Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2

Significance New therapeutic targets are urgently needed against SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic. Results in this study show that targeting the transcriptional regulation of host entry factors TMPRSS2 and ACE2 is a viable treatment strategy to prevent SARS-CoV-2 infection. In particular, inhibitors of androgen receptor (AR) or bromodomain and extraterminal domain (BET) proteins are effective against SARS-CoV-2 infection. AR inhibitors are already approved in the clinic for treatment of prostate cancer and are under investigation in COVID-19 patients; BET inhibitors are also in clinical development for other indications and could be rapidly repurposed for COVID-19.

. Additional single cell analyses of mouse lung from published datasets. Bubble plot of Ace2, Tmprss2, and Ar expression from two additional publicly available scRNAseq datasets of mouse lung (1,2). Color bar represents mean expression of each gene in specific cell types, and bubble size represents the percentage of cells in each cell type that expresses the appropriate gene. Plot is labeled below with an identifier, reflecting the name of the first author of the appropriate manuscript. Figure S3. Relative expression levels of ACE2, TMPRSS2, and AR across cell types in lung and prostate. (A) Violin plot representing relative expression of ACE2, TMPRSS2, and AR in the human lung from four publicly available single cell datasets (2)(3)(4)(5). (B) Violin plot representing relative expression of ACE2, TMPRSS2, and AR in the human prostate from one public single cell dataset (6). (C)Violin plots representing relative expression of Ace2, Tmprss2, and Ar in mouse lung from a publicly available single cell dataset (4). (D) Violin plots representing relative expression of Ace2, Tmprss2, and Ar in mouse prostate from a publicly available single cell dataset (7).  , and Ace2 ISH of murine lungs from male (intact, castrated, castrated + testosterone) and female (intact, intact + testosterone). Increased magnification of the same images for intact and castrated male are shown in Figure 2A, C, and E. (B) Representative images of Tmprss2 ISH in non-bronchial cells of murine lungs from male (intact, castrated, castrated + testosterone) and female (intact, intact + testosterone). (C) Quantitative analysis of Tmprss2 ISH H-score in non-bronchial cells of murine lungs from indicated groups. P values were calculated by unpaired t test. (D) Representative images of ACE2 IHC in non-bronchial cells of murine lungs from male (intact, castrated, castrated + testosterone) and female (intact, intact + testosterone). (E) Quantitative analysis of ACE2 IHC in non-bronchial cells of murine lungs from indicated groups.      Figure 6A.

Immunohistochemistry (IHC) scoring
For AR, the final product score was calculated by multiplying the percentage of immune-positive cells by staining intensity (based on the following criteria: low intensity = score 1, moderate intensity = score 2, high intensity = score 3) with a possible maximum score of 300. For ACE2, the total number of immune-positive cells out of 500 (n/500) in the representative non-bronchial regions (alveolar predominantly) was recorded and expressed as a percentage of positive cells for quantification purposes.

RNA in situ hybridization (RNA-ISH) probes and scoring
Target probes used in RNA-ISH and catalog numbers in parentheses are as follows: Hs-TMPRSS2 previously published scoring criteria (8,9).

Immunoblotting
Cell lysates were harvested in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo

RNA isolation and quantitative real-time PCR (qPCR)
Total RNA was extracted from cells or tissue using the miRNeasy mini kit (Qiagen), and complementary DNA (cDNA) was synthesized from 1 µg total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed using either fast SYBR green or TaqMan master mix (Applied Biosystems) on the ViiA7 Real-Time PCR System (Applied Biosystems). Target mRNA expression was quantified using the ΔΔCt method and normalized to GAPDH. Primer sequences used for SYBR green qPCR were:

JQ1 treatment in mice
Eight-week old male C57BL/6 mice were purchased from the Jackson Laboratory. Six mice were treated with vehicle [10% HPβCD (hydroxypropyl beta cyclodextrin) in D5W], and nine mice were treated with 50 mg/kg JQ1 daily for 5 days by intraperitoneal injection. JQ1 (Cat. 11187, Caymen) was first dissolved in DMSO and then diluted in 10% HPβCD daily prior to injection.
Mice were killed four hours post-last dosing, and lungs were inflated by PBS before tissue collection. Left lungs were fixed for FFPE, and right lungs were snap frozen for RNA extraction.
The University of Michigan (U-M) Institutional Animal Care and Use Committee (IACUC) approved this study.

Murine lung preparation for single cell nuclei sequencing
Eight-week old male C57BL/6 mice were purchased from the Jackson Laboratory. Four mice were surgically castrated and monitored for post-surgical health condition. Twelve days post-castration, four castrated male mice, together with four age-matched intact male mice, were killed and lungs perfused through the right ventricle with 10-20 ml PBS. After perfusion, the entire lung was washed twice with PBS and flash frozen in liquid nitrogen for use immediately or stored at -80 o C for single nuclei isolation. The U-M IACUC approved this study.

Single nucleus RNA sequencing
All buffers were pre-chilled to 4 o C, and all steps were performed on ice or at 4 o C, unless otherwise mentioned. Flash-frozen mouse lung tissues were minced with a fresh, RNase-free scalpel and added to a tube containing chilled nuclei isolation buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.16 % Nonidet™ P40, 0.2 U/µL RNase inhibitor) at a tissue:buffer ratio of 0.1g:100 µL. After incubating at 4 o C with rotation for 10 minutes, tubes were incubated at 4 o C for two minutes to settle debris, and the supernatant containing the nuclei was collected. The supernatant was centrifuged at 500g, 4 o C for five minutes to pellet the nuclei. The supernatant was discarded and the pellet was resuspended in 500 µL resuspension buffer (1x PBS, 0.4 % bovine serum albumin (BSA), 0.2 U/µl RNase inhibitor) and passed through a 70 µm filter. The above step was batch effects with integrate data function before dimension reduction with t-distributed stochastic neighbor embedding (t-SNE) (12). Cells were assigned to clusters using FindClusters function with resolution = 0.5. Known markers for major lung cell types were used to annotate identified cell clusters.

Analysis of expression levels of lung and prostate
To compare ACE2, TMPRSS2, and AR expression levels in lung and prostate, the mean expression of stably expressed genes (SEGs) across cell types and tissues were used as internal reference for each cell. SEGs were based on published gene lists which derived from early human and mouse development scRNA-seq datasets and proved to be more stable than commonly used housekeeping genes at the single cell level (13). To avoid using genes exhibiting low expression and low stability in the specific datasets we used, genes with SEG index estimated by scMerge (13) lower than the median were removed. Relative expression of ACE2, TMPRSS2, and AR was calculated by subtracting mean expression of selected stably expressed genes. All calculations were based on log-normalized UMI by Seurat.