Investigation of SARS-CoV-2 Main Protease Potential Inhibitory Activities of Some Natural Antiviral Compounds Via Molecular Docking and Dynamics Approaches

Coronaviruses caused an outbreak pandemic disease characterized by a severe acute respiratory distress syndrome leading to the infection of more than 200 million patients and the death of more than 4 million individuals.The primary treatment is either supportive or symptomatic.Natural products have an important role in the development of various drugs.Thus,screening of natural compounds with reported antiviral activities can lead to the discovery of potential inhibitory entities against coronaviruses.In the current study,an in-silico molecular docking experiment was conducted on the effects of some of these natural antiviral phytoconstituents,(e.g.,procyanidin B2,theaflavin,quercetin,ellagic acid,caffeoylquinic acid derivatives,berginin,eudesm-1β,6α,11-triol and arbutin),on the crystal structure of SARS-CoV-2 main protease(PDB ID:6w63)using AutoDock-Vina software.Many of the docked compounds revealed good binding affinity,with procyanidin B2(–8.6 Kcal/mol)and theaflavin(–8.5 Kcal/mol)showing a better or similar binding score as the ligand(–8.5 Kcal/mol).Molecular dynamics simulations were carried out at 100 ns and revealed that procyanidin B2 forms a more stable complex with SARS-CoV-2 main protease than theaflavin.Procyanidin B2,theaflavin,and 4,5-dicaffeoylquinic acid were evaluated for toxicity by ProTox-II webserver and were non-toxic according to the predicted LD50 values and safe on different organs and pathways.Additionally,these phytoconstituents showed good ADME properties and acceptable lipophilicity,as evaluated using WLOGP.Amongst the tested compounds,procyanidin B2 showed the highest lipophilic value.It is worth mentioning that these natural inhibitiors of SARS-CoV-2 main protease are components of green and black tea that can be used as a supporting supplement for COVID patients or as potential nuclei for further drug design and development campaigns.

Identification of Potential Flavonoid Inhibitors of the SARS-CoV-2 Main Protease 6YNQ:A Molecular Docking Study

Objective Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent for coronavirus disease 2019(COVID-19),is responsible for the recent global pandemic.As there are no effective drugs or vaccines available for SARS-CoV-2,we investigated the potential of flavonoids against SARS-CoV-2 main protease 6YNQ.Methods In silico molecular simulation study against SARS-CoV-2 main protease 6YNQ.Results Among the 21 selected flavonoids,rutin demonstrated the highest binding energy(−8.7 kcal/mol)and displayed perfect binding with the catalytic sites.Conclusions Our study demonstrates the inhibitory potential of flavonoids against SARS-CoV-2 main protease 6YNQ.These computational simulation studies support the hypothesis that flavonoids might be helpful for the treatment of COVID-19.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex.Four fluorogenic substrates of M^(pro),including a prototypical,internally quenched Dabcyl-EDANS peptide,were compared in terms of solubility under typical assay conditions.By exploiting the new titrant,key kinetic parameters for the M^(pro)-catalyzed cleavage of these substrates were determined.

Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specificity.To elucidate the target specificity and the cellular target engagement of the claimed M^(pro) inhibitors,we systematically characterize their mechanism of action using the cell-free FRET assay,the thermal shift-binding assay,the cell lysate Protease-Glo luciferase assay,and the cell-based FlipGFP assay.Collectively,our results have shown that majority of the M^(pro) inhibitors identified from drug repurposing including ebselen,carmofur,disulfiram,and shikonin are promiscuous cysteine inhibitors that are not specific to M^(pro),while chloroquine,oxytetracycline,montelukast,candesartan,and dipyridamole do not inhibit M^(pro) in any of the assays tested.Overall,our study highlights the need of stringent hit validation at the early stage of drug discovery.

Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 713C and SARS-CoV-2 main protease

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins.Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine,offering the opportunity to inhibit these enzymes with electrophilic small molecules.Here we describe the successful application of quantitative irreversible tethering(qIT)to identify acrylamide fragments that target the active site cysteine of the 3C protease(3Cpro)of Enterovirus 71,the causative agent of hand,foot and mouth disease in humans,altering the substrate binding region.Further,we re-purpose these hits towards the main protease(Mpro)of SARS-CoV-2 which shares the 3C-like fold and a similar active site.The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity.We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects,distorting secondary structures to disrupt the active dimeric unit.

