key: cord-0770484-co6qdzuc
authors: Yu, Shaopeng; Chen, Yao; Xiang, Yusen; Lin, He; Wang, Mengge; Ye, Wenbo; Zhang, Pei; Chen, Hongzhuan; Lin, Guoqiang; Zhu, Yuying; Chen, Lili; Zhang, Jiange
title: Pseudoephedrine and its derivatives antagonize wild and mutated severe acute respiratory syndrome‐CoV‐2 viruses through blocking virus invasion and antiinflammatory effect
date: 2021-09-01
journal: Phytother Res
DOI: 10.1002/ptr.7245
sha: fb39c2a77ce490fcba5025e41339aee47bc0da4c
doc_id: 770484
cord_uid: co6qdzuc

The coronavirus disease 2019 has infected over 150 million people worldwide and led to over 3 million deaths. Severe acute respiratory syndrome (SARS)‐CoV‐2 lineages B.1.1.7, B.1.617, B.1.351, and P.1 were reported to have higher infection rates than that of wild one. These mutations were noticed to happen in the receptor‐binding domain of spike protein (S‐RBD), especially mutations N501Y, E484Q, E484K, K417N, K417T, and L452R. Currently, there is still no specific medicine against the virus; moreover, cytokine storm is also a dangerous factor for severe infected patients. In this study, potential S‐RBD‐targeted active monomers from traditional Chinese medicine Ephedra sinica Stapf (ephedra) were discovered by virtual screening. NanoBiT assay was performed to confirm blocking activities of the screened compounds against the interaction between SARS‐CoV‐2 S‐RBD and angiotensin‐converting enzyme 2 (ACE2). We further analyzed the blocking effect of the active compounds on the interactions of mutated S‐RBD and ACE2 by computational studies. Moreover, antiinflammatory activities were evaluated using qRT‐PCR, enzyme‐linked immune sorbent assay, and Western blot analysis. As a result, pseudoephedrine (MHJ‐17) and its derivative (MHJ‐11) were found as efficient inhibitors disrupting the interactions between ACE2 and both wild and mutated S‐RBDs. In addition, they also have antiinflammatory activities, which can be potential drug candidates or lead compounds for further study.

Since late 2019, an epidemic called coronavirus disease 2019 caused by severe acute respiratory syndrome (SARS)-CoV-2 virus has swept rapidly to the world (Rubin, Baden, Morrissey, & Campion, 2020; Wu, Leung, & Leung, 2020) . To date, over 200 countries and regions have invaded by the disease. In addition, over 150 million people worldwide have been infected.

Compared with SARS-CoV in 2003, a higher mortality was observed in this disease and led to over 3 million deaths. High infectious capacity was another property in this epidemic (Chu et al., 2020) .

The world economy was also suffered in a great loss (Ayittey, Ayittey, Chiwero, Kamasah, & Dzuvor, 2020) . Moreover, it was reported that inflammatory cytokine storm was also a common symptom in patients with severe COVID-19 and usually led to higher death rate (Abou-Ismaila, Diamonda, Kapoorc, Arafaha, & Nayak, 2020; Scheen, Marre, & Thivolet, 2020) . It is worth noting that there is no specific drug yet on the market. However, drug discovery is a lengthy and arduous mission, especially for this kind of RNA viruses such as SARS-CoV-2. Mutations are always occurred during replication, and several kinds of mutant viruses were discovered independently in many countries, such as England, India, South Africa, and Japan (Frampton et al., 2021; Johansen & Nohynek, 2021; Muñoz, Patiño, Ballesteros, Paniz-Mondolfi, & Ramírez, 2021; Oshitani, 2020) . In all kinds of mutated viruses, SARS-CoV-2 lineages B.1.1.7, B.1.617, B.1.351, and P.1 are the most notably varieties, because they have higher infectious capacity than that of the wild virus, especially lineage B.1.617 has caused a great disaster in India recently. These lineages of viruses have many mutations in receptor-binding domain (RBD) of S1 subunit of SARS-CoV-2, especially mutations N501Y, E484Q, E484K, L452R, K417N, and K417T (Ostrov, 2021; Verghese et al., 2021) . Therefore, although vaccines are available worldwide, multi-target drug for anti-SARS-CoV-2 and antiinflammatory is still desirable. Furthermore, it is still urgent to search for a drug candidate or a lead compound that is effective against both wild and mutated viruses.

