Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide

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1. Version published to 10.1038/s41467-021-21171-x

   Feb 8, 2021
2. 

   ScreenIT

   Sep 16, 2020

   SciScore for 10.1101/2020.09.11.293035: (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 primary antibodies included anti-β-Actin (Sigma-Aldrich, A3854, 1:5,000), anti-AR (Abcam, ab108341, 1:2,000), anti-TMPRSS2 (Abcam, ab92323, 1:1,000), and anti-ACE2 (Proteintech, 21115-1-AP, 1:1000) antibodies.	anti-β-Actin suggested: None A3854 suggested: (Sigma-Aldrich Cat# A3854, RRID:AB_262011) anti-ACE2 suggested: (Proteintech Cat# 21115-1-AP, RRID:AB_10732845) 21115-1-AP suggested: (Proteintech Cat# 21115-1-AP, RRID:AB_10732845)
   The following primary …

   SciScore for 10.1101/2020.09.11.293035: (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 primary antibodies included anti-β-Actin (Sigma-Aldrich, A3854, 1:5,000), anti-AR (Abcam, ab108341, 1:2,000), anti-TMPRSS2 (Abcam, ab92323, 1:1,000), and anti-ACE2 (Proteintech, 21115-1-AP, 1:1000) antibodies.	anti-β-Actin suggested: None A3854 suggested: (Sigma-Aldrich Cat# A3854, RRID:AB_262011) anti-ACE2 suggested: (Proteintech Cat# 21115-1-AP, RRID:AB_10732845) 21115-1-AP suggested: (Proteintech Cat# 21115-1-AP, RRID:AB_10732845)
   The following primary antibodies were used: anti-FLAG (Abmart, M20008F, 1:250) and anti-AR (Abcam, ab108341, 1:200).	anti-FLAG suggested: (Advanced Targeting Systems Cat# IT-59-250, RRID:AB_10971726) M20008F suggested: None anti-AR suggested: (RayBiotech Cat# 130-10082-200, RRID:AB_11219819)
   The monoclonal antibody against the RBD domain of SARS-CoV-2 S protein (anti-S) was cloned from a convalescent SARS-CoV-2 individual and produced by transiently transfecting HEK293F cells.	anti-S suggested: None
   The following primary antibodies were used: anti-SPC (Sigma-Aldrich, ab3786, 1:200), anti-AR (Abcam, ab108341, 1:200), and anti-TMPRSS2 (Abcam, ab92323, 1:250).	anti-SPC suggested: None ab3786 suggested: (Millipore Cat# AB3786, RRID:AB_91588) anti-TMPRSS2 suggested: (Abcam Cat# ab92323, RRID:AB_10585592)
   Experimental Models: Cell Lines
   Sentences	Resources
   The monoclonal antibody against the RBD domain of SARS-CoV-2 S protein (anti-S) was cloned from a convalescent SARS-CoV-2 individual and produced by transiently transfecting HEK293F cells.	HEK293F suggested: RRID:CVCL_6642)
   Pseudovirus production: SARS-CoV-2 pseudovirus was produced by cotransfection of 293T cells with pNL4-3.luc.RE and PCDNA3.1 encoding the SARS-CoV-2 S protein using Vigofect transfection reagent (Vigorous Biotechnology, T001).	293T suggested: None
   SARS-CoV-2 were propagated in Vero cells.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   R package Diffbind was used to identify overlapped peaks between AR ChIP-seq-generated peaks in LNCaP cells and ATAC-seq-generated peaks in all four cell lines, respectively.	LNCaP suggested: CLS Cat# 300265/p761_LNCaP, RRID:CVCL_0395)
   We next performed GSEA to determine whether hallmark androgen response genes show significantly differences between AR-positive prostate cancer cells (LNCaP, VCaP and 22RV1) and AR-positive lung cancer cells (A549, H1437 and H2126)44.	A549 suggested: None
   Software and Algorithms
   Sentences	Resources
   Sequences of primers are listed as followed: ChIP-seq library preparation: ChIP-seq was performed as previously described36.	ChIP-seq suggested: (ChIP-seq, RRID:SCR_001237)
   DeepTools was further applied for heatmap visualization with the function of computeMatrix and plotHeatmap.	DeepTools suggested: (Deeptools, RRID:SCR_016366) plotHeatmap suggested: None
   In brief, after raw reads were trimmed with TrimGalore-0.5.0, Bowtie2 was used for mapping the reads to the hg19 genome37.	Bowtie2 suggested: (Bowtie 2, RRID:SCR_016368)
   SAMtools was further utilized for bam file sorting and indexing.	SAMtools suggested: (SAMTOOLS, RRID:SCR_002105)
   R package Diffbind was used to identify overlapped peaks between AR ChIP-seq-generated peaks in LNCaP cells and ATAC-seq-generated peaks in all four cell lines, respectively.	Diffbind suggested: (DiffBind, RRID:SCR_012918)
   To further identify specific prostate-open peaks, we employed the intersect function in bedtools 41 to exclude peaks that emerged in any of the three lung cell lines in LNCaP cells.	bedtools suggested: (BEDTools, RRID:SCR_006646)
   Statistical analysis: Two-tailed Student’s t-tests were performed in GraphPad Prism 7 to compare differences between two groups.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   However, our study had limitations. Microenvironmental components, including immune cells, nerve cells and stromal cells, are involved in viral infection and subsequent replication33–35. Although we employed Ad-ACE2-transduced in vivo mouse models, our models did not consider the human lung microenvironment. Since stromal cells in multiple human organs, including the lungs, are also characterized by AR expression, we cannot exclude the possibility that enzalutamide might display antiviral activity by altering the expression of some essential cytokines or chemokines in stromal cells. It was also noting that SARS-CoV-2 could still infect the lungs of Tmprss2-KO mice with lower effectivity. Besides, when transduced with Ad-ACE2, both TMPRSS2-negative prostate cells PC3 (data not shown) and lung cells H23 were permissive for robust SARS-CoV-2-driven entry (Extended Data Fig. 5k). These findings implied that besides TMPRSS2, other factors may also play a crucial role in promoting SARS-CoV-2 infection. Further studies to identify these factors and their precise functions in mediating SARS-CoV-2 infection will be really necessary. Finally, we took advantage of multiple models of human prostate and lung cells, patients-derived benign lung organoids and Ad-ACE2-transduced Tmprss2-KO and WT mice to comprehensively confirm the pivotal function of TMPRSS2 in SARS-CoV-2 infection. Our findings validated that enzalutamide significantly inhibits SARS-CoV-2 infection in AR and TMPRSS2 double...

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04475601	Recruiting	Enzalutamide Treatment in COVID-19
   NCT04456049	Not yet recruiting	Randomized Open Label Phase II Study to Evaluate the Efficac…

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 16, 11 and 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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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   Read the original source
3. 

   Version published to 10.1101/2020.09.11.293035v1 on bioRxiv

   Sep 12, 2020