV-2)has been identified as the etiologic agent for the COVID-19 outbreak.Currently,effective treatment options remain very limited for this disease;therefore,there is an urgent need to identify new anti-COVID-19 agents.In this study,we screened over 6,000 compounds that included approved drugs,drug candidates in clinical trials,and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease(PLpro).Together with main protease(Mpro),PLpro is responsible for processing the viral replicase polyprotein into functional units.There-fore,it is an attractive target for antiviral drug develop-ment.Here we discovered four compounds,YM155,cryptotanshinone,tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 pmol/L.These compounds also exhibit strong antiviral activities in cell-based assays.YM155,an anti-cancer drug candidate in clinical trials,has the most potent antiviral activity with an EC50 value of 170 nmol/L.In addition,we have determined the crystal structures of this enzyme and its complex with YM155,revealing a unique binding mode.YM155 simultaneously targets three"hot"spots on PLpro,including the substrate-binding pocket,the interferon stimulating gene product 15(ISG15)binding site and zinc finger motif.Our results demonstrate the efficacy of this screening and repur-posing strategy,which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

Schaftoside inhibits 3CL^(pro)and PL^(pro)of SARS-CoV-2 virus and regulates immune response and inflammation of host cells for the treatment of COVID-19

It is an urgent demand worldwide to control the coronavirus disease 2019(COVID-19)pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus.The 3-chymotrypsin-like protease(3CL^(pro))and papain-like protease(PL^(pro))are key targets to discover SARS-CoV-2 inhibitors.After screening 12 Chinese herbal medicines and 125 compounds from licorice,we found that a popular natural product schaftoside inhibited 3CL^(pro)and PL^(pro)with IC_(50)values of 1.73±0.22 and 3.91±0.19μmol/L,respectively,and inhibited SARS CoV-2 virus in Vero E6 cells with EC_(50)of 11.83±3. 23μmol/L. Hydrogen-deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of3CLpro, and K157, E167 and A246 of PLpro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The antiinflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.