冠状病毒PLpro蛋白酶对泛素样分子的DUB活性

SARS冠状病毒基因组中非结构基因nsp3编码的木瓜样蛋白酶 (PLpro) 在病毒基因组复制及逃避宿主天然免疫中发挥重要作用,是研发抗病毒药物的重要靶标．SARS冠状病毒PLpro是一种病毒编码的去泛素化酶 (DUB)．为深入研究SARS冠状病毒 PLpro对泛素样分子 (ubiquitin-like protein,UBL) 的DUB特性,本研究构建缺失 PLpro N末端泛素样结构域 (Ubl) 和下游跨膜结构域 (TM) 的PLpro构建体（constructs）,并构建3种缺失蛋白酶催化活性的突变体,检测PLpro对泛素样分子干扰素刺激基因15 (ISG15)及SUMO-1的作用．实验结果表明,PLpro和PLpro-TM 在细胞内具有很强的去ISG(DeISGylation) 活性;缺失PLpro N末端泛素样结构域(Ubl) 对PLpro 的去ISG15 活性没有影响;对PLpro蛋白酶活性位点C1651 和 H1812 突变后,PLpro-TM的去ISG15活性消失,而对D1826位点突变后不影响此活性．PLpro 不具有去SUMO (DeSUMOylation)活性,而PLpro-TM具有一定的去SUMO活性;PLpro催化活性相关的3个关键氨基酸残基 Cys-His-Asp突变后对去SUMO活性有一定的影响．研究结果提示,SARS PLpro除了具有DUB的活性,还具有体内去ISG活性和去SUMO活性;PLpro蛋白酶活性与其去ISG活性之间有一定相关性;PLpro去SUMO-1 活性具有TM 依赖性．SARS冠状病毒PLpro 对泛素样分子作用特性的研究为阐明病毒逃避宿主天然免疫机制和开发新型抗病毒药物提供重要的理论依据．     

新型冠状病毒PLpro蛋白酶结构与功能的生物信息学分析

2019年12月,由新型冠状病毒(SARS-CoV-2)引起的新型冠状病毒肺炎(COVID-19)在中国武汉暴发。SARS-CoV-2的基因组编码2种病毒蛋白酶,即木瓜样蛋白酶(Papain-like protease,PLpro)和3C样蛋白酶(3C-like protease)。其中PLpro是SARS-CoV-2复制酶复合体(RC)形成的重要调节蛋白分子,对于病毒基因组转录和复制至关重要。因此,将SARS-CoV-2 PLpro作为药物的靶点对COVID-19的治疗具有积极意义。本研究应用生物信息学工具分析新型冠状病毒的木瓜样蛋白酶的结构和功能,首先利用BLAST和BioEdit获取SARS-CoV-2 PLpro蛋白酶(SC2-PLpro)及其同源蛋白的氨基酸序列,并利用BLAST和MEGA 6.0进行同源性分析。之后,利用ProParam和Proscale分别对SC2-PLpro蛋白酶的理化性质、亲水性和疏水性进行分析。然后,通过SOMPA、ScanProsite和InterPro分别预测SC2-PLpro蛋白酶的二级结构和功能区域,进一步利用SignalP 4.0和TMHMM对SC2-PLpro蛋白酶的信号肽和跨膜区进行分析。最后,通过SWISS-MODEL对SARS-CoV-2 PLpro蛋白酶进行三级结构同源建模。结果显示,对SARS-CoV-2 PLpro蛋白酶与已报道的PLpro蛋白酶进行多序列比对后,发现SARS-CoV-2 nsp3的746~1063段氨基酸与多种冠状病毒PLpro蛋白酶氨基酸序列高度相似。同时,同源性分析发现SARS-CoV-2与蝙蝠冠状病毒的PLpro蛋白酶具有同源性,其中与QHR63299、AVP78030相似性最高。对SC2-PLpro进行理化性质预测结果显示,其由318个氨基酸所组成,为稳定亲水性蛋白。二级结构预测结果显示SC2-PLpro主要含有α-螺旋、延伸链、β-转角、无规卷曲,四种结构贯穿整条氨基酸链。进一步进行功能分析,发现其具有完整的催化三联体、锌结合域、泛素样N末端结构域,故推测该蛋白具有去泛素化的功能。然后,信号肽假说和跨膜结构域分析结果表明,SC2-PLpro既不是分泌蛋白,也不属于跨膜蛋白。本研究提示,生物信息学分析SC2-PLpro为稳定性亲水蛋白,属于非跨膜蛋白,比较保守,具有去泛素化的功能,利用此功能可以进一步规避宿主的固有免疫反应。通过制备PLpro蛋白酶小分子抑制剂,可能有助于治疗新型冠状病毒肺炎。     

