TMPRSS2 transcriptional inhibition as a therapeutic strategy for
COVID-19
Xinchen Wang, Ph.D.
1*
, Ryan S. Dhindsa, Ph.D.
1,2
, Gundula Povysil, M.D., Ph.D.
1
, Anthony
Zoghbi, M.D.
1,3,4
, Joshua E. Motelow, M.D., Ph.D.
1,5
, Joseph A. Hostyk, B.Sc.
1
, Nicholas
Nickols, M.D. Ph.D.
6,7
, Matthew Rettig, M.D.
8,9
, David B. Goldstein, Ph.D.
1,2*
1
Institute for Genomic Medicine, Columbia University Irving Medical Center, New York,
2
Department of Genetics & Development, Columbia University Irving Medical Center, New York,
3
Department of Psychiatry, Columbia University Irving Medical Center, New York
4
New York State Psychiatric Institute, New York
5
Division of Pediatric Critical Care, Department of Pediatrics, New York-Presbyterian Morgan Stanley
Children’s Hospital, Columbia University Irving Medical Center, New York
6
Department of Radiation Oncology, University of California, Los Angeles, Los Angeles
7
Department of Radiation Oncology, Veteran Affairs Greater Los Angeles Healthcare System, Los
Angeles, California
8
Division of Hematology and Oncology, David Geffen School of Medicine, University of California, Los
Angeles, Los Angeles
9
Division of Hematology and Oncology, VA Greater Los Angeles Healthcare System, Los Angeles, Los
Angeles, California
*To whom correspondence should be addressed: xw2553@cumc.columbia.edu (X.W.),
dg2875@cumc.columbia.edu (D.B.G.)
Abstract
There is an urgent need to identify effective therapies for COVID-19. The SARS-CoV-2 host
factor protease TMPRSS2 is required for viral entry and thus an attractive target for therapeutic
intervention. In mouse, knockout of tmprss2 led to protection against SARS-CoV-1 with no
deleterious phenotypes, and in human populations genetic loss of TMPRSS2 does not appear
to be selected against. Here, we mined publicly available gene expression data to identify
several compounds that down-regulate TMPRSS2. Recognizing the need for immediately
available treatment options, we focused on FDA-approved drugs. We found 20 independent
studies that implicate estrogenic and androgenic compounds as transcriptional modulators
of TMPRSS2, suggesting these classes of drugs may be promising therapeutic candidates for
clinical testing and observational studies of COVID-19. We also note that expression of
TMPRSS2 is highly variable and skewed in humans, with a minority of individuals having
extremely high expression. Combined with literature showing that inhibition of TMPRSS2
protease activity reduces SARS-CoV-2 viral entry in human cells, our results raise the
hypothesis that modulation of TMPRSS2 expression is a promising therapeutic avenue for
COVID-19.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 April 2020 doi:10.20944/preprints202003.0360.v2
© 2020 by the author(s). Distributed under a Creative Commons CC BY license.
Introduction
The rapid international spread of the novel pathogenic severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), which causes the disease known as COVID-19, poses a global
health emergency. As of April 5, 2020, there have been over 1,133,000 confirmed cases and
62,500 deaths worldwide
1
. The clinical presentation of COVID-19 ranges from mild respiratory
symptoms to severe progressive pneumonia, multiorgan failure, and death
2
. Therapeutic
interventions beyond supportive care in the literature have included oseltamivir, remdesivir,
ganciclovir, α-interferon, hydroxychloroquine and lopinavir
2-7
. Lopinavir, a protease inhibitor, is
the only drug with a completed clinical trial but failed to shorten time to improvement or viral
shedding. Any effective intervention rapidly mobilized to the frontlines could profoundly impact
resource allocation
8
. Effective treatments are therefore vital to handle the surge of COVID-19
infections.
SARS-CoV-2 host factors are attractive targets for therapeutic intervention. The SARS-CoV-2
spike (S) glycoprotein binds the angiotensin-converting enzyme 2 (ACE2), allowing the viral
particle to enter host cells
9
. Viral entry into host cells also requires cleavage of the viral S protein
by host proteases; this cleavage results in irreversible conformational changes to the S protein
that allow the virus and host cell membranes to fuse
9
. S protein cleavage, called priming, can
use the host serine protease TMPRSS2 or the cysteine proteases cathepsin B or L (CatB/L)
10-14
.
A recent single-cell RNA-sequencing study of human and non-human primate tissues revealed
three major cell types that co-express TMPRSS2 and ACE2: type II pneumocytes in the lung,
absorptive enterocytes in the terminal ileum, and nasal goblet secretory cells
15
.