SARS-COV-2 M^(pro)抑制剂2-(2-氯苯基)-7-(异喹啉-4-基)-5,7-二氮杂螺[3.4]辛-6,8-二酮的合成

目的研究SARS-COV-2 M^(pro)抑制剂2-(2-氯苯基)-7-(异喹啉-4-基)-5,7-二氮杂螺[3.4]辛-6,8-二酮(1)的合成方法,为其深入研究和其衍生物的合成提供借鉴方法。方法以(2-氯苯基)乙酸甲酯为起始原料,经亲电取代、还原、磺酰化、环化、选择性水解得到3-(2-氯苯基)-1-乙氧羰基环丁烷-1-甲酸(6),在三乙胺存在下,化合物6与叠氮磷酸二苯酯反应,生成的酰基叠氮化合物经Curtius重排生成相应的异氰酸酯;再与4-氨基异喹啉反应,并在碳酸钠的作用下进一步环合生成目标化合物。结果中间体及目标化合物的化学结构经^(1)H-NMR、^(13)C-NMR、ESI-MS确证。结论对化合物6的合成路线进行了优化;通过化合物6获得了一条更精简的化合物1的合成路线,该路线既避免了有毒的三光气的使用,也避免了副反应的发生;通过制备型HPLC得到化合物1的两个顺反异构体,并使用NOESY确定了化合物的构型。

Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart?A crystallographic,biophysical,and theoretical study

During the continuing evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November of that year.Along with its sublineages,it has maintained a prominent role ever since.The Nsp5 main protease(Mpro)of the Omicron virus is characterized by a single dominant mutation,P132H.Here we determined the X-ray crystal structures of the P132H mutant(or O-Mpro)as a free enzyme and in complex with the Mpro inhibitor,the alpha-ketoamide 13b-K,and we conducted enzymological,biophysical,as well as theoretical studies to characterize the O-Mpro.We found that O-Mpro has a similar overall structure and binding with 13b-K;however,it displays lower enzymatic activity and lower thermal stability compared to the WT-Mpro(with“WT”referring to the prototype strain).Intriguingly,the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here.Empirical folding free energy calculations suggest that the O-Mpro dimer is destabilized relative to the WT-Mpro due to less favorable van der Waals interactions and backbone conformations in the individual protomers.All-atom continuous constant-pH molecular dynamics(MD)simulations reveal that His132 and Glu240 display coupled titration.At pH 7,His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged.In order to examine whether the Omicron mutation eases the emergence of further Mpro mutations,we also analyzed the P132H+T169S double mutant,which is characteristic of the BA.1.1.2 lineage.However,we found little evidence of a correlation between the two mutation sites.

Assessing the inhibition efficacy of clinical drugs against the main proteases of SARS-CoV-2 variants and other coronaviruses

The rapid evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)mainly due to its high mutation rate and rapid viral replication,has led to new variants resistant to the available vaccines and monoclonal antibodies.In contrast,oral clinical drugs targeting viral protease and RNA polymerase remain effective against Omicron variants[1].Main protease(Mpro)plays a crucial role in the maturation and replication of viral strains,making it an attractive target for developing antiviral drugs.Nirmatrelvir(NTV)is the first-in-class Mpro peptidomimetic covalent inhibitor known as“Paxlovid”approved in 2021 by the Food and Drug Administration[2].Nevertheless,NTV-resistant Mpro mutants particularly the E166V mutation,have been characterized in the Global Initiative on Sharing Avian Influenza Data(GISAID)database[3]and reported in COVID-19 patients[4,5].

计算机模拟纳豆源多肽与DPP-IV和SARS-CoV-2Mpro的相互作用

兼具糖尿病的新型冠状病毒肺炎(Corona Virus Disease2019,COVID-19)患者病死率明显偏高,抑制Ⅱ型糖尿病关键酶二肽基肽酶4(DPP-IV)和新冠肺炎病毒主蛋白酶(SARS-CoV-2 Mpro)的活性能缓解相应的病症。该研究采用UniProt、NCBI和PDB数据库检索纳豆蛋白,基于BIOPBP-UWM数据库开展计算机模拟胃肠道蛋白酶(胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶)水解纳豆蛋白的研究,最后利用分子对接技术分别研究纳豆蛋白源多肽与DPP-IV和SARS-CoV-2 Mpro的结合能力,分析参与相互作用的氨基酸残基与分子作用力类型。结果发现,糖转运蛋白与两种酶具有良好的结合效果。特别地,序列为ISQPR、TIPVR和STVTR的多肽对两种酶都具有较高的结合分数(≤-130),被鉴定为DPP-IV和SARS-CoV-2Mpro的双重抑制肽。因此,纳豆蛋白具有缓解Ⅱ型糖尿病病症和作为新型冠状病毒感染病人营养补充剂的潜力。