Traditional Chinese medicine (TCM) was proved to be advantageous in treating COVID-19, because it has an efficacy and comprehensive therapeutic theory Liu et al., 2020) . Among reported clinically effective treatment schemes of TCM for treating COVID-19 by Zhang and co-workers (Zheng et al., 2020) , Ephedra sinica Stapf (ephedra) was used for several times. Also, it was also reported that water distillate of ephedra was found to be effective in alleviating the inflammatory and arthritis response (Yeom et al., 2006) .

As the herb had been reported to be efficacious both on and antiinflammatory, the active components and molecular mechanisms are of great significance, because it may alleviate the trouble caused by both virus invasion and inflammation. In addition, the x-ray structures of lineages B.1.1.7 and B.1.617 receptor-binding domains of spike protein (S-RBDs) and angiotensin-converting enzyme 2 (ACE2) complexes are still unreported. However, they can still be constructed using homology modeling, and mutated S-RBD-ACE2 protein-protein interactions (PPIs) can also be predicated by software.

Moreover, as spike protein of lineage B.1.351 (Protein Data Bank

[PDB] id = 7LYN) was reported by Gobeil and co-workers (Gobeil et al., 2021) , S-RBD of this variety was obtained by intercepting the x-ray structure from amino acid sequence 330-583 (S-RBD part). In addition, an x-ray structure of P.1 variety S-RBD complexed with ACE2 was reported by Dejnirattisai and co-workers , so the crystal structure was used for docking experiment without further simulations. Therefore, compounds against variant viruses can also be screening in silico.

Herein, we investigated active components derived from ephedra and evaluated their blocking activities between S-RBD and ACE2 through virtual screening. We found that ephedrine and pseudoephedrine are potent agents worthy of further study. Then, a NanoBiT assay was performed to evaluate these two compounds and their commercially available derivatives for their inhibitory activities against the interaction between S-RBD and ACE2. Moreover, to confirm whether they can disrupt the interaction between mutated S-RBDs and ACE2, S-RBD models of lineages B. were also determined to prove that they have potent activities of antiinflammatory cytokine storm, indicating that they are antiviral and antiinflammatory agents. The results revealed that pseudoephedrine (MHJ-17) and its derivative (MHJ-11) are promising and safe inhibitors that could block the interaction between ACE2 and both wild and mutated S-RBDs. In addition, they are also multi-target candidates for their antiinflammatory activities, which could be used as lead compounds for further study.

All the ephedrine derivatives were purchased from commercial suppliers or from our laboratory compound library. All these molecules were at least 99.5% purity and were used without any purification.

Chemical structures and compound ID of each molecule were shown in Figure 1.

Protein preparation and binding-site definition were performed using protocols inside the program Discovery Studio 4.0 (v 4.0.0.13258).

Small molecules were prepared under prepare ligands followed by full minimization protocols. CHARMm forcefield was used in both preparation procedures of proteins and small molecules. All compounds were tried to dock into the crystal structure of S-RBD and ACE2 complex (PDB id = 6M0J) (Lan et al., 2020) . Lineage P.1 S-RBD and ACE2 complex (PDB id = 7NXC) were downloaded from PDB and were prepared using Discovery Studio 4.0. Libdock protocol was used in high throughput screening of small molecules with potential activity of disrupting the interaction between wild S-RBD and ACE2. The physical properties and libdock scores were used to conclude active ingredients. CDOCKER protocol was used in molecular docking experiments to judge whether active components could block the interaction between mutated S-RBD and ACE2. A combination of physical properties and scores of CDOCKER ENERGY and CDOCKER INTER-ACTION ENERGY were used to select the final pose.