Computational and in vitro screens are useful to identify compounds that either act
directly against viral proteins, or that disrupt protein interactions between SARS-CoV-2 and host
proteins required for its viral life cycle. Here we propose and develop a complementary
approach seeking to identify transcriptional regulators of the host proteins most critical to viral
entry and replication within host cells. Given the aggressiveness of this pandemic and the
urgency of deploying effective treatments, our first efforts focus on the repurposing of existing
drugs as an attractive alternative to novel compound discovery. We note, however, that this
screening approach could also be applied to the discovery of new chemical entities with more
desirable properties than already available approved medicines.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 April 2020 doi:10.20944/preprints202003.0360.v2
Several lines of evidence point to the host protease TMPRSS2 that the virus employs to
gain entry into lung epithelium as a promising target for pharmacologic targeting. Pharmacologic
inhibition of TMPRSS2 prevents SARS-CoV-2 entry into cultured human lung cells
11
, and work
in mouse also supports loss of tmprss2 as being protective against SARS-CoV-1 (details in
Supplementary Materials)
16
. Furthermore, loss-of-function of TMPRSS2 is not strongly selected
against in human populations (Supplementary Fig. 1)
17
. In contrast, for different reasons
neither ACE2 nor CatB/L appear as strong candidates for therapeutic intervention based on
transcriptional inhibition (details in Supplementary Materials). For these reasons, we focus on
transcriptional modulation of TMPRSS2 as the highest priority, but have generated results for
other critical host proteins using the same framework, in particular the other entry proteins and a
broader set of proteins that interact with viral proteins
18
. The initial identification of these
compounds highlights potential therapeutic targets and pathways that could be pursued by drug
repurposing for the amelioratation of COVID-19 symptoms.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 April 2020 doi:10.20944/preprints202003.0360.v2
Results
Literature-wide screen for transcriptional inhibitors of SARS-CoV-2 host factors reveals
drug repurposing candidates
To identify compounds that transcriptionally inhibit host factors required for SARS-CoV-2 viral
entry, we performed a literature-wide screen of RNA-seq datasets in the NCBI Sequence Read
Archive (SRA) that incorporated keywords relating to drug treatments. Of 252,877 human RNA-
seq datasets in the SRA that were uniformly mapped by the Skymap project
19
, we identified
29,550 samples in 1,222 studies that involved a drug treatment (Methods). Within each study,
we manually assigned samples as case or control for each comparison group based on sample
descriptions and literature reviews, yielding 3,089 distinct case-control comparisons. We used
these comparisons to create a database of differentially expressed genes under various drug
treatments.
SARS-CoV-2 host factors for viral entry
NCBI SRA database
3,000+ treatment comparisons
RNA-seq, entire transcriptome
Connectivity Map
6,000+ compounds
1000 Landmark genes
t-SNE1
t-SNE2
-30
-30
-20
-20
-10
-10
0
0
10
10
20
20
30
30
scRNA-seq drug signatures
188 compounds
scRNA-seq, entire transcriptome
Computational screen for transcriptional inhibitors
Host factor gene
expression
0
5
10
15
20
Control Drug
treatment
Candidate transcriptional inhibitors
ACE2
TMPRSS2
Cathepsin B/L
Fig. 1: Overview of repurposing approach to iden tify transcriptional inhibitors of SARS-CoV -2
host factors
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 April 2020 doi:10.20944/preprints202003.0360.v2
Identification of TMPRSS2 transcriptional inhibitors
Next, we queried this database to identify drug treatments that led to significant differential
expression of SARS-CoV-2 host factor genes. We first focused on TMPRSS2, given its promise
as a candidate for therapeutic intervention. At a Bonferroni-corrected p-value of 0.05 (raw p <
2.06 x 10
-5
), we identified 32 treatment conditions that led to significant down-regulation of
TMPRSS2 in human cell lines, and 76 conditions that led to up-regulation. While the drugs that
down-regulate TMRPSS2 may be useful as anti-viral targets, the drugs that up-regulate are also
important to recognize because they may exacerbate viral infection. Notably, 12 of 32 drug
treatments that significantly down-regulated TMPRSS2 (seven independent studies) and 24 of
76 treatments that led to TMPRSS2 up-regulation (15 independent studies) involved estrogens,
androgens, or agonists or antagonists or their receptors. These results are consistent with
studies showing that TMPRSS2 is regulated by androgens
20
. Specifically, treatment with
estradiol (longer than 3 hours, see Supplementary Fig. 2 for time-course data), genistein (a
phytoestrogen that modulates ERα and ERβ), and MDV3100/enzalutamide (an androgen
receptor antagonist commonly used in advanced prostate cancer) led to statistically significant
down-regulation of TMPRSS2 between 1.6-fold and 14-fold, depending on experimental
conditions, cell lines and choice of controls (example in Fig. 2, data in Supplementary Table
1). Accordingly, we also replicated the enzalutamide signal in the LAPC4 prostate cancer cell
line. (Fig. 2). Conversely, TMPRSS2 expression increased between 1.4-fold and 20-fold
following treatment with androgens (e.g. testosterone, any duration of treatment, see
Supplementary Fig. 2), synthetic androgens (R1881/Metribolone) or short-term exposure to
estradiol (under 3 hours, see Supplementary Fig. 2). In addition to estrogen and androgen-
related compounds, other treatments that decrease TMPRSS2 expression include: dual TGFβ
and EGF treatment (347-fold decrease in expression in HeLa cells, single study only), and
chaetocin (non-specific histone lysine methyltransferase inhibitor, ~4-fold decrease).
To identify more compounds that modulate TMPRSS2 expression, we considered data
from the Connectivity Map
21
, which includes an unbiased screen of compounds in multiple cell
types followed by expression profiling of 978 landmark genes (L1000 platform) and statistical
imputation for the rest of the transcriptome. However, TMPRSS2 was not well-imputed by the
Connectivity Map (self-correlation = 0.56 between RNA-seq expression and imputed
expression). Consistent with a recent preprint on the lack of reproducibility for L1000-based
gene imputation values
22
, we did not observe a significant difference in TMPRSS2 expression
following estradiol treatment in the breast or prostate cancer cells used in the RNA-seq studies
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 April 2020 doi:10.20944/preprints202003.0360.v2