In Silico Evaluation of the Potential Interference of Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376 in the Binding of SARS-CoV-2 Spike Protein to Human Nanobody Nb20

Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (Mpro) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 Mpro: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.

Jun12682,a potent SARS-CoV-2 papain-like protease inhibitor with exceptional antiviral efficacy in mice

Recently,a collaborative research study published in Science,led by Jun Wang,Xufang Deng,Eddy Arnold,and Francesc Xavier Ruiz1,identified a series of potent small molecule inhibitors that specifically target SARS-CoV-2 papain-like protease(PL^(pro)).The study demonstrated nanomolar PL^(pro) inhibitory potency with K_(i) values ranging from 13.2 to 88.2 nmol/L.By employing a structure-based drug design strategy,the researchers discovered an exceptionally promising compound,named Jun12682,that effectively targets both the newly discovered ubiquitin Val70(Val70^(Ub))-binding site and the known blocking loop(BL2)groove near the S4 subsite of PL^(pro).Furthermore,studies on the mechanism of action revealed that Jun12682 inhibits the deubiquitinating and deISGylating activities of PLpro,which are crucial for antagonizing the host’s innate immune response upon viral infection.Structural biology studies confirmed the“two-pronged”binding mode of Jun12682,aligning perfectly with their drug design rationale.Importantly,Jun12682 exhibited potent antiviral activity against SARS-CoV-2 and its variants,including nirmatrelvir-resistant mutants,in Caco-2 cells(EC_(50):0.44-2.02 μmol/L).It is noteworthy that its oral administration significantly improved survival rates and alleviated both lung virus loads and histopathological lesions in a lethal SARS-CoV-2 mouse model.In conclusion。

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.

TCMSP数据库中药化学成分的SARS-CoV-2主蛋白酶抑制剂虚拟筛选

目的运用计算机模拟技术从中药系统药理学数据库与分析平台(TCMSP)的中药化学成分中挖掘SARS-CoV-2主蛋白酶(Main protease,Mpro)抑制剂。方法运用类药性评估、分子对接、ADME预测、结合自由能计算、分子动力学模拟等多轮虚拟筛选的策略,从TCMSP数据库中筛选Mpro抑制剂。结果共筛选得到10个命中分子,其中前3位最佳命中分子为MOL003392、MOL011716、MOL002966,表现出与靶标活性口袋关键氨基酸残基的强相互作用。结论该研究可为发现SARS-CoV-2 Mpro小分子抑制剂提供新的思路。

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.

Medicinal chemistry strategies towards the development of non-covalent SARS-CoV-2 Mpro inhibitors

The main protease(M^(pro))of SARS-CoV-2 is an attractive target in anti-COVID-19 therapy for its high conservation and major role in the virus life cycle.The covalent M^(pro)inhibitor nirmatrelvir(in combination with ritonavir,a pharmacokinetic enhancer)and the non-covalent inhibitor ensitrelvir have shown efficacy in clinical trials and have been approved for therapeutic use.Effective antiviral drugs are needed to fight the pandemic,while non-covalent M^(pro)inhibitors could be promising alternatives due to their high selectivity and favorable druggability.Numerous non-covalent M^(pro)inhibitors with desirable properties have been developed based on available crystal structures of M^(pro).In this article,we describe medicinal chemistry strategies applied for the discovery and optimization of non-covalent M^(pro)inhibitors,followed by a general overview and critical analysis of the available information.Prospective viewpoints and insights into current strategies for the development of non-covalent M^(pro)inhibitors are also discussed.