The amino acid sequence of lineages B.1.1.7 and B.1.617 mutated S-RBDs was downloaded from PubMed website (https://www.ncbi. nlm.nih.gov). The S-RBDs of two x-ray structures of S-RBD-ACE2 complex (PDB id = 6M0J and 6ZGG; Lan et al., 2020; Wrobel et al., 2020) were used as templates for homology modeling for their high identity to mutated S-RBDs of SARS-CoV-2 lineages B.1.1.7 and B.1.617. Mutated S-RBD sequences downloaded in the previous step were aligned to the sequences corresponding to wild S-RBD crystal structures using align sequences protocol within the program Discovery Studio 4.0. For building of a protein amino acid sequence into its 3D structure, homology modeling is a more reliable protocol than build mutants procedure (Dubreuil et al., 2005) .

Thus, build homology models protocol was used to build mutated S-RBD models. The number of models generated was set as 20.

Optimization level was set as high, and refine loops were set as true. A combination of probability density function (PDF) total energy, Ramachandran plot, and DOPE score was used to choose the final conformation.

The models of mutated S-RBD built in last step and the crystal structure of the spike protein of lineage B.1.351 (PDB id = 7LYN) downloaded from PDB as well as the ACE2 part of S-RBD-ACE2 complex (PDB id = 6M0J) were prepared and minimized using prepare protein protocol. We made an attempt to find out the strength of interaction of mutated S-RBDs and ACE2, so that their binding mode can be predicated based on their PPIs, which will also give a message about the small-molecule binding site between the two proteins.

ZDOCK protocol was used to perform protein-protein docking between the prepared proteins. The mutated S-RBDs were set as ligands, and the ACE2 was defined as receptor. Angular step size was set as 6 ; 2,000 poses were generated for each docking process, and a distance cutoff of 8 Å was used to filter poses. A RMSD cutoff value of 6 Å and an interface cutoff value of 9 Å were used to cluster poses.

The ranking of the poses was done, and electrostatic and desolvation energy were calculated during docking processes. The predicated docking conformations calculated by ZDOCK procedure were subjected to refinement and re-ranking using RDOCK protocol to minimize the complexes under CHARMm forcefield. Finally, the final poses were selected from a comprehensive consideration of ZDOCK scores, ZRank scores, and RDOCK scores among all refined conformations.

2.5 | NanoBiT-based SARS-CoV-2 S-RBD/ACE2 binding assay

The preliminary screening and 50% inhibitory concentration (IC 50 ) determination of the 17 compounds ( Figure 1 ) to block SARS-CoV-2 S-RBD/ACE2 interaction were performed by NanoBiT-based assay F I G U R E 1 Structures of all tested compounds according to our previous procedure . In brief, SARS-CoV-2 S-RBD-LgBiT and SmBiT-ACE2 fusion plasmids were transiently co-transfected into HEK293 cells in a six-well plate using FuGENE HD transfection reagent (Promega, Madison, WI) according to the manufacturer's instructions. After transfection for 6 hr, cells were detached by gentle pipetting, suspended in fresh Opti-MEM medium (Gibco-BRL/Invitrogen, Carlsbad, CA) containing 2% FBS and reseeded into a 96-well plate. To measure if the test compounds could block the interaction between SARS-CoV-2 S-RBD and ACE2, the compounds were serially diluted to indicated concentrations and added to the wells of the 96-well plate and incubated for 3 hr at 37 C. Then, Nano-Glo live Cell Assay reagent was added, and luminescence was measured on an Envision plate reader (EnVision, Perkin Elmer, Waltham, MA). False positive results were considered by determining the cytotoxicity of the compounds on the HEK293 cells and the inhibitory effects of the compounds on NanoLuc (HEK293/ Nanoluc stable cells), respectively. Data were analyzed and expressed using the inhibitory effects on SARS-CoV-2 S-RBD/ACE2 interaction (NanoBiT inh%), NanoLuc luciferase (NanoLuc inh%), and the cell proliferation (Cytotox inh%, CC 50 ) on HEK293 cells. MHJ-17 (125, 250, and 500 μM). Raw264.7 cells were treated in a 37 C, 5% CO 2 incubator for 6 hr, and total RNA was extracted using TRIzol reagent (Sangon Biotech). Total RNA was then used for reverse transcription with PrimeScript™ RT reagent kit (TaKaRa, Tokyo, Japan), and TB Green™ Premix Ex Taq™ (TaKaRa) was used for qRT-PCR analysis. The mRNA levels were normalized to the β-actin mRNA.