Geranylated flavonoids displaying SARS-CoV papain-like protease inhibition from the fruits of Paulownia tomentosa

SARS-CoV papain-like protease (PLpro) is an important antiviral target due to its key roles in SARS virus replication. The MeOH extracts of the fruits of the Paulownia tree yielded many small molecules capable of targeting PLpro. Five of these compounds were new geranylated flavonoids, tomentin A, tomentin B, tomentin C, tomentin D, tomentin E (1–5). Structure analysis of new compounds (1–5) by NMR showed that they all contain a 3,4-dihydro-2H-pyran moiety. This chemotype is very rare and is derived from cyclization of a geranyl group with a phenol functionality. Most compounds (1–12) inhibited PLpro in a dose dependent manner with IC₅₀’s raging between 5.0 and 14.4 μM. All new compounds having the dihydro-2H-pyran group showed better inhibition than their parent compounds (1 vs 11, 2 vs 9, 4 vs 12, 5 vs 6). In kinetic studies, 1–12 emerged to be reversible, mixed inhibitors.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Phytocompounds as potential inhibitors of SARS-CoV-2 Mpro and PLpro through computational studies

The inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) and papain-like protease (PLpro) prevents viral multiplications; these viral enzymes have been recognized as one of the most favorable targets for drug discovery against SARS-CoV-2. In the present study, we screened 225 phytocompounds present in 28 different Indian spices to identify compounds as potential inhibitors of SARS-CoV-2 Mpro and PLpro. Molecular docking, molecular dynamics simulation, molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and absorption, distribution, metabolism, excretion and toxicity (ADMET) studies were done. Based on binding affinity, dynamics behavior, and binding free energies, the present study identifies pentaoxahexacyclo-dotriacontanonaen-trihydroxybenzoate derivative (PDT), rutin, and dihyroxy-oxan-phenyl-chromen-4-one derivative (DOC), luteolin-7-glucoside-4′-neohesperidoside as promising inhibitors of SARS-CoV-2 Mpro and PLpro, respectively.     

Comparison of SARS and NL63 papain-like protease binding sites and binding site dynamics: inhibitor design implications

The human severe acute respiratory syndrome coronavirus (SARS-CoV) and the NL63 coronaviruses are human respiratory pathogens for which no effective antiviral treatment exists. The papain-like cysteine proteases encoded by the coronavirus (SARS-CoV: PLpro; NL63: PLP1 and PLP2) represent potential targets for antiviral drug development. Three recent inhibitor-bound PLpro structures highlight the role of an extremely flexible six-residue loop in inhibitor binding. The high binding site plasticity is a major challenge in computational drug discovery/design efforts. From conventional molecular dynamics and accelerated molecular dynamics (aMD) simulations, we find that with conventional molecular dynamics simulation, PLpro translationally samples the open and closed conformation of BL2 loop on a picosecond-nanosecond timescale but does not reproduce the peptide bond inversion between loop residues Tyr269 and Gln270 that is observed on inhibitor GRL0617 binding. Only aMD simulation, starting from the closed loop conformation, reproduced the 180° ?-ψ dihedral rotation back to the open loop state. The Tyr-Gln peptide bond inversion appears to involve a progressive conformational change of the full loop, starting at one side, and progressing to the other. We used the SARS-CoV apo X-ray structure to develop a model of the NL63-PLP2 catalytic site. Superimposition of the PLP2 model on the PLpro X-ray structure identifies binding site residues in PLP2 that contribute to the distinct substrate cleavage site specificities between the two proteases. The topological and electrostatic differences between the two protease binding sites also help explain the selectivity of non-covalent PLpro inhibitors.