Crystal structure of SARS-CoV-2 papain-like protease

The pandemic of coronavirus disease 2019(COVID-19)is changing the world like never before.This crisis is unlikely contained in the absence of effective therapeutics or vaccine.The papain-like protease(PLpro)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)plays essential roles in virus replication and immune evasion,presenting a charming drug target.Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology,inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development.In this study,we sought to provide structural frameworks for PLpro inhibitor design.We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111 S,which shares many structural features of SARS-CoV PLpro.This crystal form has unique packing,high solvent content and reasonable resolution 2.5 A°,hence provides a good possibility for fragment-based screening using crystallographic approach.We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture.We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARSCoV-2 with the IC50 of 2.2?0.3 mmol/L.We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 A°,showing the inhibitor accommodates the S3 e S4 pockets of the substrate binding cleft.The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft,whereas the binding of a tetrapeptide substrate enlarges the cleft.Hence,our results suggest a mechanism of GRL0617 inhibition,that GRL0617 not only occupies the substrate pockets,but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.

新冠病毒刺突蛋白与糖分子结合的特点

病毒的感染过程通常从病毒结合到细胞表面上的糖分子开始,这些糖分子包括以硫酸乙酰肝素为代表的糖胺聚糖和糖蛋白、糖脂上带有的含有唾液酸的糖等.本研究系统测定和比较了新冠病毒、非典病毒和中东呼吸综合征病毒的刺突蛋白及其各个亚基与这些糖分子的相互作用.结果显示这些病毒的刺突蛋白和亚基都能够与硫酸乙酰肝素结合,且结合与硫酸化程度正相关,但是它们都不与含唾液酸的糖分子结合.这一结果说明硫酸乙酰肝素在这三个病毒的感染过程中可能发挥了重要作用,它们在感染的开始阶段可能都是通过硫酸乙酰肝素与宿主细胞结合的.更为重要的是,本研究提示硫酸乙酰肝素类分子可能是可以用于研发抗冠状病毒药物的一类重要分子.

Targeting papain-like protease for broad-spectrum coronavirus inhibition

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics.Drugging the multi-functional papain-like protease(PLpro)domain of the viral nsp3 holds promise.However,none of the known coronavirus PLpro inhibitors has been shown to be in vivo active.Herein,we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity,including against the Sarbecoviruses(SARSCoV-1 and SARS-CoV-2),Merbecovirus(MERS-CoV),as well as the Alphacoronavirus(hCoV-229E and hCoVOC43).Importantly,F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice.F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage,as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity.Despite the significant difference of substrate recognition,mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue,whereas an allosteric inhibitor of MERSPLpro interacting with its 271E position.Our proof-ofconcept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anticoronavirus agents.The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.

Natural ventilation driven by a restricted heat source elevated to different levels

The thermal buoyancy generated by the difference in air density in a building can drive hot-pressed natural ventilation,which is an energy-efficient means of ventilation used to obtain higher air quality.Therefore,the effect of a single-point heat source with limited sides at different heights on stratified flow was studied in a naturally ventilated room in this paper.Based on the classical plume diffusion law of an independent point heat source and the mirroring principle,a calculation model of the thermal stratification height with a restricted source elevated to different levels was derived and validated.The quantitative effects of the heat source height from the floor,the effective opening area and other factors on the natural ventilation of hot pressure were analyzed.A threshold X_(T)for the separation between a point source and a sidewall was defined to estimate whether the thermal plume was independent or restricted by a sidewall.And a method for calculating the threshold xt was obtained.This research can provide a reference basis for designing natural ventilation for buildings with a restricted heat source at different levels to achieve a desired indoor environment.