Relative quantification was performed as 2 ÀΔΔCt . Three repeats were executed for each assay. Amplification primers were as follows: IL-6 (F: 5 0 -CTGCA AGAGA CTTCC ATCCA G-3 0 , R: 5 0 -AGTGG TATAG ACAGG TCTGT TGG-3 0 ), TNF-α (F:5 0 -CTGTA GCCCA CGTCG TAGC-3 0 , R: 5 0 -TTGAG ATCCA TGCCG TTG-3 0 ), and β-actin (F: 5 0 -GTCCC TCACC CTCCC AAAAG-3 0 , R: 5 0 -GCTGC CTCAA CACCT CAACC C-3 0 ).

Ctrl, Model, MHJ-11, and MHJ-17 groups were set as those in qRT-PCR analysis. Raw264.7 cells were treated for 24 hr, and the supernatants were collected for IL-6 and TNF-α detection. Protein levels were measured by enzyme-linked immune sorbent assay (ELISA) according to the manufacturer's instructions (NeoBioscience, Shanghai, China).

Three repeats were executed for each assay. Three repeats were executed for each assay.

The raw data were analyzed using GraphPad Prism 8.0.1 software.

Two-tailed Student's t test was used to determine the significance of differences in the above-mentioned assays. All values are presented as mean ± SD. The P value was used to indicate statistical significance.

The aim of our virtual screening was to search for active ingredients derived from TCM Ephedra sinica Stapf (ephedra), which could block the interaction between S-RBD of SARS-CoV-2 and ACE2. Therefore, a crystal structure of S-RBD and ACE2 complex (PDB id = 6M0J) was used as docking target. The long and narrow binding site was detected from receptor cavities by the function within Discovery Studio 4.0.

Among the candidates successfully docked into the cavity, we found that ephedrine (MHJ-16) and pseudoephedrine (MHJ-17) were the most potent ingredients in disrupting the interaction between SARS-CoV-2 S-RBD and ACE2, and their CDOCKER interaction energies were lower than other candidates. Therefore, it could be predicted that ephedrine, pseudoephedrine, or their derivatives may have potent activity in blocking the interaction. The binding site of the crystal structure and docking pose of ephedrine were shown in Figure 2 .

The inhibitory activities of ephedrine (MHJ-16), pseudoephedrine (MHJ-17), and 15 corresponding commercial derivatives against SARS-CoV-2 S-RBD/ACE2 interaction were investigated by NanoBiT assay. Fortunately, three compounds were active according to preliminary screening results. Then, these three active compounds were further verified in a dose-dependent manner. As shown in Figure 3 , the IC 50 values of these three compounds on S-RBD/ACE2 interaction (NanoBiT inh%), NanoLuc luciferase (NanoLuc inh%), and HEK293 cells proliferation (Cytotox inh%) were analyzed. Notably, none of the three compounds showed obvious inhibitory effects on Nanoluc luciferase (IC 50 > 100 μM), and they had low cytotoxicity to HEK293 cells (IC 50 > 100 μM; Figure 3 ).

