Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus
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Abstract
Background
The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating. The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial.
Purpose
The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection.
Methods
In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2 h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2 h cells.
Results
Results showed that HCQ is slightly more toxic to ACE2 h cells than CQ. Both CQ and HCQ could bind to ACE2 with K D =(7.31±0.62)e −7 M and (4.82±0.87)e −7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells.
Conclusions
CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.
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SciScore for 10.1101/2020.06.22.164665: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The membranes were then incubated overnight at 4°C with the following primary antibodies: anti-ACE2 (1:500, EPR4435, Abcam), anti-LC3 (1:1000, #2775, Cell Signaling Technology [CST]) and anti-GAPDH (1:2000, a#2118, CST). anti-ACE2suggested: Noneanti-LC3suggested: Noneanti-GAPDHsuggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)Experimental Models: Cell Lines Sentences Resources Annexin V-FITC/PI Apoptosis Detection Kit (Cat. No. A005-3) and Cell … SciScore for 10.1101/2020.06.22.164665: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The membranes were then incubated overnight at 4°C with the following primary antibodies: anti-ACE2 (1:500, EPR4435, Abcam), anti-LC3 (1:1000, #2775, Cell Signaling Technology [CST]) and anti-GAPDH (1:2000, a#2118, CST). anti-ACE2suggested: Noneanti-LC3suggested: Noneanti-GAPDHsuggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)Experimental Models: Cell Lines Sentences Resources Annexin V-FITC/PI Apoptosis Detection Kit (Cat. No. A005-3) and Cell Counting Kit were purchased from 7Sea Pharmatech Co., Ltd (Shanghai, China), the 2019-nCoV spike pseudotyped virus (Cat: PSV001) was purchased from Sino Biological (Beijing, China) Cell culture: HEK293T cells, human airway epithelial cells (HSAEpC), alveolar type II epithelial cells (AT2), and eosinophilic leukemia (EOL-1) cells were from ATCC. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)HSAEpC and AT2 cells were maintained in DMEM with high glucose containing 10% FBS and 1% penicillin-streptomycin; EOL-1 cells were kept in 1640 medium containing 10% FBS and 1% penicillin-streptomycin; ACE2h cells were maintained in DMEM with high glucose medium containing 10% FBS, 1% penicillin-streptomycin, and 4 μg/mL puromycin and cultured at 37°C in a 5% CO2 incubator. HSAEpCsuggested: NoneEOL-1suggested: NoneSoftware and Algorithms Sentences Resources A Lane 1 DTM transilluminator (Beijing Creation Science, Beijing, China) was used to capture the images of the developed blots, and Image-Pro Plus 5.1 software (Rockville, MD, USA) was used to quantify the protein levels. Image-Pro Plussuggested: (Image-Pro Plus, RRID:SCR_007369)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.06.22.164665: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , anti-LC3 ( 1:1000 , #2775 , Cell Signaling Technology [ CST] ) and anti-GAPDH ( 1:2000 , a#2118 , CST) . anti-GAPDHsuggested: (Cell Signaling Technology Cat# 2118, AB_561053)The cells were then continuously incubated with LC3 primary antibody at 37°C for 3 h , and the fluorescent secondary antibody at room temperature for 2 h . LC3suggested: NoneSciScore for 10.1101/2020.06.22.164665: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , anti-LC3 ( 1:1000 , #2775 , Cell Signaling Technology [ CST] ) and anti-GAPDH ( 1:2000 , a#2118 , CST) . anti-GAPDHsuggested: (Cell Signaling Technology Cat# 2118, AB_561053)The cells were then continuously incubated with LC3 primary antibody at 37°C for 3 h , and the fluorescent secondary antibody at room temperature for 2 h . LC3suggested: NoneWu Y , Wang F , Shen C , Peng W , Li D , Zhao C , Li Z , Li S , Bi Y , Yang Y et al: A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2 . ACE2suggested: NoneExperimental Models: Cell Lines Sentences Resources In our study, we used CCK-8 stain, flow cytometry and immunofluorescent stain to evaluated the toxicity and autophagy of CQ and HCQ respectively on ACE2 high expressed HEK293T cells (ACE2hi cells). HEK293Tsuggested: KCB Cat# KCB 200744YJ, CVCL_0063We also confirmed the high expression of ACE2 protein in AT2 cells, and found that EOL-1 cells express ACE2 protein for the first time (Figure 1A). EOL-1suggested: NoneCell lines HEK293T cells , HSAEpC cells ( human airway epithelial cells) , AT2 cells ( alveolar type II epithelial cells ) were kept in DMEM high glucose medium containing 10 % FBS and 1 % penicillinstreptomycin ； EOL-1 cells ( human eosinophilic Leukemia cells ) were kept in 1640 medium containing 10 % FBS and 1 % penicillin-streptomycin; ACE2 high expressing HEK293T cells ( ACE2hi cells ) were kept in DMEM high glucose medium containing 10 % FBS , 1 % penicillinstreptomycin,4μg/mL puromycin and cultured at 37°C in a 5 % CO2 incubator . HSAEpCsuggested: None2×103 ACE2 cells were seeded on the coverslip and cultured in a six-well plate. ACE2suggested: CVCL_DR94Software and Algorithms Sentences Resources A Lane 1 DTM transilluminator ( Beijing Creation Science Co . , Ltd. , Beijing , China ) was used to capture the images of the developed blots , and Image-Pro Plus 5.1 software ( Media Cybernetics , Inc . , Rockville , MD , USA ) was used to quantify the protein levels . Image-Pro Plussuggested: (Image-Pro Plus, SCR_007369)Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).
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SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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