SARS-CoV-2 Mpro抑制剂筛选体系的构建及临床常用抗病毒中药制剂的初步筛选

目的:利用荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术构建新冠病毒主蛋白酶(SARS-CoV-2 Mpro)中药抑制剂的体外筛选方法。方法:构建Mpro及筛选探针原核表达的工程质粒pGEX-4T-1-Mpro、pET-28a(+)-Ls-mK。使用荧光探针Ls-mK检测Mpro的生物活性及酶动力学参数,并对筛选条件进行优化。采用阳性药物GC-376及阴性对照PLpro(Papain-like protease)和TEV酶(Tobacco Etch Virus Proteinase)评价荧光探针Ls-mK的特异性及检测能力与商业化探针的差异。根据所构建的筛选体系考察五种中药制剂筛选热毒宁注射液、血必净注射液、抗病毒口服液和蒲地蓝口服液对Mpro的抑制能力。结果:本研究成功构建原核表达的工程质粒并获得纯度为90%左右的重组蛋白荧光探针Ls-mK及Mpro。荧光探针Ls-mK可成功产生FRET现象并具有良好的特异性。Mpro具有良好的生物活性。本研究所构建的筛选体系具有与商业化探针相同的检测能力,对五种中药制剂考察发现仅喜炎平注射液对Mpro具有良好的抑制活性,其IC_(50)值为(3.32±0.03)mg/mL。结论:本研究成功构建基于荧光蛋白SARS-CoV-2 Mpro的简便、灵敏和低成本的筛选体系,为新冠病毒主蛋白酶抑制剂的筛选与发现奠定了实验基础。

SARS-CoV-2主蛋白酶耐药机制与抗耐药性药物化学策略

严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)主蛋白酶(main protease,M^(pro))在病毒复制周期中发挥关键作用,是抗新冠病毒药物的重要靶点。然而,病毒的快速变异引起了对M^(pro)抑制剂的耐药性问题,对全球公共卫生构成严重威胁。对SARS-CoV-2 M^(pro)的耐药机制进行总结,并探讨了新型抗耐药性抑制剂的设计策略。针对M^(pro)耐药突变对现有抑制剂疗效的影响,提出了多种基于靶标结构的抗耐药性药物设计方法,如多位点占据、变构位点开发、共价结合等策略,此外还探讨了蛋白降解靶向嵌合体(proteolytic targeting chimera,PROTAC)在降解病毒靶蛋白中的应用。为解决SARS-CoV-2 M^(pro)耐药性问题提供了新思路,并为未来可能出现的冠状病毒疫情提供药物筛选和开发的参考。

基于成分-单药-组方策略的中药抑制SARS-CoV-2主蛋白酶活性研究进展

新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)自从暴发以来对人类身体健康产生了严重威胁,主蛋白酶(Mpro)作为病毒基因复制和转录的主要参与酶,是研究抗新冠病毒药物的重要靶点。对于新型冠状病毒肺炎,中药防治具有巨大潜力,以经典方剂为基础开发的一些中药复方被应用于临床,取得了良好的效果。该文以天然活性成分-单味药-组方3个层面为基础,对SARS-CoV-2 Mpro以及相关中药抑制剂的研究进展进行综述,以期为开发出抗SARSCoV-2的中药特效药及中药复方提供思路。

新型冠状病毒相关TMPRSS2蛋白结构特征和抗原表位分析

采用生物信息学方法分析预测新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)跨膜蛋白酶丝氨酸2(transmembrane protease serine 2,TMPRSS2)的理化特性、结构特征和抗原表位,为抗SARS-CoV-2药物研发提供参考。利用ProtParam、ProtScale分析预测TMPRSS2蛋白酶的理化特性;利用COILS Server、SignalP、TMPred、TargetP Server、NetPhos Server、NetNGlyc Server服务器对TMPRSS2蛋白酶结构进行功能结构的分析预测;利用SOPMA、Pfam、SWISS MODEL分析预测TMPRSS2蛋白酶高级结构;利用IEBD分析预测TMPRSS2蛋白酶B细胞、T细胞表位。TMPRSS2蛋白酶氨基酸组成数为492个,其中丝氨酸占比最高;亲水性较高,含10个跨膜螺旋区;具有4个磷酸化位点,3个糖基化修饰点;二级结构中无规则卷曲占据主导地位,三级结构能与已知的5ce.1.1.A(SMTL ID)模型同源建模;存在13个潜在的B细胞表位,12个得分较高的T细胞表位。