Among the three compounds, MHJ-17 (IC 50 = 13 μM) showed better blocking activity compared with MHJ-11 (IC 50 = 49 μM) and MHJ-16 (IC 50 = 73 μM). Therefore, these three compounds showed blocking activity against the interaction between SARS-CoV-2 S-RBD and ACE2, although MHJ-16 exhibited lower activity.

Homology modeling is a powerful and widely used tool to build and predict 3D structures of proteins whose folded forms are unknown (Waterhouse et al., 2018) . Although x-ray structure of lineage B.1.1.7 S-RBD complexed with antibody was reported , the structure of the protein docked with natural receptor may be quite different. To reveal a more accurate result, homology modeling was used in this research. Two different S-RBD parts of x-ray structures of S-RBD-ACE2 complex (PDB id = 6M0J and 6ZGG) were used as templates. Both templates were combined with their endogenous receptor protein ACE2. SARS-CoV-2 lineage B.1.1.7 has a mutation called N501Y, and lineage B.1.617 is used to refer to two mutations in S protein at the positions E484Q and L452R. These varieties caused the infection rates of these viruses increased dramatically. Fortunately, the sequences of mutated viruses can be downloaded from PubMed website. As shown in Figure 4 , sequence alignment revealed that these sequences are highly homologous, and mutations N501Y, E484Q, and L452R were highlighted in red. In the building procedure of S-RBD of lineage B.1.1.7, 20 molecular models were generated during initial procedure, and the best model ( Figure 5a,b) with the lowest PDFs total energy and PDF physical energy of 8,820.1 and 591.4, respectively, was chosen for further CHARMm forcefield refinement and research. In another hand, S-RBD of lineage B.1.617 was built under the same procedure, and the PDF total energy and PDF physical energy of the best model were 9,075.5 and 764.0, respectively. Ramachandran plots of the two generated models were also shown in Figure 5c ,d, and it was indicated that most of the amino acids in the best predicated model were considered favorable. Importantly, the highlighted mutated residues N501Y of B.1.1.7, and E484Q and L452R of B.1.617 were also in green, indicating that the built models are reliable.

ZDOCK and RDOCK protocols were used to generate and refine protein-protein docking complex, respectively. As the crystal structure of spike protein of lineage B.1.351 (PDB id = 7LYN) was reported (Gobeil et al., 2021) , the RBD part of it was directly used at this stage. As a result, eight poses for variety B.1.1.7, 12 poses for variety B.1.617, and 28 poses for variety B.1.351 complexed with ACE2 were generated, which were further refined and re-ranked using CHARMm forcefield. The best docking pose of each procedure was selected under a consideration of highest ZDOCK score and lowest E_RDOCK, which was considered as the conformation closest to the real. The best pose of lineage B.1.1.7 S-RBD-ACE2 complex was shown in Figure 6 , and the contacting surface area was observed like the previous wild S-RBD-ACE2 complex. However, in the constructed model, the interacting amino acid residues of both parts are different from the previous one (Table 1 and Figure 6b ,c,e,f,h,i). It was visualized that Arg403, Asn439, Tyr473, Gly476, Glu484, Cys488, Phe490, Pro499, Val503, and Gln506 in lineage B.1.1.7 S-RBD were interacted with ACE2 protein, which did not occur before. Residues Lys417 and Gly446 in the new model had no interaction with ACE2. Moreover, it could be observed that Ser19, Thr324, Gln325, Gly326, Glu329, Gly352, Phe356, and Ala386 were interacted with lineage B.1.1.7 S-RBD, which were not detected before. Also, Glu35, Glu37, Gln42, Arg357, and Arg393 were not interacted with mutated S-RBD as Also, Ser19, Thr20, Glu23, Lys26, Thr324, Gln325, Gly326, Phe327, Phe356, Met383, Ala384, Ala386, and Ala387 were observed to have interactions with ACE2. As a result, all the mutated S-RBDs and ACE2 had more amino acid residues interact with each other. It may be explained that mutated viruses resulted in higher transmissibility than the previous viruses (Chan et al., 2020) .

NanoBiT-based severe acute respiratory syndrome (SARS)-CoV-2 receptor-binding domain of spike protein (S-RBD)/angiotensinconverting enzyme 2 (ACE2) interaction assay for MHJ-11, MHJ-16, and MHJ-17. NanoBiT inh%: the inhibition rates against SARS-CoV-2 S-RBD/ACE2 interaction; NanoLuc inh%: the inhibition rates against NanoLuc luciferase; Cytotox inh%: the inhibition rates against the transfected HEK293 cells proliferation. n = 3 F I G U R E 4 The sequences of two templates and mutated receptor-binding domains of spike protein variety and ACE2 complex model, as shown in Figure 7a , MHJ-11 has several π relevant interactions with Gln388, Lys417, and Pro389 and has many strong hydrogen bond interactions with surrounding residues, such as Ala386, Ala387, Gln388, Asp405, and Tyr505. These T A B L E 1 Interacting amino acid residues between angiotensin-converting enzyme 2 and wild and mutated receptor-binding domains of spike protein Lys458 Glu35 

As systemic inflammatory response is a major symptom for coronavirus infections, macrophages play critical role in this process. Therefore, we detected the antiinflammation activity of MHJ-11 and MHJ-17 in Raw264.7 cells. As shown in Figure 

COVID-19 has been spreading for over 1 year and may continue for a long time (Daughton, 2020; Ouslander & Grabowski, 2020) . This kind of novel virus can spread during the asymptomatic period or form asymptomatic persons, and the number of infected people is larger than 150 million and is still increasing. Moreover, it is worth noting that patients infected with this virus are usually accompanied by an inflammatory factor storm that could make the death rate dramatically

increased. Therefore, attention should be paid to antiinflammation while treating COVID-19 . Furthermore, with the evolution of this virus, several lineages have determined as more dangerous mutated forms with high infection and low detection rates Dorpa et al., 2020) , such as lineage B. also opens a potential binding pocket at the interface of spike protein (Ostrov, 2021) . Because amino acid D614 is not located in S-RBD and may not affect S-RBD/ACE2 interaction, it is not investigated in this study. Several reports have indicated that these mutations were occurred in the S-RBDs and may allow the viruses to undergo immune escape to existing vaccines (Saghazadeh & Rezaei, 2020; Pinto et al., 2020) . Thus, searching for a broad-spectrum PPI inhibitor with antiinflammatory activity is an urgent and long-term task.

Ephedra, the traditional Chinese herb medicine, was reported to have both activities in treating COVID-19 clinically and in antiinflammatory effect in vivo Liu et al., 2020) , which is very attractive to us. Initially, we performed virtual screening to judge whether ingredients from ephedra could have activities of blocking the interaction between SARS-CoV-2 S-RBD and ACE2 pro- and MHJ-17 are reliable drug candidates or lead compounds for blocking the interactions between ACE2 and both wild and mutated S-RBDs, which could be used for further research or modification.

Immune response in severe COVID-19 is characterized by cytokine storm, which is associated with untoward clinicopathological consequences (Mahmudpour, Roozbeh, Keshavarz, Farrokhi, & Nabipour, 2020) . Studies showed that higher levels of inflammatory factors, including IL-6 and TNF-α, were found in deceased patients of SARS-CoV-2 infection compared with those recovered from the disease (Mehta et al., 2020; Huang et al., 2020) . Thus, we also detected the antiinflammatory effect of MHJ-11 and MHJ-17, which inhibited the mRNA and protein levels of IL-6 and TNF-α in LPS-activated Raw264.7 cells. Moreover, the phosphorylation levels of inflammation-related proteins NFκB p65, p44/42 MAPK, SAPK/JNK, p38, and IκBα were all reduced. Therefore, MHJ-11 and MHJ-17 might be multi-target inhibitors against SARS-CoV-2 infections. 

The hypercoagulable state in COVID-19: Incidence, pathophysiology, and management

Economic impacts of Wuhan 2019-nCoV on China and the world

Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a Golden Syrian hamster model: Implications for disease pathogenesis and transmissibility

Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: An ex vivo study with implications for the pathogenesis of COVID-19

0 (a) Details of ephedrine docked into receptor-binding domain of spike protein (S-RBD)-angiotensin-converting enzyme 2 (ACE2) complex. (b) Details of pseudoephedrine docked into S-RBD-ACE2 complex

Wastewater surveillance for population-wide Covid-19: The present and future

Antibody evasion by the P.1 strain of SARS-CoV-2

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2. Infection

Fine tuning of the specificity of an antiprogesterone antibody by first and second sphere residue engineering

Genomic characteristics and clinical effect of the emergent SARS-CoV-2 B.1.1.7 lineage in London, UK: A wholegenome sequencing and hospital-based cohort study

Effect of natural mutations of SARS-CoV-2 on spike structure

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

No country or continent is on its own in the ongoing COVID-19 pandemic

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity

Clinical practice guidelines and experts' consensuses of traditional Chinese herbal medicine for novel coronavirus (COVID-19): Protocol of a systematic review

Efficacy and safety of integrated traditional Chinese and Western medicine for Corona virus disease 2019 (COVID-19): A systematic review and meta-analysis

Screening and evaluation of anti-SARS-CoV-2 components from Ephedra sinica by ACE2/CMC-HPLC-IT-TOF-MS approach

COVID-19 cytokine storm: The anger of inflammation

COVID-19: Consider cytokine storm syndromes and immunosuppression

Characterizing SARS-CoV-2 genome diversity circulating in south American countries: Signatures of potentially emergent lineages?

Cluster-based approach to coronavirus disease 2019 (COVID-19) response in Japan

Structural consequences of variation in SARS-CoV-2 B.1.1.7

COVID-19 in nursing homes: Calming the perfect storm

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

Towards treatment planning of COVID-19: Rationale and hypothesis for the use of multiple immunosuppressive agents: Anti-antibodies, immunoglobulins, and corticosteroids

Prognostic factors in patients with diabetes hospitalized for COVID-19: Findings from the CORONADO study and other recent reports

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera

A SARS-CoV-2 variant with L452R and E484Q neutralization resistance mutations

SWISS-MODEL: Homology modelling of protein structures and complexes

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furincleavage effects

Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: A modelling study

Anti-arthritic effects of Ephedra sinica STAPF herb-acupuncture: Inhibition of lipopolysaccharide-induced inflammation and adjuvantinduced polyarthritis

Glycyrrhizic acid exerts inhibitory activity against the spike protein of SARS-CoV-2

The use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (COVID-19): The perspectives of clinical immunologists from China

Comprehensive analysis of diagnosis and treatment schemes for prevention and treatment of novel coronavirus pneumonia by traditional Chinese medicine

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein

Pseudoephedrine and its derivatives antagonize wild and mutated severe acute respiratory syndrome-CoV-2 viruses through blocking virus invasion and antiinflammatory effect

The authors thank Department of Pharmacy, Nanjing University of 

Jiange Zhang, Guoqiang Lin, Lili Chen, and Hongzhuan Chen conceived and supervised the whole study. Shaopeng Yu and Wenbo Ye performed virtual screening, homology modeling, protein-protein docking, and molecular docking. Yuying Zhu, Yao Chen, Yuseng Xiang, and Mengge Wang carried out the experiments and data analysis. Shaopeng Yu, Yuying Zhu, and Lili Chen drafted the manuscript. He Lin, Wenbo Ye, and Pei Zhang contributed to experimental consumables. Jiange Zhang, Guoqiang Lin, Lili Chen, and Hongzhuan Chen revised the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

The authors declare no competing interests.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0002-1434-6040